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Secreted Glycoprotein (secreted + glycoprotein)
Selected AbstractsProduction of a Secreted Glycoprotein from an Inducible Promoter System in a Perfusion BioreactorBIOTECHNOLOGY PROGRESS, Issue 5 2004Matthew L. Lipscomb The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated. [source] Contribution of the Reelin signaling pathways to nociceptive processingEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2008Alin L. Akopians Abstract The reeler gene encodes Reelin, a secreted glycoprotein that binds to the very-low-density lipoprotein receptor (Vldlr) and apolipoprotein E receptor 2 (Apoer,2), and induces Src- and Fyn-mediated tyrosine phosphorylation of the intracellular adaptor protein Disabled-1 (Dab1). This Reelin,Dab1 signaling pathway regulates neuronal positioning during development. A second Reelin pathway acts through Apoer,2,exon 19 to modulate synaptic plasticity in adult mice. We recently reported positioning errors in reeler dorsal horn laminae I,II and V, and the lateral spinal nucleus. Behavioral correlates of these positioning errors include a decreased mechanical and increased thermal sensitivity in reeler mice. Here we examined mice with deletions or modifications of both the Reelin,Dab1 signaling pathway and the Reelin,Apoer,2,exon 19 pathway on a Vldlr-deficient background. We detected reeler -like dorsal horn positioning errors only in Dab1 mutant and Apoer,2/Vldlr double mutant mice. Although Dab1 mutants, like reeler, showed decreased mechanical and increased thermal sensitivity, neither the single Vldlr or Apoer,2 knockouts, nor the Apoer,2,exon 19 mutants differed in their acute pain sensitivity from controls. However, despite the dramatic alterations in acute ,pain' processing in reeler and Dab1 mutants, the exacerbation of pain processing after tissue injury (hindpaw carrageenan injection) was preserved. Finally, we recapitulated the reeler dorsal horn positioning errors by inhibiting Dab1 phosphorylation in organotypic cultures. We conclude that the Reelin,Dab1 pathway differentially contributes to acute and persistent pain, and that the plasticity associated with the Reelin,Apoer,2,exon 19 pathway is distinct from that which contributes to injury-induced enhancement of ,pain' processing. [source] Cellular repressor of E1A-stimulated genes attenuates cardiac hypertrophy and fibrosisJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 7 2009Zhouyan Bian Abstract Cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein of 220 amino acids. It has been proposed that CREG acts as a ligand that enhances differentiation and/or reduces cell proliferation. CREG has been shown previously to attenuate cardiac hypertrophy in vitro. However, such a role has not been determined in vivo. In the present study, we tested the hypothesis that overexpression of CREG in the murine heart would protect against cardiac hypertrophy and fibrosis in vivo. The effects of constitutive human CREG expression on cardiac hypertrophy were investigated using both in vitro and in vivo models. Cardiac hypertrophy was produced by aortic banding and infusion of angiotensin II in CREG transgenic mice and control animals. The extent of cardiac hypertrophy was quantitated by two-dimensional and M-mode echocardiography as well as by molecular and pathological analyses of heart samples. Constitutive over-expression of human CREG in the murine heart attenuated the hypertrophic response, markedly reduced inflammation. Cardiac function was also preserved in hearts with increased CREG levels in response to hypertrophic stimuli. These beneficial effects were associated with attenuation of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase 1 (MEK-ERK1)/2-dependent signalling cascade. In addition, CREG expression blocked fibrosis and collagen synthesis through blocking MEK-ERK1/2-dependent Smad 2/3 activation in vitro and in vivo. Therefore, the expression of CREG improves cardiac functions and inhibits cardiac hypertrophy, inflammation and fibrosis through blocking MEK-ERK1/2-dependent signalling. [source] Phosphorylated osteopontin promotes migration of human choriocarcinoma cells via a p70 S6 kinase-dependent pathwayJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Rania Al-Shami Abstract This study examined the role of osteopontin (OPN), a phosphorylated secreted glycoprotein, in the promotion of trophoblastic cell migration, an early event in the embryo implantation process. Three human choriocarcinoma cell lines, namely JAR, BeWo, and JEG-3, were treated with variants of OPN differing in the extent of phosphorylation following sequential dephosphorylation with tartrate-resistant acid phosphatase (TRAP), and their migratory response was measured. The highly phosphorylated human milk form of OPN (OPN-1) strongly triggered migration in all three cell lines, whereas the less phosphorylated variants, OPN-2a and OPN-2b, failed to stimulate migration. JAR cell migration in response to OPN-1 was accompanied by a rapid rearrangement of actin filaments to the cellular membrane. Using broad spectrum protein kinase profiling, we identified p70 S6 kinase as a major signal transduction pathway activated by OPN-1 during the migratory response in JAR cells. Activation was blocked completely by rapamycin and LY294002, thus demonstrating that OPN-1-stimulated migration occurs through mTOR and PI3K pathways, respectively. Conversely, PD98059 did not affect the activation of p70 S6 kinase by OPN-1, therefore, this response does not involve the Ras/ MAPK signaling cascade. Together, these data show that the highly phosphorylated human OPN-1 can stimulate trophoblastic cell migration and provides evidence for the involvement of the PI3K/mTOR/p70 S6 kinase pathway in the JAR cells response. Because both OPN and TRAP are expressed in the uterus during early pregnancy, it is conceivable that extracellular phosphatases such as TRAP may modify OPN charge state and thus modulate cell migration. © 2005 Wiley-Liss, Inc. [source] C-terminal region-dependent change of antibody-binding to the Eighth Reelin repeat reflects the signaling activity of ReelinJOURNAL OF NEUROSCIENCE RESEARCH, Issue 14 2009Takao Kohno Abstract Reelin is a secreted glycoprotein that plays pivotal roles in the development and function of the brain, but how it activates downstream intracellular signaling is not fully understood. We have recently reported that the highly conserved C-terminal region (CTR) of Reelin is required for its full signaling activity, although the underlying mechanism remains unknown. During biochemical study of Reelin, we serendipitously found that one commercially available anti-Reelin antibody G20 can bind to CTR-lacking mutant Reelin proteins, but not wild-type Reelin, on Western blotting. The G20 epitope resides in the last 19 residues of Reelin-repeat 8 (RR8), and neither posttranslational modification nor proteolysis can explain this effect. Furthermore, when an unrelated sequence, such as FLAG-tag, is inserted between RR8 and CTR, the reactivity of the corresponding antibody greatly decreases. These results suggest that RR8 and CTR form a tight structure that makes the surrounding sequence inaccessible to an antibody. Taking advantage of this phenomenon, we show the existence of CTR-lacking Reelin isoform in vivo for the first time and estimate its contribution to the total amount of secreted Reelin. Importantly, the extent to which Reelin mutants react with G20 is inversely correlated with their signaling activity, indicating that the CTR-induced structural change of RR8 is a prerequisite for downstream signaling activation, presumably via binding to a certain neuronal membrane molecule(s). © 2009 Wiley-Liss, Inc. [source] Expression and downregulation of WNT signaling pathway genes in rhesus monkey oocytes and embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2006Ping Zheng Abstract Mammalian WNT genes encode secreted glycoproteins that are conserved homologues of the Drosophila Wingless gene, which plays a crucial role in Drosophila development. Recently, WNT pathway signaling has been implicated in ovarian development, oogenesis, and early development. We sought to evaluate whether these genes may contribute to the formation of healthy human oocytes or embryos, and whether the expression of these genes could provide informative markers of human oocyte and embryo quality. To do this, we employed the primate embryo gene expression resource (PREGER; www.preger.org) to examine expression of mRNAs encoding 38 components of the WNT signaling pathway in rhesus monkey oocytes and embryos as a nonhuman primate model. We observed considerable conservation between rhesus monkey and mouse of expression of WNT, FZD, and effector gene mRNAs, and a generalized downregulation of genes encoding key components of the WNT signaling pathway during preimplantation development. Our results support a role for WNT signaling during oocyte growth or maturation, but not during preimplantation development. Additionally, we observed differences between in vitro cultured and in vivo developing blastocysts, indicating possible effects of culture on WNT signaling during the peri-implantation period. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] | ||||||||||