Metastasis-inducing DNA Regulates the Expression of the Osteopontin Gene by Binding the Transcription Factor Tcf-4

Small 1,000-bp fragments of genomic DNA obtained from human malignant breast cancer cell lines when transfected into a benign rat mammary cell line enhance transcription of the osteopontin gene and thereby cause the cells to metastasize in syngeneic rats. To identify the molecular events underlying this process, transient cotransfections of an osteopontin promoter-reporter construct and fragments of one metastasis-inducing DNA (Met-DNA) have identified the active components in the Met-DNA as the binding sites for the T-cell factor (Tcf) family of transcription factors. Incubation of cell extracts with active DNA fragments containing the sequence CAAAG caused retardation of their mobilities on polyacrylamide gels, and Western blotting identified Tcf-4, beta-catenin, and E-cadherin in the relevant DNA complexes in vitro. Transfection of an expression vector for Tcf-4 inhibited the stimulated activity of the osteopontin promoter-reporter construct caused by transiently transfected active fragments of Met-DNA or permanently transfected Met-DNA. This stimulated activity of the osteopontin promoter-reporter construct is accompanied by an increase in endogenous osteopontin mRNA but not in fos or actin mRNAs in the transfected cells. Permanent transfection of the benign rat mammary cell line with a 20-bp fragment from the Met-DNA containing the Tcf recognition sequence CAAAG caused an enhanced permanent production of endogenous osteopontin protein in vitro and induced the cells to metastasize in syngeneic rats in vivo. The corresponding fragment without the CAAAG sequence was without either effect. Therefore, the regulatory effect of the C9-Met-DNA is exerted, at least in part, by a CAAAG sequence that can sequester the endogenous inhibitory Tcf-4 and thereby promote transcription of osteopontin, the direct effector of metastasis in this system.

Citizenship training in school from the perspective of future primary education teachers

The aim of this paper is to recognize the images that prospective elementary school teachers have about citizenship and citizenship education in school. The qualitative methodology with an ethnographic approach is used for that deep interview of ethnographic kind and analysis of institutional documents, for example, curricula, course syllabus and graduated profi le. The revision of documents helps to a better understanding of discourses. The analysis of the corpus obtained after the application of the paradigm of codifi cation proposed in the established theory. As the principal fi ndings we way point out that the concept of citizens that is common among prospective elementary school teachers is reduced to the school environment because they consider that the promotion of citizenship helps to the incorporation of children to society, thus reproducing a propaedeutic view of education.

On the road from consultation cynicism to energising e-consultation

A major concern in recent political discourse is that government has become both isolated from and unresponsive to its citizens. Democracy, by definition, demands a two-way flow of communication between government and civil society and it is now commonly argued that ICTs have the potential to facilitate such improved flows of communication--hence, e-democracy and e-consultation. The preliminary research findings presented here are part of a larger ongoing research project on e-consultation on the island of Ireland (see http://e-consultation.org). The paper initially draws on focus group discussions on the theme of (e)consultation conducted amongst activist citizens. High levels of frustration, scepticism and cynicism were expressed on the form, nature and process of extant consultation processes. The main focus addressed in this paper, however, is on how these citizens envisage ICT being used in future e-consultations. In general, most focus group participants were open to the use of ICT in future e-consultation processes but the consensus was that community groups did not currently have access to an appropriate level or range of infrastructure, technologies or skills. As a follow up to the focus group findings the research group ran a number of demonstrations on e-consultation technologies with invited activist citizens. Technologies introduced included chat room, video-conferencing, WikiPedia, WebIQ, Zing and others. The main preliminary findings and feedback from one such demonstration, and our own observations, are then presented which suggest that the potential does exist for using e-consultation technologies in local democracy and in local government to drive positive change in the government-citizen relationship. We present no na�¯ve solutions here; we merely point to some possibilities and we acknowledge that ICT alone is very unlikely to be a panacea for the declining levels of citizen participation in most democratic societies.

Detection of cathepsin S cysteine protease in human brain tumour microdialysates in vivo.

Microdialysis enables the chemistry of extracellular ?uid in body tissues to be measured. Extracellular proteases such as the cysteine protease, cathepsin S (CatS), are thought to facilitate astrocytoma invasion. Microdialysates obtained from human brain tumoursin vivo were subjected to cathepsin S activity and ELISA assays. Cathepsin S ELISA expression was detected in ?ve out of 10 tumour microdialysates, while activity was detected in ?ve out of 11 tumour microdialysates. Cathepsin S expression was also detected in microdialysate from the normal brain control although no activity was found in the same sample. While some re?nements to the technique are necessary, the authors demonstrate the feasibility and safety of microdialysis in human astrocytomasin vivo. Characterisation of the extracellular environment of brain tumoursin vivo using microdialysis may be a useful tool to identify the protease pro?le of brain tumours.

Measles virus and classical Hodgkin lymphoma: no evidence for a direct association.

A proportion of Hodgkin lymphoma (HL) cases are causally associated with the Epstein-Barr virus (EBV) but the aetiology of the remaining cases remains obscure. Over the last 3 decades several studies have found an association between HL and measles virus (MV) including a recent cohort study describing the detection of MV antigens in Hodgkin and Reed-Sternberg cells, the tumour cells in HL. In the present study we looked at the relationship between history of MV infection and risk of developing HL in a population-based, case/control study of HL. In addition we used immunohistochemistry and RT-PCR to look for direct evidence of MV in HL biopsies. There was no significant difference in the proportion of cases reporting previous measles compared to controls in the entire data set or when young adults were considered separately. Using a robust immunohistochemical assay for MV infection, we failed to find evidence of MV in biopsies from 97 cases of HL and RT-PCR studies similarly gave negative results. This study therefore provides no evidence that MV is directly involved in the development of HL. However, when age at first reported MV infection was investigated, significant differences emerged with children infected before school-age having higher risk, especially of EBV-ve HL, when compared with children infected at older ages; the interpretation of these latter results is unclear.

The in-service teacher trainer: profile, career path and duties

This article is the result of a doctoral thesis that aims to understand the reality of teacher trainers at a given moment, when their professional role changes from being a managing training advisor to a role that is more centred on process counselling. Using in-depth interviews, we defined the personal and professional profile of training counsellors in the Balearic Islands, their career path, the process for their inclusion in Teacher Centres (CEP) and, finally, the duties and skills that they perform as teacher trainers. The data that has been collected shows the coexistence of different professional roles in the group of education advisors. Moreover, it also indicates a lack of definition of the profile needed to access an advisor's position. There are indeed some coincidences determining the access route to such a position when it comes to the teaching profession since their leadership qualities and group dynamics expertise are a common indicator in most cases. The research also shows that they are subjected not only to a wide range of roles and tasks but also to a vast array of competences required to tackle the education advisor's tasks.

Speeds of sound, isentropic compressibilities, and excess molar volumes of cycloalkanes, alkanes and aromatic hydrocarbons at 303.15 K. II. Results for cycloalkanes+aromatic hydrocarbons

The speeds of sound u, isentropic compressibilities ?S, molar sound functions R, excess isentropic compressibilities ?SE and excess molar volumes VE for eight binary mixtures of cyclopentane, cyclohexane, cyclooctane and methylcyclohexane with benzene and of cyclohexane with toluene, ethyl benzene, p-xylene and propyl benzene at 303.15 K are reported. The effects of molecular sizes and shapes of the component molecules and of interaction energy in the mixture have been discussed. The Prigogine–Flory–Patterson theory has been applied to analyze the present binary mixtures along with the mixtures of cis- and trans-decalins with benzene and toluene taken from the literature.

Densities, speeds of sound, isentropic compressibilities, refractive indexes, and viscosities of tetrahydrofuran with haloalkane or alkyl ethanoate at T = 303.15 K

Isentropic compressibilities ?S, excess isentropic compressibilities image, excess molar volumes VE, viscosity deviations ??, and excess Gibbs energy of activation of viscous flow ?G*E for nine binary mixtures of C4H8O with CCl4, CHCl3, CHCl2CHCl2, 1-C6H13Cl, 1-C6H13Br, CH3CO2CH3, CH3CO2C2H5, CH3CO2C4H9, and CH3CO2C5H11 at 303.15 K have been derived from experimental densities ?, speeds of sound u, refractive indexes nD and viscosities ?. The limiting values of excess partial molar volumes of C4H8O at infinite dilution image in different solvents have been estimated. The results obtained for dynamic viscosity of binary mixtures were used to test the semi-empirical relations of Grunberg–Nissan, Tamura–Kurata, Hind–McLaughlin–Ubbelohde, Katti–Chaudhri, McAllister, Heric, and Auslaender. Finally, the experimental refractive indexes were compared with the predicted results for Lorentz–Lorenz, Dale–Gladstone, Eykman, Arago–Boit, Newton, Oster, Heller, and Wiener equations.

Speeds of sound, isentropic compressibilities, viscosities, and excess molar volumes of binary mixtures of alkanoates with tetra- and trichloromethanes at 303.15 K

Speeds of sound u, isentropic compressibilities ?S, viscosities ?, excess isentropic compressibilities ?SE, excess molar volumes VE, viscosity deviations ??, and excess Gibbs energies of activation ?G*E of viscous flow have been investigated for six binary mixtures of diethyl malonate, diethyl bromomalonate, and ethyl chloroacetate with tetra- and trichloromethane at 303.15 K. The values of ?SE, VE, ??, and ?G*E are highly dependent on the type of components involved and the composition curves are unsymmetrical. The results obtained for viscosity of binary mixtures were used to test the semi-empirical relations of Grunberg-Nissan, Tamura-Kurata, Hind-McLaughlin-Ubbelohde, Katti-Chaudhri, McAllister, Heric-Brewer and Auslaender. The experimental speeds of sound have been analyzed in terms of collision factor theory and free length theory of solutions.

Protein kinase B/Akt activity is involved in renal TGF-beta 1-driven epithelial-mesenchymal transition in vitro and in vivo

The molecular pathogenesis of diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is complex and not fully understood. Transforming growth factor-beta (TGF-beta1) plays a critical role in many fibrotic disorders, including DN. In this study, we report protein kinase B (PKB/Akt) activation as a downstream event contributing to the pathophysiology of DN. We investigated the potential of PKB/Akt to mediate the profibrotic bioactions of TGF-beta1 in kidney. Treatment of normal rat kidney epithelial cells (NRK52E) with TGF-beta1 resulted in activation of phosphatidylinositol 3-kinase (PI3K) and PKB/Akt as evidenced by increased Ser473 phosphorylation and GSK-3beta phosphorylation. TGF-beta1 also stimulated increased Smad3 phosphorylation in these cells, a response that was insensitive to inhibition of PI3K or PKB/Akt. NRK52E cells displayed a loss of zona occludins 1 and E-cadherin and a gain in vimentin and alpha-smooth muscle actin expression, consistent with the fibrotic actions of TGF-beta1. These effects were blocked with inhibitors of PI3K and PKB/Akt. Furthermore, overexpression of PTEN, the lipid phosphatase regulator of PKB/Akt activation, inhibited TGF-beta1-induced PKB/Akt activation. Interestingly, in the Goto-Kakizaki rat model of type 2 diabetes, we also detected increased phosphorylation of PKB/Akt and its downstream target, GSK-3beta, in the tubules, relative to that in control Wistar rats. Elevated Smad3 phosphorylation was also detected in kidney extracts from Goto-Kakizaki rats with chronic diabetes. Together, these data suggest that TGF-beta1-mediated PKB/Akt activation may be important in renal fibrosis during diabetic nephropathy.

Vitamin D Receptor Modulates the Neoplastic Phenotype Through Antagonistic Growth Regulatory Signals

Vitamin D receptor (VDR) can modulate functionally antagonistic growth regulatory pathways, involving beta-catenin/E-cadherin on one hand and osteopontin (OPN) on the other. This study investigates effects of VDR ligand treatment on the balance of these discordant signals and on associated cell behavior. Treatment of Rama 37 or SW480 cells by 1 alpha,25-(OH)(2) D-3 or analogs suppressed beta-catenin/Lef-1/Tcf signaling and upregulated E-cadherin, consistent with a cancer-inhibitory action. Conversely, treatment also increased transcription of OPN that may be implicated in tumor progression. Molecular crosstalk was observed between the antagonistic VDR-dependent signals, in that beta-catenin/Lef-1/Tcf molecules modulated VDR activation of OPN. Treatment effects on cell growth were related to a constitutive balance of OPN and E-cadherin expression. No growth effects were observed in Rama 37 cells that have low OPN and high E-cadherin expression. Conversely, treatment of Rama 37 stably transfected subclones that had high OPN and/or low level E-cadherin induced small but significant increases of cell attachment to fibronectin, anchorage-independent growth or invasion. This study shows that relative expression levels of key VDR downstream genes may influence growth regulation by 1 alpha,25-(OH)(2) D-3 or analogs. These findings may be relevant to the cell- or tissue-specificity of vitamin D growth regulation. (C) 2009 Wiley-Liss, Inc.

Development of selective media for the isolation of yeasts and filamentous fungi from the sputum of adult patients with cystic fibrosis (CF)

Yeasts and filamentous fungi are beginning to emerge as significant microbial pathogens in patients with cystic fibrosis (CF), particularly in relation to allergic-type responses, as seen in patients with allergic bronchopulmonary aspergillosis (ABPA), Aspergillus bronchitis and in invasive fungal disease in lung transplant patients. Four fungal media were compared in this study, including Sabouraud Dextrose Agar (SDA) and Medium B, with and without the addition of selective antibiotics, where antibiotic-supplemented media were designated with (+). These media were compared for their ability to suppress contaminating, mainly Gram-ve pathogens, in CF sputa (Pseudomonas aeruginosa, Burkholderia cepacia complex [BCC] organisms) and to enhance the growth of fungi present in CF sputum. Medium B consisted of glucose (16.7 g/l), agar (20 g/l), yeast extract (30 g/l) and peptone (6.8 g/l) at pH 6.3 and both SDA(+) and Medium B+ were supplemented with cotrimethoxazole, 128 mg/l; chloramphenicol, 50 mg/l; ceftazidime, 32 mg/l; colistin, 24 mg/l). Employment of SDA(+) or Medium B+ allowed an increase in specificity in the detection of yeasts and moulds, by 42.8% and 39.3%, respectively, over SDA when used solely. SDA(+) had a greater ability than Medium B+ to suppress bacterial growth from predominantly Gram-ve co-colonisers. This is a significant benefit when attempting to detect and isolate fungi from the sputum of CF patients, as it largely suppressed any bacterial growth, with the exception of the BCC organisms, thus allowing for an increased opportunity to detect target fungal organisms in sputum and represented a significant improvement over the commercial medium (SDA), which is currently used. Overall, both novel selective media were superior in their ability to suppress bacteria in comparison with the commercially available SDA medium, which is routinely employed in most clinical microbiology diagnostic laboratories presently. Alternatively, Medium B+ had a great ability to grow fungi than SDA(+) and when employed together, the specificity of combined use was 82%, with a sensitivity for yeasts, filamentous fungi, and combined overall fungi of 96.0%, 92.3% and 96.0%, respectively. Overall, when employing one fungal selective medium for the routine detection of yeasts and filamentous fungi in the sputum of CF patients, we would recommend employment of Medium B+. However, we would recommend the combined employment of SDA(+) and Medium B+, in order to synergistically isolate and detect the greatest number of fungi present in CF sputa. (C) 2008 European Cystic Fibrosis Society. Published by Elsevier B.V All rights reserved.

Novel in vitro assays for the characterization of EMT in tumourigenesis

Background Two novel assays quantifying Epithelial to Mesenchymal Transition (EMT) were compared to traditional motility and migration assays. TGF-ß1 treatment of AY-27 rat bladder cancer cells acted as a model of EMT in tumourigenesis. Methods AY-27 rat bladder cancer cells incubated with 3ng/ml TGF-ß1 or control media for 24 or 48h were assessed using novel and traditional assays. The Spindle Index, a novel measure of spindle phenotype, was derived from the ratio of maximum length to maximum width of cells. The area covered by cells which migrated from a fixed coverslip towards supplemented agarose was measured in a novel chemoattractant assay. Motility, migration and immunoreactivity for E-cadherin, Vimentin and cytokeratin were assessed. Results TGF-ß1 treated cells had increased “spindle” phenotype together with decreased E-cadherin, decreased Cytokeratin-18 and increased Vimentin immunoreactivity. After 48h, the mean Spindle Index of TGF-ß1 treated cells was significantly higher than Mock (p=0.02 Bonferroni test) and there were significant differences in migration across treatment groups measured using the novel chemoattractant assay (p = 0.02, Chi-Square). TGF-ß1 significantly increased matrigel invasion. Conclusion The Spindle Index and the novel chemoattractant assay are valuable adjunctive assays for objective characterization of EMT changes during tumourigenesis.

Jagged/Notch signalling is required for a subset of TGFβ1 responses in human kidney epithelial cells.

The Jagged/Notch pathway has been implicated in TGFß1 responses in epithelial cells in diabetic nephropathy and other fibrotic conditions in vivo. Here, we identify that Jagged/Notch signalling is required for a subset of TGFß1-stimulated gene responses in human kidney epithelial cells in vitro. TGFß1 treatment of HK-2 and RPTEC cells for 24 h increased Jagged1 (a Notch ligand) and Hes1 (a Notch target) mRNA. This response was inhibited by co-incubation with Compound E, an inhibitor of ?-secretase (GSI), an enzyme required for Notch receptor cleavage and transcription regulation. In both cell types, TGFß1-responsive genes associated with epithelial–mesenchymal transition such as E-cadherin and vimentin were also affected by ?-secretase inhibition, but other TGFß1 targets such as connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1) were not. TGFß1-induced changes in Jagged1 expression preceded EMT-associated gene changes, and co-incubation with GSI altered TGFß1-induced changes in cell shape and cytoskeleton. Transfection of cells with the activated, cleaved form of Notch (NICD) triggered decreased expression of E-cadherin in the absence of TGFß1, but did not affect a-smooth muscle actin expression, suggesting differential requirements for Notch signalling within the TGFß1-responsive gene subset. Increased Jagged1 expression upon TGFß1 exposure required Smad3 signalling, and was also regulated by PI3K and ERK. These data suggest that Jagged/Notch signalling is required for a subset of TGFß1-responsive genes, and that complex signalling pathways are involved in the crosstalk between TGFß1 and Notch cascades in kidney epithelia.


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