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See detailKu proteins interact with activator protein-2 transcription factors and contribute to ERBB2 overexpression in breast cancer cell lines.
Nolens, Grégory ULg; Pignon, Jean-Christophe ULg; Koopmansch, Benjamin ULg et al

in Breast Cancer Research [=BCR] (2009), 11(6),

INTRODUCTION: Activator protein-2 (AP-2) alpha and AP-2 gamma transcription factors contribute to ERBB2 gene overexpression in breast cancer. In order to understand the mechanism by which the ERBB2 gene ... [more ▼]

INTRODUCTION: Activator protein-2 (AP-2) alpha and AP-2 gamma transcription factors contribute to ERBB2 gene overexpression in breast cancer. In order to understand the mechanism by which the ERBB2 gene is overexpressed we searched for novel AP-2 interacting factors that contribute to its activity. METHODS: Ku proteins were identified as AP-2 alpha interacting proteins by glutathione serine transferase (GST)-pull down followed by mass spectrometry. Transfection of the cells with siRNA, expression vectors and reporter vectors as well as chromatin immunoprecipitation (ChIP) assay were used to ascertain the implication of Ku proteins on ERBB2 expression. RESULTS: Nuclear proteins from BT-474 cells overexpressing AP-2 alpha and AP-2 gamma were incubated with GST-AP2 or GST coated beads. Among the proteins retained specifically on GST-AP2 coated beads Ku70 and Ku80 proteins were identified by mass spectrometry. The contribution of Ku proteins to ERBB2 gene expression in BT-474 and SKBR3 cell lines was investigated by downregulating Ku proteins through the use of specific siRNAs. Depletion of Ku proteins led to downregulation of ERBB2 mRNA and protein levels. Furthermore, reduction of Ku80 in HCT116 cell line decreased the AP-2 alpha activity on a reporter vector containing an AP-2 binding site linked to the ERBB2 core promoter, and transfection of Ku80 increased the activity of AP-2 alpha on this promoter. Ku siRNAs also inhibited the activity of this reporter vector in BT-474 and SKBR3 cell lines and the activity of the ERBB2 promoter was further reduced by combining Ku siRNAs with AP-2 alpha and AP-2 gamma siRNAs. ChIP experiments with chromatin extracted from wild type or AP-2 alpha and AP-2 gamma or Ku70 siRNA transfected BT-474 cells demonstrated Ku70 recruitment to the ERBB2 proximal promoter in association with AP-2 alpha and AP-2 gamma. Moreover, Ku70 siRNA like AP-2 siRNAs, greatly reduced PolII recruitment to the ERBB2 proximal promoter. CONCLUSIONS: Ku proteins in interaction with AP-2 (alpha and gamma) contribute to increased ERBB2 mRNA and protein levels in breast cancer cells. [less ▲]

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See detailLa décontamination des prions selon les méthodes plasmas
Elmoualij, Benaïssa ULg; Thellin, Olivier ULg; Zorzi, Willy ULg

in Bioadh 2009 (2009, November 03)

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See detailPrion: Biosafety Level 3-unconventional transmissible agent
Zorzi, Willy ULg; Elmoualij, Benaïssa ULg

Conference (2009, April 22)

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See detailRisk assessment of laboratories involving the manipulation of unconventional agents causing TSE
Leunda-Casi, A.; Pauwels, K.; Herman, Philippe et al

Report (2009)

The present document aims at summarizing the biosafety recommendations and the containment level required for laboratories where animal and human tissues potentially contaminated by a TSE (Transmissible ... [more ▼]

The present document aims at summarizing the biosafety recommendations and the containment level required for laboratories where animal and human tissues potentially contaminated by a TSE (Transmissible Spongiform Encephalitis) causing agent are manipulated. A particular attention will be paid to decontamination procedures, as the prion protein1 is remarkably resistant to conventional inactivation methods and may stay infectious for long periods of time. We will discuss large surface decontamination procedures of facilities handling TSE causing agents. This is of specific concern for laboratories that have been manipulating TSE causing agents, sometimes for years, but wish today dedicate the facilities to another activity. [less ▲]

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See detailA decade of improvements in quantification of gene expression and internal standard selection.
Thellin, Olivier ULg; Elmoualij, Benaïssa ULg; Heinen, Ernst ULg et al

in Biotechnology Advances (2009)

Major improvements have been made in mRNA quantification and internal standard selection over the last decade. Our aim in this paper is to present the main developments that are of interest for practical ... [more ▼]

Major improvements have been made in mRNA quantification and internal standard selection over the last decade. Our aim in this paper is to present the main developments that are of interest for practical laboratory work, contrasting the situation as it is now with the one of ten years ago, and presenting some excellent examples of what can be done today. Specifically, we will mainly discuss Real-Time RT-PCR major improvements that have been performed in the following areas: the most commonly used quantification techniques, the mathematical and software tools created to help researchers in their work on internal standard selection, the availability of detection chemistries and technical information and of commercial tools and services. In addition to mRNA quantification, we will also discuss some aspects of non-coding RNA and protein quantification. In addition to technical improvements, the development of international cooperation and the creation of technical databases are likely to represent a major tool for the future in the standardization of gene expression quantification. [less ▲]

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See detailCreutzfeldt-jakob, Parkinson, lewy body dementia and Alzheimer diseases: from diagnosis to therapy.
Dupiereux-Fettweis, Ingrid ULg; Zorzi, Willy ULg; Quadrio, Isabelle et al

in Central Nervous System Agents in Medicinal Chemistry (2009), 9(1), 2-11

Depositions of proteins in form of amyloid and non-amyloid plaques are common pathogenic signs of more than 20 degenerative diseases affecting the central nervous system or a variety of peripheral tissues ... [more ▼]

Depositions of proteins in form of amyloid and non-amyloid plaques are common pathogenic signs of more than 20 degenerative diseases affecting the central nervous system or a variety of peripheral tissues. Among the neuropathological conditions, Alzheimer's, Parkinson's and the prion diseases, such as Creutzfeldt-Jakob disease (CJD), present ambiguities as regarding their differential diagnosis. At present, their diagnosis must be confirmed by post-mortem examination of the brain. Currently the ante-mortem diagnosis is still based on the integration of multiple data (clinical, paraclinical and biological analyses) because no unique marker exists for such diseases. The detection of specific biomarkers would be useful to develop a differential diagnostic, distinguishing not only different neurodegenerative diseases but also the disease from the non-pathological effects of aging. Several neurodegenerative biomarkers are present at very low levels during the early stages of the disease development and their ultra-low detection is needed for early diagnosis, which should permit more effective therapeutic interventions, before the disease concerned can progress to a stage where considerable damage to the brain has already occurred. In the case of prion diseases, there are concerns regarding not only patient care, but the wider community too, with regard to the risk of transmission of prions, especially during blood transfusion, for which, four cases of variant CJD infection associated with transfusion of non-leukocyte-depleted blood components have been confirmed. Therefore the development of techniques with high sensitivity and specificity represent the major challenge in the field of the protein misfolding diseases. In this paper we review the current analytical and/or biochemical diagnostic technologies used mainly in prion, but also in Alzheimer and Parkinson diseases and emphasizing work on the protein detection as a surrogates and specific biomarker in the body fluid of patients (urine, CSF and blood). This review highlights the urgency of the development of early and sensitive diagnostics in terms of therapeutic challenge. [less ▲]

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See detailImmuno PCR : a new tool for the ultra-sensitive detection of biomolecules
Zorzi, Willy ULg; Elmoualij, Benaïssa ULg

Conference (2008, December 11)

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See detailProtective effect of prion protein via the N-terminal region in mediating a protective effect on paraquat-induced oxidative injury in neuronal cells.
Dupiereux-Fettweis, Ingrid ULg; Falisse-Poirier, Nandini; Zorzi, Willy ULg et al

in Journal of Neuroscience Research (2008), 86(3), 653-9

Transmissible spongiform encephalopathies are a group of neurodegenerative disorders caused by a posttranslational, conformational change in the cellular isoform of the prion protein (PrP(C)) into an ... [more ▼]

Transmissible spongiform encephalopathies are a group of neurodegenerative disorders caused by a posttranslational, conformational change in the cellular isoform of the prion protein (PrP(C)) into an infectious, disease-associated form (PrP(Sc)). Increasing evidence supports a role for PrP(C) in the cellular response to oxidative stress. We investigated the effect of oxidative stress mediated by paraquat exposure on SH-SY5Y neuroblastoma cells. A loss of mitochondrial membrane potential and subsequent reduction in ATP production were demonstrated in untransfected SH-SY5Y cells, an effect that was ameliorated by the expression of PrP(C). Cells expressing either PrP-DeltaOct, which lacks the octapeptide repeats, or PrP-DA, in which the N-terminus is tethered to the membrane, showed increased sensitivity to paraquat compared with cells expressing wild-type PrP(C) as shown by reduced viability, loss of their membrane integrity, and reduced mitochondrial bioenergetic measurements. Exposure of prion-infected mouse SMB15S cells to paraquat resulted in a reduction in viability to levels similar to those seen in the untransfected SH-SY5Y cells. However, "curing" the cells with pentosan sulfate restored the viability to the level observed in the SH-SY5Y cells expressing PrP(C). These data would indicate that the molecular mechanism promoting cellular resistance to oxidative stress had been compromised in the infected SMB15S cells, which could be reinstated upon curing. Our study supports the hypothesis that PrP(C) expression protects cells against paraquat-induced oxidative injury, demonstrates the significance of the N-terminal region of the protein in mediating this protective effect, and also shows that the biochemical consequences of prion infection may be reversed with therapeutic intervention. [less ▲]

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See detailAn immuno-PF2D-MS/MS proteomic approach for bacterial antigenic characterization: To Bacillus and beyond
Ruelle, Virginie ULg; Falisse-Poirier, Nandini; Elmoualij, Benaïssa ULg et al

in Journal of Proteome Research (2007), 6(6), 2168-2175

We are confronted daily to unknown microorganisms that have yet to be characterized, detected, and/ or analyzed. We propose, in this study, a multidimensional strategy using polyclonal antibodies ... [more ▼]

We are confronted daily to unknown microorganisms that have yet to be characterized, detected, and/ or analyzed. We propose, in this study, a multidimensional strategy using polyclonal antibodies, consisting of a novel proteomic tool, the ProteomeLab PF2D, coupled to immunological techniques and mass spectrometry ( i-PF2D-MS/MS). To evaluate this strategy, we have applied it to Bacillus subtilis, considered here as our unknown bacterial model. [less ▲]

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See detailTRANSMISSION ET RÉSISTANCE DES PRIONS : LA PRATIQUE DE LA MÉDECINE DENTAIRE EN SERA T-ELLEAFFECTÉE ?
Elmoualij, Benaïssa ULg; Heinen, Ernst ULg; Zorzi, Willy ULg et al

in Journal de l'Ordre des Dentistes du Québec (2006), 43(9), 461-467

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See detailAdvances in immunoproteomics for serological characterization of microbial antigens
Falisse-Poirier, Nandini; Ruelle, Virginie ULg; Elmoualij, Benaïssa ULg et al

in Journal of Microbiological Methods (2006), 67(3), 593-596

We propose a multi-dimensional strategy, associating immunodetection to a protein fractionating two-dimensional liquid chromatography tool, for serological characterization of microbial antigens. The ... [more ▼]

We propose a multi-dimensional strategy, associating immunodetection to a protein fractionating two-dimensional liquid chromatography tool, for serological characterization of microbial antigens. The originality of such immunoproteomic approaches resides in their application in large-scale studies for rapid serotyping of micro-organisms, evaluation of immunomes and could be proposed in the development and monitoring of vaccines. (c) 2006 Elsevier B.V. All rights reserved. [less ▲]

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See detailImmunoquantitative real-time PCR for detection and quantification of Staphylococcus aureus enterotoxin B in foods
Rajkovic, A.; Elmoualij, Benaïssa ULg; Uyttendaele, M. et al

in Applied and Environmental Microbiology (2006), 72(10), 6593-6599

A real-time immunoquantitative PCR (iqPCR) method for detection of Staphylococcus aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The assay consisted of ... [more ▼]

A real-time immunoquantitative PCR (iqPCR) method for detection of Staphylococcus aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The assay consisted of immunocapture of SEB and real-time PCR amplification of the DNA probe linked to the detection antibody. iqPCR was compared to an in-house enzyme-linked immunosorbent assay (ELISA) using the same couple of capture-detection antibodies and to commercial kits for detection of S. aureus enterotoxins (SE). The iqPCR was approximately 1,000 times more sensitive (< 10 pg ml(-1)) than the in-house ELISA and had a dynamic range of approximately 10 pg ml(-1) to approximately 30,000 pg ml(-1). iqPCR was not inhibited by any of the foods tested and was able to detect SEB present in these foods. No cross-reactivity with SE other than SEB was observed. Application of iqPCR for detection of SEB in cultures of S. aureus revealed the onset of SEB production after 4 It of incubation at 22, 37, and 42 degrees C, which was in the first half of the exponential growth phase. The total amounts of SEB produced by the two strains tested were larger at 42 degrees C than at 37 degrees C and were strain dependent. [less ▲]

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See detailStudy on the toxic mechanism of prion protein peptide 106-126 in neuronal and non neuronal cells
Dupiereux-Fettweis, Ingrid ULg; Zorzi, Willy ULg; Rachidi, W. et al

in Journal of Neuroscience Research (2006), 84(3), 637-646

A synthetic peptide corresponding to the 106-126 amyloidogenic region of the cellular human prion protein (PrPc) is useful for in vitro study of prion-induced neuronal cell death. The aim of the present ... [more ▼]

A synthetic peptide corresponding to the 106-126 amyloidogenic region of the cellular human prion protein (PrPc) is useful for in vitro study of prion-induced neuronal cell death. The aim of the present work was to examine the implication of the cellular prion protein in the toxicity mechanism induced by PrP 106-126. The effect of PrP 106-126 was investigated both on human neuroblastoma SH-SY5Y cells and on SH-SY5Y over-expressing murine cellular prions (wtPrP). We show by metabolic assay tests and ATP assays that PrPc expression does not modulate the toxicity of the prion peptide. Moreover, we investigated the effect of this peptide on an established non neuronal model, rabbit kidney epithelial A74 cells that express a doxycycline-inducible murine PrPc gene. We show for the first time that the prion peptide 106-126 does not exert any toxic effect on this cell line in the presence or absence of doxycycline. Our results show that the PrP 106-126-induced cell alteration is independent of PrPc expression. Rather, it seems to act via an interaction with lipidic components of the plasma membrane as strengthened by our results showing the differential susceptibility of neuronal and non neuronal cell lines that significantly differ by their membrane fatty acid composition. (c) 2006 Wiley-Liss, Inc. [less ▲]

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