Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis.

International audienceHuman cdc25C protein, a specific tyrosine phosphatase that activates the p34cdc2 protein kinase at mitosis, is itself a phosphoprotein that shows increased phosphorylation during the G2-M transition. In vitro, cdc25C protein is substantially phosphorylated by purified p34cdc2-cyclin B protein kinase. Of seven putative phosphorylation sites for p34cdc2 protein kinase present in human cdc25C, five are phosphorylated by p34cdc2 protein kinase in vitro, as assessed by tryptic phosphopeptide mapping and peptide sequencing. These same sites are also phosphorylated in vivo during the G2-M transition in normal mammalian fibroblasts and have been precisely mapped. The cdc25C phosphorylated in vitro by p34cdc2 protein kinase exhibits a 2-3-fold higher activity than the nonphosphorylated cdc25C, as assayed by activation of inactive cdc2 prokinase. Microinjection of purified cdc25C proteins into living fibroblasts reveals that only the phosphorylated form of cdc25 is highly effective in activating G2 cells into premature prophase in a manner similar to microinjection of purified active p34cdc2 protein kinase. Together these data show that multisite phosphorylation of cdc25C by p34cdc2-cyclin B protein kinase occurs at the G2-M transition and is sufficient to induce the autoamplification of cdc2/M-phase promoting factor necessary to drive somatic mammalian cells into mitosis

Sheep Updates 2005 - Part 5

This session covers eleven papers from different authors; MARKET INFORMATION 1. Crystal Spring - Crystal clear and consistant, Geoff Duddy, Livestock Officer (Sheep & Wool) Yanco, NSW, Brent McLoud, (Product Development Officer) Cowra, NSW, John Sullivan, J.J Dresser and Co (Agent), Woodstock, NSW 2. An overview of Recent Developments in Dark and Medullated Fibre Testing, T.J. Mahar, A. Balasingam, AWTA Ltd 3. Opportunities and Implications for Wool Producers of the TEAM3 Prediction Equations, J.H. Stanton12 K.M.S. Curtis1 , 1Department of Agriculture Western Australia, 2 Curtin University, WA 4. Premiums and Discounts for Fibre Properties in Superfine Wool, Now and in the Future?, K.M.S. Curtis1, P.R. Lamb2, 1 Department of Agriculture Western Australia, 2Lambshift Consulting, Geelong VIC FEEDLOTTING 5. Manure in sheep feedlots: problem or opportunity?, Eliza Dowling, Ned Crossley Department of Agriculture , Western Australia, Surrender Mann, Chemistry Centre (WA), East Perth WA, 6. The State of Lamb Confinement Feeding in WA, Ned Crossley, Department of Agriculture, Western Australia 7. Finishing lambs in a feed lot - Is it profitable?, Lucy Anderton, Department of Agriculture, Western Australia 8. Repeated live weights can mardinally improve prediction of compliance to markey specifications, Mattew Kelly, Andrew Swan, CSIRO livestock industries, Ian McFarland, Department of Agriculture Western Australia. WELFARE 9. Mulesing accreditation - to be or not to be? Di Evans, Department of Agriculture, Western Australia. 10. The Economic and Research Implications of managing Merino Sheep with out Mulesing, K. Bell, Sheep Management and Production Consultants, North Fremantle WA, D. Sackett, Homes Sackett and Associates, Wagga Wagga NSW 11. How do lambs fare during curfew, Dr Robin Jacob, Department of Agriculture, Western Australi

Combination of metformin with sodium selenite induces a functional phenotypic switch of human GM-CSF monocyte-derived macrophages

International audienc

Méthode computationnelle pour la prédiction de la mobilité des peptides et l'identification de leurs sites de phosphorylation par empreinte phospho-peptidique bidimensionnelle sur couche mince de cellulose

Actuellement, un des plus importants challenges en biologie est la compréhension des mécanismes moléculaires modulant et régulant les fonctions cellulaires, et plus particulièrement le rôle des modifications post-traductionnelles des protéines. Contrairement au génome, qui est le même pour chacun des cellules d'un organisme, les protéomes varient dynamiquement en fonction du type cellulaire, de la physiologie cellulaire et au travers de modifications post-traductionnelles intervenant sur plusieurs sites simultanément. Pour les protéines eucaryotes, la phosphorylation réversible est parmi les plus importantes modifications et agit en modulant de nombreux évènements cellulaires incluant la progression du cycle cellulaire, la croissance et la différentiation. Approximativement 30% des protéines cellulaires sont susceptibles d'être phosphorylées et environ 30% de ces protéines sont phosphorylées à tout moment. Bien que le marquage métabolique avec [32P] permette d'accroître le seuil de détection des phospho-protéines, ce dernier n'est pas compatible avec la spectrométrie de masse. Nous avons donc développé une méthode alternative pour l'identification des protéines et de leurs modifications post-traductionnelles à partir de la séparation de peptides trypsiques en deux dimensions sur couche mince de cellulose. Les peptides sont, dans un premier temps, séparés par électrophorèse à haut voltage en fonction de leurs charges nettes et de leurs masses moléculaires avant d'être séparés en deuxième dimension en fonction de leurs propriétés physico-chimiques par chromatographie ascendante. La mobilité est mesurée pour chacun des peptides dans les deux dimensions en co-migrant des peptides marqueurs pour calibrer la migration, et leur identification est établie à partir de leurs scores d'identité dérivés des modèles calculant les mobilités électrophorétiques et chromatographiques théoriques à partir de la séquence primaire des peptides. Le taux de réussite de notre approche est estimé à 98% pour un mélange peptidique simple et à 80% sur l'ensemble des digestions protéiques. De plus, nous sommes capable d'identifier spécifiquement une même séquence peptidique contenant des sites phosphorylés, oxydés et/ou d'exception pour la trypsineTo date, one of the most important challenges in biology is to understand the molecular mechanisms which modulate and regulate cellular functions and in particular the role of protein post-translational modification. Contrary to the genome with is the same for each cell within an organism, proteomes vary dynamically according to cell type, cell physiology and through multi-site simultaneous post-translational modifications. In eukaryotic proteins, reversible phosphorylation is among the most important modifications and acts in modulating many cellular events including cell cycle transit, growth and differentiation. Approximately 30% of cellular proteins are susceptible to phosphorylation and around 30% of these proteins are phosphorylated at any given time. While metabolic labelling with [32P] provides a powerful and highly sensitive means to increase phospho-protein detection threshold, it is presently incompatible with mass spectrometry. We have therefore developed an alternative method for identifying proteins and post-translational modifications based on analysis of two dimensional tryptic peptide separation on thin layer cellulose plates. Peptides are separated by high voltage electrophoresis according to net charge and molecular mass before second dimension separation by physical-chemical characteristics in a water solvent exchange system using ascending chromatography. Phospho-peptide mobility is measured for each peptide in each dimension with the use of co-migrated marker peptides to calibrate peptide migrations and peptide identity established from an identity score derived from models calculating the predicted electrophoretic and chromatographic mobilities from the primary aminoacid sequence of the peptides. The success rate of this approach, which exceeds 98% on simple peptide mixtures, is estimated at 80% on whole protein digests. Moreover, we are able to specifically differentiate a same peptidic sequence containing phosphorylated, oxidized amino acids and/or exception trypsic sitesMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Dietary apigenin in the prevention of endothelial cell dysfunction

International audienc

Étude des mécanismes d'aneuploïdie dans les gamètes humains (apport de la cytogénétique moléculaire)

MONTPELLIER-BU Médecine UPM (341722108) / SudocMONTPELLIER-BU Médecine (341722104) / SudocSudocFranceF

Des cellules souches dérivées de muscles adultes se greffent et se différencient en cellules exprimant l'insuline dans les îlots pancréatiques de souris diabétiques soulageant l'hyperglycémie.

International audienceStatement of the Problem: Pancreatic beta cells are unique effectors in the control of glucose homeostasis and their deficiency results in impaired insulin production leading to severe diabetic diseases. Here, we investigated the potential of a population of non-adherent Muscle-Derived Stem Cells (MDSC) from adult mouse or human muscle to differentiate in vitro into beta cells and when transplanted in vivo, as undifferentiated stem cells, differentiate in vivo and compensate for beta cell deficiency. Methodology & Theoretical Orientation: In vitro, MDSC were isolated on the basis of their poor adherence by serial preplating for 8 days. MDSC cultured for several weeks, spontaneously differentiated into insulin-expressing islet-like cell clusters as revealed using MDSC from transgenic mice expressing GFP or mCherry under the control of an insulin promoter. Differentiated clusters of beta-like cells co-expressed insulin with the transcription factors Pdx1, Nkx2.2, Nkx6.1 and MafA, and secreted significant levels of insulin in response to glucose challenges. In vivo, undifferentiated MDSC injected intraperitoneal into streptozotocin (STZ)-treated mice, engrafted within 48h specifically into damaged pancreatic islets and are shown to differentiate and express insulin 2-12 days after injection. In addition injection of MDSC to hyperglycemic diabetic STZ treated mice reduced their blood glucose levels for 2 to 10 weeks. Conclusion & Significance: These data show that muscle stem cells, MDSC, are capable of differentiating into mature pancreatic beta islet-like cells not only upon culture in vitro but also in vivo after systemic injection in STZ-induced diabetic mouse models. Being non teratogenic, MDSC can be used directly by systemic injection and this potential reveals a promising alternative avenue in stem cell-based treatment of beta cell deficiencies. ; Ned Lamb et al., Endocrinol Diabetes Res 2019, Volume 05Exposé du problème: Les cellules bêta du pancréas sont des effecteurs uniques dans le contrôle de l'homéostasie du glucose et leur carence entraîne une production insuffisante d'insuline menant à de graves maladies du diabète. Ici, nous avons étudié le potentiel d’une population de cellules souches dérivées des muscles non adhérentes (MDSC) provenant de muscles de souris ou humains adultes à se différencier in vitro en cellules bêta et lorsqu’une greffe in vivo, en tant que cellules souches indifférenciées, différencie in vivo et compense pour le déficit en cellules bêta. Méthodologie et orientation théorique: In vitro, les CDEM ont été isolés sur la base de leur faible adhérence par prélaquage en série pendant 8 jours. MDSC en culture pendant plusieurs semaines, spontanément différenciée en grappes de cellules ressemblant à des îlots exprimant l'insuline, révélée à l'aide de MDSC provenant de souris transgéniques exprimant la GFP ou mCherry sous le contrôle d'un promoteur de l'insuline. Des groupes différenciés de cellules de type bêta co-exprimaient l'insuline avec les facteurs de transcription Pdx1, Nkx2.2, Nkx6.1 et MafA, et sécrétaient des taux importants d'insuline en réponse à des problèmes de glucose. MDSC non différenciée in vivo, injectée par voie intrapéritonéale à des souris traitées à la streptozotocine (STZ), greffée 48 heures plus tard dans des îlots pancréatiques endommagés, et capable de différencier et d'exprimer l'insuline 2 à 12 jours après l'injection. De plus, l'injection de MDSC à des souris hyperglycémiques diabétiques traitées à la STZ a réduit leur glycémie pendant 2 à 10 semaines. Conclusion et signification: Ces données montrent que les cellules souches musculaires, MDSC, sont capables de se différencier en cellules matures ressemblant à des îlots bêta du pancréas, non seulement lors d'une culture in vitro, mais également in vivo après une injection systémique chez des modèles de souris diabétiques induites par STZ. Étant non tératogène, le MDSC peut être utilisé directement par injection systémique et ce potentiel révèle une alternative prometteuse dans le traitement par cellules souches des carences en cellules bêta. ; Ned Lamb et al., Endocrinol Diabetes Res 2019, Volume 0

Adult Muscle-derived stem cells engraft and differentiate into insulin-expressing cells in pancreatic islets of diabetic mice relieving hyperglycemia

International audiencePancreatic beta cells are unique effectors in the control of glucose homeostasis and their deficiency results in impaired insulin production leading to severe diabetic diseases. Here, we investigated the potential of a population of non-adherent Muscle-Derived Stem Cells (MDSC) from adult mouse or human muscle to differentiate in vitro into beta cells and when transplanted in vivo, as undifferentiated stem cells, differentiate in vivo and compensate for beta cell deficiency

Linking PCNA-dependent replication and ATR by human Claspin.

Recent studies in Xenopus have identified a new checkpoint protein called Claspin that is believed to transduce the checkpoint DNA damage signals to Chk1 kinase. Here we show that the human Claspin homolog is a chromatin bound protein either in the absence or in the presence of damaged DNA, independent of its association with ATR. Furthermore, we show that human Claspin is found in complex with PCNA, an essential component of the DNA replication machinery, and is released upon DNA replication arrest. Interfering with PCNA function by overexpression of p21 mutant, impaired in its interaction with Cdks but not with PCNA, leads to ATR-dependent Chk1 activation. These findings suggest that the dissociation of Claspin-PCNA could be part of the signal leading to Chk1 activation