MALDI/MS peptide mass fingerprinting for proteome analysis: identification of hydrophobic proteins attached to eucaryote keratinocyte cytoplasmic membrane using different matrices in concert

BACKGROUND: MALDI-TOF-MS has become an important analytical tool in the identification of proteins and evaluation of their role in biological processes. A typical protocol consists of sample purification, separation of proteins by 2D-PAGE, enzymatic digestion and identification of proteins by peptide mass fingerprint. Unfortunately, this approach is not appropriate for the identification of membrane or low or high pI proteins. An alternative technique uses 1D-PAGE, which results in a mixture of proteins in each gel band. The direct analysis of the proteolytic digestion of this mixture is often problematic because of poor peptide detection and consequent poor sequence coverage in databases. Sequence coverage can be improved through the combination of several matrices. RESULTS: The aim of this study was to trust the MALDI analysis of complex biological samples, in order to identify proteins that interact with the membrane network of keratinocytes. Peptides obtained from protein trypsin digestions may have either hydrophobic or hydrophilic sections, in which case, the direct analysis of such a mixture by MALDI does not allow desorbing of all peptides. In this work, MALDI/MS experiments were thus performed using four different matrices in concert. The data were analysed with three algorithms in order to test each of them. We observed that the use of at least two matrices in concert leads to a twofold increase of the coverage of each protein. Considering data obtained in this study, we recommend the use of HCCA in concert with the SA matrix in order to obtain a good coverage of hydrophilic proteins, and DHB in concert with the SA matrix to obtain a good coverage of hydrophobic proteins. CONCLUSION: In this work, experiments were performed directly on complex biological samples, in order to see systematic comparison between different matrices for real-life samples and to show a correlation that will be applicable to similar studies. When 1D gel is needed, each band may contain a great number of proteins, each present in small amounts. To improve the proteins coverage, we have performed experiments with some matrices in concert. These experiments enabled reliable identification of proteins, without the use of Nanospray MS/MS experiments

Connexin30 mutations responsible for hidrotic ectodermal dysplasia cause abnormal hemichannel activity

Clouston syndrome or hidrotic ectodermal dysplasia (HED) is a rare dominant genodermatosis characterized by palmoplantar hyperkeratosis, generalized alopecia and nail defects. The disease is caused by mutations in the human GJB6 gene which encodes the gap junction protein connexin30 (Cx30). To gain insight into the molecular mechanisms underlying HED, we have analyzed the consequences of two of these mutations (G11R Cx30 and A88V Cx30) on the functional properties of the connexons they form. Here, we show that the distribution of Cx30 is similar in affected palmoplantar skin and in normal epidermis. We further demonstrate that the presence of the wild-type protein (wt Cx30) improves the trafficking of mutated Cx30 to the plasma membrane where both G11R and A88V Cx30 co-localize with wt Cx30 and form functional intercellular channels. The electrophysiological properties of channels made of G11R and A88V Cx30 differ slightly from those of wt Cx30 but allow for dye transfer between transfected HeLa cells. Finally, we document a gain of function of G11R and A88V Cx30, which form functional hemichannels at the cell surface and, when expressed in HeLa cells, generate a leakage of ATP into the extracellular medium. Such increased ATP levels might act as a paracrine messenger that, by altering the epidermal factors which control the proliferation and differentiation of keratinocytes, may play an important role in the pathophysiological processes leading to the HED phenotyp

International Expert Consensus on Switching Platelet P2Y(12) Receptor-Inhibiting Therapies

Dual antiplatelet therapy with aspirin and a P2Y(12) inhibitor is the treatment of choice for the prevention of atherothrombotic events in patients with acute coronary syndromes and for those undergoing percutaneous coronary interventions. The availability of different oral P2Y(12) inhibitors (clopidogrel, prasugrel, ticagrelor) has enabled physicians to contemplate switching among therapies because of specific clinical scenarios. The recent introduction of an intravenous P2Y(12) inhibitor (cangrelor) further adds to the multitude of modalities and settings in which switching therapies may occur. In clinical practice, it is not uncommon to switch P2Y(12) inhibitor, and switching may be attributed to a variety of factors. However, concerns about the safety of switching between these agents have emerged. Practice guidelines have not fully elaborated on how to switch therapies, leaving clinicians with limited guidance on when and how to switch therapies when needed. This prompted the development of this expert consensus document by key leaders from North America and Europe with expertise in basic, translational, and clinical sciences in the field of antiplatelet therapy. This expert consensus provides an overview of the pharmacology of P2Y(12) inhibitors, different modalities and definitions of switching, and available literature and recommendations for switching between P2Y(12) inhibitors

Etude des proprietes fonctionnelles de l'endopeptidase neutre cerebrale

SIGLECNRS TD Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Identification de marqueurs de prolifération et de différenciation des kératinocytes de l'épiderme humain

Les kératinocytes souches de la couche basale de l'épiderme humain assurent l'auto-renouvellement de ce tissu. Lors de leur différenciation, les kératinocytes migrent vers les couches supérieures de l'épiderme jusqu'à la desquamation. Les mécanismes de différenciation des kératinocytes en culture sont mal connus. L'apparition de nouvelles technologies comme les puces à ADN ou l'analyse protéomique nous a permis d'identifier des gènes et des protéines exprimés au cours de la différenciation des kératinocytes. Dans le but de mieux caractériser les cellules souches, nous avons entrepris l'identification de protéines membranaires des kératinocytes de la couche basale. En isolant des microdomaines membranaires de ces cellules, nous avons pu identifier par analyse protéomique des protéines représentant des marqueurs potentiels des kératinocytes de la couche basale. Cette nouvelle approche a permis d'isoler des kératinocytes de la couche basale et par conséquent, les cellules souches.Keratinocyte stem cells of the basal layer of the epidermis ensure the self-renewal of this tissue. Their progeny differentiate as they migrate towards the squamous layer. The mechanisms of human keratinocyte differentiation are still poorly known. The emergence of new technologies, such as cDNA microarrays or proteomic analysis, allowed us to identify, in tissue culture, genes and proteins expressed in proliferating versus differentiating keratinocytes. In order to further characterize keratinocyte stem cells, the identification of plasma membrane markers of basal keratinocytes was performed by isolating membrane microdomains from keratinocytes. By proteomic approach, we identified proteins representing potential cell surface markers of the epidermis. Altogether, these results allowed us to define a new strategy to isolate progenitors of basal cells and consequently keratinocyte stem cells.EVRY-BU (912282101) / SudocSudocFranceF

Caractérisation fonctionnelle et moléculaire de progéniteurs épidermiques humains

L'épiderme est un tissu en constant renouvellement dont la régénération est due à la présence de cellules souches à sa base. En raison du manque de marqueurs spécifiques des cellules souches épidermiques qui permettraient de les purifier, la biologie de ces cellules est mal connue. Or, le phénotype d'exclusion du Hœchst 33342, appelé SP, apparaît comme une caractéristique commune aux cellules souches de nombreux tissus adultes. Cette technique a donc été adaptée pour isoler une population SP à partir de kératinocytes humains en culture dont les identités cellulaire et moléculaire ont ensuite été étudiées. Ainsi, un kératinocyte SP possède un potentiel de prolifération suffisamment élevé pour régénérer la totalité de l'épiderme, propriété attribuée aux cellules souches de ce tissu. Parallèlement, une expérience d'hybridation sur puces à ADN a permis de mettre en évidence 41 gènes différentiellement exprimés, 37 gènes étant surexprimés dans les kératinocytes SP contre 4 gènes réprimés.The aim of the present study was to characterize human progenitor cells isolated from primary keratinocyte cultures. For that purpose, the Hoechst exclusion assay described for haematopoietic cells was adapted to keratinocytes to isolate SP cells that exclude the dye. We show that SP cells represented an average of 0.16 % of the total population. These keratinocytes exhibited a larger expansion potential in long-term cultures ; indeed, one SP cell can generate enough keratinocytes to cover the whole body. To further characterize SP cells, cDNA microarrays spotted with 9120 probes were used to identify their molecular signature. Transcriptome analysis showed that 41 genes were differentially expressed, with 37 up-regulated genes and only 4 down-regulated genes in SP cells. In conclusion, SP keratinocytes can be isolated from primary cultures of adult human epidermis. These cells exhibit a high proliferative potential and a specific gene expression profile.EVRY-BU (912282101) / SudocSudocFranceF

Inégalités sociales d'accès aux soins primaires (l'exemple des consultations inappropriées aux urgences)

PARIS6-Bibl. St Antoine CHU (751122104) / SudocSudocFranceF

Analyse fonctionnelle des mutations du gène GJB6 codant la connexine30 humaine responsable du syndrome de Clouston

La dysplasie ectodermique hidrotique ou syndrome de Clouston est une génodermatose rare qui se caractérise par une hyperkératose palmoplantaire, une alopécie généralisée et une dystrophie des ongles. Nous avons démontré que ce syndrome est dû à des mutations dans le gène GJB6, qui code la connexine30 humaine, pour lequel nous avons déterminé la structure génomique et analysé la région promotrice. L'analyse fonctionnelle des formes mutées de la protéine a permis de démontrer que leur transport bénéficie de la présence de la Cx30 sauvage et que les jonctions communicantes formées de Cx30 mutées sont fonctionnelles. Cependant, les Cx30 mutées se sont avérées capables de former des hemicanaux non-appariés constitutivement ouverts à la surface de la cellule qui entraînent une fuite d'ATP vers le milieu extracellulaire. Cette observation nous a permis d'émettre l'hypothèse que cette libération d'ATP est à l'origine du phénotype observé au niveau de l'épiderme atteint.EVRY-BU (912282101) / SudocSudocFranceF