3 resultados para ORGANIZATION AND MANAGEMENT OF ENTERPRISES

em DigitalCommons@The Texas Medical Center


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Glycoprotein (GP) Ib-IX complex, the second most abundant receptor expressed on the platelet surface, plays critical roles in haemostasis and thrombosis by binding to its ligand, von Willebrand factor (vWF). Defect or malfunction of the complex leads to severe bleeding disorders, heart attack or stroke. Comprised of three type I transmembrane subunits—GPIbα, GPIbβ and GPIX, efficient expression of the GPIb-IX complex requires all three subunits, as evident from genetic mutations identified in the patients and reproduced in transfected Chinese hamster ovary (CHO) cells. However, how the subunits are assembled together and how the complex function is regulated is not fully clear. By probing the interactions among the three subunits in transfected cells, we have demonstrated that the transmembrane domains of the three subunits interact with one another, facilitating formation of the two membrane-proximal disulfide bonds between GPIbα and GPIbβ. We have also identified the interface between extracellular domains of GPIbβ and GPIX, and provided evidence suggesting a direct interaction between extracellular domains of GPIbα and GPIX. All of these interactions are not only critical for correct assembly and consequently efficient expression of the GPIb-IX complex on the cell surface, but also for its function, such as the proper ligand binding, since removing the two inter-subunit disulfide bonds significantly hampers vWF binding to the complex under both static and physiological flow conditions. The two inter-subunit disulfide bonds are also critical for regulating the ectodomain shedding of GPIbα by the GPIbβ cytoplasmic domain. Mutations in the juxtamembrane region of the GPIbβ cytoplasmic domain deregulate GPIbα shedding, and such deregulation is further enhanced when the two inter-subunit disulfide bonds are removed. In summary, we have established the overall organization of the GPIb-IX complex, and the importance of proper organization on its function. ^

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Genes of the basic helix-loop-helix transcription factor family have been implicated in many different developmental processes from neurogenesis to myogenesis. The recently cloned bHLH transcription factor, paraxis, has been found to be expressed in the paraxial mesoderm of the mouse suggesting a role for paraxis in the development of this mesodermal subtype which gives rise to the axial muscle, skeleton, and dermis of the embryo. In order to perform in vivo gain of function assays and obtain a better understanding of the possible roles of paraxis in mesodermal and somitic development, we have successfully identified homologues of paraxis in the frog, Xenopus laevis, where the process of mesodermal induction and development is best understood. The two homologues, Xparaxis-a and Xparaxis-b, are conserved with respect to their murine homologue in structure and expression within the embryo. Xparaxis genes are expressed immediately after gastrulation in the paraxial mesoderm of Xenopus embryos and are down regulated in the myotome of the mature somite with continued expression in the undifferentiated dermatome. Overexpression of Xparaxis-b in Xenopus embryos caused defects in the organization and morphology of the somites. This effect was not dependent on DNA binding of Xparaxis but is likely due to its dimerization with other bHLH factors. Co-injections with XE12 did not diminish the effects indicating that the defects were not the result of limiting amounts of XE12. We also demonstrated that Xparaxis does not cause obvious defects in the cell adhesions and movements required for proper mesoderm patterning during gastrulation. The paraxis proteins also lacked the ability to activate transcription as GAL4 fusion proteins in a GAL4 reporter assay, indicating that the genes may function more as modulators of the activity of dimerization partners than as positively acting cell determination factors. In agreement with this, Xparaxis is regulated in response to other pathways of bHLH gene action, in that XE12 can activate Xparaxis-b, in vivo. In addition we show regulation of Xparaxis in response to mMyoD induced myogenesis pathways, again suggesting Xparaxis plays an important role in the patterning and organization of the paraxial mesoderm. ^

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Transmembrane segments of polytopic membrane proteins once inserted are generally considered stably oriented due to the large free energy barrier for topological reorientation of adjacent extra-membrane domains. However, proper topology and function of the polytopic membrane protein lactose permease (LacY) of Escherichia coli is dependent on the membrane phospholipid composition revealing topological dynamics of transmembrane domains (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107–2116). The high affinity phenylalanine permease PheP shares many topological similarities with LacY. In this study, mutant E. coli cells lacking phosphatidylethanolamine (PE) as a membrane component were used to evaluate the role of PE in the function and assembly of PheP. Active transport of phenylalanine by cells lacking PE was severely inhibited (both Vmax and Km were altered), whereas the PheP protein level in membranes was unaffected. Cysteine residues were introduced into predicted periplasmic or cytoplasmic segments of cysteine-less PheP, and the topology of the protein was explored using a membrane-impermeable thiol-specific biotinylated probe. Based on the biotinylation patterns of PheP in whole cells, the N-terminus and adjoining transmembrane hairpin of PheP adopted an inverted topological orientation in PE-lacking cells. Introduction of PE following the assembly of PheP triggered a reorientation of the N-terminus and adjacent hairpin to their native orientation associated with regain of wild type transport function. These results coupled with the results for LacY support a specific role for membrane lipid composition in determining topological organization and function of membrane proteins. Several other secondary symporters are compromised for activity in PE-lacking cells suggesting that lipid-assisted topogenesis is a general property of such transporters. The reversible orientation of these secondary transport proteins in response to a change of phospholipid composition might be a result of inherent conformational flexibility necessary for transport function or during protein assembly. ^