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Biomedical subjects

M D Esposti

Publications and source records attributed to M D Esposti.

15 recordsLinked to original sources
PubMed1999

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol.

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.

Adenylate Kinase↗
PubMed1999

Bcl-2 and mitochondrial oxygen radicals. New approaches with reactive oxygen species-sensitive probes.

Investigations into the capacity of the Bcl-2 protein to prevent apoptosis have targeted mitochondria as key sites of the preventative action accorded by Bcl-2 to cells. Using novel approaches with fluorescence probes and autofluorescence detection of endogenous NAD(P)H, we have examined the effects of expressing Bcl-2 in the Bcl-2 negative Burkitt's lymphoma cell line Daudi. We evaluated for the first time the effect of Bcl-2 expression on the intracellular distribution and production of hydrogen peroxide, under basal conditions and after treatment with apoptosis inducing agents, ceramide analogs and tumor necrosis factor (TNF)-alpha. Increased availability of mitochondrial NAD(P)H was detected in Bcl-2-expressing cells and was correlated with an increased constitutive mitochondrial production of hydrogen peroxide. Although production of hydrogen peroxide was increased by either C(6)-ceramide or TNF-alpha in Bcl-2 negative Daudi cells commensurate with the early phases of apoptosis, this increase did not occur in Bcl-2-expressing cells. Thus, Bcl-2 appears to allow cells to adapt to an increased state of oxidative stress, fortifying the cellular anti-oxidant defenses and counteracting the radical overproduction imposed by different cell death stimuli. Furthermore, we report altered cytological features of mitochondria during the early phases of apoptosis induced by C(6)-ceramide and TNF-alpha. In particular, mitochondria changed in appearance, clustering in the perinuclear region and Bcl-2 expression prevented these changes from occurring.

Ceramides↗
PubMed1999

Inhibition of mitochondrial oxidative phosphorylation induces hyper-expression of glutamic acid decarboxylase in pancreatic islet cells.

It has been hypothesised that mitochondrial dysfunction in pancreatic beta cells could produce hyper-expression of glutamic acid decarboxylase (GAD), a major autoantigen in insulin-dependent diabetes mellitus (IDDM) (Degli Esposti, M. and Mackay, I.R. Diabetologia 40: 352-356, 1997). Here we report that specific inhibition of mitochondrial respiration enhances the expression of GAD in both foetal mouse pancreatic tissue and hamster HIT-T15 cells. Inhibitors of NADH-ubiquinone oxidoreductase (complex I) seem to be particularly effective in increasing the expression of GAD in both foetal mouse pancreas and HIT-T15 hamster beta cells, especially in the presence of nutrients such as arginine and glucose. These results represent the first evidence that GAD expression is enhanced under conditions that are toxic to pancreatic beta cells, and establish a link between mitochondrial dysfunction and expression of IDDM autoantigens.

Animals↗
PubMed1997

Measurement of the membrane potential generated by complex I in submitochondrial particles.

To investigate the energy-conserving function of the NADH:ubiquinone reductase (complex I), we have selected oxonol VI [bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol] as the most sensitive probe for measuring the reactions of membrane potential generation in submitochondrial particles. Calibration of the oxonol signals with potassium diffusion potentials shows a non-linear response after a threshold around -50 mV. Thermodynamic evaluations indicate that the upper limit of the oxonol response to the potential generated by complex I is around -220 mV, which is close to the maximal protonmotive force in coupled submitochondrial particles. NADH addition to particles in which ubiquinol oxidation is blocked by inhibitors of other respiratory complexes generates oxonol signals corresponding to membrane potentials of -130 to -180 mV. These signals are produced by about four turnovers of the complex reducing endogenous ubiquinone (i.e. non-steady-state conditions) and are equivalent to a charge separation similar to that of the antimycin-sensitive reactions of ubiquinol:cytochrome c reductase (complex III). The transient oxonol signals under non-steady-state conditions are thus informative of crucial steps in the electrogenic reactions catalyzed by complex I. The possible nature of these electrogenic reactions is discussed in relation to proposed mechanisms for complex I.

Animals↗
PubMed1996

The interaction of Q analogs, particularly hydroxydecyl benzoquinone (idebenone), with the respiratory complexes of heart mitochondria.

We have studied the interaction of idebenone (2,3-dimethoxy-5-methy-6-(10-hydroxy)decyl-1,4-benzoquinone) with the energy-conserving complexes of the respiratory chain in beef heart mitochondria and compared its energetic efficiency with that of other analogs of coenzyme Q. Idebenone is a very effective substrate for succinate:Q reductase and ubiquinol:cytochrome c reductase, but it is clearly a poor substrate for NADH:Q reductase (complex I). Indeed, idebenone is a strong inhibitor of both the redox and proton pumping activity of complex I, showing effects in part similar to those of coenzyme Q-2. However, the mechanism of idebenone interaction with complex I may be different from that of Q-2 because of its different sensitivity to inhibitors. The possible relevance of the present findings to the therapeutic use of idebenone is discussed.

Animals↗
PubMed1996

Inhibition of mitochondrial complex I may account for IDDM induced by intoxication with the rodenticide Vacor.

Human intoxication with the rodenticide Vacor [N-3-pyridylmethyl-N'-p-nitrophenyl urea or 1-(4-nitrophenyl)-3-(3-pyridylmethyl) urea] induces acute IDDM. We report here that Vacor specifically inhibits the NADH:ubiquinone reductase activity of complex I in mammalian mitochondria. The activity of other respiratory enzymes of mitochondria is unaffected by Vacor at concentrations that completely inhibit the redox and energetic function of complex I. Vacor inhibition of complex I activity quantitatively correlates with the inhibition of insulin release in insulinoma cells and pancreatic islets and is also consistent with the doses reported in cases of human poisoning. These results indicate that the toxic and diabetogenic action of Vacor primarily derives from the inhibition of mitochondrial respiration of NAD-linked substrates in the high-energy demanding cells of the pancreatic islets. This newly identified mechanism of the pathological effects resulting from Vacor intoxication could constitute a paradigm in which to understand environmental or metabolic causes of IDDM.

Animals↗
PubMed1993

Mitochondrial cytochrome b: evolution and structure of the protein.

Cytochrome b is the central redox catalytic subunit of the quinol: cytochrome c or plastocyanin oxidoreductases. It is involved in the binding of the quinone substrate and it is responsible for the transmembrane electron transfer by which redox energy is converted into a protonmotive force. Cytochrome b also contains the sites to which various inhibitors and quinone antagonists bind and, consequently, inhibit the oxidoreductase. Ten partial primary sequences of cytochrome b are presented here and they are compared with sequence data from over 800 species for a detailed analysis of the natural variation in the protein. This sequence information has been used to predict some aspects of the structure of the protein, in particular the folding of the transmembrane helices and the location of the quinone- and heme-binding pockets. We have observed that inhibitor sensitivity varies greatly among species. The comparison of inhibition titrations in combination with the analysis of the primary structures has enabled us to identify amino acid residues in cytochrome b that may be involved in the binding of the inhibitors and, by extrapolation, quinone/quinol. The information on the quinone-binding sites obtained in this way is expected to be both complementary and supplementary to that which will be obtained in the future by mutagenesis and X-ray crystallography.

Amino Acid Sequence↗
PubMed1991

The kinetic mechanism of ubiquinol: cytochrome c reductase at steady state.

The steady-state kinetics of ubiquinol: cytochrome c reductase (cytochrome bc1 complex) is analyzed in this work. The graphical pattern of the titrations is clearly indicative of a ping-pong mechanism, but the two products ubiquinone and reduced cytochrome c behave competitively with their substrate and noncompetitively with the other substrate. Hence, the mechanism of the reductase is of a ping-pong two-site type. A minimal reaction scheme for the enzymatic mechanism is proposed and approximate values of its rate constants are deduced on the assumption that each substrate is in rapid equilibrium at its catalytic site. This has been substantiated by presteady-state measurements of the reduction and oxidation of cytochrome b by a short-chain homolog of ubiquinol. Values of the rate constants of the reaction scheme have been deduced from the steady-state titrations for a series of 2,3-dimethoxy-5-methyl quinols having different hydrophobic substituents in position 6 of the ring. The results provide a quantitative estimation of the specificity of the quinol catalytic site in the transmembrane portion of the bc1 complex. In particular, a reasonable correlation is found between the rate of the second-order reaction of quinols with the enzyme and their solubility in lipids.

Animals↗
PubMed1989

Prediction and comparison of the haem-binding sites in membrane haemoproteins.

This article contains a comparative review of the structural properties of membrane haemoproteins, with particular emphasis on the possible similarities of the haem-binding peptides. A procedure is suggested for identifying the peptides which may bind membrane-buried haems on the basis of the primary sequences of the proteins. The integration of this procedure with the information deduced by refined hydropathy analysis indicates that the basic structural model for the haemoproteins which interact with quinones may be a transmembrane helical bundle containing the haem(s) at its centre. Structural similarities exist in the sequence of hydrophobic segments that are predicted to bind the membrane-buried haems of b-cytochromes which interact with quinones. The predicted haem-binding sites show similarities also with the peptides that bind the non-haem iron in the bacterial reaction centres, and this may be correlated to the common function of interacting with quinones and their intermediates. The analysis of the amino-acid composition of the proposed ligand peptides in the membrane haemoproteins examined has provided a molecular rationale for explaining the highly anisotropic low-spin EPR signal which is characteristic of many membrane-bound b-cytochromes.

Amino Acid Sequence↗