Importance of the redox state of cytochrome c during caspase activation in cytosolic extracts

Hampton, Blake M; Zhivotovsky, Boris; Slater, F. G. Andrew; Burgess, H. David; Orrenius, Sten

doi:10.1042/bj3290095

Cited by 118 publications

(81 citation statements)

References 14 publications

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“…Secondly, cyanide will be directly toxic to airway cells and, finally, cyanide may also contribute to the relative inability of neutrophils to clear P. aeruginosa infection in the CF lung [15,16]. In the concentrations reported, cyanide has been shown to inhibit the function of the enzyme myeloperoxidase and prevent production of hypochlorous acid during the oxidative burst [17]. Cyanide may also impair neutrophil migration and cause morphological changes related to damage to the cell cytoskeleton [18].…”

Section: Discussionmentioning

confidence: 99%

Bacterial cyanogenesis occurs in the cystic fibrosis lung

Sanderson¹,

Wescombe²,

Kirov³

et al. 2008

Eur Respir J

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The cystic fibrosis (CF) lung environment is poorly defined, but data suggest that bacteria may encounter reduced oxygen tensions and possibly an anaerobic environment. Pseudomonas aeruginosa produces the potent toxin cyanide under strictly microaerobic conditions. Evidence of bacterial cyanogenesis in the CF lung was investigated in the present study by measuring sputum cyanide concentrations.Sputum cyanide was measured in seven stable CF patients, as well as before and after intravenous antibiotic therapy during a hospital admission in a further eight patients experiencing acute exacerbations. All patients were chronically infected with P. aeruginosa. Comparative sputum data were obtained from nine CF patients with no documented P. aeruginosa infection and 10 healthy, nonsmoking normal controls.High levels of cyanide were detected in all the P. aeruginosa-infected stable CF patients (median (range) 0.56 (0.37-2.81) mg?mL -1 ), and in seven out of eight acute sputum samples (0.73 (0-1.43) mg?mL -1 ). In contrast, cyanide was not detectable in sputum from eight out of nine CF patients without P. aeruginosa infection or in any of the normal controls. Intravenous antibiotic treatment significantly reduced sputum cyanide levels (median 0.73 to median 0.0 mg?mL -1 ).The cyanide detected indicates that the cystic fibrosis lung provides a predominantly microaerobic environment for Pseudomonas aeruginosa. Cyanide is likely to be a potentially important virulence factor in Pseudomonas aeruginosa-infected cystic fibrosis patients.

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Section: Discussionmentioning

confidence: 99%

Bacterial cyanogenesis occurs in the cystic fibrosis lung

Sanderson¹,

Wescombe²,

Kirov³

et al. 2008

Eur Respir J

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“…We have been unable to do this due mainly to the difficulty of maintaining the injected cytochrome c in the oxidized state in vivo. Hampton et al 14 in their in vitro experiments used minimal quantities of cyanide to maintain the protein in its oxidized state to demonstrate its efficacy in activating caspases. We are currently grappling with the technical difficulties of using a similar approach in vivo.…”

Section: Membrane Potential In Xenopus Oocytes During Cell Deathmentioning

confidence: 99%

“…An in vitro model indeed indicated that either oxidation state is effective in the activation of caspases. 14 15,16 In our experiments we compared the kinetics of plasma membrane depolarization induced by oxidized cytochrome c and dithionite-reduced cytochrome c added to the cytosol by microinjection. In the time dependence of the change of DC, shown in Figure 3A, we observe a time lag of *20 min for the oxidized state of cytochrome c relative to the reduced one.…”

Section: Membrane Potential In Xenopus Oocytes During Cell Deathmentioning

confidence: 99%

Resting membrane potential as a marker of apoptosis: studies on Xenopus oocytes microinjected with cytochrome c

2001

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Observation of the electrical potential difference across the cell membrane is described as a new method for monitoring apoptosis of a single cell. The resting membrane potential (DC) of Xenopus oocytes has been recorded in real time following microinjection of cytochrome c. Soon after microinjection, DC becomes less negative and attains a new constant value with a half time, t m , of about 35 (+5) min at all cytochrome c concentrations greater than 1 mM. The cytosol extract of cytochrome c-injected oocytes shows DEVD proteolytic activity characteristic of aspartate specific proteases, implicating an apoptotic death pathway. In response to the delivery of cytochrome c into the cytosol, caspases are activated within 7 min while the changes in DC begin to occur after about 30 min. The method described here will be potentially useful to assess the effectiveness of cell death regulators and modulators of synthetic and biological origin, and the results presented shed light on the currently debated issue of the importance of the redox state of cytochrome c in the initiation of apoptosis. Cell Death and Differentiation (2001) 8, 63 ± 69.

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“…6,13 In this manner S-100 cytosols from various cell sources can be shown to generate specific type-II caspase activity (DEVD-ase) dependent upon holoCyt-c and dATP additions. 10,14 Therefore, we choose to determine if the capacity of human skeletal muscle cytosols qualitatively paralleled the holoCyt-c/dATP-mediated activation of type-II caspases seen in haematopoietic cells. Our observations suggest that these cytosols were devoid of holoCyt-c-inducible caspase activity and that this result is indicative of a necessary endogenous mechanism for the regulation and maintainance of skeletal muscle integrity.…”

Section: Introductionmentioning

confidence: 99%

Human skeletal muscle cytosols are refractory to cytochrome c-dependent activation of type-II caspases and lack APAF-1

et al. 1999

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Apoptotic regulatory mechanisms in skeletal muscle have not been revealed. This is despite indications that remnant apoptotic events are detected following exercise, muscle injury and the progression of dystrophinopathies. The recent elicitation of a cytochrome c-mediated induction of caspases has led to speculation regarding a cytochrome c mechanism in muscle. We demonstrate that cytosols from skeletal muscle biopsies from healthy human volunteers lack the ability to activate type-II caspases by a cytochrome c-mediated pathway despite the confirmed presence of both procaspase-3 and -9. This was not due to the presence of an endogenous inhibitor, as the muscle cytosols enhanced caspase activity when added to a control cytosol, subsequently activated by cytochrome c and dATP. In addition, we demonstrate that muscle cytosols lack the apoptosis protease activator protein-1 (APAF-1), both at the protein and mRNA levels. These data indicate that human skeletal muscle cells will be refractory to mitochondrial-mediated events leading to apoptosis and thus can escape a major pro-apoptotic regulatory mechanism. This may reflect an evolutionary adaptation of cell survival in the presence of the profusion of mitochondria required for energy generation in motility.

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Importance of the redox state of cytochrome c during caspase activation in cytosolic extracts

Cited by 118 publications

References 14 publications

Bacterial cyanogenesis occurs in the cystic fibrosis lung

Bacterial cyanogenesis occurs in the cystic fibrosis lung

Resting membrane potential as a marker of apoptosis: studies on Xenopus oocytes microinjected with cytochrome c

Human skeletal muscle cytosols are refractory to cytochrome c-dependent activation of type-II caspases and lack APAF-1

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