Pleiotropy of Glycogen Synthase Kinase-3 Inhibition by CHIR99021 Promotes Self-Renewal of Embryonic Stem Cells from Refractory Mouse Strains

Background: Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. b-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains. Methodology/Principal Findings: We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of b-catenin and c-Myc protein levels. Stabilized b-catenin promoted ES self-renewal through two mechanisms. First, b-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor–independent manner. Second, b-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. b-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice. Conclusions/Significance: Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cell

Targeted Knockdown of RNA-Binding Protein TIAR for Promoting Self-Renewal and Attenuating Differentiation of Mouse Embryonic Stem Cells

RNA-binding protein TIAR has been suggested to mediate the translational silencing of ARE-containing mRNAs. To analyze the functions of TIAR, we established RNAi and genetic rescue assays. We evaluated the expression of neuroectoderm markers Pax6 and nestin, mesoderm markers brachyury and Flk1, and hypoblast and definitive endoderm markers Sox17 and Gata6 during EB differentiation and found that knockdown TIAR expression restrained the differentiation of E14 cells. We assessed gene expression levels of Flk-1 and VE-cadherin and observed attenuated differentiation of E14 cells into endothelial cells upon downregulation of TIAR gene expression. As such, we hypothesized an essential role of TIAR related to EB differentiation. As TIAR inhibits the translation of c-myc, we proposed that downregulation of TIAR results in restrained differentiation of E14 cells, due in part to the function of c-myc. We found that TIAR inhibited c-myc expression at the translational level in E14 cells; accordingly, a reduction of TIAR expression promoted self-renewal of pluripotent cells and attenuated differentiation. Additionally, we established that TIAR inhibited TIA-1 expression at the translational level in E14 cells. Taken together, we have contributed to the understanding of the regulatory relationships between TIAR and both c-myc and TIA-1

Targeted Knockdown of RNA-Binding Protein TIAR for Promoting Self-Renewal and Attenuating Differentiation of Mouse Embryonic Stem Cells

RNA-binding protein TIAR has been suggested to mediate the translational silencing of ARE-containing mRNAs. To analyze the functions of TIAR, we established RNAi and genetic rescue assays. We evaluated the expression of neuroectoderm markers Pax6 and nestin, mesoderm markers brachyury and Flk1, and hypoblast and definitive endoderm markers Sox17 and Gata6 during EB differentiation and found that knockdown TIAR expression restrained the differentiation of E14 cells. We assessed gene expression levels of Flk-1 and VE-cadherin and observed attenuated differentiation of E14 cells into endothelial cells upon downregulation of TIAR gene expression. As such, we hypothesized an essential role of TIAR related to EB differentiation. As TIAR inhibits the translation of c-myc, we proposed that downregulation of TIAR results in restrained differentiation of E14 cells, due in part to the function of c-myc. We found that TIAR inhibited c-myc expression at the translational level in E14 cells; accordingly, a reduction of TIAR expression promoted self-renewal of pluripotent cells and attenuated differentiation. Additionally, we established that TIAR inhibited TIA-1 expression at the translational level in E14 cells. Taken together, we have contributed to the understanding of the regulatory relationships between TIAR and both c-myc and TIA-1

Yesassociated protein (yap) is required for early embryonic development in zebrafish (Danio rerio

Abstract The hippo (Hpo) signaling pathway plays a critical role in regulation of organ size. The kinase cascade ultimately antagonizes the transcriptional co-activator Yki/YAP, which is a key regulator of cell proliferation and apoptosis. In this study, we performed a knocking down study using antisense morpholino (MO) reagents and found that zebrafish YAP, a key transcriptional co-activator of Hpo pathway, plays a critical role in early embryonic development. At the cellular level, yap inhibition increases apoptosis and decreases cell proliferation. Reduction of yap function severely delays several developmental events, including gastrulation, cardiogenesis and hematopoiesis. Knockdown of yap showed some evidence of ventralization, including reduction of dorsally expressed marker goosecoid (gsc), expansion of ventral marker gata2, disruption of the somites, and reduction in head size. Finally, we performed a preliminary analysis with real-time polymerase chain reaction (qPCR) for the candidate targets of zebrafish Hpo pathway. In conclusion, our results revealed that zebrafish yap coordinately regulates cell proliferation and apoptosis and is required for dorsoventral axis formation, gastrulation, cardiogenesis, hematopoiesis, and somitogenesis

Characterization of a novel bifunctional mutant of staphylokinase with platelet-targeted thrombolysis and antiplatelet aggregation activities-1

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a novel bifunctional mutant of staphylokinase with platelet-targeted thrombolysis and antiplatelet aggregation activities"</p><p>http://www.biomedcentral.com/1471-2199/8/88</p><p>BMC Molecular Biology 2007;8():88-88.</p><p>Published online 7 Oct 2007</p><p>PMCID:PMC2194782.</p><p></p>ermination of purified RGD-SAK by MALDF-TOF mass spectrometry. Peaks corresponding to various protonated RGD-SAK species are marked. The mass analysis determined that the RGD-SAK has a molecular weight of 15.4 KDa. (C) The secondary structure contents of RGD-SAK and SAK analyzed by far-UV CD spectrum. The secondary structure revealed by the curve of the two proteins were very similar. (D) RGD-SAK fibrinolysis activity assayed on fibrin plate. The numbers above the well represent various concentration of samples. 1–5 represent various concentrations of standard SAK (6.5 IU, 12.5 IU, 25 IU, 50 IU, 100 IU/well, respectively). Duplicate wells (6–10) corresponding to 1–5 were included. 11–13 represent various diluted RGD-SAK determined by comparing with the standard SAK. Three duplicated sets (14–16, 17–19, 20–22) of RGD-SAK corresponding to 11–13 were also shown

Characterization of a novel bifunctional mutant of staphylokinase with platelet-targeted thrombolysis and antiplatelet aggregation activities-2

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a novel bifunctional mutant of staphylokinase with platelet-targeted thrombolysis and antiplatelet aggregation activities"</p><p>http://www.biomedcentral.com/1471-2199/8/88</p><p>BMC Molecular Biology 2007;8():88-88.</p><p>Published online 7 Oct 2007</p><p>PMCID:PMC2194782.</p><p></p>ts. (B) ELISA was taken to measure the OD450, which represented the quantity of SAK or RGD-SAK bound to the active platelet. The statistical differences are significant for each tested concentration (RGD-SAK vs SAK, < 0.01). The means ± SD came from three separate experiments

Characterization of a novel bifunctional mutant of staphylokinase with platelet-targeted thrombolysis and antiplatelet aggregation activities-4

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a novel bifunctional mutant of staphylokinase with platelet-targeted thrombolysis and antiplatelet aggregation activities"</p><p>http://www.biomedcentral.com/1471-2199/8/88</p><p>BMC Molecular Biology 2007;8():88-88.</p><p>Published online 7 Oct 2007</p><p>PMCID:PMC2194782.</p><p></p>o recombinant proteins taken as 100%. The statistical differences between the inhibitory effects of RGD-SAK and SAK for each tested concentration are significant (< 0.01)