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P Nagley

Publications and source records attributed to P Nagley.

At least 19 recordsLinked to original sources
PubMed2005

Smac/DIABLO is not released from mitochondria during apoptotic signalling in cells deficient in cytochrome c.

We have characterised the apoptotic defects in cells null for cytochrome c (cyt c-/-). Such cells treated with staurosporine (STS) exhibited translocation to the mitochondria and activation of the proapoptotic signalling molecule Bax but failed to release Smac/DIABLO from these organelles, judged by both confocal microscopy and Western blotting. While reference cells expressing cytochrome c released both it and Smac/DIABLO under a variety of conditions of apoptotic induction, we have never observed release of Smac/DIABLO from cyt c-/- cells. We eliminate the possibility that proteasomal degradation of cytosolically localised Smac/DIABLO is responsible for our failure to visualise the protein outside the mitochondria. Our findings indicate an unanticipated nexus between release of cytochrome c and Smac/DIABLO from mitochondria, previously thought to be a more or less synchronised event early in apoptosis. We suggest that the failure of cyt c-/- cells to release Smac/DIABLO after recruitment of Bax to mitochondria represents an extreme manifestation of some inherent difference in the regulation of release of these two proteins from mitochondria.

Animals↗
PubMed2003

High frequency of mitochondrial genome instability in human endometrial carcinomas.

To investigate the occurrence of somatic mitochondrial DNA (mtDNA) mutations in human primary endometrial carcinomas, we sequenced the D-loop region, the 12S and 16S rRNA genes of mtDNA of cancer tissues and their matched normal controls. About 56% (28 out of 50) of cases carry one or more somatic changes in mtDNA including deletion, point mutation and mitochondrial microsatellite instability (mtMSI), namely the change in length of short base-repetitive sequences of mtDNA. In particular, mtMSI was frequently detected in 89% (25 out of 28) of all the cases carrying somatic changes followed by point mutations (25%; seven out of 28) and deletion (3.5%; one out of 28). The CCCCCTCCCC sequences located in the Hypervariable Regions I and II of the D-loop and 12S rRNA gene are instability hot spot regions in endometrial carcinomas. It is suggested that errors in replication may account for the high frequency of mtMSI in human endometrial carcinomas. The relatively high prevalence of mtMSI may be a potential new tool for detection of endometrial cancer.

Case-Control Studies↗
PubMed2001

High incidence of somatic mitochondrial DNA mutations in human ovarian carcinomas.

To investigate the potential role of somatic mitochondrial DNA (mtDNA) mutations in tumorigenesis, the occurrence of mutations in mtDNA of ovarian carcinomas was studied. We sequenced the D-loop region of mtDNA of 15 primary ovarian carcinomas and their matched normal controls. Somatic mtDNA mutations were detected in 20% (3 of 15) tumor samples carrying single or multiple changes. Complete sequence analysis of the mtDNA genomes of another 10 pairs of primary ovarian carcinomas and control tissues revealed somatic mtDNA mutations in 60% (6 of 10) of tumor samples. Most of these mutations were homoplasmic, and most were T-->C or G-->A transitions, but one represented a differential length within a run of identical C residues. A region of mtDNA sequence including the 16S and 12S rRNA genes, the D-loop and the cytochrome b gene, may represent the zone of preferred mtDNA mutation in ovarian cancer. The high incidence of mtDNA mutations found in ovarian carcinomas and other human cancers suggests that genetic instability of mtDNA might play a significant role in tumorigenesis.

DNA, Mitochondrial↗
PubMed2001

The protonophore CCCP induces mitochondrial permeability transition without cytochrome c release in human osteosarcoma cells.

Mitochondrial permeability transition (MPT) and cytochrome c redistribution from mitochondria are two events associated with apoptosis. We investigated whether an MPT event obligatorily leads to cytochrome c release in vivo. We have previously shown that treatment of human osteosarcoma cells with the protonophore m-chlorophenylhydrazone (CCCP) for 6 h induces MPT and mitochondrial swelling without significant cell death. Here we demonstrate that release of cytochrome c does not occur and the cells remain viable even after 72 h of treatment with CCCP. Bax is not mobilized to mitochondria under these conditions. However, subsequent exposure of CCCP-treated cells to etoposide or staurosporine for 48 h results in rapid cell death and cytochrome c release that is accompanied by Bax association with mitochondria, demonstrating competency of these mitochondria to release cytochrome c with additional triggers. Our findings suggest that MPT is not a sufficient condition, in itself, to effect cytochrome c release.

Bone Neoplasms↗
PubMed2000

Insights into ATP synthase assembly and function through the molecular genetic manipulation of subunits of the yeast mitochondrial enzyme complex.

Development of an increasingly detailed understanding of the eucaryotic mitochondrial ATP synthase requires a detailed knowledge of the stoichiometry, structure and function of F(0) sector subunits in the contexts of the proton channel and the stator stalk. Still to be resolved are the precise locations and roles of other supernumerary subunits present in mitochondrial ATP synthase complexes, but not found in the bacterial or chloroplast enzymes. The highly developed system of molecular genetic manipulation available in the yeast Saccharomyces cerevisiae, a unicellular eucaryote, permits testing for gene function based on the effects of gene disruption or deletion. In addition, the genes encoding ATP synthase subunits can be manipulated to introduce specific amino acids at desired positions within a subunit, or to add epitope or affinity tags at the C-terminus, enabling questions of stoichiometry, structure and function to be addressed. Newly emerging technologies, such as fusions of subunits with GFP are being applied to probe the dynamic interactions within mitochondrial ATP synthase, between ATP synthase complexes, and between ATP synthase and other mitochondrial enzyme complexes.

Genes, Fungal↗
PubMed2000

A cytochrome c-GFP fusion is not released from mitochondria into the cytoplasm upon expression of Bax in yeast cells.

To study Bax-induced release of cytochrome c in vivo, we have expressed a cytochrome c-GFP (green fluorescent protein) fusion in Saccharomyces cerevisiae cells null for the expression of the endogenous cytochrome. We show here that cytochrome c-GFP is efficiently localised to mitochondria and able to function as an electron carrier between complexes III and IV of the respiratory chain. Strikingly, while natural cytochrome c is released into the cytoplasm upon expression of Bax, the cytochrome c-GFP fusion is not. Nevertheless, cells co-expressing Bax and the cytochrome c-GFP fusion die, indicating that mitochondrial release of cytochrome c is not essential for cell death to occur in yeast. The failure to release cytochrome c-GFP is presumed to arise from increased bulk due to the GFP moiety. We propose that in intact yeast cells, Bax-induced release of cytochrome c into the cytoplasm occurs through a selective pore and not as a consequence of the non-specific breakage of the mitochondrial outer membrane.

Animals↗
PubMed2000

The oligomycin axis of mitochondrial ATP synthase: OSCP and the proton channel.

Oligomycin has long been known as an inhibitor of mitochondrial ATP synthase, putatively binding the F(o) subunits 9 and 6 that contribute to proton channel function of the complex. As its name implies, OSCP is the oligomycin sensitivity-conferring protein necessary for the intact enzyme complex to display sensitivity to oligomycin. Recent advances concerning the structure and mechanism of mitochondrial ATP synthase have led to OSCP now being considered a component of the peripheral stator stalk rather than a central stalk component. How OSCP confers oligomycin sensitivity on the enzyme is unknown, but probably reflects important protein-protein interactions made within the assembled complex and transmitted down the stator stalk, thereby influencing proton channel function. We review here our studies directed toward establishing the stoichiometry, assembly, and function of OSCP in the context of knowledge of the organization of the stator stalk and the proton channel.

Adenosine Triphosphatases↗
PubMed2000

Modulation at a distance of proton conductance through the Saccharomyces cerevisiae mitochondrial F1F0-ATP synthase by variants of the oligomycin sensitivity-conferring protein containing substitutions near the C-terminus.

We have sought to elucidate how the oligomycin sensitivity-conferring protein (OSCP) of the mitochondrial F(1)F(0)-ATP synthase (mtATPase) can influence proton channel function. Variants of OSCP, from the yeast Saccharomyces cerevisiae, having amino acid substitutions at a strictly conserved residue (Gly166) were expressed in place of normal OSCP. Cells expressing the OSCP variants were able to grow on nonfermentable substrates, albeit with some increase in generation time. Moreover, these strains exhibited increased sensitivity to oligomycin, suggestive of modification in functional interactions between the F(1) and F(0) sectors mediated by OSCP. Bioenergetic analysis of mitochondria from cells expressing OSCP variants indicated an increased respiratory rate under conditions of no net ATP synthesis. Using specific inhibitors of mtATPase, in conjunction with measurement of changes in mitochondrial transmembrane potential, it was revealed that this increased respiratory rate was a result of increased proton flux through the F(0) sector. This proton conductance, which is not coupled to phosphorylation, is exquisitely sensitive to inhibition by oligomycin. Nevertheless, the oxidative phosphorylation capacity of these mitochondria from cells expressing OSCP variants was no different to that of the control. These results suggest that the incorporation of OSCP variants into functional ATP synthase complexes can display effects in the control of proton flux through the F(0) sector, most likely mediated through altered protein-protein contacts within the enzyme complex. This conclusion is supported by data indicating impaired stability of solubilized mtATPase complexes that is not, however, reflected in the assembly of functional enzyme complexes in vivo. Given a location for OSCP atop the F(1)-alpha(3)beta(3) hexamer that is distant from the proton channel, then the modulation of proton flux by OSCP must occur "at a distance." We consider how subtle conformational changes in OSCP may be transmitted to F(0).

Adenosine Triphosphatases↗
PubMed2000

Topology and proximity relationships of yeast mitochondrial ATP synthase subunit 8 determined by unique introduced cysteine residues.

We have used site-directed chemical labelling to demonstrate the membrane topology and to identify neighbouring subunits of subunit 8 (Y8) in yeast mitochondrial ATP synthase (mtATPase). Unique cysteine residues were introduced at the N or C-terminus of Y8 by site-directed mutagenesis. Expression and targeting to mitochondria in vivo of each of these variants in a yeast Y8 null mutant was able to restore activity to an otherwise nonfunctional ATP synthase complex. The position of each introduced cysteine relative to the inner mitochondrial membrane was probed with thiol-specific nonpermeant and permeant reagents in both intact and lysed mitochondria. The data indicate that the N-terminus of Y8 is located in the intermembrane space of mitochondria whereas the C-terminus is located within the mitochondrial matrix. The proximity of Y8 to other proteins of mtATPase was tested using heterobifunctional cross-linking reagents, each with one thiol-specific reactive group and one nonspecific, photoactivatible reactive group. These experiments revealed the proximity of the C-terminal domain of Y8 to subunits d and f, and that of the N-terminal domain to subunit f. It is concluded that Y8 possesses a single transmembrane domain which extends across the inner membrane of intact mitochondria. As subunit d is a likely component of the stator stalk of mitochondrial ATP synthase, we propose, on the basis of the observed cross-links, that Y8 may also be part of the stator stalk.

Amino Acid Sequence↗
PubMed2000

Tolerance to ischemia and hypoxia is reduced in aged human myocardium.

BACKGROUND: Recovery of cardiac function after cardiac surgery and other interventional cardiac procedures in elderly patients is inferior to that in younger patients, suggesting that the aged myocardium is more sensitive to ischemia and other stresses. Although convincing data from animal studies of senescence now exist, there is a dearth of controlled in vitro studies that examine the specific response of aged human myocardium to the stress of hypoxia or ischemia. OBJECTIVE: We sought to determine the effect of age on the capacity of human atrial trabeculae to recover contractile function after in vitro hypoxic or ischemic stress. METHODS: Atrial pectinate trabeculae were dissected from the tip of 58 right atrial appendages harvested during an operation in patients aged between 34 and 89 years and electrically stimulated at 1 Hz in oxygenated Ringer's solution at 37 degrees C. Tissues experienced 30 minutes of either hypoxia (N(2) and perfusate) or simulated ischemia (humidified N(2) without perfusate) and were returned to normoxia for recovery of function for 30 minutes. Developed force and other contractile variables were determined during each period. RESULTS: Under normoxic conditions, no significant age difference was observed for any contractile function variable. However, after hypoxia, the old (70-89 years) and intermediate age groups (60-69 years) showed reduced recovery of developed force (48.5% +/- 22.2% [n = 11] and 44.9% +/- 19% [n = 12], respectively) compared with that found (66.4% +/- 19.7% [n = 15]) in the younger (34-59 years) group (mean +/- SD, P =.02). Similarly, after simulated ischemia, the groups of 70- to 89-year-old and 60- to 69-year-old subjects showed reduced recovery of developed force (35.7% +/- 17% [n = 5] and 51.1% +/- 11.8% [n = 9], respectively) compared with that found (68.2% +/- 10.4% [n = 6]) in the group of 34- to 59-year-old subjects (P =.01). Multivariable analysis, comparing 20 factors of surgical patient characteristics and recovery of developed force, found that only age (P =.01) and hypertension (P =.01) were predictors of reduced recovery of developed force after either hypoxia or simulated ischemia. CONCLUSIONS: In aged human atrial myocardium, the capacity to recover contractile function after in vitro hypoxia or simulated ischemia is reduced compared with the younger myocardium of mature adults. These findings suggest that enhanced myocardial protective strategies may be indicated for elderly patients undergoing cardiac surgery.

Adult↗
PubMed1999

Single copies of subunits d, oligomycin-sensitivity conferring protein, and b are present in the Saccharomyces cerevisiae mitochondrial ATP synthase.

In the mitochondrial ATP synthase (mtATPase) of the yeast Saccharomyces cerevisiae, the stoichiometry of subunits d, oligomycin-sensitivity conferring protein (OSCP), and b is poorly defined. We have investigated the stoichiometry of these subunits by the application of hexahistidine affinity purification technology. We have previously demonstrated that intact mtATPase complexes incorporating a Hex6-tagged subunit can be isolated via Ni2+-nitrilotriacetic acid affinity chromatography (Bateson, M., Devenish, R. J., Nagley, P., and Prescott, M. (1996) Anal. Biochem. 238, 14-18). Strains were constructed in which Hex6-tagged versions of subunits d, OSCP, and b were coexpressed with the corresponding wild-type subunit. This coexpression resulted in a mixed population of mtATPase complexes containing untagged wild-type and Hex6-tagged subunits. The stoichiometry of each subunit was then assessed by determining whether or not the untagged wild-type subunit could be recovered from Ni2+-nitrilotriacetic acid purifications as an integral component of those complexes absorbed by virtue of the Hex6-tagged subunit. As only the Hex6-tagged subunit was recovered from such purifications, we demonstrate that the stoichiometry of subunits d, OSCP, and b in yeast is 1 in each case.

Adenosine Triphosphatases↗
PubMed1999

Concurrent increase of oxidative DNA damage and lipid peroxidation together with mitochondrial DNA mutation in human lung tissues during aging--smoking enhances oxidative stress on the aged tissues.

Although mutation of mitochondrial DNA (mtDNA) in human tissues has been established to associate with intrinsic aging, the impact of environmental factors on the formation and accumulation of mtDNA mutations and oxidative DNA damage in human tissues is poorly understood. We have investigated the levels of mtDNA with the 4977-bp deletion and A3243G point mutation, oxidative DNA damage (indicated by the formation of 8-hydroxy-2'-deoxyguanosine, 8-OH-dG), and lipid peroxides in lung tissues from smokers and nonsmokers of subjects of different ages. The results showed concurrent age-dependent increase of the 4977-bp deleted mtDNA (P < 0.001), 8-OH-dG (P < 0.05), and lipid peroxides (P < 0.05) in the human lung. In the group of subjects above 60 years old, smokers had more extensive DNA damage and lipid peroxidation than did the nonsmokers. However, the levels of mtDNA with the 4977-bp deletion and A3243G point mutation in the lung of smokers were not significantly different from those of the age-matched nonsmokers. Taken together, these results suggest that accumulation of mtDNA with the 4977-bp deletion together with oxidative DNA damage and lipid peroxides is associated with aging and that smoking enhances oxidative damage in human lung tissues.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Mitochondrial permeability transition and swelling can occur reversibly without inducing cell death in intact human cells.

Severe disruption of mitochondrial function is generally considered to provide a powerful trigger for apoptosis in mammalian cells. We report here that intact cells may undergo the mitochondrial permeability transition and mitochondria swell in a fully reversible manner, without inducing cell death. Cultured human osteosarcoma cells (143B TK-) stained with JC-1, MitoTracker dyes, or calcein plus Co2+ were imaged by confocal microscopy to visualize changes of mitochondrial membrane potential (DeltaPsim), morphology, and permeability transition, respectively, during treatment with a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells rapidly exhibited mitochondrial permeability transition and swelling after addition of CCCP, but the swelling subsided within hours, leaving mitochondria that appeared in punctate form, not filamentous as before CCCP treatment. Cyclosporin A impeded the permeability transition and swelling, although complete inhibition was not observed. Cells survived the dissipation of DeltaPsim by CCCP for up to 6 h without developing any obvious cell damage or signs of apoptosis. With the restoration of DeltaPsim after removal of CCCP (following 6 h of CCCP treatment), permeability transition pores were closed. These results suggest that none of the following events represent a point of no return in the process of apoptotic cell death: loss of DeltaPsim, mitochondrial permeability transition, or mitochondrial swelling.

Aldehydes↗
PubMed1999

Mitochondrial DNA deletions in human cardiac tissue show a gross mosaic distribution.

The variability of mitochondrial DNA (mtDNA) deletional patterns has been investigated in adjacent slices of human heart atrium. Using quantitative PCR we found differential abundances of one particular mtDNA deletion, that of 4977 bp (mtDNA4977), among sets of adjacent slices of right atrial trabeculae pectinatae from 10 subjects. Some subjects had relatively constant abundance of mtDNA4977 among the tissue slices, while others covered a wide range. A qualitative PCR procedure was used to visualize the patterns of multiple deletions within an 8.64-kb segment of the mtDNA genome, in the same set of atrial trabeculae samples. Some subjects showed completely different multiple deletional patterns in each of the trabeculae slices analyzed. There was no correlation between the variation of the abundance of mtDNA4977 and that of the multiple deletions. The results are consistent with the notion that the occurrence of mtDNA deletions during aging is a random process, involving their production throughout the lifetime of an individual. In this view, the patterns of new deletions are superimposed on those already accumulated by propagation and segregation of mutations formed earlier in life.

Adult↗
PubMed1999

Coenzyme Q10 improves the tolerance of the senescent myocardium to aerobic and ischemic stress: studies in rats and in human atrial tissue.

The inferior recovery of cardiac function after interventional cardiac procedures in elderly patients compared to younger patients suggests that the aged myocardium is more sensitive to stress. We report two studies that demonstrate an age-related deficit in myocardial performance after aerobic and ischemic stress and the capacity of CoQ10 treatment to correct age-specific diminished recovery of function. In Study 1 the functional recovery of young (4 mo) and senescent (35 mo) isolated working rat hearts after aerobic stress produced by rapid electrical pacing was examined. After pacing, the senescent hearts, compared to young, showed reduced recovery of pre-stress work performance. CoQ10 pretreatment (daily intraperitoneal injections of 4 mg/kg CoQ10 for 6 weeks) in senescent hearts improved their recovery to match that of young hearts. Study 2 tested whether the capacity of human atrial trabeculae (obtained during surgery) to recover contractile function, following ischemic stress in vitro (60 min), is decreased with age and whether this decrease can be reversed by CoQ10. Trabeculae from older individuals (> or = 70 yr) showed reduced recovery of developed force after simulated ischemia compared to younger counterparts (< 70 yr). Notably, this age-associated effect was prevented in trabeculae pretreated in vitro (30 min at 24 degrees C) with CoQ10 (400 MicroM). We measured significantly lower CoQ10 content in trabeculae from > or = 70 yr patients. In vitro pretreatment raised trabecular CoQ10 content to similar levels in all groups. We conclude that, compared to younger counterparts, the senescent myocardium of rats and humans has a reduced capacity to tolerate ischemic or aerobic stress and recover pre-stress contractile performance, however, this reduction is attenuated by CoQ10 pretreatment.

Aerobiosis↗
PubMed1999

Bioenergetic and structural consequences of allotopic expression of subunit 8 of yeast mitochondrial ATP synthase. The hydrophobic character of residues 23 and 24 is essential for maximal activity and structural stability of the enzyme complex.

Subunit 8 (Y8), a mitochondrially encoded subunit of the F0 sector of the F1F0-ATP synthase is essential for oxidative phosphorylation. We have previously introduced the technique of allotopic expression to study the structure/function of Y8, whereby an artificial Y8 gene is expressed in the nucleus of cells lacking a functional mitochondrial Y8, thus generating assembly of a functional F1F0-ATPase complex. In this paper we show that when a gene encoding an essentially unmodified version of Y8 is allotopically expressed, ATP synthesis and hydrolysis rates, as well as efficiency of oxidative phosphorylation, were similar to those of the parental wild-type strain in which Y8 is naturally expressed in mitochondria. We then tested the requirement for the hydrophobicity of the central domain (residues 14-32), which possibly represents a transmembrane stem, by introducing adjacent negative charges at different positions of Y8. One of the variants thus generated, which carries the double substitution Leu23-->Asp, Leu24-->Asp, when expressed in a strain lacking endogenous Y8, gave rise to cells which grew very slowly by oxidative phosphorylation. Measurement of bioenergetic parameters showed two major defects in these cells relative to control cells allotopically expressing unmodified Y8. First, the activity of the F1F0-ATP synthase was significantly decreased. ATP synthesis and state 3 of respiration were reduced by approximately 30-40%. ATP hydrolysis was reduced by approximately 30% and was almost insensitive to the F0 inhibitor oligomycin. Second, the physical coupling between the two sectors of the enzyme, as well as the stability of the F1 sector itself, were affected as shown by decreased recovery of F0 sector [8, 9, b, oligomycin sensitivity-conferring protein (OSCP), d, h and f] and F1 sector (alpha, gamma, delta) subunits in immunoprecipitates of ATP synthase. This study indicates that Y8 not only performs an important role in the structure of the mitochondrial complex but also in its activity. We conclude that the hydrophobic character of amino acids 23 and 24 in the middle of the putative transmembrane stem of Y8 is essential for coupling proton transport through F0 to ATP synthesis on F1.

Adenosine Triphosphate↗