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PubMed1970

Role of Pyridine Nucleotides in the Control of Respiration in Ultraviolet-Irradiated Escherichia coli B/r Cells.

Escherichia coli B/r cells grown on glycerol-containing medium and irradiated with ultraviolet light to about 1% survival respire for about 1 hr and then cease completely for several hours. The results of studies on cell-free respiration and analyses of pyridine nucleotide levels at various times after ultraviolet irradiation show that the cessation of respiration is associated with two changes-loss of glycerol kinase activity and complete disappearance of pyridine nucleotides. Under other cultural conditions in which respiratory inhibition is less complete and more transitory, the losses of pyridine nucleotides are smaller and the rises which follow are correlated with increases in respiratory activity.

Journal Article↗

PubMed1971

Endogenous dark respiration of the blue-green alga, Plectonema boryanum.

Endogenous dark respiration in the blue-green alga Plectonema boryanum is markedly affected by preincubation in the light: it can be increased from a basal rate of 5 nmoles of O(2) to 55 nmoles of O(2) per mg of cell protein per min after exposure of the cells to light for 8 to 10 hr. Under conditions of enhanced dark respiration, cyanophage multiplication in the dark increases drastically and approaches the cyanophage yields obtained in photosynthesizing Plectonema cells. This implies that the biosynthetic capabilities of the algal cells, at least with respect to viral synthesis, can be similar in the dark to those in the light. The enhanced endogenous respiration rate was found to be dependent on photoassimilation of CO(2) and on protein synthesis. The implications of these findings with respect to obligate photoautotrophic metabolism in blue-green algae are discussed.

Carbon Dioxide↗

PubMed1973

Altered mitochondrial respiration in a chromosomal mutant of Neurospora crassa.

A mutant of Neurospora crassa (cni-1) has been isolated that has two pathways of mitochondrial respiration. One pathway is sensitive to cyanide and antimycin A, the other is sensitive only to salicyl hydroxamic acid. Respiration can proceed through either pathway and both pathways together in this mutant account for greater than 90% of all mitochondrial respiration. The cni-1 mutation segregates as a nuclear gene in crosses to other strains of Neurospora. Absorption spectra of isolated mitochondria from cni-1 show typical b- and c-type cytochromes but the absorption peaks corresponding to cytochrome aa(3) are not detectable. Extraction of soluble cytochrome c-546 from these mitochondria followed by reduction with ascorbate reveals a new absorption peak at 426 nm that is not present in wild-type mitochondria. This peak may be due to an altered cytochrome oxidase with abnormal spectral properties. Mitochondria from cni-1 have elevated levels of succinate-cytochrome c reductase but reduced levels of nicotinamide adenine dinucleotide reduced form cytochrome c reductase and of cyanide- and azide-sensitive cytochrome c oxidase. These studies suggest that the cni-1 mutation results in the abnormal assembly of cytochrome c oxidase so that the typical cytochrome aa(3) spectrum is lost and the enzyme activity is reduced. As a consequence of this alteration, a cyanide-insensitive respiratory pathway is elaborated by these mitochondria which may serve to stimulate adenosine 5'-triphosphate production via substrate level phosphorylation by glycolysis and the Krebs cycle.

Antimycin A↗

PubMed1973

Respiration of wild type and extrachromosomal mutants of Neurospora crassa.

The specific rates of respiration of cells of wild type and four extrachromosomal mutants of Neurospora crassa were measured throughout the vegetative growth cycle. Two forms of respiration were observed: (i) cyanide sensitive; and (ii) cyanide resistant, salicyl hydroxamate sensitive. These two forms are called terminal and alternate, respectively. The former proceeds by the mitochondrial electron transfer chain and involves the cytochromes; the latter apparently proceeds by the initial portion of the electron transfer chain and does not involve cytochromes. Large and rapid changes of both the terminal and alternate respiratory activities occurred during the vegetative growth cycle. The kinetics of these changes in wild type were compared under some conditions which inhibit protein synthesis and others in which the nitrogen source was varied. The kinetics of the changes of the two forms of respiration of mutants differed from those normally exhibited by wild type, but with varied experimental conditions wild type could be made to resemble the mutants. The results of these studies are discussed in terms of a dynamic model of regulation of mitochondrial biogenesis in the coordination of the synthesis of mitochondrial proteins encoded by nuclear and mitochondrial genomes.

Chloramphenicol↗

PubMed1980

Are growth rates of Escherichia coli in batch cultures limited by respiration?

Batch cultures of Escherichia coli were grown in minimal media supplemented with various carbon sources which supported growth at specific growth rates from 0.2 to 1.3/h. The respiration rates of the cultures were measured continuously. With few exceptions, the specific rate of oxygen consumption was about 20 mmol of O2/h per g (dry weight), suggesting that the respiratory capacity was limited at this value. The adenosine triphosphate (ATP) required for the production of cell material from the different carbon sources was calculated on the basis of known ATP requirements in the biochemical pathways and routes of macromolecular synthesis. The calculated ATP requirements, together with the measured growth rates and growth yields on the different carbon sources, were used to calculate the rate of ATP synthesis by oxidative phosphorylation. This rate was closely related to the respiration rate. We suggest that aerobic growth of E. coli in batch cultures is limited by the rate of respiration and the concomitant rate of ATP generation through oxidative phosphorylation.

Adenosine Triphosphate↗

PubMed1996

Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids.

The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O2 coincided with an increase of the cellular ATP/ADP ratio. When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity. To explain these observations, the effects of nitrogenase activity and CCCP on the O2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 microM O2, a decline in the O2 consumption rate was observed to 50 to 70% of the maximal O2 consumption rate. A determination of the redox state of the cytochromes during an O2 consumption experiment indicated that at O2 concentrations above 5 microM, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O2 concentrations (if >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (Km of 45 +/- 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O2.

Adenosine Diphosphate↗

PubMed1998

Toxicity of linoleic acid hydroperoxide to Saccharomyces cerevisiae: involvement of a respiration-related process for maximal sensitivity and adaptive response.

Linoleic acid hydroperoxide (LoaOOH) formed during free radical attack on long-chain unsaturated fatty acids is an important source of biomembrane damage and is implicated in the onset of atherosclerosis, hepatic diseases, and food rancidity. LoaOOH is toxic to wild-type Saccharomyces cerevisiae at a very low concentration (0.2 mM) relative to other peroxides. By using isogenic mutant strains, the possible roles of glutathione (gsh1 and gsh2), glutathione reductase (glr1), respiratory competence ([rho0] petite), and yAP-1p-mediated expression (yap1) in conferring LoaOOH resistance have been examined. Respiration-related processes were essential for maximal toxicity and adaptation, as evidenced by the fact that the [rho0] petite mutant was most resistant to LoaOOH but could not adapt. Furthermore, when respiration was blocked by using inhibitors of respiration and mutants defective in respiratory-chain components, cells became more resistant. An important role for reduced glutathione and yAP-1 in the cellular response to LoaOOH was shown, since the yap1 and glr1 mutants were more sensitive than the wild type. In addition, total glutathione peroxidase activity increased following treatment with LoaOOH, indicating a possible detoxification role for this enzyme. Yeast also showed an adaptive response when pretreated with a nonlethal dose of LoaOOH (0.05 mM) and subsequently treated with a lethal dose (0.2 mM), and de novo protein synthesis was required, since adaptation was abolished upon treatment of cells with cycloheximide (25 microg ml-1). The wild-type adaptive response to LoaOOH was independent of those for the superoxide-generating agents paraquat and menadione and also of those for the organic hydroperoxides cumene hydroperoxide and tert-butyl hydroperoxide. Pretreatment with LoaOOH induced resistance to hydrogen peroxide, while pretreatment of cells with malondialdehyde (a lipid peroxidation product) and heat shock (37 degrees C) gave cross-adaptation to LoaOOH, indicating that yeast has effective overlapping defense systems that can detoxify fatty acid hydroperoxides directly or indirectly.

Adaptation, Physiological↗

PubMed2002

Periplasmic nitrate reductase (NapABC enzyme) supports anaerobic respiration by Escherichia coli K-12.

Periplasmic nitrate reductase (NapABC enzyme) has been characterized from a variety of proteobacteria, especially Paracoccus pantotrophus. Whole-genome sequencing of Escherichia coli revealed the structural genes napFDAGHBC, which encode NapABC enzyme and associated electron transfer components. E. coli also expresses two membrane-bound proton-translocating nitrate reductases, encoded by the narGHJI and narZYWV operons. We measured reduced viologen-dependent nitrate reductase activity in a series of strains with combinations of nar and nap null alleles. The napF operon-encoded nitrate reductase activity was not sensitive to azide, as shown previously for the P. pantotrophus NapA enzyme. A strain carrying null alleles of narG and narZ grew exponentially on glycerol with nitrate as the respiratory oxidant (anaerobic respiration), whereas a strain also carrying a null allele of napA did not. By contrast, the presence of napA+ had no influence on the more rapid growth of narG+ strains. These results indicate that periplasmic nitrate reductase, like fumarate reductase, can function in anaerobic respiration but does not constitute a site for generating proton motive force. The time course of phi(napF-lacZ) expression during growth in batch culture displayed a complex pattern in response to the dynamic nitrate/nitrite ratio. Our results are consistent with the observation that phi(napF-lacZ) is expressed preferentially at relatively low nitrate concentrations in continuous cultures (H. Wang, C.-P. Tseng, and R. P. Gunsalus, J. Bacteriol. 181:5303-5308, 1999). This finding and other considerations support the hypothesis that NapABC enzyme may function in E. coli when low nitrate concentrations limit the bioenergetic efficiency of nitrate respiration via NarGHI enzyme.

Anaerobiosis↗

PubMed1965

Differential Reduction of Tellurite by Growing Colonies of Normal Yeast and Respiration-Deficient Mutants.

Nagai, Susumu (National Women's University, Nara, Japan). Differential reduction of tellurite by growing colonies of normal yeast and respiration-deficient mutants. J. Bacteriol. 90:220-222. 1965.-A differential reduction of sodium tellurite was observed between normal and respiration-deficient mutant colonies of several species of Saccharomyces. Normal colonies turned black in contrast to mutant colonies which remained nearly white when grown on an agar medium containing 30 to 40 mg per liter of tellurite. Schopfer's medium enriched with yeast extract and a mixture of vitamins was most suitable to develop such black-and-white contrast. The difference was far less obvious when the asparagine of this medium was replaced by other nitrogen sources such as glutamate, peptone, or Casamino Acids. Addition of ammonium sulfate to the medium weakened and sometimes completely reversed the contrast. The usefulness of tellurite medium for diagnostic color differentiation of respiration deficiency was considered.

Journal Article↗

PubMed2006

Defective mitochondrial gene expression results in reactive oxygen species-mediated inhibition of respiration and reduction of yeast life span.

Mitochondrial dysfunction causes numerous human diseases and is widely believed to be involved in aging. However, mechanisms through which compromised mitochondrial gene expression elicits the reported variety of cellular defects remain unclear. The amino-terminal domain (ATD) of yeast mitochondrial RNA polymerase is required to couple transcription to translation during expression of mitochondrial DNA-encoded oxidative phosphorylation subunits. Here we report that several ATD mutants exhibit reduced chronological life span. The most severe of these (harboring the rpo41-R129D mutation) displays imbalanced mitochondrial translation, conditional inactivation of respiration, elevated production of reactive oxygen species (ROS), and increased oxidative stress. Reduction of ROS, via overexpression of superoxide dismutase (SOD1 or SOD2 product), not only greatly extends the life span of this mutant but also increases its ability to respire. Another ATD mutant with similarly reduced respiration (rpo41-D152A/D154A) accumulates only intermediate levels of ROS and has a less severe life span defect that is not rescued by SOD. Altogether, our results provide compelling evidence for the "vicious cycle" of mitochondrial ROS production and lead us to propose that the amount of ROS generated depends on the precise nature of the mitochondrial gene expression defect and initiates a downward spiral of oxidative stress only if a critical threshold is crossed.

DNA-Binding Proteins↗

PubMed1977

Cuirass respirator treatment of chronic respiratory failure in scoliotic patients.

The results are reported of domiciliary cuirass respirator treatment, using tailor-made shells, in four patients with severe thoracic scoliosis. Three of the patients had suffered from poliomyelitis. All complained of increasing dyspnoea on exertion, ultimately interfering with almost every activity of daily life; three patients had severe acute respiratory failure necessitating urgent admission to the Respiratory Care Unit. Right heart failure was present in two. Two patients required mechanical treatment via an endotracheal tube. All the patients were discharged home with a cuirass respirator. Standard type shells were used initially with low efficiency due to the poor fit of the cuirass shell to the deformed thoracic cage. Tailor-made shells were constructed from polyester reinforced with glass fibre, modelled on plaster casts of the thoracic cage. Subjectively the patients improved greatly and were able to resume and increase many activities. One patient committed suicide for reasons unconnected with treatment but the other three patients have been doing well from the time the cuirass respirator treatment was started, respectively, 3, 6, and 10 years ago. This treatment seems particularly effective in younger patients with severe paralytic scoliosis and cardiorespiratory failure, although the possibility of using it in older patients suffering from scoliosis of other aetiology should certainly be explored.

Adolescent↗

PubMed1981

Protective value of dust respirators in extrinsic allergic alveolitis: clinical assessment using inhalation provocation tests.

Six subjects with budgerigar fancier's lung, pigeon breeder's lung, or mushroom worker's lung underwent identical paired inhalation provocation tests of 20 minutes to five hours duration (mean 2.3 hours) using a natural method of antigen exposure. For one test of each pair, the subjects were protected by the use of industrial dust respirators that incorporate electrostatically charged, resin-impregnated merino wool filters. The unprotected challenges provoked late responses of mild to moderate severity, that were characteristic of extrinsic allergic alveolitis. These were monitored by methods described previously, using four subjective and six objective monitoring tests. A score of 1 was allowed for each monitoring test showing a significant change. A combined monitoring score of 32/57 was associated with the unprotected challenges-that is, 56% of the maximum score possible. The respirators successfully protected four of the subjects from any suspicion of a positive response. The remaining two subjects reported symptoms of minimal severity, but there was no independent confirmatory evidence of positive reactions. The combined monitoring score associated with all six protected challenges was 2/60 (3%), while that associated with corresponding control challenges was 3/73 (4%). We conclude that respirators of this type can offer substantial, and in most cases complete, protection against single exposures to environmental dusts that may provoke extrinsic allergic alveolitis.

Adult↗

PubMed2000

Oxygen respiration by desulfovibrio species.

Throughout the first 90 years after their discovery, sulfate-reducing bacteria were thought to be strict anaerobes. During the last 15 years, however, it has turned out that they have manifold properties that enable them to cope with oxygen. Sulfate-reducing bacteria not only survive oxygen exposure for at least days, but many of them even reduce oxygen to water. This process can be a true respiration process when it is coupled to energy conservation. Various oxygen-reducing systems are present in Desulfovibrio species. In Desulfovibrio vulgaris and Desulfovibrio desulfuricans, oxygen reduction was coupled to proton translocation and ATP conservation. In these species, the periplasmic fraction, which contains hydrogenase and cytochrome c3, was found to catalyze oxygen reduction with high rates. In Desulfovibrio gigas, a cytoplasmic rubredoxin oxidase was identified as an oxygen-reducing terminal oxidase. Generally, the same substrates as with sulfate are oxidized with oxygen. As additional electron donors, reduced sulfur compounds can be oxidized to sulfate. Sulfate-reducing bacteria are thus able to catalyze all reactions of a complete sulfur cycle. Despite a high respiration rate and energy coupling, aerobic growth of pure cultures is poor or absent. Instead, the respiration capacity appears to have a protective function. High numbers of sulfate-reducing bacteria are present in the oxic zones and near the oxic-anoxic boundaries of sediments and in stratified water bodies, microbial mats and termite guts. Community structure analyses and microbiological studies have shown that the populations in those zones are especially adapted to oxygen. How dissimilatory sulfate reduction can occur in the presence of oxygen is still enigmatic, because in pure culture oxygen blocks sulfate reduction. Behavioral responses to oxygen include aggregation, migration to anoxic zones, and aerotaxis. The latter leads to band formation in oxygen-containing zones at concentrations of </=20% air saturation.

Adaptation, Biological↗

PubMed1984

Role of adenosine as an endogenous regulator of respiration in hamster brown adipocytes.

The action of endogeneous adenosine on isolated hamster brown adipocytes was examined. Adenosine production from brown adipocytes was measured after labeling of the intracellular nucleotide pool with [3H]adenine. Accumulation of [3H]adenosine in the incubation medium was maximum after 5 min of incubation and was still present after 20 min. When adenosine accumulation was prevented by addition of adenosine deaminase, the stimulatory effects of isoproterenol on oxygen uptake, lipolysis, and adenosine 3',5'-cyclic monophosphate (cAMP) generation were enhanced. However, basal rates of lipolysis and oxygen consumption and levels of cAMP were not affected on addition of adenosine deaminase. A similar potentiation of isoproterenol responses was produced by the adenosine receptor antagonist, 3-isobutyl-1-methylxanthine, present at a concentration (10 microM) which did not change basal levels of respiration or lipolysis. Addition of the adenosine analogue 2-chloroadenosine antagonized isoproterenol-stimulated respiration and lipolysis and prevented potentiation of isoproterenol responses with 3-isobutyl-1-methylxanthine. To localize the site of adenosine action, activity of adenylate cyclase in membrane preparations from brown adipocytes was measured. Isoproterenol-stimulated adenylate cyclase activity was partially inhibited by 2-chloroadenosine in a GTP-dependent manner. Addition of Na+ enhanced the inhibitory effect of 2-chloroadenosine, and 3-isobutyl-1-methylxanthine blocked it. The calculated 50% effective dose for 2-chloroadenosine inhibition was between 10 and 15 nM. These data suggest that adenosine produced by brown adipocytes is an endogenous regulator of respiration in these cells acting at the level of the adenylate cyclase enzyme.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Oxygen dependence of respiration in coupled and uncoupled endothelial cells.

We studied the oxygen dependence of respiration in cultured human umbilical vein endothelial cells by use of high-resolution respirometry. The rate of oxygen consumption varied from 30 to 50 pmol O2.s-1.(10(6) cells)-1 over a sixfold range of cell densities. Respiration was stimulated up to 3.5-fold by uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhydrazone or 2,4-dinitrophenol, and the PO2 at half-maximal respiration (P50) increased from 0.05 to 0.12 kPa (0.3 to 0.9 Torr) with respiratory rate. P50 decreased to a minimum of 0.02 kPa when uncoupled cells were inhibited to control levels. Differences in cell size explained a variation of approximately 0.015 kPa in P50 at similar respiratory rates per cell. Oxygen diffusion to mitochondria contributed maximally 30% to the regulation of P50 in coupled cells, as deduced from the shallow slope of the flux dependence of P50 in uncoupled-inhibited cells compared with the slope in coupled cells. Therefore 70% of the flux dependence of P50 in coupled cells was caused by changes in metabolic state, which correlated with respiratory rate.

Cell Hypoxia↗

PubMed1999

Contribution of mitochondrial proton leak to respiration rate in working skeletal muscle and liver and to SMR.

Proton pumping across the mitochondrial inner membrane and proton leak back through the natural proton conductance pathway make up a futile cycle that dissipates redox energy. We measured respiration and average mitochondrial membrane potential in perfused rat hindquarter with maximal tetanic contraction of the left gastrocnemius-soleus-plantaris muscle group, and we estimate that the mitochondrial proton cycle accounted for 34% of the respiration rate of the preparation. Similar measurements in rat hepatocytes given substrates to cause a high rate of gluconeogenesis and ureagenesis showed that the proton cycle accounted for 22% of the respiration rate of these cells. Combining these in vitro values with literature values for the contribution of skeletal muscle and liver to standard metabolic rate (SMR), we calculate that the proton cycle in working muscle and liver may account for 15% of SMR in vivo. Although this value is less than the 20% of SMR we calculated previously using data from resting skeletal muscle and hepatocytes, it is still large, and we conclude that the futile proton cycle is a major contributor to SMR.

Adenosine Triphosphate↗

PubMed1986

Prostaglandins stimulate calcium-linked changes in heart mitochondrial respiration.

This study was done to determine the influence of prostaglandins (PGs) on oxidative phosphorylation and Ca-related changes in cardiac mitochondria. For most of the experiments mitochondria were isolated by using a nagarse-homogenization combination method that yields both subsarcolemmal and interfibrillar populations. When Ca (30-420 microM) was added a progressive stimulation in resting respiration was observed. Similarly, preaddition of Ca inhibited subsequent oxidative phosphorylation when ADP was added to the same preparation. The effect of calcium was enhanced by pretreating mitochondria with PGs E2, F2 alpha, and I2 (prostacyclin). Three PG concentrations, 100 pg/ml, 1 ng/ml, and 10 ng/ml were evaluated. For the most part the effect of PGs was concentration dependent with the exception of PGE2, which showed no effect at the highest concentration. The effects of PGs were observed when both NADH (pyruvate-malate) and succinate-linked respiratory substrates were used. Similar effects were evident when mitochondria were harvested using a non-nagarse homogenization method that yields a primarily subsarcolemmal population, although this preparation exhibited substantially higher sensitivity to Ca. None of the PGs had any direct effect on either resting respiration rate or oxidative phosphorylation in the absence of Ca, irrespective of isolation procedure or choice of substrate. The study demonstrates that PGs can augment Ca-related changes in cardiac mitochondrial respiration through an as yet unknown mechanism. The results may be important in understanding the role of endogenously synthesized PGs in cardiac pathology associated with defective intracellular Ca homeostasis.

Animals↗

PubMed2003

Mitochondrial respiration in muscle and liver from cold-acclimated hypothyroid rats.

The effects of long-term cold exposure on muscle and liver mitochondrial oxygen consumption in hypothyroid and normal rats were examined. Thyroid ablation was performed after 8-wk acclimation to 4 degrees C. Hypothyroid and normal controls remained in the cold for an additional 8 wk. At the end of 16-wk cold exposure, all hypothyroid rats were alive and normothermic and had normal body weight. At ambient temperature (24 degrees C), thyroid ablation induced a 65% fall in muscle mitochondrial oxygen consumption, which was reversed by thyroxine but not by norepinephrine administration. After cold acclimation was reached, suppression of thyroid function reduced muscle mitochondrial respiration by 30%, but the hypothyroid values remained about threefold higher than those in hypothyroid muscle in the warm. Blockade of beta- and alpha1-adrenergic receptors in both hypothyroid and normal rats produced hypothermia in vivo and a fall in muscle, liver, and brown adipose tissue mitochondria respiration in vitro. In normal rats, cold acclimation enhanced muscle respiration by 35%, in liver 18%, and in brown adipose tissue 450% over values in the warm. The results demonstrate that thyroid hormones, in the presence of norepinephrine, are major determinants of thermogenic activity in muscle and liver of cold-acclimated rats. After thyroid ablation, cold-induced nonshivering thermogenesis replaced 3,5,3'-triiodothyronine-induced thermogenesis, and normal body temperature was maintained.

Adipose Tissue, Brown↗

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