Search PubMed⌕ Search

Biomedical subjects

A F Miranda

Publications and source records attributed to A F Miranda.

At least 37 records · Page 2Linked to original sources
PubMed1995

Myoblast fusion and innervation with rat motor nerve alter distribution of acetylcholinesterase and its mRNA in cultures of human muscle.

To elucidate the mechanisms underlying acetylcholinesterase (AChE) localization, we analyzed the distribution of AChE and Ache mRNA during myogenesis in cocultures of human muscle and fetal rat spinal cord. We observed a temporal coincidence in alterations of AChE localization and nuclei expressing the message, suggesting developmental regulation at the mRNA level. Nonuniform mRNA staining among nuclei suggests asynchronous regulation, also supporting an earlier proposal that transcription proceeds intermittently. Asynchrony seems to be overridden by generally acting factors during myoblast fusion, when message is up-regulated, and at the onset of muscle contractions, when it becomes restricted to some nuclei in the junctional region and focal patches of AChE appear near nerve contacts. Coincidence of mRNA down-regulation and synthesis of stable basal lamina-bound AChE suggests coordinated adaptation, so that sufficient enzyme may be derived from low message levels.

Acetylcholinesterase↗
PubMed1994

Evidence for embryonic peroxidase-catalyzed bioactivation and glutathione-dependent cytoprotection in phenytoin teratogenicity: modulation by eicosatetraynoic acid and buthionine sulfoximine in murine embryo culture.

Phenytoin teratogenicity may result from embryonic, peroxidase-catalyzed bioactivation of phenytoin to a toxic reactive free radical intermediate for which embryonic glutathione (GSH) is cytoprotective. This hypothesis was tested in embryo culture using 5,8,11,14-eicosatetraynoic acid (ETYA), a dual inhibitor of two peroxidase systems, prostaglandin synthetase, and lipoxygenases. Embryos from CD-1 mice were explanted on Gestational Day 9.5 (vaginal plug, Day 1) and incubated for 24 hr at 37 degrees C in culture medium (35% male rat serum, 15% fetal bovine serum, and 50% Waymouth's medium) saturated with 5% CO2 in air. Initially, a nonembryotoxic concentration of ETYA (0,40,80, or 100 microM) was established within its peroxidase inhibitory range (Ki = 4-8 microM). Subsequently, embryos were incubated with vehicle alone, a therapeutic concentration of phenytoin alone (20 micrograms/ml or 80 microM), ETYA alone (40 microM), or phenytoin and ETYA combined. ETYA alone below 100 microM had no effect on yolk sac diameter (YSD), crown-rump length (CRL), somite development (SD), anterior neuropore closure (ANPC), or turning, but at 100 microM reduced CRL, YSD, and SD (p < or = 0.05). Phenytoin alone was embryotoxic, causing reduced CRL, YSD, and SD (p < or = 0.0001). Phenytoin and ETYA (40 microM) together resulted in an increase in YSD, SD, and CRL relative to those with phenytoin alone (p < or = 0.01), indicating that inhibition by ETYA of embryonic, peroxidase-catalyzed bioactivation of phenytoin is cytoprotective. GSH may play a critical role in detoxifying a phenytoin free radical or subsequent activated oxygen species, thereby reducing covalent binding, lipid peroxidation, and oxidative stress that may initiate embryotoxicity or death. To test this hypothesis, embryos were cultured in the presence or absence of 1 mM buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, for 3 hr, at which time BSO was washed out and the embryos were incubated for 24 hr in fresh culture medium containing 80 microM phenytoin or its vehicle. Soluble thiols, including GSH, and disulfides, including oxidized GSH (GSSG), were measured using high-performance liquid chromatography. Immediately after BSO treatment, there were no differences in the concentrations of GSH or GSSG between BSO-exposed embryos and controls. However, at 24 hr, GSH concentrations in untreated embryos increased almost 17-fold over those at 3 hr concentrations, while GSH in BSO-exposed embryos were reduced to 15% of control values (p = 0.0008).(ABSTRACT TRUNCATED AT 400 WORDS)

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1994

Innervation of MyoD-converted human amniocytes and fibroblasts by fetal rodent spinal cord neurons.

MyoD is one member of a gene family involved in the regulation of myogenesis. MyoD transfection induces myogenesis in a variety of non-muscle cells. Human amniocytes and fibroblasts were infected with a MyoD-retrovirus vector, to determine whether the converted cells can mature normally to form functional muscle fibers. MyoD-converted cells were cocultured with fetal rat spinal cord. After 2-3 weeks of co-culture cross-striated, contracting muscle fibers were observed. Combined acetylcholinesterase cytochemistry and acetylcholine receptor labeling showed prominent staining at nerve-muscle contacts. Approximately half of the total creatine kinase activity was due to the muscle-specific isozyme. Innervated MyoD-converted cells might represent a new source of muscle cells for studying the molecular events leading toward the formation of functional muscle. This system also appears suitable for studying the pathogenesis of hereditary, often rare, myopathies affecting muscle-specific proteins, for which muscle tissue is frequently unavailable for in vitro analysis.

Adult↗
PubMed1993

Analysis of dystrophin expression after activation of myogenesis in amniocytes, chorionic-villus cells, and fibroblasts. A new method for diagnosing Duchenne's muscular dystrophy.

BACKGROUND: DNA analysis of peripheral-blood leukocytes is routinely used to demonstrate mutations in the dystrophin gene in patients with Duchenne's or Becker's muscular dystrophy. In approximately 35 percent of patients, DNA studies are not informative; in these patients immunochemical analysis of a muscle-biopsy specimen can determine whether dystrophin, the protein product of the gene for Duchenne's dystrophy, is present at reduced levels or absent. DNA analysis can be performed in amniocytes or chorionic-villus cells to identify mutations of the dystrophic gene prenatally, but immunochemical testing for dystrophin cannot be performed because the protein is not expressed in these cells. METHODS: To circumvent this limitation in prenatal diagnosis, we induced myogenesis in 21 cultures of skin fibroblasts, 49 amniocyte cultures, and 6 chorionic-villus cell cultures by infecting the cells with a retrovirus vector containing MyoD, a gene regulating myogenesis. Transfection of MyoD into cells that do not normally develop into muscle cells results in the production of a protein that switches on myogenesis. We performed immunocytochemical analysis for dystrophin in the MyoD-converted muscle cells. RESULTS: We found that 60 of 61 myotube cultures from subjects with no family history of Duchenne's dystrophy expressed dystrophin. Both myotube cultures from the two patients with Becker's dystrophy also expressed dystrophin, but all cultures from nine patients and two fetuses with Duchenne's dystrophy were dystrophin-deficient. CONCLUSIONS: Immunocytochemical analysis of dystrophin in genetically altered non-muscle cells is feasible and may be applicable to the prenatal and postnatal diagnosis of Duchenne's muscular dystrophy when conventional DNA analysis is not informative.

Amniotic Fluid↗
PubMed1993

Phosphoglycerate kinase deficiency: biochemical and molecular genetic studies in a new myopathic variant (PGK Alberta)

Biochemical analysis of muscle in a 37-year-old man with exercise intolerance, myalgia, recurrent myoglobinuria, and retinitis pigmentosa showed phosphoglycerate kinase (PGK) deficiency. Kinetic and physical characteristics of the mutant enzyme differed from those of two previously reported cases, suggesting a distinct mutation. Southern blot analysis showed similar bands in patient and control, but Northern blot analysis of muscle mRNA showed an abnormally large message. These data demonstrate that PGK deficiency is clinically, biochemically, and genetically heterogeneous.

Adult↗
PubMed1992

Structural and functional mitochondrial abnormalities associated with high levels of partially deleted mitochondrial DNAs in somatic cell hybrids.

Kearns-Sayre syndrome (KSS) is a progressive and ultimately fatal human encephalomyopathy that is associated with large-scale deletions of mitochondrial DNA (mtDNA). To gain new insights into the developmental pathobiology of this disease, we studied the maintenance and expression of deleted mtDNAs (delta-mtDNAs) in somatic cell hybrids generated by fusion of HeLacot cells with a KSS fibroblast clone containing both wild-type and delta-mtDNAs. We observed that delta-mtDNAs were preferentially maintained over the KSS wild-type mtDNAs (wt-mtDNAs) in almost all isolated hybrid clones. Mitochondrial metabolism was not compromised in hybrids containing as much as 70-79% delta-mtDNAs. Two clones containing more than 99% delta-mtDNA were severely deficient in oxidative phosphorylation and exhibited abnormal, enlarged mitochondria. These clones had undetectable levels of mtDNA-encoded polypeptides, but contained normal amounts of a nuclear DNA-encoded mitochondrial protein. The data suggest a nonrandom pattern of mtDNA segregation in the triplasmic hybrids and a correlation among delta-mtDNA, structural mitochondrial abnormalities, and mitochondrial dysfunction.

Cell Division↗
PubMed1992

Propofol and methohexitone for elective caesarean--a comparative study.

Two identical groups of females underwent caesarean operations. One group was induced with propofol 2.04 (SD 0.023) mg per kilogram and the other group induced with methohexitone 1.05 (SD 0.15) mg per kilogram body weight. Maintenance of anaesthesia was identical in both groups. Post-intubation blood pressure in the methohexitone group was significantly raised whereas with propofol the changes were not significant. There were no significant differences in the Apgar scores, uterine contractility and umbilical venous or arterial blood gases. There was a significant difference in the analgesic requirement in the first hour of the post-operative period; in the propofol group, patients needed less analgesia compared to the methohexitone group. There was no maternal awareness in both groups.

Adult↗
PubMed1991

Differential diagnosis of fatal and benign cytochrome c oxidase-deficient myopathies of infancy: an immunohistochemical approach.

To differentiate the 2 major myopathies of infancy due to cytochrome c oxidase (COX) deficiency, we studied muscle biopsies from 4 patients with fatal myopathy and 4 with benign myopathy using biochemical, histochemical, and immunohistochemical techniques. Immunohistochemistry with antibodies directed against individual subunits of COX differentiated the 2 phenotypes: the fatal infantile myopathy was characterized by absence of the nuclear DNA (nDNA)-encoded subunit VIIa,b of COX, while in the benign myopathy both VIIa,b and the mitochondrial DNA (mtDNA)-encoded subunit II were absent. Early differential diagnosis between fatal and benign COX-deficient myopathies is of critical importance for prognosis and management of these infants, because the benign form is initially life-threatening but ultimately reversible.

Biopsy↗
PubMed1991

Mitochondrial encephalomyopathies: biochemical approach.

Thanks to recent advances in the molecular genetics of mitochondrial encephalomyopathies, we can now begin to correlate genetic lesions with biochemical defects. In the fatal infantile myopathy due to cytochrome c oxidase (COX) deficiency, an autosomal recessive condition, immunocytochemical studies have shown an isolated defect of subunit VIIa, which is 1 of the only 2 tissue-specific subunits of human COX. In muscle biopsies from patients with Kearns-Sayre syndrome, a multisystem disorder characterized by deletions of the mitochondrial DNA (mtDNA), the activities of all mitochondrial enzymes containing mtDNA-encoded subunits are decreased. The results of Northern analysis, in situ hybridization, and immunocytochemistry in muscle, and of mitochondrial protein synthesis in cultured fibroblasts suggest that partially deleted mtDNAs are transcribed but not translated, probably due to lack of indispensable tRNAs.

Brain Diseases↗
PubMed1991

Quality of life and longterm survival after intensive care discharge.

From 1st January 1986 till 31st December 1986; 273 patients were treated in the Intensive Care Ward. The mortality in the Intensive Care Unit was 24.5%, mortality of patients 60 years and above was 35%. Of 187 patients who had survived, only 105 (56.2%) responded to the questionnaire, 39 (20.9%) did not respond and 43 (23.0%) could not be traced. Of the total discharged alive, 95 (51.9%) survived two years and eight (4.6%) died over the two years. Forty (41%) have returned to normal routine and are satisfied with their life style; 57 (59%) were not satisfied with their life style for various reasons, ill health being one. As regards patients above 60 years; 21 (53.8%) are alive and 10 (47.6%) are happy and satisfied with their life style.

Adolescent↗
PubMed1990

Cytochrome c oxidase deficiency.

Cytochrome c oxidase (COX) is a complex enzyme composed of 13 subunits, three of which are encoded by the mitochondrial DNA (mtDNA). The other 10 subunits are encoded by the nuclear DNA, synthesized in the cytoplasm, and transported into the mitochondria. The complexity of the enzyme and its dual genetic control explain the heterogeneity of clinical phenotypes associated with COX deficiency. There are two major syndromes, one characterized by muscle involvement (fatal infantile or benign infantile myopathy), the other dominated by brain disease (Leigh syndrome, myoclonic epilepsy with ragged red fibers, Menkes' disease). Partial defects of COX have been shown in muscle of patients with progressive external ophthalmoplegia, either alone (ocular myopathy) or as part of Kearns-Sayre syndrome. Biochemical studies have documented either muscle-specific or generalized defects of COX; COX deficiency is reversible in the benign infantile myopathy. Immunologically detectable protein may be normal (benign myopathy) or variably decreased (fatal myopathy, Leigh syndrome). The subunit pattern of COX is normal by immunoblot in patients with fatal myopathy and Leigh syndrome; a disproportionate decrease of subunit II was seen in a patient with myoclonic epilepsy with ragged red fibers. Availability of the three mtDNA genes and of complementary DNA probes for eight of the 10 nuclear DNA-encoded subunits makes it possible to investigate the different diseases at the molecular level. Large deletions of mtDNA have been found in patients with ocular myopathy and Kearns-Sayre syndrome: the deleted mtDNA appear to be transcribed but not translated, thus explaining the partial COX deficiency.

Brain Diseases↗
PubMed1990

Widespread tissue distribution of mitochondrial DNA deletions in Kearns-Sayre syndrome.

We performed Southern analysis of mitochondrial DNA (mtDNA) in 6 tissues from a patient with Kearns-Sayre syndrome and found a single deletion of 4.9 kb in all tissues. The percentage of deleted mtDNAs varied widely between tissues, from only 4% in smooth muscle to approximately 50% in skeletal muscle. Samples of DNA obtained from 3 different skeletal muscles and from separate areas of individual tissues showed little variation in percentage of deleted mtDNA. Biochemical analysis showed no clear correlation between mitochondrial enzyme activity and deleted mtDNAs.

Blotting, Southern↗
PubMed1990

Partial dystrophin deficiency in monozygous twin carriers of the Duchenne gene discordant for clinical myopathy.

We studied monozygous twin women, age 63. One, asymptomatic, had a serum creatine kinase (CK) level of 191 units (normal, 1 to 50); her son died of typical Duchenne muscular dystrophy (DMD) at age 18. Her twin sister had symptomatic limb weakness from about age 40. Her serum CK was 495 units. EMG and muscle biopsy were compatible with myopathy. In the asymptomatic twin, the peripheral blood lymphocyte karyotype was 46,XX. In the affected twin, 18% of cells were 45,X, and the others 46,XX, without X/autosome translocation. DNA analysis did not reveal a deletion at the DMD locus. Immunologic studies of dystrophin showed a partial deficiency of the protein that was more severe in the symptomatic twin. The clinical discordance and the different severity of dystrophin deficiency may have resulted from the effects of lyonization.

Creatine Kinase↗
PubMed1990

Controlled ventilation with Brain laryngeal mask.

A Brain laryngeal mask was assessed in fifty patients undergoing general anaesthesia who required controlled ventilation. The mask was inserted in all patients without any difficulty and the satisfactory seal obtained enabled ventilation in all patients in a wide range of positions. Airway obstruction occurred in seven patients secondary to downfolding of the epiglottis and this was rectified by reinsertion. The incidence of sore throat was 10%. The Brain laryngeal mask is a safe alternative to the tracheal tube for controlled ventilation during general anaesthesia.

Adult↗
PubMed1989

Mitochondrial DNA deletions in progressive external ophthalmoplegia and Kearns-Sayre syndrome.

We investigated the correlations of deletions of mitochondrial DNA in skeletal muscle with clinical manifestations of mitochondrial myopathies, a group of disorders defined either by biochemical abnormalities of mitochondria or by morphologic changes causing a ragged red appearance of the muscle fibers histochemically. We performed genomic Southern blot analysis of muscle mitochondrial DNA from 123 patients with different mitochondrial myopathies or encephalomyopathies. Deletions were found in the mitochondrial DNA of 32 patients, all of whom had progressive external ophthalmoplegia. Some patients had only ocular myopathy, whereas others had Kearns-Sayre syndrome, a multisystem disorder characterized by ophthalmoplegia, pigmentary retinopathy, heart block, and cerebellar ataxia. The deletions ranged in size from 1.3 to 7.6 kilobases and were mapped to different sites in the mitochondrial DNA, but an identical 4.9-kilobase deletion was found in the same location in 11 patients. Biochemical analysis showed decreased activities of NADH dehydrogenase, rotenone-sensitive NADH-cytochrome c reductase, succinate-cytochrome c reductase, and cytochrome c oxidase, four enzymes of the mitochondrial respiratory chain containing subunits encoded by mitochondrial DNA. We conclude that deletions of muscle mitochondrial DNA are associated with ophthalmoplegia and may result in impaired mitochondrial function. However, the precise relation between clinical and biochemical phenotypes and deletions remains to be defined.

Blotting, Southern↗