Nuclear activation as a high dynamic range diagnostic of laser-plasma interactions

Proton imaging has become a common diagnostic technique for use in laser-plasma research experiments due to their ability to diagnose electric field effects and to resolve small density differences caused through shock effects. These interactions are highly dependent on the use of radiochromic film (RCF) as a detection system for the particle probe, and produces very high-resolution images. However, saturation effects, and in many cases, damage to the film limits the usefulness of this technique for high-flux particle probing. This paper outlines the use of a new technique using contact radiography of (p,n)-generated isotopes in activation samples to produce high dynamic range 2D images with high spatial resolution and extremely high dynamic range, whilst maintaining both energy resolution and absolute flux measurements. (C)007 Elsevier B.V. All rights reserved.

The purification and properties of phosphonoacetate hydrolase, a novel carbon-phosphorus bond-cleavage enzyme from Pseudomonas fluorescens 23F

A novel, inducible, carbon-phosphorus bond-cleavage enzyme, phosphonoacetate hydrolase, was purified from cells of Pseudomonas fluorescens 23F grown phosphonoacetate. The native enzyme had a molecular mass of approximately 80 kDa and, upon SDS/PAGE, yielded a homogenous protein band with an apparent molecular mass of about 38 kDa. Activity of purified phosphonoacetate hydrolase was Zn2+ dependent and showed pH and temperature optima of approximately 7.8 and 37 degrees C, respectively. The purified enzyme had an apparent K-m of 1.25 mM for its sole substrate phosphonoacetate, and was inhibited by the structural analogues 3-phosphonopropionate and phosphonoformate. The NH2-terminal sequence of the first 19 amino acids displayed no significant similarity to other databank sequences.

EFFECT OF IN-VIVO AND IN-VITRO LOVASTATIN TREATMENT ON MAST-CELL ACTIVATION

The hydroxymethylglutaryl coenzmye A (HMG CoA) reductase inhibitor lovastatin is used to treat hyperlipidaemia. This agent prevents the isoprenylation of some proteins involved in signal transduction processes and inhibits IgE-receptor-linked mediator release from RBL-2H3 cells. In this study the effect of in vivo and in vitro administration of lovastatin on histamine release from rat peritoneal mast cells was examined. Lovastatin (4 mg/kg/day for 2 weeks) inhibited histamine release induced by concanavalin A (con A) from rat peritoneal mast cells of Hooded-Lister rats and both homozygous lean and obese Zucker rats. In contrast, release induced by antirat IgE (anti-IgE) was only significantly inhibited in cells derived from Hooded-Lister rats and that induced by compound 48/ 80 was not altered. Lovastatin (20 mu M, 24 h, in vitro) caused a significant inhibition of the subsequent histamine release to con A, anti-IgE and compound 48/80 but not to the calcium ionophore A 23187. It is important to determine whether such inhibitory effects are also observed after the chronic, clinical administration of lovastatin and other HMG CoA reductase inhibitors.

Histamine dilutions modulate basophil activation

Background:In order to demonstrate that high dilutions of histamine are able to inhibit basophil activation in a reproducible fashion, several techniques were used in different research laboratories.

Ran Is a Potential Therapeutic Target for Cancer Cells with Molecular Changes Associated with Activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK Pathways

Purpose: Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We nowinvestigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities.

Strength development of mortars containing ground granulated blast-furnace slag: Effect of curing temperature and determination of apparent activation energies

The strength development of mortars containing ground granulated blast-furnace slag (ggbs) and portland cement was investigated. Variables were the level of ggbs in the binder, water-binder ratio and curing temperature. All mortars gain strength more rapidly at higher temperatures and have a lower calculated ultimate strength. The early age strength is much more sensitive to temperature for higher levels of ground granulated blast-furnace slag. The calculated ultimate strength is affected to a similar degree for all ggbs levels and water-binder ratios, with only the curing temperature having a significant effect. Apparent activation energies were determined according to ASTM C1074 and were found to vary approximately linearly with ggbs level from 34 kJ/mol for portland cement mortars to around 60 kJ/mol for mortars containing 70% ggbs. The water-binder ratio appears to have little or no effect oil the apparent activation energy. (c) 2005 Elsevier Ltd. All rights reserved.

Protein kinases C: potential targets for intervention in diabetic nephropathy

Protein kinases C are a family of serine threonine protein kinases that play key roles in extracellular signal transduction. Inappropriate activation of protein kinase C has been implicated in the pathophysiology of many diseases, including diabetes mellitus. Indeed, protein kinase C activation may contribute not only to the pathogenesis of diabetic complications such as nephropathy and retinopathy, but also to insulin resistance. Growing awareness that protein kinase C isoforms subserve specific subcellular functions has led to the development of isoform-specific inhibitors, which may be useful investigational tools and therapeutic agents for attenuating the effects of inappropriate protein kinase C activity. Here we review the role played by protein kinases C in diabetic nephropathy and the recent progress that has been made to modulate its activity using specific inhibitors. Curr Opin Nephrol Hypertens 7:563-570. (C) 1998 Lippincott Wiiliams & Wilkins.

Selective Orthosteric Free Fatty Acid Receptor 2 (FFA2) Agonists IDENTIFICATION OF THE STRUCTURAL AND CHEMICAL REQUIREMENTS FOR SELECTIVE ACTIVATION OF FFA2 VERSUS FFA3

Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [S-35] guanosine 5'-(3-O-thio) triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp(3)-hybridized alpha-carbons preferentially activate FFA3, whereas ligands with sp(2)- or sp-hybridized alpha-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors.

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up-regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF-beta function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP-2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C-zeta to the leading edge of migrating cells. Inhibition of CTGF-induced protein kinase C-zeta activity with a myristolated PKC-zeta inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC-zeta kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF-induced migration compared with wild-type. Furthermore, CTGF stimulated phosphorylation and activation of GSK-3beta. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC-zeta and GSK-3beta. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.

Selective Hydrogenation of alpha,beta-Unsaturated Aldehydes and Ketones using Novel Manganese Oxide and Platinum Supported on Manganese Oxide Octahedral Molecular Sieves as Catalysts

The selective hydrogenation of ,-unsaturated aldehydes and ketones has been studied using ketoisophorone and cinnamaldehyde as model substrates using manganese oxide octahedral molecular sieve (OMS-2) based catalysts. For the first time, OMS-2 has been shown to be an efficient and selective hydrogenation catalyst. High selectivities for either the CC or CO double bond at approximate to 100% conversion were achieved by using OMS-2 and platinum supported on OMS-2 catalysts. Density functional theory (DFT) calculations showed that the dissociation of H2 on OMS-2 was water assisted and occurred on the surface Mn of OMS-2(001) that had been modified by an adsorbed H2O molecule. The theoretically calculated activation barrier was in good agreement with the experimentally determined value for the hydrogenation reactions, indicating that H2 dissociation on OMS-2 is likely to be the rate-determining step. A significant increase in the rate of reaction was observed in the presence of Pt as a result of the enhancement of H2 dissociative adsorption and subsequent reaction on the Pt or spillover of the hydrogen to the OMS-2 support. The relative adsorption strengths of ketoisophorone and cinnamaldehyde on the OMS-2 support compared with the Pt were found to determine the product selectivity.

XBP1 mRNA splicing triggers an autophagic response in endothelial cells through BECLIN-1 transcriptional activation

Sustained activation of X-box-binding protein 1 (XBP1) results in endothelial cell (EC) apoptosis and atherosclerosis development. The present study provides evidence that XBP1 mRNA splicing triggered an autophagic response in ECs by inducing autophagic vesicle formation and markers of autophagy BECLIN-1 and microtubule-associated protein 1 light chain 3ß (LC3-ßII). Endostatin activated autophagic gene expression through XBP1 mRNA splicing in an inositol-requiring enzyme 1a (IRE1a)-dependent manner. Knockdown of XBP1 or IRE1a by shRNA in ECs ablated endostatin-induced autophagosome formation. Importantly, data from arterial vessels from XBP1 EC conditional knock-out (XBP1eko) mice demonstrated that XBP1 deficiency in ECs reduced the basal level of LC3ß expression and ablated response to endostatin. Chromatin immunoprecipitation assays further revealed that the spliced XBP1 isoform bound directly to the BECLIN-1 promoter at the region from nt -537 to -755. BECLIN-1 deficiency in ECs abolished the XBP1-induced autophagy response, whereas spliced XBP1 did not induce transcriptional activation of a truncated BECLIN-1 promoter. These results suggest that XBP1 mRNA splicing triggers an autophagic signal pathway through transcriptional regulation of BECLIN-1.

Klebsiella pneumoniae subverts the activation of inflammatory responses in a NOD1-dependent manner

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Subversion of inflammation is essential for pathogen survival during infection. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response although the molecular bases are currently unknown. Here we unveil a novel strategy employed by a pathogen to counteract the activation of inflammatory responses. K. pneumoniae attenuates pro-inflammatory mediators-induced IL-8 secretion. Klebsiella antagonizes the activation of NF-?B via the deubiquitinase CYLD and blocks the phosphorylation of mitogen-activated protein kinases (MAPKs) via the MAPK phosphatase MKP-1. Our studies demonstrate that K. pneumoniae has evolved the capacity to manipulate host systems dedicated to control the immune balance. To exert this anti-inflammatory effect, Klebsiella engages NOD1. In NOD1 knock-down cells, Klebsiella neither induces the expression of CYLD and MKP-1 nor blocks the activation of NF-?B and MAPKs. Klebsiella inhibits Rac1 activation; and inhibition of Rac1 activity triggers a NOD1-mediated CYLD and MKP-1 expression which in turn attenuates IL-1ß-induced IL-8 secretion. A capsule (CPS) mutant does not attenuate the inflammatory response. However, purified CPS neither reduces IL-1ß-induced IL-8 secretion nor induces the expression of CYLD and MKP-1 thereby indicating that CPS is necessary but not sufficient to attenuate inflammation.

Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells

Outer membrane protein A (OmpA) is a class of proteins highly conserved among the Enterobacteriaceae family and throughout evolution. Klebsiella pneumoniae is a capsulated Gram-negative pathogen. It is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by a lack of an early inflammatory response. Data from our laboratory indicate that K. pneumoniae CPS helps to suppress the host inflammatory response. However, it is unknown whether K. pneumoniae employs additional factors to modulate host inflammatory responses. Here, we report that K. pneumoniae OmpA is important for immune evasion in vitro and in vivo. Infection of A549 and normal human bronchial cells with 52OmpA2, an ompA mutant, increased the levels of IL-8. 52145-?wca ompA, which does not express CPS and ompA, induced the highest levels of IL-8. Both mutants could be complemented. In vivo, 52OmpA2 induced higher levels of tnfa, kc, and il6 than the wild type. ompA mutants activated NF-?B, and the phosphorylation of p38, p44/42, and JNK MAPKs and IL-8 induction was via NF-?B-dependent and p38- and p44/42-dependent pathways. 52OmpA2 engaged TLR2 and -4 to activate NF-?B, whereas 52145-?wca ompA activated not only TLR2 and TLR4 but also NOD1. Finally, we demonstrate that the ompA mutant is attenuated in the pneumonia mouse model. The results of this study indicate that K. pneumoniae OmpA contributes to attenuate airway cell responses. This may facilitate pathogen survival in the hostile environment of the lung. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Endocrine disrupting effects of ochratoxin A at the level of nuclear receptor activation and steroidogenesis

Ochratoxin A (OTA) is a mycotoxin and extrolite of fungi which has been reported in a range of foods. This study uses mammalian reporter gene assays (RGAs) with natural steroid receptors and the H295R steroidogenesis assay to assess the endocrine disrupting activity of OTA.

At the receptor level, OTA (within a concentration range of 0.25–2500 ng/ml) did not induce an agonistic response in an oestrogen, androgen, progestagen or glucocorticoid RGA. An antagonistic effect was observed in all of the RGAs at the highest concentration tested (2500 ng/ml). However, while there was no significant cytotoxic effect observed in the MTT (thiazolyl blue tetrazolium bromide) cell viability assay at this concentration, there was a corresponding change in cell morphology which may be related to the resulting antagonistic effect.

At the hormone production level, H295R cells were used as a steroidogenesis model and exposed to OTA (within a concentration range of 0.1–1000 ng/ml). Treatment of the cells with 1000 ng/ml OTA increased the production of estradiol (117 ± 14 ng/ml) over 3 times that of the solvent control (36 ± 9 pg/ml). Western blotting confirmed an increase in aromatase protein.

Overall the results indicate that OTA does not appear to interact with steroid receptors but has the potential to cause endocrine disruption by interfering with steroidogenesis. This is the first study identifying the effect OTA may have on production of the steroid hormone estradiol.