Conformational Change of Mitochondrial Complex I Increases ROS Sensitivity During Ischemia

Aims: Myocardial ischemia/reperfusion (I/R) is associated with mitochondrial dysfunction and subsequent cardiomyocyte death. The generation of excessive quantities of reactive oxygen species (ROS) and resultant damage to mitochondrial enzymes is considered an important mechanism underlying reperfusion injury. Mitochondrial complex I can exist in two interconvertible states: active (A) and deactive or dormant (D). We have studied the active/deactive (A/D) equilibrium in several tissues under ischemic conditions in vivo and investigated the sensitivity of both forms of the heart enzyme to ROS.

Results: We found that in the heart, t½ of complex I deactivation during ischemia was 10?min, and that reperfusion resulted in the return of A/D equilibrium to its initial level. The rate of superoxide generation by complex I was higher in ischemic samples where content of the D-form was higher. Only the D-form was susceptible to inhibition by H2O2 or superoxide, whereas turnover-dependent activation of the enzyme resulted in formation of the A-form, which was much less sensitive to ROS. The mitochondrial-encoded subunit ND3, most likely responsible for the sensitivity of the D-form to ROS, was identified by redox difference gel electrophoresis.

Innovation: A combined in vivo and biochemical approach suggests that sensitivity of the mitochondrial system to ROS during myocardial I/R can be significantly affected by the conformational state of complex I, which may therefore represent a new therapeutic target in this setting.

Conclusion: The presented data suggest that transition of complex I into the D-form in the absence of oxygen may represent a key event in promoting cardiac injury during I/R.

Ischemic A/D transition of mitochondrial complex I and its role in ROS generation

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) is a key enzyme in cellular energy metabolism and provides approximately 40% of the proton-motive force that is utilized during mitochondrial ATP production. The dysregulation of complex I function – either genetically, pharmacologically, or metabolically induced – has severe pathophysiological consequences that often involve an imbalance in the production of reactive oxygen species (ROS). Slow transition of the active (A) enzyme to the deactive, dormant (D) form takes place during ischemia in metabolically active organs such as the heart and brain. The reactivation of complex I occurs upon reoxygenation of ischemic tissue, a process that is usually accompanied by an increase in cellular ROS production. Complex I in the D-form serves as a protective mechanism preventing the oxidative burst upon reperfusion. Conversely, however, the D-form is more vulnerable to oxidative/nitrosative damage. Understanding the so-called active/deactive (A/D) transition may contribute to the development of new therapeutic interventions for conditions like stroke, cardiac infarction, and other ischemia-associated pathologies. In this review, we summarize current knowledge on the mechanism of A/D transition of mitochondrial complex I considering recently available structural data and site-specific labeling experiments. In addition, this review discusses in detail the impact of the A/D transition on ROS production by complex I and the S-nitrosation of a critical cysteine residue of subunit ND3 as a strategy to prevent oxidative damage and tissue damage during ischemia–reperfusion injury.

EFFECTS OF ENVIRONMENTAL ANTIOXIDANTS ON PRO-ANGIOGENIC ROS SIGNALLING IN ENDOTHELIAL COLONY FORMING CELLS IN VITRO

Introduction. Endothelial colony-forming cells (ECFCs) hold great cytotherapeutic potential for ischaemic disease. Whilst increasing evidence supports a key role for reactive oxygen species (ROS), specifically those derived from Nox NADPH oxidases, in the underlying angiogenic processes of these and other endothelial cells, such studies investigating the role of redox signalling may be hampered by the standard inclusion of antioxidant agents in endothelial cell media, such as phenol red. Aims. To study the effects of antioxidants present in culture media on pro-angiogenic function of ECFCs in vitro. Methods. Human ECFCs isolated from umbilical cord blood were maintained in media with and without antioxidant components (EGM2 and phenol red-free DMEM, respectively) prior to treatment with pro-oxidant PMA and assessment of their in vitro migratory capacity using a scratch-wound assay to measure pro-angiogenic activity. Results. Our previous work in our group indicated that PMA (500nM) increased ECFC migration in a both a superoxide and NADPH oxidase-dependent manner (control 18.6±2.8, PMA 32.7±6.6% wound closure; n=6, P<0.05), as indicated by attenuation with PEG-SOD and VAS2870. However, inconsistencies in the data generated under varying experimental conditions led us to hypothesise that antioxidant agents in the standard ECFC media may be influencing these effects. Indeed, a direct comparison of cell migration between ECFCs incubated in EGM2 DMEM demonstrated a clear trend towards higher migration in the latter (EGM2 9.0±4.5, DMEM 22.7±6.4%; n=3, P=NS). Similar to our previous EGM2 studies, cell migration was potentiated by PMA (control 11.6±1.6, PMA 25.1±2.8%; n=3, P<0.05), but at a lower dose (100nM), which is consistent with a reduction in media antioxidants. Notably, this response was attenuated by VAS2870 (PMA 37.6±7.3, PMA+VAS2870 10.3±2.9%; n=6, P<0.05), underlining a likely role for Nox NADPH oxidases. Conclusion. Taken together, these data indicate that ECFC migration is sensitive to different endothelial cell growth media, which appears to be dependent upon their antioxidant content. Although further experiments, such as quantification of cellular superoxide generation by dihydroethidium fluorescence may be required to confirm a specific role for antioxidants, such blunting of ROS signalling in vitro is clearly an important consideration which may significantly impact upon data interpretation.

Andrew Marvell's Latin Poetry: From Text to Context (356pp.)

David Norbrook, Review of English Studies 56 (Sept. 2005), 675-6.
‘We have waited a long time for a study of Marvell’s Latin poetry; fortunately, Estelle Haan’s monograph generously makes good the loss ... One of her most intriguing suggestions … is that Marvell may have presented paired poems like ‘Ros’ and ‘On a Drop of Dew’, and the poems to the obligingly named Dr Witty, to his student Maria Fairfax as his own patterns for the pedagogical practice of double translation. Perhaps the most original parts of the book, however, move beyond the familiar canon to cover the generic range of the Latin verse. Haan offers a very full contextualization of the early Horatian Ode to Charles I in seventeenth-century exercises in parodia. In a rewarding reading of the poem to Dr Ingelo she shows how Marvell deploys the language of Ovid’s Tristia to present Sweden as a place of shivering exile, only to subvert this model with a neo-Virgilian celebration of Christina as a virtuous, city-building Dido. She draws extensively on historical as well as literary sources to offer very detailed contextualizations of the poem to Maniban and ‘Scaevola Scotto-Britannus’... This monograph opens up many new ways into the Latin verse, not least because it is rounded off with new texts and prose translations of the Latin poems. These make a substantial contribution in their own right. They are the best and most accurate translations to date (those in Smith’s edition having some lapses); they avoid poeticisms but bring out the structure of the poems' wordplay very clearly. This book brings us a lot closer to seeing Marvell whole.'

ATR-dependent radiation-induced gammaH2AX foci in bystander primary human astrocytes and glioma cells.

Radiotherapy is an important treatment for patients suffering from high-grade malignant gliomas. Non-targeted (bystander) effects may influence these cells' response to radiation and the investigation of these effects may therefore provide new insights into mechanisms of radiosensitivity and responses to radiotherapy as well as define new targets for therapeutic approaches. Normal primary human astrocytes (NHA) and T98G glioma cells were irradiated with helium ions using the Gray Cancer Institute microbeam facility targeting individual cells. Irradiated NHA and T98G glioma cells generated signals that induced gammaH2AX foci in neighbouring non-targeted bystander cells up to 48 h after irradiation. gammaH2AX bystander foci were also observed in co-cultures targeting either NHA or T98G cells and in medium transfer experiments. Dimethyl sulphoxide, Filipin and anti-transforming growth factor (TGF)-beta 1 could suppress gammaH2AX foci in bystander cells, confirming that reactive oxygen species (ROS) and membrane-mediated signals are involved in the bystander signalling pathways. Also, TGF-beta 1 induced gammaH2AX in an ROS-dependent manner similar to bystander foci. ROS and membrane signalling-dependent differences in bystander foci induction between T98G glioma cells and normal human astrocytes have been observed. Inhibition of ataxia telangiectasia mutated (ATM) protein and DNA-PK could not suppress the induction of bystander gammaH2AX foci whereas the mutation of ATM- and rad3-related (ATR) abrogated bystander foci induction. Furthermore, ATR-dependent bystander foci induction was restricted to S-phase cells. These observations may provide additional therapeutic targets for the exploitation of the bystander effect.

Stress resistance and disease resistance in seaweeds: The role of reactive oxygen metabolism

It has become clear over the last 15-20 years that the immediate effect of a wide range of environmental stresses, and of infection, on vascular plants is to increase the formation of reactive oxygen species (ROS) and to impose oxidative stress on the cells. Since 1994, sufficient examples of similar responses in a broad range of marine macroalgae have been described to show that reactive oxygen metabolism also underlies the mechanisms by which seaweeds respond (and become resistant) to stress and infection. Desiccation, freezing, low temperatures, high light, ultraviolet radiation, and heavy metals all tend to result in a gradual and continued buildup of ROS because photosynthesis is inhibited and excess energy results in the formation of singlet oxygen. The response to other stresses (infection or oligosaccharides which signal that infection is occurring, mechanical stress, hyperosmotic shock) is quite different-a more rapid and intense, but short-lived production of ROS, described as an

Signaling factors for irradiated glioma cells induced bystander responses in fibroblasts

The aim of this study was to investigate the signaling factor and its pathway involved in the targeted irradiation-induced bystander response from glioblastoma cells to primary fibroblasts. After co-culturing with a glioblastoma T98G population where a fraction of cells had been individually irradiated with a precise number of helium particles, additional micronucleus (MN) were induced in the non-irradiated human fibroblasts AG01522 cells and its yield was independent of irradiation dose. This bystander MN induction was eliminated by treating the cells with either aminoguanidine (AG), an iNOS inhibitor, or anti-transforming growth factor-beta 1 (anti-TGF-beta 1). In addition, TGF-beta 1 could be released from irradiated T98G cells but this release was inhibited by AG. In consistent, TGF-beta 1 could also be induced from T98G cells treated with diethylamine nitric oxide (DEANO), a donor of nitric oxide (NO). Moreover, the effect of TGF-beta 1 on bystander AG01522 cells was investigated. It was found that reactive oxygen species (ROS) and MN were induced in AG01522 cells after TGF-beta 1 treatment. Our results indicate that, downstream of NO, TGF-beta 1 plays an important role in the targeted T98G cells induced bystander response to AGO cells by further causing DNA damage in vicinal fibroblasts through a ROS related pathway. This study may have implications for properly evaluating the secondary effects of radiotherapy. (C) 2007 Elsevier B.V. All rights reserved.

Real-time imaging of novel spatial and temporal responses to photodynamic stress.

Cells subjected to various forms of stress have been shown to induce bystander responses in nontargeted cells, thus extending the stress response to a larger population. However, the mechanism(s) of bystander responses remains to be clearly identified, particularly for photodynamic stress. Oxidative stress and cell viability were studied on the spatial and temporal levels after photodynamic targeting of a subpopulation of EMT6 murine mammary cancer cells in a multiwell plate by computerized time-lapse fluorescence microscopy. In the targeted population a dose-dependent loss of cell viability was observed in accordance with increased oxidative stress. This was accompanied by increased oxidative stress in bystander populations but on different time scales, reaching a maximum more rapidly in targeted cells. Treatment with extracellular catalase, or the NADPH oxidase inhibitor diphenyleneiodinium, decreased production of reactive oxygen species (ROS) in both populations. These effects are ascribed to photodynamic activation of NADPH-oxidase in the targeted cells, resulting in a rapid burst of ROS formation with hydrogen peroxide acting as the signaling molecule responsible for initiation of these photodynamic bystander responses. The consequences of increased oxidative stress in bystander cells should be considered in the overall framework of photodynamic stress.

A tip-high, Ca2+-interdependent, reactive oxygen species gradient is associated with polarized growth in Fucus serratus zygotes

We report the existence of a tip-high reactive oxygen species (ROS) gradient in growing Fucus serratus zygotes, using both 5-(and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein and nitroblue tetrazolium staining to report ROS generation. Suppression of the ROS gradient inhibits polarized zygotic growth; conversely, exogenous ROS generation can redirect zygotic polarization following inhibition of endogenous ROS. Confocal imaging of fluo-4 dextran distributions suggests that the ROS gradient is interdependent on the tip-high [Ca2+](cyt) gradient which is known to be associated with polarized growth. Our data support a model in which localized production of ROS at the rhizoid tip stimulates formation of a localized tip-high [Ca2+](cyt) gradient. Such modulation of intracellular [Ca2+](cyt) signals by ROS is a common motif in many plant and algal systems and this study extends this mechanism to embryogenesis.

The `Chicken and Egg' of Subjective and Social Factors in Desistance from Crime

It is now widely acknowledged that progression from persistent offending to desistance from crime is the outcome of a complex interaction between subjective/ agency factors and social/environmental factors. A methodological challenge for desistance researchers is to unravel the differential impacts of these internal and external factors and the sequence in which they come into play. Towards this, the present investigation draws on a prospective study of 130 male property offenders, interviewed in the 1990s (the Oxford Recidivism Study), and followed up 10 years later. The analysis supports a `subjective—social model' in which subjective states measured before release have a direct effect on recidivism as well as indirect effects through their impact on social circumstances experienced after release from prison.

Measurement of the duration of X-ray lasing pumped by an optical laser pulse of picosecond duration

Measurements of the duration of X-ray lasing pumped with picosecond pulses from the VULCAN optical laser are obtained using a streak camera with 700 fs temporal resolution. Combined with a temporal smearing due to the spectrometer employed, we have measured X-ray laser pulse durations for Ni-like silver at 13.9 nm with a total time resolution of 1.1 ps. For Ni-like silver, the X-ray laser output has a steep rise followed by an approximately exponential temporal decay with measured full-width at half-maximum (FWHM) of 3.7 (+/-0.5) ps. For Ne-like nickel lasing at 23.1 nm, the measured duration of lasing is approximate to10.7 (+/-1) ps (FWHM). An estimate of the duration of the X-ray laser gain has been obtained by temporally resolving spectrally integrated continuum and resonance line emission. For Ni-like silver, this time of emission is approximate to22 (+/-2) ps (FWHM), while for Ne-like nickel we measure approximate to35 (+/-2) ps (FWHM). Assuming that these times of emission correspond to the gain duration, we show that a simple model consistently relates the gain durations to the measured durations of X-ray lasing. (C) 2002 Elsevier Science B.V. All rights reserved.

A review of X-ray laser development at Rutherford Appleton Laboratory

Recent experiments undertaken at the Rutherford Appleton Laboratory to produce X-ray lasing over the 5-30 nm wavelength range are reviewed. The efficiency of lasing is optimized when the main pumping pulse interacts with a preformed plasma. Experiments using double 75-ps pulses and picosecond pulses superimposed on 300-ps background pulses are described. The use of travelling wave pumping with the approximately picosecond pulse experiments is necessary as the gain duration becomes comparable to the time for the X-ray laser pulse to propagate along the target length. Results from a model taking account of laser saturation and deviations from the speed of light c of the travelling wave and X-ray laser group velocity are presented. We show that X-ray laser pulses as short as 2-3 ps can be produced with optical pumping pulses of approximate to1-ps.