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PubMed1989

Respiration in the filarial nematode Brugia pahangi.

Glucose-supported O2 uptake in the filarial nematode Brugia pahangi was partially inhibited by antimycin A (30-40%), with the remaining activity being sensitive to o-hydroxydiphenyl or salicylhydroxamic acid (SHAM). The production of CO2 by B. pahangi in the presence of D-glucose was stimulated by O2; the stimulation of CO2; the stimulation of CO2 production was sensitive to antimycin A. The O2 dependencies of respiration showed that the apparent O2 affinity for B. pahangi was diminished in the presence of antimycin A; O2 thresholds for inhibition of respiration were observed which showed that the alternative electron transport pathway was less sensitive to inhibition at elevated O2 concentrations. H2O2 production and its excretion could be detected in whole B. pahangi; higher rates were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. The effects of inhibitors on H2O2 production suggest two sites of H2O2 production, one associated with the classical antimycin A-sensitive pathway, the other with the alternative respiratory pathway. The similarity in the O2 dependencies of H2O2 production and respiration may indicate that H2O2 production is involved in O2-mediated toxicity. Succinate and malate respiring sub-mitochondrial particles of B. pahangi produced O2.- radicals at a site on the antimycin A-sensitive respiratory pathway. Inhibition of the alternative electron pathway by SHAM was unusual; sub-millimolar concentrations markedly stimulated respiration, H2O2 production and O2.- production by 30, 20 and 25%, respectively, whereas higher concentrations (greater than 2.5 mM) inhibited respiration by 75% and H2O2 and O2.- production by up to 85%.

Animals↗

PubMed1986

Lack of UV-induced respiration shutoff in a recF strain of Escherichia coli: temperature conditional suppression at 30 degrees C by the sfrA mutation.

A mutation in the recF gene of Escherichia coli results in a radiation-sensitive strain. The RecF pathway and the RecBC pathway account for nearly all of the conjugative recombination occurring in E. coli. recBC cells are radiation-sensitive and carry only out a small amount of recombination but these deficiencies are suppressed by an sbcB as recombination is shunted to the RecF pathway. A recBC sbcB recF strain is very radiation-sensitive and is devoid of recombination ability. These deficiencies are suppressed by the srfA mutation; srfA is a recA allele. UV-induced respiration shutoff is a recA+, lexA+ and recBC+ dependent. We report in this paper that respiration does not shutoff in a recF strain at 37 and 30 degrees C. an srfA mutation suppresses this lack of respiration shutoff effect in a recF srfA mutant at 30 degrees C but not at 37 degrees C; no suppression by this mutation occurs at either temperature in a recF recBC sbcB strain. An srfA strain also does not shut off its respiration at 37 degrees C and shows a temperature conditional UV-induced respiration shutoff response at 30 degrees C. The srfA mutation is thought to cause an altered RecA protein to be produced and we suggest that at 37 degrees This altered protein is temperature sensitive. We conclude from the results in this paper that the recF gene product is required for UV-induced respiration shutoff and that the RecA protein plays a special role in the induction process.

Bacterial Proteins↗

PubMed1989

Inhibition of mitochondrial respiration by cationic rhodamines as a possible teratogenicity mechanism.

Exposure of mice to cationic rhodamines, Rh 123 and Rh 6G, has been found to be associated with developmental toxicity, while neutral rhodamines (e.g., Rh B) had no such effect. When mouse embryos from dams given ip injections of Rh 123, Rh 6G (15 mg/kg), or Rh B (30 mg/kg) on gestation Day (GD) 10 were examined, Rh 123, Rh 6G were present in embryonic tissue in fluorescent bodies within the average dimensions of mitochondria. Rh B was evenly distributed in the cytoplasm. With in vitro exposure of isolated mitochondria to rhodamines on GD 12, 3-4 times more Rh 123 was associated with mitochondria under energized conditions than under nonenergized conditions; the amount of Rh 6G associated with mitochondria was much less under either condition. Treatment of pregnant mice (ip) with Rh 123 (15 mg/kg/day) or Rh 6G (0.5 mg/kg/day) on GD 7-10 resulted in inhibition of state 3 respiration of embryonic mitochondria isolated on GD 12. When isolated embryonic mitochondria were exposed to the cationic rhodamines, inhibition of state 3 respiration was dose dependent. With 5 micrograms of Rh 123/mg mitochondrial protein, state 3 respiration decreased by 31%, while Rh 6G (1 microgram/mg) decreased state 3 respiration by 27%. In vivo exposure of maternal liver mitochondria to cationic rhodamines did not result in inhibition of respiration 2 days later, whereas in vitro results were similar to those for embryonic mitochondria. In vivo or in vitro exposure to Rh B had no effects on mitochondrial respiration. These results indicate that interference with embryonic energy metabolism is a possible mechanism by which cationic rhodamines exert adverse effects on embryogenesis.

Animals↗

PubMed1980

Modification of mitochondrial respiration by aging and dietary restriction.

Effects of aging and of dietary restriction on mitochondrial recovery and respiratory capacities have been assessed in mice. Old mice (23-26 months) did not differ from adult mice (9-12 months) in amounts of protein recovered in mitochondrial fractions of liver, brain and spleen, but did show a decline in specific activity of cytochrome c oxidase (cyt. c ox.) in liver and spleen. Age effects on in vitro respiration by mitochondria occurred in liver and spleen. In liver, only one substrate (beta-hydroxybutyrate) of four tested was respired at a different rate by old than by young mitochondria. Depression of state 3 respiration and 2,4-dinitrophenol (DNP)-uncoupled rates was observed for this substrate; however, this effect depended on expressing respiration on the basis of mitochondrial protein and was less overt if data were expressed per unit of cyt. c ox. activity. Old spleen mitochondria exhibited a grosser defect, showing a 40% decrease in the respiratory control index (RCI) for (succinate + rotenone)- supported respiration (the only substrate tested) due to a possible increase in state 4 rates. Effects of dietary restriction were assessed in liver and brain of 3-7-month-old mice underfed since weaning. Dietary restriction reduced recovery of total liver mitochondrial protein and liver cyt. c ox. specific activity. Liver mitochondria from restricted mice generally showed increased state 3 rates with no differences from controls in state 4 rates for respiration supported by glutamate or pyruvate + malate, resulting in an increased RCI for these substrates. DNP-uncoupled rates were also raised by dietary restriction. Unlike effects observed in old versus young mice, these differences obtained whether the data were expressed on the basis of mitochondrial protein or on cyt. c ox. activity. Electron microscopy of liver mitochondrial preparations revealed more non-mitochondrial contaminants in old mice and larger mitochondria in dietarily restricted mice. These findings are compatible with reports of age-dependent losses of liver mitochondria and suggest that dietary restriction may retard this loss.

Aging↗

PubMed1990

Microbial respiration in fly ash/sewage sludge-amended soils.

Microbial respiration within a Fox sandy loam and a Blount silt loam was assessed after addition of acidic (pH 3.5) and alkaline (pH 12.2) fly ash (FoxAC, BlountAC, FoxAK and BlountAK, respectively). At the 20% alkaline ash rate, respiration was completely inhibited in the FoxAK and reduced by 97% in the BlountAK. In contrast, the 20% acidic ash rate reduced respiration in the FoxAC by 28% and in the BlountAC by 33%. Co-application of 5% composted sewage sludge to the ash-soil mixtures (FoxSSAC, BlountSSAC, FoxSSAK and BlountSSAK, respectively) improved respiration in both soils except for the 20% alkaline ash rate. Soil electrical conductivity, and concentrations of B, Mo, exchangeable Al and soluble anions were not closely associated with inhibition of respiration. In contrast, high pH appeared associated with decrease in respiration. Ecological Dose 50% (EcD(50)) values of the sewage-amended treatments were greater than for those of the non-sludge treatments for each sampling date as a result of the ameliorating properties of the sludge.

Journal Article↗

PubMed1995

Effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on mitochondrial respiration in ischemic rat hearts.

The aim of the present study was to examine the effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on mitochondrial respiration in ischemic rat hearts, and to compare the effects between water-soluble pravastatin and lipid-soluble simvastatin. Either vehicle (0.5% carboxymethyl cellulose), pravastatin (2 or 4 mg/kg per day), or simvastatin (1 or 2 mg/kg per day) was orally administered for 3 weeks. Ischemia was induced by ligating the aorta for 60 min in anesthetized open chest rats under artificial respiration. The hearts were removed, mitochondria were isolated, and the respiration was determined by polarography using glutamate and succinate as substrates. When succinate was used as a substrate, the ADP-stimulated respiration (QO3) and ATP production per unit oxygen (ADP/O ratio) were decreased by ischemia. The decreases in QO3 and ADP/O ratio in the pravastatin- and simvastatin-treated groups appeared to be more prominent than those in the vehicle-treated group. This was especially true in the simvastatin-treated group. The ADP-limited respiration (QO4) with succinate in the vehicle-treated heart was slightly increased by ischemia, while that in the pravastatin- or simvastatin-treated hearts was decreased. In conclusion, HMG-CoA reductase inhibitors may result in worsening of myocardial mitochondrial respiration during ischemia.

Adenosine Diphosphate↗

PubMed2006

Do N95 respirators provide 95% protection level against airborne viruses, and how adequate are surgical masks?

BACKGROUND: Respiratory protection devices are used to protect the wearers from inhaling particles suspended in the air. Filtering face piece respirators are usually tested utilizing nonbiologic particles, whereas their use often aims at reducing exposure to biologic aerosols, including infectious agents such as viruses and bacteria. METHODS: The performance of 2 types of N95 half-mask, filtering face piece respirators and 2 types of surgical masks were determined. The collection efficiency of these respiratory protection devices was investigated using MS2 virus (a nonharmful simulant of several pathogens). The virions were detected in the particle size range of 10 to 80 nm. RESULTS: The results indicate that the penetration of virions through the National Institute for Occupational Safety and Health (NIOSH)-certified N95 respirators can exceed an expected level of 5%. As anticipated, the tested surgical masks showed a much higher particle penetration because they are known to be less efficient than the N95 respirators. The 2 surgical masks, which originated from the same manufacturer, showed tremendously different penetration levels of the MS2 virions: 20.5% and 84.5%, respectively, at an inhalation flow rate of 85 L/min. CONCLUSION: The N95 filtering face piece respirators may not provide the expected protection level against small virions. Some surgical masks may let a significant fraction of airborne viruses penetrate through their filters, providing very low protection against aerosolized infectious agents in the size range of 10 to 80 nm. It should be noted that the surgical masks are primarily designed to protect the environment from the wearer, whereas the respirators are supposed to protect the wearer from the environment.

Air Microbiology↗

PubMed2005

Cyanide-resistant respiration in Taenia crassiceps metacestode (cysticerci) is explained by the H2O2-producing side-reaction of respiratory complex I with O2.

The nature of the cyanide-resistant respiration of Taenia crassiceps metacestode was studied. Mitochondrial respiration with NADH as substrate was partially inhibited by rotenone, cyanide and antimycin in decreasing order of effectiveness. In contrast, respiration with succinate or ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was more sensitive to antimycin and cyanide. The saturation kinetics for O2 with NADH as substrate showed two components, which exhibited different oxygen affinities. The high-O2-affinity system (Km app=1.5 microM) was abolished by low cyanide concentration; it corresponded to cytochrome aa3. The low-O2-affinity system (Km app=120 microM) was resistant to cyanide. Similar O2 saturation kinetics, using succinate or ascorbate-TMPD as electron donor, showed only the high-O2-affinity cyanide-sensitive component. Horse cytochrome c increased 2-3 times the rate of electron flow across the cyanide-sensitive pathway and the contribution of the cyanide-resistant route became negligible. Mitochondrial NADH respiration produced significant amounts of H2O2 (at least 10% of the total O2 uptake). Bovine catalase and horse heart cytochrome c prevented the production and/or accumulation of H2O2. Production of H2O2 by endogenous respiration was detected in whole cysticerci using rhodamine as fluorescent sensor. Thus, the CN-resistant and low-O2-affinity respiration results mainly from a spurious reaction of the respiratory complex I with O2, producing H2O2. The meaning of this reaction in the microaerobic habitat of the parasite is discussed.

Animals↗

PubMed2000

Influence of bronchial C fibre receptors on respiration in cats: possible role in humans.

In order to find out whether the bronchial C fibre receptors influence respiration in cats, as the J receptors do, two drugs known to stimulate them consistently, i.e. phenyl diguanide (PDG) and capsaicin were injected into the left atrium (LA). The influence of these drugs, in suprathreshold doses, was seen on the intrapleural pressure (IPP), frequency of respiration (f(R)), systemic blood pressure (BP) and cardiac frequency (f(H)). PDG LA produced an inhibitory influence on respiration of a majority of cats, accompanied by a fall in BP and f(H). With capsaicin, a stimulation of respiration and a notable increase in BP (sometimes preceded by a fall also of f(H)) was seen. After blocking the epicardial/cardiac receptors with xylocaine, a local anaesthetic, the effects of PDG LA on the above variables were blocked, whereas, those of capsaicin survived--which they did, also after a bilateral vagotomy. Similar responses were obtained from injecting the drugs into the distal part of the aorta i.e. a notable stimulation of respiration by capsaicin, which survived vagotomy and hardly any effect, by PDG. It is concluded that activation of bronchial C fibre receptors does not influence respiration or BP in cats.

Anesthetics, Local↗

PubMed1998

Growth, respiration and nitrogen content in needles of Scots pine exposed to elevated ozone and carbon dioxide in the field.

Single Scots pine (Pinus sylvestris L.) trees, aged 30 years, were grown in open-top chambers and exposed to two atmospheric concentrations of ozone (O3; ambient and elevation) and carbon dioxide (CO2) as single variables or in combination for 3 years (1994-1996). Needle growth, respiration and nitrogen content were measured simultaneously over the period of needle expansion. Compared to ambient treatment (33 nmol mol(-1) O3 and 350 micromol mol(-1) CO2) doubled ambient O3 (69 nmol mol(-1)) significantly reduced the specific growth rates (SGRs) of the needles in the early stage of needle expansion and needle nitrogen concentration (N1) in the late stage, but increased apparent respiration rates (ARRs) in the late stage. Doubled ambient CO2 (about 650 micromol mol(-1)) significantly increased maximum SGR but reduced ARR and N1 in the late stage of needle expansion. The changes in ARR induced by the different treatments may be associated with treatment-induced changes in needle growth, metabolic activities and turnover of nitrogenous compounds. When ARR was partitioned into its two functional components, growth and maintenance respiration, the results showed that neither doubled ambient O3 nor doubled ambient CO2 influenced the growth respiration coefficients (Rg). However, doubled ambient O3 significantly increased the maintenance respiration coefficients (Rm) regardless of the needle development stage, while doubled ambient CO2 significantly reduced Rm only in the late stage of needle expansion. The increase in Rm under doubled ambient O3 conditions appeared to be related to an increase in metabolic activities, whereas the decrease in Rm under doubled ambient CO2 conditions may be attributed to the reduced N1 and turnover rate of nitrogenous compounds per unit. The combination of elevated O3 and CO2 had very similar effects on growth, respiration and N1 to doubled ambient O3 alone, but the interactive mechanism of the two gases is still not clear.

Journal Article↗

PubMed2006

Abnormalities in the coordination of respiration and swallow in preterm infants with bronchopulmonary dysplasia.

Individual rhythms of suck, swallow, and respiration are disrupted in preterm infants with bronchopulmonary dysplasia (BPD). Integration of respiration into suck-swallow efforts is critical for establishing coordinated suckle feeding. This study quantitatively assessed the coordination of respiration and swallow in infants with and without BPD. Thirty-four preterm infants of 26 to 33 weeks' gestational age were included: 14 participants with BPD (eight males, six females)and 20 comparison participants without BDP (10 males, 10 females). Participants were studied at postmenstrual age 32 to 40 weeks and postnatal age 2 to 12 weeks using digital recordings of pharyngeal pressure, nasal thermistor flow, and thoraco-abdominal plethysmography. The coefficients of variation (COV; standard deviation/mean) of the swallow-breath (SW-BR) and breath-breath (BR-BR) intervals during swallow runs, the percentage of 'apneic swallows' (runs of >or=3 swallows without interposed breaths), and phase relationships of respiration and swallow were used to quantify rhythmic coordination and integration of respiration into feeding episodes. Apneic swallows were significantly increased after 35 weeks in infants with BPD (mean 13.4% [SE 2.4]) compared with non-BDP infants (6.7% [SE 1.8]; p<0.05), as were SW-BR phase relationships involving apnea. The BPD cohort also had significantly higher SW-BR COV and BR-BR COV than non-BPD infants, indicating less rhythmic coordination of swallowing and respiration during feeding. Results emphasize the need for frequent rests and closer monitoring when feeding infants with respiratory compromise. Quantitative assessment of the underlying rhythms involved in feeding may be predictive of longer-term feeding and neurological problems.

Analysis of Variance↗

PubMed1996

Mg2+ control of respiration in isolated rat liver mitochondria.

The role of endogenous mitochondrial Mg2+ as a potential regulator of mitochondrial dehydrogenase activity, and therefore of cellular respiration, was measured in isolated mitochondria containing matrix Ca2+ and Mg2+ levels resembling those occurring in vivo. Ca2+ and Mg2+ depletion was carried out using the cation ionophore A23187 in the presence or absence of the Ca2+ uniporter inhibitor ruthenium red (RR). Divalent cation depletion inhibits the oxidation of alpha-ketoglutarate or pyruvate in states 4 and 3, slows uncoupled respiration and results in decreased membrane potential. Since the addition of Mg2+ could not restore respiration, these dehydrogenases appear not to be regulated by Mg2+. In contrast, similar cation depletion stimulates succinate dehydrogenase (or glutamate dehydrogenase) in state 4 without decreasing membrane potential. The addition of RR caused authentic uncoupling, accompanied by a decrease in membrane potential and an increase in membrane permeability. These effects could be completely reversed by Mg2+. These and other data, showing that Mg2+ depletion results in a change of respiration depending on the substrate oxidized and the metabolic state, indicate that Mg2+ removal may have direct and indirect effects on mitochondrial respiration. A clear direct effect is the stimulation of succinate or glutamate dehydrogenase by decreasing matrix Mg2+. Hence, changes in matrix Mg2+ (in addition to those of Ca2+) could be of great consequence, not only for the control of respiration but also for metabolic pathways affected by changes in concentrations of matrix substrates.

Animals↗

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

PubMed1965

THE RESPIRATION OF ISOLATED RAT-HEPATIC CELLS IN SUSPENSION.

1. Rat-hepatic cells in suspension have been shown to have an endogenous respiration of 5.6+/-0.17 when suspended in 0.1 m-sucrose and 0.02 m-tris-hydrochloric acid buffer. The respiration in 0.25 m-sucrose and 0.02 m-tris-hydrochloric acid buffer is 30-40% less. 2. Potassium chloride (0.05 m) is slightly inhibitory and calcium chloride (0.0025 m) highly inhibitory to endogenous respiration of the hepatic cells in suspension. The cells do not respire in Krebs-Ringer phosphate buffer. 3. The respiration of the hepatic cells in suspension is stimulated by pyruvate, citrate, isocitrate, oxoglutarate, succinate, fumarate, malate and glutamate; there is no significant stimulation (or inhibition) by glucose, fructose, acetate and butyrate. In almost all the cases where stimulation was observed, it was found that the higher the endogenous respiration the lower is the stimulation.

Acetates↗

PubMed1978

The microbial metabolism of Cl compounds. The stoicheiometry of respiration-driven proton translocation in Pseudomonas AM1 and in a mutant lacking cytochrome c.

This paper clarifies the role of cytochrome c in Pseudomonas AM1 by measuring the stoicheiometry of proton translocation driven by respiration of endogenous or added substrates in wild-type bacteria and in a mutant lacking cytochrome c (mutant PCT76). The maximum -->H(+)/O ratio (protons translocated out of the bacteria per atom of oxygen consumed during respiration) was about 4 and, except when respiration was markedly affected, this ratio was similar in mutant and wild-type bacteria. The -->H(+)/O ratios were unaltered when the usual oxidase (cytochrome a(3)) was inhibited by 300mum-KCN and respiration involved the single cytochrome b functioning as an alternative oxidase. Ratios measured in cells respiring endogenous substrate and in cells loaded with malate or 3-hydroxybutyrate suggest that there are two proton-translocating segments operating during the oxidation of NADH. By contrast, during oxidation of formaldehyde or methylamine only one pair of protons is translocated. Proton translocation could not be measured with methanol as substrate, because its oxidation was inhibited (90-95%) by 5mm-KSCN. It is tentatively proposed that the electron-transport chain for NADH oxidation in Pseudomonas AM1 is arranged such that the NADH-ubiquinone oxidoreductase forms one proton-translocating segment and the second segment consists of ubiquinone and cytochromes b and a/a(3). The cytochrome c appears to be essential only for respiration and proton translocation from methanol (and possibly from methylamine); there is no conclusive evidence that cytochrome c ever mediates between cytochromes b and a/a(3) in Pseudomonas AM1.

Cyanides↗

PubMed1992

Control of respiration and ATP synthesis in mammalian mitochondria and cells.

We have seen that there is no simple answer to the question 'what controls respiration?' The answer varies with (a) the size of the system examined (mitochondria, cell or organ), (b) the conditions (rate of ATP use, level of hormonal stimulation), and (c) the particular organ examined. Of the various theories of control of respiration outlined in the introduction the ideas of Chance & Williams (1955, 1956) give the basic mechanism of how respiration is regulated. Increased ATP usage can cause increased respiration and ATP synthesis by mass action in all the main tissues. Superimposed on this basic mechanism is calcium control of matrix dehydrogenases (at least in heart and liver), and possibly also of the respiratory chain (at least in liver) and ATP synthase (at least in heart). In many tissues calcium also stimulates ATP usage directly; thus calcium may stimulate energy metabolism at (at least) four possible sites, the importance of each regulation varying with tissue. Regulation of multiple sites may occur (from a teleological point of view) because: (a) energy metabolism is branched and thus proportionate regulation of branches is required in order to maintain constant fluxes to branches (e.g. to proton leak or different ATP uses); and/or (b) control over fluxes is shared by a number of reactions, so that large increases in flux requires stimulation at multiple sites because each site has relatively little control. Control may be distributed throughout energy metabolism, possibly due to the necessity of minimizing cell protein levels (see Brown, 1991). The idea that energy metabolism is regulated by energy charge (as proposed by Atkinson, 1968, 1977) is misleading in mammals. Neither mitochondrial ATP synthesis nor cellular ATP usage is a unique function of energy charge as AMP is not a significant regulator (see for example Erecinska et al., 1977). The near-equilibrium hypothesis of Klingenberg (1961) and Erecinska & Wilson (1982) is partially correct in that oxidative phosphorylation is often close to equilibrium (apart from cytochrome oxidase) and as a consequence respiration and ATP synthesis are mainly regulated by (a) the phosphorylation potential, and (b) the NADH/NAD+ ratio. However, oxidative phosphorylation is not always close to equilibrium, at least in isolated mitochondria, and relative proximity to equilibrium does not prevent the respiratory chain, the proton leak, the ATP synthase and ANC having significant control over the fluxes. Thus in some conditions respiration rate correlates better with [ADP] than with phosphorylation potential, and may be relatively insensitive to mitochondrial NADH/NAD+ ratio.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1994

Cytoplasmic transfer of the mtDNA nt 8993 T-->G (ATP6) point mutation associated with Leigh syndrome into mtDNA-less cells demonstrates cosegregation with a decrease in state III respiration and ADP/O ratio.

A point mutation in the mtDNA-encoded ATP6 gene (T-->G at nt 8993) associated with Leigh syndrome in two pedigrees was found to decrease ADP-stimulated (state III) respiration and the ratio of ADP molecules phosphorylated to oxygen atoms reduced (ADP/O ratio) but did not affect 2,4-dinitrophenol (DNP)-uncoupled respiration, suggesting a defective mitochondrial H(+)-translocating ATP synthase. Intact mitochondria isolated from patient and control lymphoblastoid cell lines were tested for state III, ADP-limited (state IV), and DNP-uncoupled respiration with various substrates. Mitochondria isolated from patient lymphoblasts harboring 95-100% of mtDNAs carrying the nt 8993 T-->G mutation showed state III respiration rates 26-50% lower than controls while having normal DNP-uncoupled rates. This resulted in state III/DNP ratios of 0.52-0.70 in patient mitochondria versus 0.88-0.97 in controls. The ADP/O ratio was also decreased 30-40% in patient mitochondria. Patient lymphoblasts heteroplasmic for the nt 8993 mutation were enucleated by using Percoll gradients and the cytoplasts were fused to mtDNA-deficient (rho 0) cells by electric shock. Cybrid clones homoplasmic for the wild-type nucleotide (T) at nt 8993 gave state III/DNP and ADP/O ratios similar to those of control cybrids, whereas cybrid clones homoplasmic for the mutant nucleotide (G) showed a 24-53% reduction in state III respiration, a state III/DNP ratio of 0.53-0.64, and a 30% decrease in the ADP/O ratio. Thus, the reduced state III respiration rates and ADP/O ratios are linked to the T-->G mutation at nt 8993.

DNA, Mitochondrial↗

PubMed1997

In vivo control of respiration by cytochrome c oxidase in wild-type and mitochondrial DNA mutation-carrying human cells.

The metabolic control of respiration is still poorly understood, due mainly to the lack of suitable approaches for studying it in vivo. Experiments on isolated mammalian mitochondria have indicated that a relatively small fraction of each of several components of the electron transport chain is sufficient to sustain a normal O2 consumption rate. These experiments, however, may not reflect accurately the in vivo situation, due to the lack in the mitochondrial fraction of essential cytosolic components and to the use of excess of substrates in the in vitro assays. An approach is described here whereby the control of respiration by cytochrome c oxidase (COX; EC 1.9.3.1) was analyzed in intact cultured human osteosarcoma 143B.TK- cells and other wild-type cells and in mitochondrial DNA mutation-carrying human cell lines. Surprisingly, in wild-type cells, only a slightly higher COX capacity was detected than required to support the endogenous respiration rate, pointing to a tighter in vivo control of respiration by COX than generally assumed. Cell lines carrying the MERRF mitochondrial tRNA(Lys) gene mutation, which causes a pronounced decrease in mitochondrial protein synthesis and respiration rates, revealed, in comparison, a significantly greater COX capacity relative to the residual endogenous respiration rate, and, correspondingly, a higher COX inhibition threshold above which the overall respiratory flux was affected. The observed relationship between COX respiratory threshold and relative COX capacity and the potential extension of the present analysis to other respiratory complexes have significant general implications for understanding the pathogenetic role of mutations in mtDNA-linked diseases and the tissue specificity of the mutation-associated phenotype.

Antimycin A↗