Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.

Magnetic cloud distortion resulting from propagation through a structured solar wind: Models and observations

Numerical simulations of magnetic clouds (MCs) propagating through a structured solar wind suggest that MC-associated magnetic flux ropes are highly distorted by inhomogeneities in the ambient medium. In particular, a solar wind configuration of fast wind from high latitudes and slow wind at low latitudes, common at periods close to solar minimum, should distort the cross section of magnetic clouds into concave-outward structures. This phenomenon has been reported in observations of shock front orientations, but not in the body of magnetic clouds. In this study an analytical magnetic cloud model based upon a kinematically distorted flux rope is modified to simulate propagation through a structured medium. This new model is then used to identify specific time series signatures of the resulting concave-outward flux ropes. In situ observations of three well studied magnetic clouds are examined with comparison to the model, but the expected concave-outward signatures are not present. Indeed, the observations are better described by the convex-outward flux rope model. This may be due to a sharp latitudinal transition from fast to slow wind, resulting in a globally concave-outward flux rope, but with convex-outward signatures on a local scale.

Shock persistence in a dual economy model of India

This article examines shock persistence in agricultural and industrial output in India. Drawing on the dual economy literature, the linkages between the sectors through the terms of trade are emphasised. However different dual economy models make differing assumptions regarding the categorisation of variables as being either endogenous or exogenous and this distinction is crucial in explaining the pattern of shock persistence. Using annual data for 1955-95, our results show that shocks to both output series are permanent while those to the terms of trade are transient.

Transcriptomic analysis of rhizobium leguminosarum biovar viciae in symbiosis with host plants pisum sativum and Vicia cracca

Rhizobium leguminosarum bv. viciae forms nitrogen-fixing nodules on several legumes, including pea (Pisum sativum) and vetch (Vicia cracca), and has been widely used as a model to study nodule biochemistry. To understand the complex biochemical and developmental changes undergone by R. leguminosarum bv. viciae during bacteroid development, microarray experiments were first performed with cultured bacteria grown on a variety of carbon substrates (glucose, pyruvate, succinate, inositol, acetate, and acetoacetate) and then compared to bacteroids. Bacteroid metabolism is essentially that of dicarboxylate-grown cells (i.e., induction of dicarboxylate transport, gluconeogenesis and alanine synthesis, and repression of sugar utilization). The decarboxylating arm of the tricarboxylic acid cycle is highly induced, as is gamma-aminobutyrate metabolism, particularly in bacteroids from early (7-day) nodules. To investigate bacteroid development, gene expression in bacteroids was analyzed at 7, 15, and 21 days postinoculation of peas. This revealed that bacterial rRNA isolated from pea, but not vetch, is extensively processed in mature bacteroids. In early development (7 days), there were large changes in the expression of regulators, exported and cell surface molecules, multidrug exporters, and heat and cold shock proteins. fix genes were induced early but continued to increase in mature bacteroids, while nif genes were induced strongly in older bacteroids. Mutation of 37 genes that were strongly upregulated in mature bacteroids revealed that none were essential for nitrogen fixation. However, screening of 3,072 mini-Tn5 mutants on peas revealed previously uncharacterized genes essential for nitrogen fixation. These encoded a potential magnesium transporter, an AAA domain protein, and proteins involved in cytochrome synthesis.

Heat-shock response in Arabidopsis thaliana explored by multiplexed quantitative proteomics using differential metabolic labeling

We have developed a general method for multiplexed quantitative proteomics using differential metabolic stable isotope labeling and mass spectrometry. The method was successfully used to study the dynamics of heat-shock response in Arabidopsis thaliana. A number of known heat-shock proteins were confirmed, and some proteins not previously associated with heat shock were discovered. The method is applicable in stable isotope labeling and allows for high degrees of multiplexing.

A similarity solution for a shock reflection problem in isentropic gas dynamics

A one-dimensional shock-reflection test problem in the case of slab, cylindrical, or spherical symmetry is discussed. The differential equations for a similarity solution are derived and solved numerically in conjunction with the Rankie-Hugoniot shock relations.

Efficient shock capturing for isentropic flows using arithmetic averaging

A shock capturing scheme is presented for the equations of isentropic flow based on upwind differencing applied to a locally linearized set of Riemann problems. This includes the two-dimensional shallow water equations using the familiar gas dynamics analogy. An average of the flow variables across the interface between cells is required, and this average is chosen to be the arithmetic mean for computational efficiency, leading to arithmetic averaging. This is in contrast to usual ‘square root’ averages found in this type of Riemann solver where the computational expense can be prohibitive. The scheme is applied to a two-dimensional dam-break problem and the approximate solution compares well with those given by other authors.

An efficient shock capturing scheme for the pseudo-unsteady equations representing steady inviscid flow

An efficient algorithm is presented for the solution of the steady Euler equations of gas dynamics. The scheme is based on solving linearised Riemann problems approximately and in more than one dimension incorporates operator splitting. The scheme is applied to a standard test problem of flow down a channel containing a circular arc bump for three different mesh sizes.

An efficient shock capturing algorithm for compressible flows in a duct of variable cross-section

A numerical scheme is presented for the solution of the Euler equations of compressible flow of a gas in a single spatial co-ordinate. This includes flow in a duct of variable cross-section as well as flow with slab, cylindrical or spherical symmetry and can prove useful when testing codes for the two-dimensional equations governing compressible flow of a gas. The resulting scheme requires an average of the flow variables across the interface between cells and for computational efficiency this average is chosen to be the arithmetic mean, which is in contrast to the usual ‘square root’ averages found in this type of scheme. The scheme is applied with success to five problems with either slab or cylindrical symmetry and a comparison is made in the cylindrical case with results from a two-dimensional problem with no sources.

A shock capturing scheme for steady, supercritical, free-surface flow

Abstract A finite difference scheme is presented for the solution of the two-dimensional shallow water equations in steady, supercritical flow. The scheme incorporates numerical characteristic decomposition, is shock capturing by design and incorporates space-marching as a result of the assumption that the flow is wholly supercritical in at least one space dimension. Results are shown for problems involving oblique hydraulic jumps and reflection from a wall.

Shock capturing scheme for steady, supersonic, isentropic flow

A finite difference scheme is presented for the solution of the two-dimensional equations of steady, supersonic, isentropic flow. The scheme incorporates numerical characteristic decomposition, is shock-capturing by design and incorporates space marching as a result of the assumption that the flow is wholly supersonic in at least one space dimension. Results are shown for problems involving oblique hydraulic jumps and reflection from a wall.

Physiological and proteomic approaches to address heat tolerance during anthesis in rice (Oryza sativa L.)

Episodes of high temperature at anthesis, which in rice is the most sensitive stage to temperature, are expected to occur more frequently in future climates. The morphology of the reproductive organs and pollen number, and changes in anther protein expression, were studied in response to high temperature at anthesis in three rice (Oryza sativa L.) genotypes. Plants were exposed to 6 h of high (38 °C) and control (29 °C) temperature at anthesis and spikelets collected for morphological and proteomic analysis. Moroberekan was the most heat-sensitive genotype (18% spikelet fertility at 38 °C), while IR64 (48%) and N22 (71%) were moderately and highly heat tolerant, respectively. There were significant differences among the genotypes in anther length and width, apical and basal pore lengths, apical pore area, and stigma and pistil length. Temperature also affected some of these traits, increasing anther pore size and reducing stigma length. Nonetheless, variation in the number of pollen on the stigma could not be related to measured morphological traits. Variation in spikelet fertility was highly correlated (r=0.97, n=6) with the proportion of spikelets with ≥20 germinated pollen grains on the stigma. A 2D-gel electrophoresis showed 46 protein spots changing in abundance, of which 13 differentially expressed protein spots were analysed by MS/MALDI-TOF. A cold and a heat shock protein were found significantly up-regulated in N22, and this may have contributed to the greater heat tolerance of N22. The role of differentially expressed proteins and morphology during anther dehiscence and pollination in shaping heat tolerance and susceptibility is discussed.

Nuclear Dbf2-related protein kinases (NDRs) in isolated cardiac myocytes and the myocardium: activation by cellular stresses and by phosphoprotein serine-/threonine-phosphatase inhibitors

The nuclear Dbf2-related protein kinases 1 and 2 (NDR1/2) are closely-related AGC family kinases that are strongly conserved through evolution. In mammals, they are activated inter alia by phosphorylation of an hydrophobic domain threonine-residue [NDR1(Thr-444)/NDR2(Thr-442)] by an extrinsic protein kinase followed by autophosphorylation of a catalytic domain serine-residue [NDR1(Ser-281)/NDR2(Ser-282)]. We examined NDR1/2 expression and regulation in primary cultures of neonatal rat cardiac myocytes and in perfused adult rat hearts. In myocytes, transcripts for NDR2, but not NDR1, were induced by the hypertrophic agonist, endothelin-1. NDR1(Thr-444) and NDR2(Thr-442) were rapidly phosphorylated (maximal in 15-30 min) in myocytes exposed to some phosphoprotein Ser-/Thr-phosphatase 1/2 inhibitors (calyculin A, okadaic acid) and, to a lesser extent, by hyperosmotic shock, low concentrations of H(2)O(2), or chelerythrine. In myocytes adenovirally-transduced to express FLAG-NDR2 (which exhibited a mainly-cytoplasmic localisation), the same agents increased FLAG-NDR2 activity as assessed by in vitro protein kinase assays, indicative of FLAG-NDR2(Ser-282/Thr-442) phosphorylation. Calyculin A-induced phosphorylation of NDR1(Thr-444)/NDR2(Thr-442) and activation of FLAG-NDR2 were inhibited by staurosporine, but not by other protein kinase inhibitors tested. In ex vivo rat hearts, NDR1(Thr-444)/NDR2(Thr-442) were phosphorylated in response to ischaemia-reperfusion or calyculin A. From a pathological viewpoint, we conclude that activities of NDR1 and NDR2 are responsive to cytotoxic stresses in heart preparations and this may represent a previously-unidentified response to myocardial ischaemia in vivo.

The decomposition of some RDX and HMX based materials in the one dimensional time to explosion apparatus. Part 2. Estimating the violence of the cook-off event

Various methods of assessment have been applied to the One Dimensional Time to Explosion (ODTX) apparatus and experiments with the aim of allowing an estimate of the comparative violence of the explosion event to be made. Non-mechanical methods used were a simple visual inspection, measuring the increase in the void volume of the anvils following an explosion and measuring the velocity of the sound produced by the explosion over 1 metre. Mechanical methods used included monitoring piezo-electric devices inserted in the frame of the machine and measuring the rotational velocity of a rotating bar placed on the top of the anvils after it had been displaced by the shock wave. This last method, which resembles original Hopkinson Bar experiments, seemed the easiest to apply and analyse, giving relative rankings of violence and the possibility of the calculation of a “detonation” pressure.

The experience of immediacy: emotion and enlistment in fact-based theatre

This article discusses emotion as a strategy of political agency in post-Thatcherite documentary theatre. The 1990s saw a renaissance in theatre writing based in directness and immediacy but based in two quite different forms of drama, In-Yer-Face theatre and fact-based drama. There are clear distinctions between these forms: the new brutalist writing was aggressively provocative; documentary theatre engaged the audience by revealing an urgent truth. Both claimed a kind of realism that confronted actuality, be that of situation or experience, through forms of theatre that cultivated emotional engagement. In-Yer-Face theatre used emotional shock to penetrate the numb cynicism that its creators perceived. Documentary theatre used observation and the cultivation of sympathy to enlist its audience in a shared understanding of what was hidden, not understood or not noticed. The article analyses the functioning of emotional enlistment to engage the audience politically in two examples of documentary theatre, Black Watch and Guantanamo