Role of the Netrin-like Domain of Procollagen C-Proteinase Enhancer-1 in the Control of Metalloproteinase Activity

The netrin-like (NTR) domain is a feature of several extracellular proteins, most notably the N-terminal domain of tissue inhibitors of metalloproteinases (TIMPs), where it functions as a strong inhibitor of matrix metalloproteinases and some other members of the metzincin superfamily. The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate. These observations point to a new mechanism whereby binding to cell surface-associated or extracellular heparin-like sulfated glycosaminoglycans might provide a means to accelerate procollagen processing in specific cellular and extracellular microenvironments

WEIBEL INSTABILITY IN A BI-MAXWELLIAN LASER FUSION PLASMA

We are interested in this paper to analyse the Weibel instability driven by the plasma temperature anisotropy in the corona of a high intense laser created plasma. The unperturbed electronic distribution function, of the anisotropic corona is supposed to be a bi

Radiation Hardness Studies in a CCD with High-Speed Column Parallel Readout

Charge Coupled Devices (CCDs) have been successfully used in several high energy physics experiments over the past two decades. Their high spatial resolution and thin sensitive layers make them an excellent tool for studying short-lived particles. The Linear Collider Flavour Identification (LCFI) collaboration is developing Column-Parallel CCDs (CPCCDs) for the vertex detector of the International Linear Collider (ILC). The CPCCDs can be read out many times faster than standard CCDs, significantly increasing their operating speed. The results of detailed simulations of the charge transfer inefficiency (CTI) of a prototype CPCCD are reported and studies of the influence of gate voltage on the CTI described. The effects of bulk radiation damage on the CTI of a CPCCD are studied by simulating the effects of two electron trap levels, 0.17 and 0.44 eV, at different concentrations and operating temperatures. The dependence of the CTI on different occupancy levels (percentage of hit pixels) and readout frequencies is also studied. The optimal operating temperature for the CPCCD, where the effects of the charge trapping are at a minimum, is found to be about 230 K for the range of readout speeds proposed for the ILC. The results of the full simulation have been compared with a simple analytic model.Comment: 3 pages, 6 figures; presented at IEEE'07, ALCPG'07, ICATPP'0

Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1

Aberrant transforming growth factor–β (TGF-β) signaling is a hallmark of the stromal microenvironment in cancer. Dickkopf-3 (Dkk-3), shown to inhibit TGF-β signaling, is downregulated in prostate cancer and upregulated in the stroma in benign prostatic hyperplasia, but the function of stromal Dkk-3 is unclear. Here we show that DKK3 silencing in WPMY-1 prostate stromal cells increases TGF-β signaling activity and that stromal cellconditioned media inhibit prostate cancer cell invasion in a Dkk-3-dependent manner. DKK3 silencing increased the level of the cell-adhesion regulator TGF-β–induced protein (TGFBI) in stromal and epithelial cell-conditioned media, and recombinant TGFBI increased prostate cancer cell invasion. Reduced expression of Dkk-3 in patient tumors was associated with increased expression of TGFBI. DKK3 silencing reduced the level of extracellular matrix protein-1 (ECM-1) in prostate stromal cell-conditioned media but increased it in epithelial cell-conditioned media, and recombinant ECM-1 inhibited TGFBI-induced prostate cancer cell invasion. Increased ECM1 and DKK3 mRNA expression in prostate tumors was associated with increased relapse-free survival. These observations are consistent with a model in which the loss of Dkk-3 in prostate cancer leads to increased secretion of TGFBI and ECM-1, which have tumor-promoting and tumor-protective roles, respectively. Determining how the balance between the opposing roles of extracellular factors influences prostate carcinogenesis will be key to developing therapies that target the tumor microenvironment

Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k2 = 21±1 s−1) was much higher than the HNE deacylation step (k3 = 0.57±0.05 s−1). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k1 2.4-fold and reducing k−1 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k2 value, whereas the k3 value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs

Genome profiling of ERBB2-amplified breast cancers

<p>Abstract</p> <p>Background</p> <p>Around 20% of breast cancers (BC) show <it>ERBB2 </it>gene amplification and overexpression of the ERBB2 tyrosine kinase receptor. They are associated with a poor prognosis but can benefit from targeted therapy. A better knowledge of these BCs, genomically and biologically heterogeneous, may help understand their behavior and design new therapeutic strategies.</p> <p>Methods</p> <p>We defined the high resolution genome and gene expression profiles of 54 <it>ERBB2</it>-amplified BCs using 244K oligonucleotide array-comparative genomic hybridization and whole-genome DNA microarrays. Expression of ERBB2, phosphorylated ERBB2, EGFR, IGF1R and FOXA1 proteins was assessed by immunohistochemistry to evaluate the functional ERBB2 status and identify co-expressions.</p> <p>Results</p> <p>First, we identified the <it>ERBB2</it>-<it>C17orf37</it>-<it>GRB7 </it>genomic segment as the minimal common 17q12-q21 amplicon, and <it>CRKRS </it>and <it>IKZF3 </it>as the most frequent centromeric and telomeric amplicon borders, respectively. Second, GISTIC analysis identified 17 other genome regions affected by copy number aberration (CNA) (amplifications, gains, losses). The expression of 37 genes of these regions was deregulated. Third, two types of heterogeneity were observed in <it>ERBB2</it>-amplified BCs. The genomic profiles of estrogen receptor-postive (ER+) and negative (ER-) <it>ERBB2</it>-amplified BCs were different. The WNT/β-catenin signaling pathway was involved in ER- <it>ERBB2</it>-amplified BCs, and <it>PVT1 </it>and <it>TRPS1 </it>were candidate oncogenes associated with ER+ <it>ERBB2</it>-amplified BCs. The size of the <it>ERBB2 </it>amplicon was different in inflammatory (IBC) and non-inflammatory BCs. <it>ERBB2</it>-amplified IBCs were characterized by the downregulated and upregulated mRNA expression of ten and two genes in proportion to CNA, respectively. IHC results showed (i) a linear relationship between <it>ERBB2 </it>gene amplification and its gene and protein expressions with a good correlation between ERBB2 expression and phosphorylation status; (ii) a potential signaling cross-talk between EGFR or IGF1R and ERBB2, which could influence response of <it>ERBB2</it>-positive BCs to inhibitors. FOXA1 was frequently coexpressed with ERBB2 but its expression did not impact on the outcome of patients with <it>ERBB2</it>-amplified tumors.</p> <p>Conclusion</p> <p>We have shown that ER+ and ER- <it>ERBB2</it>-amplified BCs are different, distinguished <it>ERBB2 </it>amplicons in IBC and non-IBC, and identified genomic features that may be useful in the design of alternative therapeutical strategies.</p

Chemical modifications by ionic liquid-inspired cations improve the activity and the stability of formate dehydrogenase in [MMIm][Me2PO4]

International audienceThe formate dehydrogenase (FDH, EC: 1.2. 1.2) from Candida boidinii was found to be inactivated and unstable in the presence of high concentration (>50%) of the water soluble dimethylimidazolium dimethyl phosphate ([MMIm][Me2PO4]) ionic liquid. In order to circumvent this problem, the enzyme was chemically modified by cations usually present in ionic liquids: cholinium (1), hydroxyethyl-methylimidazolium (2) and hydroxypropyl-methylimidazolium (3) cations were activated with carbonyldiimidazole before being reacted with the FDH leading to a heterogeneous population of 6–7 biocatalysts. FDH modified by (1) or (3) led to 3–9 modifications while FDH modified by (2) led to 6 proteins presenting 7–12 grafted cations. Specific activity of the modified enzymes was decreased by a 2.5–3-fold factor (0.10–0.15 μmol min−1 mg−1) compared to the non-modified FDH (0.33 μmol min−1 mg−1) when assayed in carbonate buffer (pH 9.7, 25 mM). After modification, the FDH still present 0.06 μmol min−1 mg−1 in 70% [MMIm][Me2PO4] (v:v) (30–45% of their activity in aqueous buffer) while the native enzyme is inactive at this ionic liquid concentration, proving the efficiency of this strategy. The half-life of the modified enzyme is also increased by a 5-fold factor after modification by (1) (t1/2 of 9 days) and by a 3-fold factor after modification by (2) or (3) (t1/2 of 6 and 5 days respectively) in aqueous solution. When stored in 37.5% [MMIm][Me2PO4] (v:v), both modified and unmodified FDH have an increased half-life (t1/2 of 6–9 days). This grafting strategy is found to be good methods to mimic and study the stabilizing effect of ionic liquids on enzymes