Transcriptional profiling of the LPS induced NF-kappaB response in macrophages

BACKGROUND: Exposure of macrophages to bacterial products such as lipopolysaccharide (LPS) results in activation of the NF-kappaB transcription factor, which orchestrates a gene expression programme that underpins the macrophage-dependent immune response. These changes include the induction or repression of a wide range of genes that regulate inflammation, cell proliferation, migration and cell survival. This process is tightly regulated and loss of control is associated with conditions such as septic shock, inflammatory diseases and cancer. To study this response, it is important to have in vitro model systems that reflect the behaviour of cells in vivo. In addition, it is necessary to understand the natural differences that can occur between individuals. In this report, we have investigated and compared the LPS response in macrophage derived cell lines and peripheral blood mononuclear cell (PBMC) derived macrophages. RESULTS: Gene expression profiles were determined following LPS treatment of THP-1 cells for 1 and 4 hours. LPS significantly induced or repressed 72 out of 465 genes selected as being known or putative NF-kappaB target genes, which exhibited 4 temporal patterns of expression. Results for 34 of these genes, including several genes not previously identified as LPS target genes, were validated using real time PCR. A high correlation between microarray and real time PCR data was found. Significantly, the LPS induced expression profile of THP-1 cells, as determined using real time PCR, was found to be very similar to that of human PBMC derived macrophages. Interestingly, some differences were observed in the LPS response between the two donor PBMC macrophage populations. Surprisingly, we found that the LPS response in U937 cells was dramatically different to both THP-1 and PBMC derived macrophages. CONCLUSION: This study revealed a dynamic and diverse transcriptional response to LPS in macrophages, involving both the induction and repression of gene expression in a time dependent manner. Moreover, we demonstrated that the LPS induced transcriptional response in the THP-1 cell line is very similar to primary PBMC derived macrophages. Therefore, THP-1 cells represent a good model system for studying the mechanisms of LPS and NF-kappaB dependent gene expression.

Forward modelling to determine the observational signatures of white-light imaging and interplanetary scintillation for the propagation of an interplanetary shock in the ecliptic plane

Recent coordinated observations of interplanetary scintillation (IPS) from the EISCAT, MERLIN, and STELab, and stereoscopic white-light imaging from the two heliospheric imagers (HIs) onboard the twin STEREO spacecraft are significant to continuously track the propagation and evolution of solar eruptions throughout interplanetary space. In order to obtain a better understanding of the observational signatures in these two remote-sensing techniques, the magnetohydrodynamics of the macro-scale interplanetary disturbance and the radio-wave scattering of the micro-scale electron-density fluctuation are coupled and investigated using a newly constructed multi-scale numerical model. This model is then applied to a case of an interplanetary shock propagation within the ecliptic plane. The shock could be nearly invisible to an HI, once entering the Thomson-scattering sphere of the HI. The asymmetry in the optical images between the western and eastern HIs suggests the shock propagation off the Sun–Earth line. Meanwhile, an IPS signal, strongly dependent on the local electron density, is insensitive to the density cavity far downstream of the shock front. When this cavity (or the shock nose) is cut through by an IPS ray-path, a single speed component at the flank (or the nose) of the shock can be recorded; when an IPS ray-path penetrates the sheath between the shock nose and this cavity, two speed components at the sheath and flank can be detected. Moreover, once a shock front touches an IPS ray-path, the derived position and speed at the irregularity source of this IPS signal, together with an assumption of a radial and constant propagation of the shock, can be used to estimate the later appearance of the shock front in the elongation of the HI field of view. The results of synthetic measurements from forward modelling are helpful in inferring the in-situ properties of coronal mass ejection from real observational data via an inverse approach.

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.

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.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine ( DCVC) resulted in a > 1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Characterisation of amyloid fibril formation by small heat-shock chaperone proteins human alpha A-, alpha beta- and R120G alpha B-Crystallins

alpha B-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alpha B-crystallin in detail, and also that of alpha A-crystallin and the disease-related mutant R120G (alpha B-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alpha B-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. H-1 NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alpha B-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils. (C) 2007 Elsevier Ltd. All rights reserved.

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.