Proteolytic processing of the beta-amyloid precursor protein (APP) in membranes of the human neuroblastoma SH-SY5Y cells

Bibliothèques et publics, bibliothèques publiques dans l’Europe d’Ancien Régime (XVIe-XVIIIe siècle) - Yann Sordet (Bibliothèque Mazarine) 18hLiège, ULg, Grand Physique (Bât. A1) Organisation : Cycle de conférences de Transitions En savoir plus. Source de l'information : http://web.philo.ulg.ac.be/transitions/agenda-general

STRUCTURAL AND FUNCTIONAL ANALYSIS OF COMPLEMENT FACTOR H: A CRUCIAL PROTEIN IN SEVERAL DISORDERS

Il Fattore H del complemento (FH) \ue8 un importante regolatore della via alternativa del complemento: protegge infatti le cellule dell\u2019ospite dall\u2019attacco del sistema del complemento e carenze di FH sia qualitative e quantitative dovute a mutazioni nel gene CFH sono spesso associate ad una serie di malattie umane, come la glomerulonefrite membranoproliferativa (MPGN), la sindrome emolitico-uremica atipica (aHUS) e la degenerazione maculare della retina legata all\u2019et\ue0 (AMD). Mentre esiste una caratterizzazione genetica per tutte queste malattie, i dati funzionali a livello di proteine sono spesso carenti. Inoltre, il FH gioca un ruolo significativo nelle malattie infettive: molti agenti patogeni sono infatti in grado di reclutare il FH sulla loro superficie sfruttandolo per proteggersi dagli attacchi del complemento. Mentre per alcuni agenti patogeni l'interazione con il FH \ue8 stata ben descritta, per gli altri gli "interattori" diretti sono ancora sconosciuti. Tuttavia, lo studio del FH \ue8 complicato dalla presenza di proteine FH-related (FHRs) che posseggono un elevato grado di somiglianza con il FH e ne rendono quindi difficile la purificazione e la analisi diretta. Il primo obiettivo di questo progetto \ue8 stato lo sviluppo di saggi quantitativi e funzionali FH-specifici, utilizzando un anticorpo monoclonale (Mab 5H5) prodotto nel nostro laboratorio, che si \ue8 dimostrato essere specifico per FH. Il secondo era la produzione del FH intero e di tre frammenti, contenenti diverse porzioni della proteina, sia in un sistema di espressione eucariotico che procariotico. L\u2019anticorpo 5H5 \ue8 stato prodotto in grandi quantit\ue0 utilizzando un mini-fermentatore ed \ue8 stato utilizzato per purificare il FH mediante cromatografia di affinit\ue0. La purezza e l\u2019attivit\ue0 funzionale del FH purificato sono state valutate e confrontate con gli altri metodi di purificazione (ad es. affinit\ue0 su Eparina, HPLC, HIC). \uc8 stato inoltre sviluppato un test ELISA per la corretta quantificazione del FH da fluidi biologici, utilizzando il 5H5 insieme ad un anticorpo policlonale anti-FH, anch\u2019esso prodotto nel nostro laboratorio, e al FH purificato per affinit\ue0. Questo test di quantificazione \ue8 risultato affidabile ed \ue8 stato utilizzato per valutare diversi campioni di pazienti aHUS e AMD. Inoltre, sono stati sviluppati anche due micro-metodi di purificazione del FH. Questi metodi consentono di purificare una buona quantit\ue0 di FH altamente purificato partendo da una piccola quantit\ue0 di campione e sono quindi uno strumento utile per studiare il FH specialmente in quelle patologie, quali aHUS, che colpiscono soprattutto i bambini. Infine, sono stati sviluppati due saggi funzionali per valutare sia l'attivit\ue0 cofattoriale N-terminale che l\u2019attivit\ue0 protettiva C-terminale del FH. Il FH \ue8 una grossa glicoproteina solubile che possiede funzioni diverse in differenti parti della molecola. Quindi diversi test sono necessari per valutare la completa funzionalit\ue0 del FH. Il primo test, il "cofactor assay", basato sul fatto che il FH possiede attivit\ue0 cofattoriale per il taglio del C3b mediato dal FI, \ue8 completamente funzionante ed \ue8 gi\ue0 stato utilizzato per lo screening di diversi campioni. Il secondo, il test di emolisi, si basa sul fatto che il FH \ue8 in grado di proteggere gli eritrociti di pecora dalla emolisi mediata dal complemento. In questo test il FH purificato dai pazienti verr\ue0 utilizzato insieme ad un siero di controllo depleto di FH, al fine di avere un saggio in cui l'unica variabile sia il FH. Questo test \ue8 stato gi\ue0 messo a punto utilizzando un siero di controllo al fine di verificare tutte le condizioni, ed \ue8 ora nella fase finale di sviluppo, dopo di che sar\ue0 utilizzabile su materiale dei pazienti. I quattro cloni contenenti la sequenza completa del FH e i frammenti parziali (SCRs 1-7, SCRs 8-14, SCRs 15-20) sono stati prodotti e saranno utilizzati per esprimere FH sia in forma glicosilata che non glicosilata. Sar\ue0 quindi analizzata la capacit\ue0 di queste proteine ricombinanti di interagire con le proteine dei patogeni o di ripristinare la funzione di un FH difettoso.Complement Factor H (FH) is a major regulator of the Alternative Pathway of Complement: it protects host cells from complement attack and qualitative and quantitative deficiencies of FH due to mutations in the CFH gene are frequently associated with a number of human diseases, such as membranoproliferative glomerulonephritis (MPGN), atypical haemolytic uraemic syndrome (aHUS) and age-related macular degeneration (AMD). A genetic characterisation is available for all these diseases, whereas functional data on protein levels are often missing. Furthermore, FH plays a significant role in infectious diseases, as many pathogens are able to recruit FH on their surface, thus protecting themselves from complement attack. Whereas interaction of FH with certain pathogens has been well described, for others the direct \u201cinteractors\u201d still remain unknown. However, the study of FH is complicated by the presence of FH-related proteins (FHRs) that share a high degree of similarity rendering its purification and direct analysis challenging. The first focus of this project was the development of quantitative and functional FH-specific assays, using a monoclonal antibody (MAb 5H5) produced in our laboratory, which has been shown to be specific for FH. The second aim was the production of the whole recombinant FH and of three fragments, containing different parts of the protein, in both a eukaryotic and a prokaryotic expression system. MAb 5H5 was produced in high amounts using a mini-fermentator and used to purify FH by affinity chromatography. Purity and functional activity of this affinitypurified FH were assessed and compared with FH obtained by other purification methods (eg. Heparin affinity, HPLC, HIC). An home made ELISA for the correct quantification of FH from biological fluids, making use of 5H5 together with a polyclonal anti-FH antibody, also produced in our lab, and affinity purified FH was also developed. This reliable quantification assay was used to screen samples from aHUS and AMD patients. Moreover, two micro-methods to purify FH were also developed. These methods enable to purify a good quantity of highly purified FH from a small amount of sample and are therefore a useful tool to study FH especially in those pathologies, such as aHUS, which mainly affect children. Finally, two functional assays to assess both the N-terminal cofactorial activity and C-terminal protective activity of FH have been set up. FH is a big soluble glycoprotein that bears different functions in different parts of the molecule. Therefore different tests are needed to assess full functionality of FH. The first test, the \u201ccofactor assay\u201d, based on the fact that FH possesses cofactor activity for the FI-mediated cleavage of C3b, is now fully implemented and has already been used to screen different samples. The second one, the \u201chaemolysys test\u201d is based on the fact that FH is able to protect sheep erythrocytes from haemolysis mediated by complement. In this test FH purified from patients will be used together with a control FH-depleted serum, in order to set up an assay where the only variable is FH. This test has already been set up with a control serum in order to test all the conditions, and is now in its fine tuning phase, after which it will be applicable to patient\u2019s material. The four clones containing the complete FH sequence and partial fragments (SCRs 1-7, SCRs 8-14, SCRs 15-20) have been produced and will be used to express FH in a glycosylated or unglycosylated form. These recombinant proteins will be screened for their ability to interact with proteins from pathogens or to restore the function of a defective FH

Stratigraphic evolution of the Triassic\u2013Jurassic succession in the Western Southern Alps (Italy) : the record of the two-stage rifting on the distal passive margin of Adria

The Triassic-Lower Jurassic succession of the Southern Alps is characterized by rapid thickness changes, from an average of about 5000m east of Lago Maggiore to about 500m in the Western Southern Alps. The stratigraphy reflects the Triassic evolution of the Tethyan Gulf and the Early Jurassic rifting responsible for the Middle Jurassic break-up of Adria from Europe. The succession of the Western Southern Alps starts with Lower Permian volcanics directly covered by Anisian sandstones. The top of the overlying Ladinian dolostones (300m) records subaerial exposure and karstification. Locally (Gozzano), Upper Sinemurian sediments cover the Permian volcanics, documenting pre-Sinemurian erosion. New biostratigraphic data indicate a latest Pliensbachian-Toarcian age for the Jurassic synrift deposits that unconformably cover Ladinian or Sinemurian sediments. Therefore, in the Western Southern Alps, the major rifting stage that directly evolved into the opening of the Penninic Ocean began in the latest Pliensbachian-Toarcian. New data allowed us to refine the evolution of the two previously recognized Jurassic extensional events in the Southern Alps. The youngest extensional event (Western Southern Alps) occurred as tectonic activity decreased in the Lombardy Basin. During the Sinemurian the Gozzano high represents the western shoulder of a rift basin located to the east (Lombardy). This evolution documents a transition from diffuse early rifting (Late Hettangian-Sinemurian), controlled by older discontinuities, to rifting focused along a rift valley close to the Pliensbachian-Toarcian boundary. This younger rift bridges the gap between the Hettangian-Sinemurian diffuse rifting and the Callovian-Bathonian break-up. The late Pliensbachian-Toarcian rift, which eventually lead to continental break-up, is interpreted as the major extensional episode in the evolution of the passive margin of Adria. The transition from diffuse to focused extension in the Southern Alps is comparable to the evolution of the Central Austroalpine during the Early Jurassic and of the Central and Northern Atlantic margins

CLONING OF THE GM3 SYNTHASE cDNA FROM HUMAN PLACENTA AND GENOMIC ORGANISATION OF THE GENE

INTRODUCTION: Gangliosides are a large family of sialic acid-containing glycosphingolipids that play important roles in a large variety of biological processes. Both their functions and their biosynthetic pathways are currently clarified (1, 2). In vertebrates, almost all the ganglio-series gangliosides are synthesized from a common precursor, ganglioside GM3, which has the simplest structure among the major ganglioside. GM3 itself is known to participate in induction of differentiation, modulation of proliferation, signal transduction and integrin-mediated cell adhesion. GM3 synthase (EC 2.4.99.9, ST3Gal V) is the enzyme involved in the last step of GM3 biosynthesis: it catalyses the transfer of a sialic acid moiety from CMP-sialic acid onto lactosylceramide, forming an a2-3 linkage. Whereas GM3 is ubiquitously distributed in the plasma membranes of all eukaryotic cells, GM3 synthase results expressed in a tissue-specific manner, especially in brain, placenta, muscle and testis (3). Although its cDNA has been cloned from some mouse (4, 5) and human tissues (3, 6), studies on the genomic structure (7, 8) and on its transcriptional regulation (8, 9) provides contrasting results. MATERIALS AND METHODS: To isolate the complete coding sequence of the gene of GM3 synthase from human placenta we used the 5\u2019- and 3\u2019-Rapid Amplification of cDNA Ends technology (SMART RACE cDNA Amplification Kit, Clontech) using, as specific primers, oligonucleotides derived from the human GM3 synthase cDNA sequence from differentiated HL60 cells (3). The identity of some amplified DNA fragments was confirmed by Southern blot analysis (Gene ImagesTM AlkPhos DirectTM labelling and detection system, Amersham Pharmacia Biotech). The different PCR products were cloned into the pCR2.1 vector (TA Cloning Kit, InVitrogen) and the nucleotide sequence was determined (\u201cProgetto Camilla\u201d, M-Medical). The genomic structure of the human GM3 synthase gene has been determined through a human genome BLAST homology search of the public database (GenBank) using the GM3 synthase cDNA from human placenta as the query sequence. RESULTS: A cDNA, consisting of 2149 bp and showing high sequence homology with those encoding the human GM3 synthase from other human tissues (3, 6), has been successfully isolated and cloned from human placenta. Notwithstanding our approach, our cDNA has not the poli(A) tail. Between our cDNA and the other published ones, the major difference is in the 5\u2019-end, according to the existence of different promoter regions, responsible for tissue-specific expression of the gene. Furthermore, the cDNA from the human placenta contains, upstream and in frame with the ATG indicated as translation initiation site for the GM3 synthase of HL60 cells, another ATG codon inserted in a sequence compatible with Kozak\u2019s rule, suggesting that the protein of the human placenta could have an additional portion in NH2-terminus. The complete and partial coding regions of the human placenta cDNA are going to be cloned in an expression vector, under the control of the CMV promoter, in order to evaluate their GM3 synthase activity. The results of the human genome BLAST homology search of the public database using the GM3 synthase cDNA from human placenta as the query sequence showed that the gene consists of seven exons which span over 28.5 kb, with exons ranging in size up to 1242 bp. All exon-intron boundaries follows the GT-AG rule (10). 1) Hakomori S.I. (2000) Glycoconj. J. 17, 627-647 2) Kolter T. et al. (2002) J. Biol. Chem. 277, 25859-25862 3) Ishii A. et al. (1998) J. Biol. Chem. 273, 31652-31655 4) Kono M. et al. (1998) Biochem. Biophys. Res. Comm. 253, 170-175 5) Fukumoto S. et al. (1999) J. Biol. Chem. 274, 9271-9276 6) Kapitonov D. et al.(1999) Glycoconj J. 16, 337-350 7) Kim K.W. et al. (2001) Gene 273, 163-171 8) Kim S.W. et al. (2002) ) Biochim. Biophys. Acta 1578, 84-89 9) Zeng G. et al. (2003) Biochim. Biophys. Acta 1625, 30-35 10) Breathnach R. and Chambon P. (1981) Annu. Rev. Biochem. 50, 349-38

GM3 IN THE REGULATION OF THE EXPRESSION AND ACTIVATION OF ErbB-2/EGFR HETERODIMERS

Gangliosides are a large and heterogeneous family of sialic acid containing glycosphingolipids, ubiquitous components of all eukaryotic cell membranes. They can partially segregate, together with cholesterol and specific signaling transduction proteins, such as receptor tyrosine-kinases, into unique more or less stable clusters or microdomains, indicated as \u201cglycolipid-enriched domains\u201d, \u201clipid rafts\u201d, \u201ccaveolae\u201d, contributing to the membrane structure, organization and function (1). Gangliosides are well-characterized modulators of receptor tyrosine-kinase (RTKs) phophorylation and dimerization (2). Our interest is particularly directed to study the relationship between gangliosides and two members of the tyrosine kinase ErbB receptor family: the epidermal growth factor receptor, EGFR or ErbB-1, and the structurally related protein ErbB-2. Unlike EGFR, ErbB-2 is a ligandless receptor: it can be activated by heterodimerization and cross-phosphorylation by other ligand-activated ErbB receptors (3,4). Our previous experimental evidence supports the functional relationship between ErbB-2 and gangliosides, in particular GM3 (5,6). In the present study, using the HC11 mouse mammary epithelial cell line, we investigated the ErbB-2 activation state and its tendency to form stable molecular complexes with EGFR and with ganglioside GM3, before and after EGF stimulation, by co-immunoprecipitation experiments with anti-ErbB-2 antibody and Western blot analyses. As HC11 cells express different ganglioside species, the exclusive association of GM3 with ErbB-2 and EGFR was ascertained by high performance-thin layer chromatography (HP-TLC) and TLC-immunostaining analyses of gangliosides extracted from the immunoprecipitates. Results display that in EGF-stimulated HC11 cells stable and tyrosine-phosphorylated ErbB2/EGFR dimers are formed and that GM3 is the unique ganglioside tightly associated to ErbB-2/EGFR dimers and to EGFR monomers, but not to ErbB2 monomers. In cells not stimulated with EGF a spontaneous but unproductive dimerization of ErbB2 and EGFR takes place and no ganglioside is found associated to the receptors. These observations indicate that the modulation of ErbB2 activation by GM3 may be mediated by EGFR, but that EGF stimulation is indispensable. After ganglioside depletion by [D]-PDMP, phosphorylated EGFR monomers are observed both before and after EGF stimulation, whereas ErbB-2 is present in the monomeric and unphosphorylated state even after EGF stimulation, suggesting that GM3 might have a bivalent key role in modulating the activation of ErbB-2 and EGFR. References 1. Fivaz, M., Abrami, L., Van Der Goot. F.G. Trends Cell Biol. 9(6), 212-213 (1999); 2. Bremer, E.G., Current topics in membranes 40, 387-411(1999); 3. Qian, X., LeVea, C.M., Freeman, J.K., Dougall, W.C., Greene, M.I., Proc. Natl. Acad. Sci. USA 91, 1500-1504 (1994); 4. Gulliford, T.J., Huang, G.C., Ouyang, X., Epstein, R.J., Oncogene 15, 2219-2223 (1997); 5. Sottocornola E., Berra B., and Colombo I., Biochim. Biophys. Acta-Molecolar and cell Biology of Lipids, 1635, 55-66 (2003); 6. Sottocornola E., Misasi R., Mattei V., Ciarlo L., Gradini R., Garofalo T., Berra B., Colombo I., and Sorice M., FEBS J. 273, 1821-1830 (2006)

ISOLATION AND CHARACTERIZATION OF THE GM3 SYNTHASE cDNA FROM HUMAN PLACENTA

It is known that gangliosides have various important biological functions, and their functions as well as their biosynthesis are currently clarified (1, 2). In vertebrates, almost all the ganglio-series gangliosides are synthesized from a common precursor, ganglioside GM3, which has the simplest structure among the major gangliosides. GM3 itself is known to participate in induction of differentiation, modulation of proliferation, signal transduction and integrin-mediated cell adhesion. GM3 synthase (EC 2.4.99.9, ST3Gal V) is the enzyme involved in the last step of GM3 biosynthesis: it catalyses the transfer of a sialic acid moiety from CMP-sialic acid onto lactosylceramide, forming an a2-3 linkage. Whereas GM3 is ubiquitously distributed in the plasma membranes of all eukaryotic cells, GM3 synthase results expressed in a tissue specific manner, especially in brain, placenta, muscle and testis (3). Many important issues, such as human cDNA identification and characterization, genomic structure and regulation of gene expression, are still open. To isolate the coding sequence of the gene of GM3 synthase from human placenta we used the 5\u2019- and 3\u2019-Rapid Amplification of cDNA Ends technology (SMART RACE cDNA Amplification Kit, Clontech) using, as specific primers, oligonucleotides derived from the human GM3 synthase cDNA sequence from differentiated HL60 cells (3). The different PCR products were cloned into the pCR2.1 vector (TA Cloning Kit, InVitrogen) and the nucleotide sequence was determined. A cDNA, showing high sequence homology with that encoding the human GM3 synthase from TPA-differentiated HL60 cells (3), has been successfully isolated and cloned from human placenta. The major difference between these two cDNAs is in the 5\u2019-end, according to the existence of different promoter regions, responsible for tissue-specific expression of the gene. Furthermore, the cDNA from the human placenta contains, upstream and in frame with the ATG indicated as translation initiation site for the GM3 synthase of HL60 cells, another ATG codon inserted in a sequence compatible with Kozak\u2019s rule, suggesting that the protein of the human placenta has an additional portion in NH2-terminus. The complete coding region of the human placenta cDNA is going to be cloned in an expression vector, under the control of the CMV promoter, in order to evaluate its activity. On the other hand, in vitro translation experiments are going to be carried out to define the first start codon. 1) Hakomori S.I. (2000): Glycoconj. J. 17, 627-647 2) Kolter T. et al. (2002): J.Biol.Chem. 277, 25859-25862 3) Ishii A. et al. (1998): J.B.C. 273, 31652-3165

Risk genes in head and neck cancer: a systematic review and meta-analysis of last 5 years

Head and Neck Carcinoma (HNC), of which the majority are squamous cell carcinomas of the head and neck (SCCHN), is the sixth most prevalent cancers in mankind and, presents high morbidity and low rates of survival.1 It is known that the apoptotic and proliferation genes are involved in cancer development and these could be useful as a biomarker of this pathology. Systematic reviews and meta-analysis allow stronger and more generalized conclusions for identifying some models of risk markers. These models may help in screening, early diagnosis and/or therapy in the clinic.2-5 In recent decades, there has been increased interest in genetic predisposition studies in complex disease. This has led to the production of an enormous number of epidemiologic papers about the relationship between genetic polymorphism and disease. However, the magnitude of association between specific polymorphism and disease is still not established. The identification of a predictive model of risk polymorphisms could help in early diagnosis, and in understanding disease recurrence and/or progression in the subset of patientssubmittedVersio

Implementation of case management to reduce cardiovascular disease risk in the Stanford and San Mateo Heart to Heart randomized controlled trial: study protocol and baseline characteristics

BACKGROUND: Case management has emerged as a promising alternative approach to supplement traditional one-on-one sessions between patients and doctors for improving the quality of care in chronic diseases such as coronary heart disease (CHD). However, data are lacking in terms of its efficacy and cost-effectiveness when implemented in ethnic and low-income populations. METHODS: The Stanford and San Mateo Heart to Heart (HTH) project is a randomized controlled clinical trial designed to rigorously evaluate the efficacy and cost-effectiveness of a multi-risk cardiovascular case management program in low-income, primarily ethnic minority patients served by a local county health care system in California. Randomization occurred at the patient level. The primary outcome measure is the absolute CHD risk over 10 years. Secondary outcome measures include adherence to guidelines on CHD prevention practice. We documented the study design, methodology, and baseline sociodemographic, clinical and lifestyle characteristics of 419 participants. RESULTS: We achieved equal distributions of the sociodemographic, biophysical and lifestyle characteristics between the two randomization groups. HTH participants had a mean age of 56 years, 63% were Latinos/Hispanics, 65% female, 61% less educated, and 62% were not employed. Twenty percent of participants reported having a prior cardiovascular event. 10-year CHD risk averaged 18% in men and 13% in women despite a modest low-density lipoprotein cholesterol level and a high on-treatment percentage at baseline. Sixty-three percent of participants were diagnosed with diabetes and an additional 22% had metabolic syndrome. In addition, many participants had depressed high-density lipoprotein (HDL) cholesterol levels and elevated values of total cholesterol-to-HDL ratio, triglycerides, triglyceride-to-HDL ratio, and blood pressure. Furthermore, nearly 70% of participants were obese, 45% had a family history of CHD or stroke, and 16% were current smokers. CONCLUSION: We have recruited an ethnically diverse, low-income cohort in which to implement a case management approach and test its efficacy and cost-effectiveness. HTH will advance the scientific understanding of better strategies for CHD prevention among these priority subpopulations and aid in guiding future practice that will reduce health disparities