Biomedical subjects

A F Miranda

Publications and source records attributed to A F Miranda.

At least 19 recordsLinked to original sources

PubMed2003

Functional innervation of cultured human skeletal muscle proceeds by two modes with regard to agrin effects.

Nerve-derived agrin is a specific isoform of agrin that promotes clustering of nicotinic acetylcholine receptors (AChR) and other components of the neuromuscular junction (NMJ). We investigated the effects of agrin on functional maturation of NMJs at the early stages of synaptogenesis in human muscle. Specifically, we assessed the importance of agrin for the differentiation of developing NMJs to the stage where they are able to transmit signals that result in contractions of myotubes. We utilized an in vitro model in which human myotubes are innervated by neurons extending from spinal cord explants of fetal rat. This model is suitable for functional studies because all muscle contractions are the result of neuromuscular transmission and can be quantitated. An anti-agrin antibody, Agr 33, was applied to co-cultures during de novo NMJ formation. Quantitative analyses demonstrated that Agr 33 reduced the number of AChR clusters to 20% and their long axes to 50% of control, yet still permitted early, NMJ-mediated muscle contractions that are normally observed in 7-10-day-old co-cultures. However, at later times of development, the same treatment completely prevented the increase in the number of contracting units as compared with untreated co-cultures. It is concluded that there are two modes of functional maturation of NMJs with regard to agrin effects: one that is insensitive and the other that is sensitive to agrin blockade. Agrin-insensitive mode is limited to the small population of NMJs that become functional at the earlier stages of functional innervation. However, most of the NMJs become contraction-competent at the later stages of the innervation process. These NMJs become functional only if agrin action is uncompromised. This is the first characterization of the contribution of agrin to NMJ development on human muscle.

Acetylcholine↗

PubMed2001

Differentiation of glial cells and motor neurons during the formation of neuromuscular junctions in cocultures of rat spinal cord explant and human muscle.

Motor axons extending from embryonic rat spinal cord explants form fully mature neuromuscular junctions with cocultured human muscle. This degree of maturation is not observed in muscle innervated by dissociated motor neurons. Glial cells present in the spinal cord explants seem to be, besides remaining interneurons, the major difference between the two culture systems. In light of this observation and the well documented role of glia in neuronal development, it can be hypothesized that differentiated and long-lived neuromuscular junctions form in vitro only if their formation is accompanied by codifferentiation of neuronal and glial cells and if this codifferentiation follows the spatial and temporal pattern observed in vivo. Investigation of this hypothesis necessitates the characterization of neuronal and glial cell development in spinal cord explant-muscle cocultures. No such study has been reported, although these cocultures have been used in numerous studies of neuromuscular junction formation. The aim of this work was therefore to investigate the temporal relationship between neuromuscular junction formation and the differentiation of neuronal and glial cells during the first 3 weeks of coculture, when formation and development of the neuromuscular junction occurs in vitro. The expression of stage-specific markers of neuronal and glial differentiation in these cocultures was characterized by immunocytochemical and biochemical analyses. Differentiation of astrocytes, Schwann cells, and oligodendrocytes proceeded in concert with the differentiation of motor neurons and neuromuscular junction formation. The temporal coincidence between maturation of the neuromuscular junction and lineage progression of neurons and glial cells was similar to that observed in vivo. These findings support the hypothesis that glial cells are a major contributor to maturity of the neuromuscular junction formed in vitro in spinal cord explant-muscle cocultures.

ATP-Binding Cassette Transporters↗

PubMed2001

Mechanisms of cell-mediated myocytotoxicity in the peripheral blood of patients with inflammatory myopathies.

Cell-mediated cytotoxicity is thought to play an important role in the immunopathogenesis of inflammatory myopathies. We determined whether lymphocytes circulating in patient peripheral blood contain automyocytotoxic precursors. Peripheral blood mononuclear cells, sheep red blood cell rosetting (E+) and nonrosetting (E-) cells, were isolated from patients with polymyositis or dermatomyositis and tested for their ability to kill autologous-cultured myotubes derived from diseased muscle biopsies. Patient-derived as well as normal allogeneic mononuclear cells lysed both polymyositis-dermatomyositis and nonrelated myotubes. However, whereas patient E+ cells were preferentially cytotoxic to autologous myotubes, their E-cells did not discriminate autologous from allogeneic targets. Furthermore, cultures of patient E+ cells triggered by phytohemagglutinin and interleukin-2 maintained myocytotoxic potential. In these cultures, virtually all autologous but only 50% of allogeneic killing was mediated by CD3+ T cells. Moreover, autologous cell-mediated killing was abrogated by anti-CD3 monoclonal antibodies. In conclusion, both myocytotoxic CD3+ T-cell clones specific for autologous myotubes, as well as non-T cells, which are nonspecifically myocytotoxic, are present in the peripheral blood of patients with inflammatory myopathies.

Cell Separation↗

PubMed2000

Excitotoxicity of quinolinic acid: modulation by endogenous antagonists.

Quinolinic acid (QUIN), a product of tryptophan metabolism by the kynurenine pathway, produces excitotoxicity by activation of NMDA receptors. Focal injections of QUIN can deplete the biochemical markers for dopaminergic, cholinergic, gabaergic, enkephalinergic and NADPH diaphorase neurons, which differ in their sensitivity to its neurotoxic action. This effect of QUIN differs from that of other NMDA receptor agonists in terms of its dependency on the afferent glutamatergic input and its sensitivity to the receptor antagonists. The enzymatic pathway yielding QUIN produces metabolites that inhibit QUIN-induced neurotoxicity. The most active of these metabolites, kynurenic acid (KYNA), blocks NMDA and non-NMDA receptor activity. Treatment with kynurenine hydroxylase and kynureinase inhibitors increases levels of endogenous KYNA in the brain and protects against QUIN-induced neurotoxicity. Other neuroprotective strategies involve reduction in QUIN synthesis from its immediate precursor, or endogenous synthesis of 7-chloro-kynurenic acid, a NMDA antagonist, from its halogenated precursor. Several other tryptophan metabolites--quinaldic acid, hydroxyquinaldic acid and picolinic acid--also inhibit excitotoxic damage but their presence in the brain is uncertain. Picolinic acid is of interest since it inhibits excitotoxic but not neuroexcitatory responses. The mechanism of its anti-excitotoxic action is unclear but might involve zinc chelation. Neurotoxic actions of QUIN are modulated by nitric oxide (NO). Treatment with inhibitors of NO synthase can augment QUIN toxicity in some models of excitotoxicity suggesting a neuroprotective potential of endogenous NO. In recent studies, certain nitroso compounds which could be NO donors, have been reported to reduce the NMDA receptor-mediated neurotoxicity. The existence of endogenous compounds which inhibit excitotoxicity provides a basis for future development of novel and effective neuroprotectants.

Journal Article↗

PubMed2000

Decreased antioxidant defence in individuals infected by the human immunodeficiency virus.

BACKGROUND: The oxidative stress associated with HIV infection may be important for the progression of the disease because reactive oxygen species activate the nuclear transcription factor NF-kappaB, which is obligatory for HIV replication. PATIENTS AND METHODS: The activities of the antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1) and glutathione peroxidase (GPx, EC 1.11.1.9) of blood plasma and peripheral blood mononuclear cells, as well as the plasma levels of ascorbate, alpha-tocopherol and beta-carotene, were measured in 75 subjects with HIV infection and in 26 controls. The HIV-infected patients were classified according to the Walter Reed Army Institute criteria. RESULTS: The extracellular SOD (EC-SOD) of blood plasma activity was decreased in HIV-infected patients compared to controls, while the SOD activity of mononuclear cells decreased with the HIV-associated disease progression. GPx activities and alpha-tocopherol concentration of HIV-infected patients neither differed as compared to controls nor in relation to disease progression. Lower concentrations of ascorbate and beta-carotene were found in HIV-infected patients than in controls. A positive correlation between CD4 lymphocyte counts and the SOD activities of plasma and mononuclear cells was found. CONCLUSION: These results suggest that abnormalities of antioxidant defence, mainly of SOD activity, are related to the progression of the HIV infection.

Adult↗

PubMed2000

Heparin blocks functional innervation of cultured human muscle by rat motor nerve.

In vitro innervated human muscle is the only experimental model to study synaptogenesis of the neuromuscular junction in humans. Cultured human muscle never contracts spontaneously but will if innervated and therefore is a suitable model to study the effects of specific neural factors on the formation of functional neuromuscular contacts. Here, we tested the hypothesis that nerve derived factor agrin is essential for the formation of functional synapses between human myotubes and motoneurons growing from the explant of embryonic rat spinal cord. Agrin actions were blocked by heparin and the formation of functional neuromuscular contacts was quantitated. At a heparin concentration of 25 microg/ml, the number of functional contacts was significantly reduced. At higher concentrations, formation of such contacts was blocked completely. Except at the highest heparin concentrations (150 microg/ml) neuronal outgrowth was normal indicating that blockade of neuromuscular junction formation was not due to neuronal dysfunction. Our results are in accord with the concept that binding of neural agrin to the synaptic basal lamina is essential for the formation of functional neuromuscular junctions in the human muscle.

Agrin↗

PubMed1999

Long-term analysis of differentiation in human myoblasts repopulated with mitochondria harboring mtDNA mutations.

Short-term analysis of myogenesis in respiration-deficient myoblasts demonstrated that respiratory chain dysfunction impairs muscle differentiation. To investigate long-term consequences of a deficiency in oxidative phosphorylation on myogenesis, we quantitated myoblast fusion and expression of sarcomeric myosin in respiration-deficient myogenic cybrids. We produced viable myoblasts harboring exclusively mtDNA with large-scale deletions by treating wild-type myoblasts with rhodamine 6G and fusing them with cytoplasts homoplasmic for two different mutated mtDNAs. Recovery of growth in transmitochondrial myoblasts demonstrated that respiratory chain function is not required for recovery of rhodamine 6G-treated cells. Both transmitochondrial respiration-deficient cultures exhibited impaired myoblast fusion. Expression of sarcomeric myosin was also delayed in deficient myoblasts. However, 4 weeks after induction of differentiation, one cell line was able to quantitatively recover its capacity to form postmitotic muscle cells. This indicates that while oxidative phosphorylation is an important source of ATP for muscle development, myoblast differentiation can be supported entirely by glycolysis.

Biomarkers↗

PubMed1999

Cellular and molecular studies in muscle and cultures from patients with multiple mitochondrial DNA deletions.

In the last decade, several mitochondrial encephalomyopathies have been pathogenically associated with large-scale mitochondrial DNA deletions that are sporadic, or with point mutations that are maternally inherited. The mutations were also demonstrated in cultures of muscle satellite cells obtained from the patients. Subsequently, multiple deletions in mitochondrial DNA were found in several families. The affected members had progressive external ophthalmoplegia, cataracts and limb weakness, inherited as an autosomal dominant trait, or progressive external ophthalmoplegia with neurogastrointestinal encephalomyopathy or with cardiomyopathy, inherited as an autosomal recessive trait. To better understand the developmental pathobiology and localization of the multiple deletions, we performed comparative molecular genetic studies in muscle and cultures from patients. Whereas multiple deletions were found in muscle fragments from which muscle satellite cells were removed by enzymatic digestion, no deletions were found in the satellite cells or their cultured progeny. Our results suggest that multiple mitochondrial DNA deletions arise as somatic mutations during later stages of muscle development, or in terminally differentiated myofibers.

Adolescent↗

PubMed1999

Control levels of acetylcholinesterase expression in the mammalian skeletal muscle.

Protein expression can be controled at different levels. Understanding acetylcholinesterase (EC. 3.1.1.7, AChE) expression in the living organisms therefore necessitates: (1) determination and mapping of control levels of AChE metabolism; (2) identification of the regulatory factors acting at these levels; and (3) detailed insight into the mechanisms of action of these factors. Here we summarize the results of our studies on the regulation of AChE expression in the mammalian skeletal muscle. Three experimental models were employed: in vitro innervated human muscle, mechanically denervated adult fast rat muscle, and the glucocorticoid treated fast rat muscle. In situ hybridization of AChE mRNA, combined with AChE histochemistry, revealed that different distribution patterns of AChE, observed during in vitro ontogenesis and synaptogenesis of human skeletal muscle, reflect alterations in the distribution of AChE mRNA (Z. Grubic, R. Komel, W.F. Walker, A.F. Miranda, Myoblast fusion and innervation with rat motor nerve alter the distribution of acetylcholinesterase and its mRNA in human muscle cultures, Neuron 14 (1995) 317-327). To study the mechanisms of AChE mRNA loss in denervated adult rat skeletal muscle, we exposed deproteinated AChE mRNA to various subcellular fractions in vitro. Fractions were isolated from the normal and denervated rat sternomastoideus muscle. We found significantly increased, but non-specific AChE mRNA degradation capacities in the three fractions studied, suggesting that increased susceptibility of muscle mRNA to degradation might be at least partly responsible for the decreased AChE mRNA observed under such conditions (K. Zajc-Kreft, S. Kreft, Z. Grubic, Degradation of AChE mRNA in the normal and denervated rat skeletal muscle, Book of Abstracts, The Sixth International Meeting on Cholinesterases, La Jolla, CA, March 20-24, 1998, p. A3.). In adult fast rat muscle, treated chronically with glucocorticoids, we found the fraction of early synthesized AChE molecular forms to be reduced and AChE mRNA unchanged. This observation is consistent with the explanation that translation and/or early post-translational processes are impaired under such conditions (M. Brank, K. Zajc-Kreft, S. Kreft, R. Komel, Z. Grubic, Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid-treated rat skeletal muscle, Eur. J. Biochem. 251 (1998) 374-381). The AChE mRNA level is therefore important but not the only control level of AChE expression in the mammalian skeletal muscle.

Acetylcholinesterase↗

PubMed1999

Quinolinic acid lesion of the nigrostriatal pathway: effect on turning behaviour and protection by elevation of endogenous kynurenic acid in Rattus norvegicus.

Endogenous excitotoxins have been implicated in degeneration of nigral dopaminergic neurons in Parkinson's disease. It may be possible to reduce neurodegeneration by blocking the effects of these endogenous agents. The present study shows that contralateral turning seen following quinolinic acid-induced lesions of the nigrostriatal dopaminergic pathway was reversed by a treatment that increased brain levels of kynurenic acid, an endogenous excitatory amino acid antagonist. The treatment consisted of nicotinylalanine (5.6 nmol/5 microl i.c.v.), an inhibitor of kynureninase and kynurenine hydroxylase plus the precursor kynurenine (450 mg/kg i.p.) plus probenencid (200 mg/kg i.p.), an inhibitor of organic acid transport. Thus, neuroprotection by increasing brain kynurenic acid in vivo may be useful in retarding cell loss in Parkinson's and other neurodegenerative diseases involving excitotoxicity.

Alanine↗

PubMed1999

[The patients' perception during their stay in the intensive care unit].

The purpose of this study is to identify the patients's perception during their stay in the ICU. The sample was composed by ten patients, who had gone to cardiac surgery. They received psychological assistance only after their discharge from the ICU. The data was obtained throw a qualitative approach, using a content analysis. The results suggest that the patients have a stereotyped view about the ICU, linked with the idea of suffering and death; the nurses play an important role during fragility movements, physical and emotional dependence; the pain, by its subjective nature, individually and emotionally, is inevitable, because it is related to procedure and usually it is associated to physical suffering.

Adult↗

PubMed1998

Modulation of striatal quinolinate neurotoxicity by elevation of endogenous brain kynurenic acid.

1. Nicotinylalanine, an inhibitor of kynurenine metabolism, has been shown to elevate brain levels of endogenous kynurenic acid, an excitatory amino acid receptor antagonist. This study examined the potential of nicotinylalanine to influence excitotoxic damage to striatal NADPH diaphorase (NADPH-d) and gamma-aminobutyric acid (GABA)ergic neurones that are selectively lost in Huntington's disease. 2. A unilateral injection of the N-methyl-D-aspartate (NMDA) receptor agonist, quinolinic acid, into the rat striatum produced an 88% depletion of NADPH-d neurones. Intrastriatal infusion of quinolinic acid also produced a dose-dependent reduction in striatal GABA content. 3. Nicotinylalanine (2.3, 3.2, 4.6, 6.4 nmol 5 microl(-1), i.c.v.) administered with L-kynurenine (450 mg kg(-1)), a precursor of kynurenic acid, and probenecid (200 mg kg(-1)), an inhibitor of organic acid transport, 3 h before the injection of quinolinic acid (15 nmol) produced a dose-related attenuation of the quinolinic acid-induced loss of NADPH-d neurones. Nicotinylalanine (5.6 nmol 5 microl(-1)) in combination with L-kynurenine and probenecid also attenuated quinolinic acid-induced reductions in striatal GABA content. 4. Nicotinylalanine (4.6 nmol, i.c.v.), L-kynurenine alone or L-kynurenine administered with probenecid did not attenuate quinolinic acid-induced depletion of striatal NADPH-d neurones. However, combined administration of kynurenine and probenecid did prevent quinolinic acid-induced reductions in ipsilateral striatal GABA content. 5. Injection of nicotinylalanine, at doses (4.6 nmol and 5.6 nmol i.c.v.) which attenuated quinolinic acid-induced striatal neurotoxicity, when combined with L-kynurenine and probenecid produced increases in both whole brain and striatal kynurenic acid levels. Administration of L-kynurenine and probenecid without nicotinylalanine also elevated kynurenic acid, but to a lesser extent. 6. The results of this study demonstrate that nicotinylalanine has the potential to attenuate quinolinic acid-induced striatal neurotoxicity. It is suggested that nicotinylalanine exerts its effect by increasing levels of endogenous kynurenic acid in the brain. The results of this study suggest that agents which influence levels of endogenous excitatory amino acid antagonists such as kynurenic acid may be useful in preventing excitotoxic damage to neurones in the CNS.

Alanine↗

PubMed1998

Multiple mtDNA deletions features in autosomal dominant and recessive diseases suggest distinct pathogeneses.

Multiple mitochondrial DNA (mtDNA) deletions have been described in patients with autosomal dominant progressive external ophthalmoplegia (AD-PEO) and in autosomal recessive disorders including mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and autosomal recessive cardiomyopathy ophthalmoplegia (ARCO). The pathogenic bases of these disorders are unknown. We studied three patients with AD-PEO and three patients with autosomal recessive (AR)-PEO (two patients with MNGIE and one patient with ARCO). Histochemistry and Southern blot analyses of DNA were performed in skeletal muscle from the patients. Muscle mtDNA was used to characterize the pattern and amounts of the multiple mtDNA rearrangements; PCR analysis was performed to obtain finer maps of the deleted regions in both conditions. The patients with AD-PEO had myopathic features; the patients with AR-PEO had multisystem disorders. The percentage of ragged-red and cytochrome c oxidase-negative fibers tended to be higher in muscle from the patients with AD-PEO (19% +/- 13.9, 29.7 +/- 26.3) than in muscle from the patients with AR-PEO (1.4% +/- 1.4, 3.3% +/- 3.2; p < 0.10). The sizes of the multiple mtDNA deletions ranged from approximately 4.0 to 10.0 kilobases in muscle from both groups of patients, and in both groups, we identified only deleted and no duplicated mtDNA molecules. Patients with AD-PEO harbored a greater proportion of deleted mtDNA species in muscle (31% +/- 5.3) than did patients with AR-PEO (9.7% +/- 9.1; p < 0.05). In the patients with AD-PEO, we identified a deletion that included the mtDNA heavy strand promoter (HSP) region, which had been previously described as the HSP deletion. The HSP deletion was not present in the patients with AR-PEO. Our findings show the clinical, histologic, and molecular genetic heterogeneity of these complex disorders. In particular, the proportions of multiple mtDNA deletions were higher in muscle samples from patients with AD-PEO than in those from patients with AR-PEO.

Adult↗

PubMed1997

Mitochondrial mobility in differentiating muscle heterokaryons.

Ragged-red fibers, a morphological hallmark of many patients with mitochondrial encephalomyopathies who harbor mitochondrial DNA (mtDNA) mutations, usually contain varying ratios of mutated and wild-type mtDNAs. Deficient respiratory function in muscle is almost invariably segmental. To investigate whether this observation may be explained by restricted lateral movement of mitochondria within myofibers, we studied the spatial and temporal behavior of two different mitochondrial populations within multinucleate myotubes. We co-cultured normal human and mouse myoblasts, allowed them to fuse into muscle heterokaryons and investigated whether the mitochondria remained segregated, or migrated and intermixed. Human and mouse nuclei were identified by their differential staining pattern with the dye Hoechst 33 258 and mitochondria were distinguished immunologically and by in situ hybridization. Although we observed some territoriality at very early time points after myoblast fusion, there was rapid intermixing of the mitochondrial populations, as early as 48 h after myoblast fusion. We conclude that mitochondria, unlike many other muscle components, lack territorial organization in cultured, differentiating heterokaryons.

Animals↗

PubMed1997

Protection against quinolinic acid-mediated excitotoxicity in nigrostriatal dopaminergic neurons by endogenous kynurenic acid.

Endogenous excitotoxins have been implicated in the degeneration of dopaminergic neurons in the substantia nigra compacta of patients with Parkinson's disease. One such agent quinolinic acid is an endogenous excitatory amino acid receptor agonist. This study examined whether an increased level of endogenous kynurenic acid, an excitatory amino acid receptor antagonist, can protect nigrostriatal dopamine neurons against quinolinic acid-induced excitotoxic damage. Nigral infusion of quinolinic acid (60 nmoles) or N-methyl-D- aspartate (15 nmoles) produced a significant depletion in striatal tyrosine hydroxylase activity, a biochemical marker for dopaminergic neurons. Three hours following the intraventricular infusion of nicotinylalanine (5.6 nmoles), an agent that inhibits kynureninase and kynurenine hydroxylase activity, when combined with kynurenine (450 mg/kg i.p.), the precursor of kynurenic acid, and probenecid (200 mg/kg i.p.), an inhibitor of organic acid transport, the kynurenic acid in the whole brain and substantia nigra was increased 3.3-fold and 1.5-fold respectively when compared to rats that received saline, probenecid and kynurenine. This elevation in endogenous kynurenic acid prevented the quinolinic acid-induced reduction in striatal tyrosine hydroxylase. However, 9 h following the administration of nicotinylalanine with kynurenine and probenecid, a time when whole brain kynurenic acid levels had decreased 12-fold, quinolinic acid injections produced a significant depletion in striatal tyrosine hydroxylase. Intranigral infusion of quinolinic acid in rats that received saline with kynurenine and probenecid resulted in a significant depletion of ipsilateral striatal tyrosine hydroxylase. Administration of nicotinylalanine in combination with kynurenine and probenecid also blocked N-methyl-D-aspartate-induced depletion of tyrosine hydroxylase. Tyrosine hydroxylase immunohistochemical assessment of the substantia nigra confirmed quinolinic acid-induced neuronal cell loss and the ability of nicotinylalanine in combination with kynurenine and probenecid to protect neurons from quinolinic acid-induced toxicity. The present study demonstrates that increases in endogenous kynurenic acid can prevent the loss of nigrostriatal dopaminergic neurons resulting from a focal infusion of quinolinic acid or N-methyl-D-aspartate. The strategy of neuronal protection by increasing the brain kynurenic acid may be useful in retarding cell loss in Parkinson's disease and other neurodegenerative diseases where excitotoxic mechanisms have been implicated.

Alanine↗

PubMed1997

Deficient muscle carnitine transport in primary carnitine deficiency.

Primary carnitine deficiency is associated with deficient blood and tissue carnitine concentrations. The clinical syndrome is dominated by heart and skeletal muscle symptoms, and the clinical response to oral carnitine supplementation is life-saving. Carnitine uptake has been shown to be defective in cultured skin fibroblasts and leukocytes obtained from patients with this condition. We report a new case of primary carnitine deficiency and offer direct evidence consistent with an impairment of carnitine uptake in differentiating muscle culture. The patient presented with severe and progressive cardiomyopathy and moderate proximal limb weakness. Plasma and muscle carnitine levels were very low, and the maximal rate of carnitine transport in cultured fibroblasts was deficient. An asymptomatic sister with intermediate levels of carnitine in plasma showed partially deficient carnitine uptake in fibroblasts, indicating heterozygosity. The patient's condition improved dramatically with oral carnitine therapy. Further studies were performed in cultured muscle cells at different stages of maturation, which demonstrated deficient maximal rates of carnitine uptake. Our findings are consistent with the concept that primary carnitine deficiency is the result of a generalized defect involving carnitine transport across tissue membranes.

Biological Transport↗

PubMed1997

Mitochondrial encephalomyopathy with coenzyme Q10 deficiency.

Coenzyme Q10 (CoQ10) transfers electrons from complexes I and II of the mitochondrial respiratory chain to complex III. There is one published report of human CoQ10 deficiency describing two sisters with encephalopathy, proximal weakness, myoglobinuria, and lactic acidosis. We report a patient who had delayed motor milestones, proximal weakness, premature exertional fatigue, and episodes of exercise-induced pigmenturia. She also developed partial-complex seizures. Serum creatine kinase was approximately four times the upper limit of normal and venous lactate was mildly elevated. Skeletal muscle biopsy revealed many ragged-red fibers, cytochrome c oxidase-deficient fibers, and excess lipid. In isolated muscle mitochondria, impaired oxygen consumption was corrected by the addition of decylubiquinone. During standardized exercise, ventilatory and circulatory responses were compatible with a defect of oxidation-phosphorylation, which was confirmed by near-infrared spectroscopy analysis. Biochemical analysis of muscle extracts revealed decreased activities of complexes I+II and I+III, while CoQ10 concentration was less than 25% of normal. With a brief course of CoQ10 (150 mg daily), the patient reported subjective improvement. The triad of CNS involvement, recurrent myoglobinuria, and ragged-red fibers should alert clinicians to the possibility of CoQ10 deficiency.

Adult↗

PubMed1996

Dihydrorhodamine 123 identifies impaired mitochondrial respiratory chain function in cultured cells harboring mitochondrial DNA mutations.

Several human diseases have been found to be caused by mitochondrial DNA (mtDNA) mutations. Pathogenic mutated (mut) mtDNAs are usually "heteroplasmic," coexisting intracellularly with wild-type (wt) mtDNAs. For some mtDNA mutations, cells have normal levels of respiratory chain function unless the percentage of mut-mtDNA is very high. Although progress in understanding the molecular basis of mitochondrial diseases has been remarkable, the heterogeneity of mut-mtDNA distribution, even among cells of the same tissue, makes it difficult to clearly delineate the relationships between mtDNA mutations, gene dosage, and clinical phenotypes. In a search for screening methods for identifying cultured cells with deficient mitochondrial function, we incubated living cells harboring mut-mtDNAs with dihydrorhodamine 123 (DHR123), an uncharged, nonfluorescent agent that can be converted by oxidation to the fluorescent laser dye rhodamine 123 (R123). Bright mitochondrial staining was observed in cells that respired normally. Fluorescence was significantly reduced in cells with mitochondrial respiratory chain dysfunction resulting from very high levels of mut-mtDNAs. The data show that DHR123 is useful for assessing mitochondrial function in single cells, and can be used for isolating viable, respiratory chain-deficient cells from heterogeneous cultures.

Cell Survival↗