PubMed Health⌕ Search

Biomedical subjects

A Barrientos

Publications and source records attributed to A Barrientos.

At least 55 records · Page 3Linked to original sources
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

Functional glomerular reserve in recipients of en bloc pediatric transplant kidneys.

BACKGROUND: The transplantation of an adequate renal mass is increasingly recognized to be of importance. The improved graft survival is probably due to a lesser risk of developing hyperfiltration-associated lesions. METHODS: We have reviewed the glomerular reserve in our recipients of en bloc pediatric transplant kidneys after an intravenous amino acid overload and compared them to single adult kidney transplant recipients. RESULTS: En bloc transplants evidenced increased glomerular filtration rate as compared with baseline as from the second hour of amino acid infusion (from 71+/-14 to 84.9+/-17 ml/min, 1.73 m2, P<0.05) and increased renal plasma flow as from the third hour (from 335+/-116 to 402+/-155 ml/min, 1.73 m2, P<0.05). In the single adult kidney recipient group, no change was seen either in the glomerular filtration rate (from 62.5+/-13 to 58.1+/-13 ml/min, 1.73 m2, P=NS) nor in renal plasma flow (from 354+/-125 to 304+/-98 ml/min, 1.73 m2, P=NS). CONCLUSIONS: These results show that patients receiving en bloc pediatric kidney transplantations have a greater renal functional reserve and show a lesser risk of hyperfiltration.

Adult↗
PubMed1998

Cytochrome c oxidase assay in minute amounts of human skeletal muscle using single wavelength spectrophotometers.

Determination of cytochrome c oxidase (COX; EC 1.9.3.1) activity in human mitochondria presents several technical difficulties which result in a large intra- and interlaboratory variability, especially when a single wavelength spectrophotometer (SWS) is used, as is generally done in most laboratories in the context of screening procedures for the detection of respiratory chain deficiencies. We studied the experimental conditions of COX assay in human skeletal muscle mitochondria using a SWS in order to define the optimal conditions for the assay and compared these results with those obtained using a double wavelength spectrophotometer (DWS). We demonstrate that a low intra-individual variability of COX assay can be obtained with SWS by: (1) using manual stirrers to avoid the formation of bubbles in the mixture; (2) preincubating mitochondria and laurylmaltoside before the addition of cytochrome c, which prevents light scattering secondary to mitochondrial swelling; and (3) using low amounts (1-2 microg) of mitochondrial protein to extend and linearize the reaction rate. Under these experimental conditions, the concordance between SWS and DWS was very good (R=0.975).

Electron Transport↗
PubMed1998

Biochemical parameters for the diagnosis of mitochondrial respiratory chain deficiency in humans, and their lack of age-related changes.

It is now widely acknowledged that a large number of human diseases originate from respiratory-chain dysfunctions. Because the molecular bases of these diseases are still poorly known, a biochemical approach has to be used in the screening procedures for the diagnoses of these conditions. Assessment of respiratory-chain function in human samples faces several problems: (i) the small size of available samples, (ii) the determination of discriminating parameters, and (iii) the interfering factors, such as age and physical activity. The present study focuses on isolated mitochondria prepared from a minute amount (100-200 mg) of skeletal-muscle biopsies from 201 patients between 0 and 65 years. Whereas 42 patients presented an isolated complex (C)I, CII, CIII or CIV deficiency, no respiratory-chain dysfunction or indirect evidence for a mitochondrial disorder could be attested in 159 of these patients. In this reference group, there was little correlation between enzyme activities and age, whatever the age class considered, 0-3 or 0-65 years of age. However, a confident handling of data points was largely hampered by the marked scattering of enzyme activities measured in the reference population. Activity ratios between the various respiratory-chain complexes presenting a much reduced scattering may be considered as diagnostic tools. As to the effect of age, no correlation with any of the enzyme-activity ratios could be shown. Use of age-matched controls for the diagnosis of respiratory-chain disorders may therefore be avoided, enzyme-activity ratios being highly discriminating and age-independent parameters.

Adolescent↗
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 cytochrome c oxidase inhibition during acute carbon monoxide poisoning.

Clinical symptoms of acute carbon monoxide (CO) poisoning are mainly related to the capability of haemoglobin to bind CO. However, the persistence of some clinical alterations after carboxyhaemoglobin normalization suggests that other heme containing proteins, like cytochrome c oxidase, could play a role in its pathogenesis. We studied mitochondrial enzyme activities of lymphocytes from three patients suffering from acute CO poisoning. HbCO levels were 11.6%. 19.6% and 22.3% in the acute phase, 2.3%, 2.4% and 1.5% on day 3 after admission, and 1.2%, 3.3% and 1.1% on day 12. Complex II, III and glycerol-3-phosphate dehydrogenase activities remained normal along the study, while cytochrome c oxidase (complex IV) activity showed a 76% inhibition compared to controls during acute poisoning (P < 0.01) and 48% at day 3 (P < 0.05). The activity was normal already on day 12 after the complete disappearance of symptomatology. Our results suggest that mitochondrial cytochrome c oxidase is also a target site in human acute CO poisoning, and its extended and generalized inhibition could explain the persistence of different symptoms after the normalization of HbCO levels.

Acute-Phase Proteins↗
PubMed1997

Reduced steady-state levels of mitochondrial RNA and increased mitochondrial DNA amount in human brain with aging.

The contribution of the mitochondrial genetic system in the degenerative processes of senescence remains unclear. This study deals with age-related changes in brain mtDNA expression in humans. Brain tissue from the frontal lobe cortex was obtained from autopsy of 13 humans aged between 21 and 84 years. No structural changes were detected in mtDNA, increased mtDNA content and reduced steady-state level of mitochondrial transcripts and transcription ratio (mtRNA/mtDNA) were associated with aging. These findings suggest that the increase of the mtDNA levels could be considered as an inefficient compensatory mechanism to maintain the normal levels of mtRNA transcripts. This unbalanced mitochondrial condition could play a role in the process of senescence in human brain.

Adult↗
PubMed1997

Determination of hydroxyurea in plasma and peritoneal fluid by high-performance liquid chromatography using electrochemical detection.

A sensitive method has been developed for the determination of hydroxyurea in plasma and peritoneal fluid using reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection. Plasma or peritoneal fluid samples were treated with acetonitrile to precipitate proteins then injected to the HPLC. A C18 analytical column was used to separate hydroxyurea from interfering substances in the biological matrix. The mobile phase, consisting of 0.2 M sodium perchlorate-methanol (95:5, v/v) adjusted to pH 5.0, was delivered isocratically at a flow-rate of 1 ml/min and hydroxyurea was detected using a glassy-carbon electrode operating at an applied potential of +800 mV. Hydroxyurea eluted with a retention time of 3 min. The cycle time for analysis is short and the assay precision is acceptable (C.V. plasma=1.4-3.9%. C.V. peritoneal fluid=2.1-9.7%). The method has been validated and is linear from 25 to 400 ng/ml in plasma and 5 to 30 ng/ml in peritoneal fluid. The method has been shown to be applicable for pharmacokinetic studies.

Antineoplastic Agents↗
PubMed1997

Qualitative and quantitative changes in skeletal muscle mtDNA and expression of mitochondrial-encoded genes in the human aging process.

It has been widely postulated that age-dependent changes in the mitochondrial genetic system may contribute to the human aging process. We recently reported unchanged specific activities of mitochondrial respiratory chain enzymes and a decrease in oxidation capacity of different substrates with aging, due, in part, to some confounding variables such as physical activity or tobacco consumption. The present study deals with age-related changes in muscle mtDNA structure and its biogenesis in humans. We found a low prevalence of mtDNA rearrangements with aging, only detected by PCR. The mtDNA content increased significantly with age (b = 0.0115, P < 0.0001). Also, an unchanged steady-state level of mitochondrial transcripts, a reduced transcription rate (P < 0.0001), and an increase in mitochondrial membrane lipid peroxidation (P < 0.0001) were observed in aging. These data demonstrate that minor structural mtDNA changes appear during the human aging process. By contrast, alterations in mitochondrial homeostasis ultimately producing modifications in mitochondrial biogenesis rates could play a role in the process of human senescence.

Adult↗
PubMed1997

Respiratory chain activity and mitochondrial DNA content of nonpurified and purified pancreatic islet cells.

Considerable interest has recently focused on the possible role of alterations in mitochondrial activity and mutations in the mitochondrial genome for the development of non-insulin-dependent diabetes. Our study aimed at investigating the normal mitochondrial respiratory chain activity of nonpurified and purified islet cells to further explore whether some diabetic states are associated with alterations of mitochondrial oxidative processes. For this purpose, pancreatic islets were isolated from Wistar rats. Unpurified islet cells were obtained in the presence of trypsin and DNAse, and purified beta and non-beta cells were prepared by autofluorescence-activated sorting using a flowcytometer. Intact cell respiration and substrate oxidation in digitonin-permeabilized cells were measured polarographically with a Clark oxygen electrode in a micro-water-jacketed cell. Specific activity of the individual complexes of the respiratory chain was determined spectrophotometrically in unpurified islet cells. The relative amount of mitochondrial (mtDNA) and nuclear (nDNA) DNA in all three cell populations and in rat brain and skeletal muscle was estimated by dot blotting. The intact cell respiration of unpurified islet cells corresponds to the mean of values obtained for beta and non-beta islet cells. Oxidation rates of different substrates by permeabilized beta cells were lower than those for unpurified and non-beta cells. The amount of mtDNA relative to nDNA was similar in all three groups of cells, and was also similar to that obtained from brain and skeletal muscle. In summary, we have described mitochondrial respiratory chain activity in unpurified, beta, and non-beta islet cells. Our results represent an initial step in investigating the potential pathogenic role that alterations in oxidative phosphorylation could play in some diabetic states.

Animals↗
PubMed1997

Cardiovascular effects of recombinant human erythropoietin in predialysis patients.

Treatment with recombinant human erythropoietin (rHuEPO) has solved the problem of anemia in patients on dialysis. However, its application to predialysis patients has raised some doubts about its effects on the progression of renal disease and on blood pressure (BP) and hemodynamic regulation. We have prospectively studied over at least 6 months a group of 11 predialysis patients receiving rHuEPO treatment (initial dose, 1,000 U subcutaneously three times a week). Clinical assessment and biochemical and hematologic measurements were made once every 2 weeks. Twenty-four-hour ambulatory BP monitoring, echocardiography, and determination of neurohumoral mediators of hemodynamics were performed once every 3 months. An adequate hematologic response was found (hemoglobin, 11.7 +/- 0.4 g/dL v 9 +/- 0.3 g/dL) without changes in the progression of renal disease. A decrease in cardiac output and an increase in total peripheral resistance was seen as anemia improved. A trend toward decreased left ventricular (LV) thickness and a significant decrease in LV mass index (from 178.2 +/- 20.6 g/m2 to 147.3 +/- 20.6 g/m2) were observed. Blood pressure control did not improve; moreover, in some patients an increase in systolic values was detected by ambulatory BP. Casual BP remained seemingly stable. Sequential determinations of neurohumoral mediators of hemodynamic substances (endothelin, renin, norepinephrine, epinephrine, dopamine) failed to explain these results. Ambulatory BP reveals a worse control in some patients who were previously hypertensive and confirms the utility of this technique in the assessment of patients under erythropoietin treatment. The trend toward LV hypertrophy regression without improved BP control confirms the role of anemia among the multiple factors leading to LV hypertrophy in end-stage renal disease (ESRD), and opens therapeutic possibilities. Better control of BP may avoid a potential offsetting of beneficial effects that correcting anemia would have on the cardiovascular system.

Adult↗