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Carlos T Moraes

Publications and source records attributed to Carlos T Moraes.

12 recordsLinked to original sources
PubMed2004

An ALS mouse model with a permeable blood-brain barrier benefits from systemic cyclosporine A treatment.

To test potentially beneficial drugs to amyotrophic lateral sclerosis (ALS), we created an ALS mouse model with a permeable blood-brain barrier, by crossing the G93A-SOD1 transgenic mouse with a multiple drug resistance type 1a/b (mdr1a/b) gene knockout mouse. To validate the model, we administered cyclosporine A intraperitoneally to the mice. Cyclosporine A accumulated in the brain and spinal cord of this mouse model, whereas it was unable to penetrate the CNS of mdr1a/b wild-type animals. Systemic administration of cyclosporine A extended the life of the double-mutant male mice by approximately 12%. Surprisingly, the effect was more robust in male mice and only marginal in female mice. These results demonstrate the usefulness of this combined mouse model for the testing of potentially therapeutic drugs and support the role of mitochondrial-mediated apoptosis in the pathway to motor neuron death in SOD1-associated ALS.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Decreased mitochondrial tRNALys steady-state levels and aminoacylation are associated with the pathogenic G8313A mitochondrial DNA mutation.

Mutations in human mitochondrial tRNA genes cause a number of multisystemic disorders. A G-to-A transition at position 8313 (G8313A) transition in the mitochondrial tRNALys gene has been associated with a childhood syndrome characterized by gastrointestinal-system involvement and encephaloneuropathy. We have used transmitochondrial cybrid clones harbouring patient-derived mitochondrial DNA with the G8313A mutation for the study of the molecular pathogenesis. Our results showed that mutant mitochondrial cybrids respired poorly, and had severely defective mitochondrial protein synthesis and respiratory-chain-enzyme activity. Mutant cybrids also showed a marked decrease in tRNALys steady-state levels and aminoacylation, suggesting that these molecular abnormalities may underlie the pathogenesis of the mitochondrial G8313A mutation.

Acylation↗
PubMed2003

A chemical enucleation method for the transfer of mitochondrial DNA to rho(o) cells.

The study of pathogenic mitochondrial DNA mutations has, in most cases, relied on the production of transmitochondrial cybrids. Although the procedure to produce such cybrids is well established, it is laborious and cumbersome. Moreover, the mechanical enucleation procedure is inefficient and different techniques have to be used depending on the adherence properties of the cell. To circumvent these difficulties, we developed a chemical enucleation method that can have wide applicability for the production of transmitochondrial cybrids. The method is based on the use of actinomycin D to render the nuclear genome transcription/replication inactive and unable to recover after treatment. Such treated cells are fused to cells devoid of mitochondrial DNA and selected for the presence of a functional oxidative phosphorylation system. Our results showed that 95% of the clones recovered by this procedure are true transmitochondrial cybrids. This method greatly facilitates the production of transmitochondrial cybrids, thereby increasing the number of mtDNA mutations and the recipient cell types that can be studied by this system.

Animals↗
PubMed2003

Regular exercise is beneficial to a mouse model of amyotrophic lateral sclerosis.

We tested whether a regular exercise regimen was associated with a change in the life span of G93A-SOD1 transgenic mice, a model of familial ALS. Regular treadmill running for 10 weeks led to a significant increase in the life span of G93A-SOD1 mice. The effect was stronger in male mice, whereas there was only a trend between exercised and sedentary female G93A-SOD1 mice. The data suggest that regular exercise has a beneficial effect on the progression of ALS.

Amyotrophic Lateral Sclerosis↗
PubMed2003

Techniques and pitfalls in the detection of pathogenic mitochondrial DNA mutations.

Mutations in the mitochondrial DNA (mtDNA) are now recognized as major contributors to human pathologies and possibly to normal aging. A large number of rearrangements and point mutations in protein coding and tRNA genes have been identified in patients with mitochondrial disorders. In this review, we discuss genotype-phenotype correlations in mitochondrial diseases and common techniques used to identify pathogenic mtDNA mutations in human tissues. Although most of these approaches employ standard molecular biology tools, the co-existence of wild-type and mutated mtDNA (mtDNA heteroplasmy) in diseased tissues complicates both the detection and accurate determination of the size of the mutated fractions. To address these problems, novel approaches were developed and are discussed in this review.

DNA Mutational Analysis↗
PubMed2003

Limitations of allotopic expression of mitochondrial genes in mammalian cells.

The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria.

Animals↗
PubMed2002

BCL-2 improves oxidative phosphorylation and modulates adenine nucleotide translocation in mitochondria of cells harboring mutant mtDNA.

Members of the BCL-2-related antiapoptotic family of proteins have been shown previously to regulate ATP/ADP exchange across the mitochondrial membranes and to prevent the loss of coupled mitochondrial respiration during apoptosis. We have found that BCL-2/BCL-x(L) can also improve mitochondrial oxidative phosphorylation in cells harboring pathogenic mutations in mitochondrial tRNA genes. The effect of BCL-2 overexpression in mutated cells was independent from apoptosis and was presumably associated with a modulation of adenine nucleotide exchange between mitochondria and cytosol. These results suggest that BCL-2 can regulate respiratory functions in response to mitochondrial distress by regulating the levels of adenine nucleotides.

Adenine Nucleotides↗
PubMed2002

Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control.

Partially-deleted mitochondrial DNA (DeltamtDNA) accumulates during aging of postmitotic tissues. This accumulation has been linked to decreased metabolic activity, increased reactive oxygen species formation and the aging process. Taking advantage of cell lines with heteroplasmic mtDNA mutations, we showed that, after severe mtDNA depletion, organelles are quickly and predominantly repopulated with DeltamtDNA, whereas repopulation with the wild-type counterpart is slower. This behavior was not observed for full-length genomes with pathogenic point mutations. The faster repopulation of smaller molecules was supported by metabolic labeling of mtDNA with [3H]thymidine during relaxed copy number control conditions. We also showed that hybrid cells containing two defective mtDNA haplotypes tend to retain the smaller one as they adjust their normal mtDNA copy number. Taken together, our results indicate that, under relaxed copy number control, DeltamtDNAs repopulate mitochondria more efficiently than full-length genomes.

Aging↗
PubMed2002

Generation of histocompatible tissues using nuclear transplantation.

Nuclear transplantation (therapeutic cloning) could theoretically provide a limitless source of cells for regenerative therapy. Although the cloned cells would carry the nuclear genome of the patient, the presence of mitochondria inherited from the recipient oocyte raises questions about the histocompatibility of the resulting cells. In this study, we created bioengineered tissues from cardiac, skeletal muscle, and renal cells cloned from adult bovine fibroblasts. Long-term viability was demonstrated after transplantation of the grafts into the nuclear donor animals. Reverse transcription-PCR (RT-PCR) and western blot analysis confirmed that the cloned tissues expressed tissue-specific mRNA and proteins while expressing a different mitochondrial DNA (mtDNA) haplotype. In addition to creating skeletal muscle and cardiac "patches", nuclear transplantation was used to generate functioning renal units that produced urinelike fluid and demonstrated unidirectional secretion and concentration of urea nitrogen and creatinine. Examination of the explanted renal devices revealed formation of organized glomeruli- and tubule-like structures. Delayed-type hypersensitivity (DTH) testing in vivo and Elispot analysis in vitro suggested that there was no rejection response to the cloned renal cells. The ability to generate histocompatible cells using cloning techniques addresses one of the major challenges in transplantation medicine.

Animals↗
PubMed2002

Pattern of organization of human mitochondrial pseudogenes in the nuclear genome.

Mitochondrial pseudogenes in the human nuclear genome have been previously described, mostly as a source of artifacts during the analysis of the mitochondrial genome. With the availability of the complete human genome sequence, we performed a comprehensive analysis of mtDNA insertions into the nucleus. We found 612 independent integrations that are evenly distributed among all chromosomes as well as within each individual chromosome. The identified pseudogenes account for a content of at least 0.016% of the human nuclear DNA. Up to 30% of a chromosome's mtDNA pseudogene content is composed of fragments that encompass two or more adjacent mitochondrial genes, and we found no correlation between the abundance of mitochondrial transcripts and the multiplicity of integrations. These observations indicate that the migrations of mitochondrial DNA sequences to the nucleus were predominantly DNA mediated. Phylogenetic analysis of the mtDNA pseudogenes and mtDNA sequences of primates indicate a continuous transfer into the nucleus. Because of the limited window of opportunity for mtDNA transfer to the germline, sperm mtDNA, which is released from degenerating mitochondria after fertilization, could be an important source of nuclear mtDNA pseudogenes.

Biological Transport↗