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Cytokine signalling functions of human soluble IgE receptors in peripheral blood mononuclear cells from normal and hyper-allergic individuals and in B-lymphoblastoid and monocytic cell linesAskew, Sandra Lyn January 2006 (has links)
CD23 is a multifunctional receptor/ligand, found in a variety of cell types, such as human peripheral blood mononuclear cells (PBMCs), B-lymphoblastoid cell lines, mast cells and basophils. It is also found on a variety of haematopoietic cell lines. As the low-affinity receptor for immunoglobulin E (IgE), CD23 plays a role in antigen-presentation and macrophage activation. As a surface molecule cleaved from the cell membrane, soluble CD23 (sCD23) can act as an adhesion molecule and a cytokine. Perturbances of such molecular interactions may lead to various diseases such as allergies and other inflammatory diseases. It has been speculated that elevated levels of sCD23 may be used to bind secreted IgE, thus preventing it from binding to membrane CD23 on haematopoietic cells, preventing B cells from being activated into IgE producing cells. Signal transduction by sCD23 is dependent on cell subsets, ligands and co-factors required for its function. sCD23 plays a direct role in inducing tumour necrosis factor alpha (TNFα), interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1β) and soluble IL-1 receptor from activated human monocytes and PBMCs in vitro. Recombinant forms of 25 and 37 kDa human sCD23 were produced by polymerase chain reaction (PCR)-cloning into pET23a, a bacterial expression vector. The proteins were expressed and refolded, followed by purification by gel filtration chromatography. The purified proteins were biochemically characterized to ensure purity and biological activity, by observing the binding to human IgE both in enzyme-linked immunosorbant assay (ELISA) and surface plasmon resonance (SPR) spectroscopy. ELISA showed KD values of 7.23 x 10-9M and 8.12 x 10-9M for the 25 and 37 kDa proteins, respectively. These values were significantly lower than that of Hibbert et al., (2005). SPR data obtained for the 25 kDa CD23 was not of reliable quality but SPR for the 33kDa sCD23 showed a KD of 1.18 x 10-7M, close to that of Hibbert et al., (2005), J. Exp. Med, 202: 751-760. To test the therapeutic potential of the recombinant molecule, a B-lymphoblastoid cell line (Raji), a pre-monocytic cell line (U937), and PBMCs from normal and hyper-allergic individuals were used. All cells showed no change in production of cytokines. It is essential to investigate further cytokine functions and production implicated by recombinant forms of sCD23, as well as binding of sCD23 to CD21 and CD11b/c, and in vivo IgE regulation before a conclusion can be drawn as to whether recombinant sCD23 is a potential therapeutic target against allergic disease.
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An investigation on the role and regulation of signal transduction pathways during embryonic wound healingLi, Jingjing January 2013 (has links)
For years, it has been appreciated that embryos have remarkable abilities to heal wounds efficiently and perfectly, without scar formation. However, the molecular mechanisms underlying embryonic wound healing, especially how they coordinate and function in an efficient way, remains poorly understood. The primary aim of my PhD thesis was to use Xenopus as a model system to investigate the molecular and cellular mechanisms which are responsible for the regulation and coordination of embryonic wound healing. More specifically, my thesis includes the study of three signalling pathways during embryonic wound healing; namely the Erk MAPK pathway, PI3K pathway and inositol phosphate pathways. Erk and PI3K signalling are sequentially activated post injury, during separate phases of wound closure. The initial activation of Erk signalling governs the initial stage of wound closure, by mediating myosin-2 phosphorylation and actomyosin contraction through Rho activity. PI3K signalling increases in the late stage of wound closure, promotes leading edge migration and zippering via Rac and Cdc42 activity (Manuscript #1). From the findings of this study, I proposed a novel model, which suggests a cooperation of these two signalling pathways in orchestrating distinct cytoskeletal events during in tissue morphogenesis. In the second part of my thesis, I studied the role of inositol phosphate signalling during wound healing. In particular, I studied the role of the enzyme Itpkb and its product InsP4, in promoting rapid wound healing (Manuscript #2). Itpkb colocalizes with F-actin cable and promotes its formation at the wound edge in both single cell and multicellular wounds, enhancing the activity of three Rho GTPases Rac, Cdc42 and Rho at the same time. In addition, itpkb is required for calcium propagation from the wound edge to distant cells, suggesting a role in transmitting the wound signal across the tissue, resulting in the coordination of healing in multicellular wounds. Together, these PhD work provided more insights into the in vivo regulation of intracellular and intercellular signals in coordinating cell behavior in tissue movement during embryonic wound healing.
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Structural and Biophysical Studies of Hair Cell Mechanotransduction ProteinsDionne, Gilman January 2020 (has links)
Our senses of hearing, balance, and motion are the result of hair cells that act as cellular accelerometers to transmute mechanical forces into electrical signals for decoding by the central nervous system. To accomplish this task of mechanoelectrical transduction (MET), hair cells use an array of stereocilia that conduct electrical currents in response to mechanical stimuli. At the tips of these stereocilia, hair cells assemble over a dozen protein components to construct a sophisticated nanomachine that couples the movement of their stereocilia with the opening of mechanically gated ion channels. When forces impinge on stereocilia, they pivot at their base, slide past each other, and impart tension on the tip link, a protein linkage that connects the side of one stereocilium to the mechanically gated ion channel of an adjacent, shorter stereocilium.
While this conceptual framework of hair cell of mechanoelectrical transduction has been established, a precise molecular description of the proteins that comprise the machinery is still lacking. A structural understanding of the molecular components of the MET complex and how they function in mechanoelectrical transduction are limited. While previous structural studies on MET-related molecules have been performed, they have not yet produced a clear understanding of the molecular mechanisms underlying MET. The work presented in this thesis seeks to expand our structural descriptions of different components of the transduction machinery, and to validate the functional mechanisms suggested by these descriptions.
The tip link that couples adjacent stereocilia is composed of two large proteins – cadherin 23 and protocadherin 15 (PCDH15). It had previously been demonstrated that the extracellular region of the tip link protein PCDH15 was cis-dimeric, yet the molecular details of PCDH15 self-interaction remained elusive despite the existence of structural information for two-thirds of the molecule. Using a series of biophysical experiments and electron microscopy, we revealed that dimerization of the PCDH15 ectodomain is mediated by two distinct interfaces at opposite ends of the molecule. We determined the crystal structure of one of these interfaces, allowing us to engineer monomeric versions of PCDH15 through structure-guided mutations. By expressing these monomeric versions of PCDH15 in hair cells, we were able to demonstrate the functional importance of PCDH15 cis-dimerization for transduction.
Within the MET machinery is an elastic element that is in series with the mechanically gated ion channel. While some have postulated the molecular identity of this so-called gating spring to be the tip link proteins, others have cast doubts on this idea due to an apparent mismatch in their stiffness. Along with our collaborators, we performed single-molecule photonic force microscopy studies to directly measure the elasticity of monomeric PCDH15. By analyzing unfolding events observed in these single-molecule experiments, we determined the unfolding behavior of PCDH15 domains. Our results suggested that individual domains of PCDH15 unfold in the force regime of native sound response, and suggest that the domains of PCDH15 and CDH23 can be unfolded in functional hair bundles.
On the cytoplasmic side of the MET machinery, it was recently shown that the cytosolic calcium and integrin-binding proteins CIB2 and CIB3 interact with the MET associated membrane protein TMC1. This interaction has been shown to be critical for MET, but the molecular functions of these proteins remained unknown. Using a series of co- immunoprecipitation experiments and peptide binding assays, we defined the CIB2 binding region of TMC1. By determining the crystal structures of CIB3 and its complex with a TMC1- peptide, we were able to visualize the molecular determinants that allow CIB3 to interact with itself and TMC1. We demonstrate that CIB2 mutations affect the single channel conductance of the MET channel, indicating that CIB2 is a regulator of the MET complex.
Lastly, I sought to develop methods and protocols for producing samples of MET associated membrane proteins TMIE and TMC1 for structural studies, ultimately producing milligram quantities of TMIE. Using multi-angle light scattering, I found evidence that TMIE forms a hexamer. I also explored the potential of using Caenorhabditis elegans to generate a sample of native-like TMC protein. Both of these lines of work require continuing experimentation.
Overall, the works presented here provide molecular descriptions of various MET related proteins. Our structural and biophysical studies of PCDH15 revealed the molecular determinates of PCDH15 cis-dimerization and enabled us to engineer PCDH15 mutants with novel properties. The helical nature of PCDH15 suggests a mechanism for tip link extension through helical unwinding. Our single molecule investigations of PCDH15 strongly implicate it and CDH23 as the gating spring molecules. Our work with CIB2 establishes it as regulator of TMC1 function, and thus MET properties. The crystal structure of the CIB3:TMC1-peptide complex suggests potential mechanisms for this regulation that need to be investigated further.
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T Cell Rescue of Monocytes From Apoptosis: Role of the CD40-CD40L Interaction and Requirement for CD40-Mediated Induction of Protein Tyrosine Kinase ActivitySuttles, Jill, Evans, Mike, Miller, Robert W., Poe, Jonathan C., Stout, Robert D., Wahl, Larry M. 01 January 1996 (has links)
Circulating monocytes have a limited life span and will undergo apoptosis in the absence of specific stimuli. Recent studies have demonstrated that monocytes can be rescued from apoptosis via lipopolysaccharide (LPS) activation or stimulation with interleukin-1 or tumor necrosis factor-α. Based on previous studies from our laboratory, we hypothesized that, in nonseptic (e.g., autoimmune) inflammation, the presence of activated T cells may enhance monocyte longevity through T cell contact-dependent signaling. Plasma membranes prepared from 6 h activated (Tm(A)) and resting (Tm(R)) purified CD4+ T cells were added to resting elutriation-purified monocytes cultured in serum-free medium. Cells were assayed for degree of apoptosis occurring over a 72-h incubation using both agarose gel electrophoresis and flow cytometry. The addition of Tm(A) (but not Tm(R)) was capable of blocking monocyte apoptosis and the ability of Tm(A) to rescue monocytes was abrogated by the addition of anti-CD40L antibodies. Rescue of monocytes from apoptosis could also be mediated by direct cross-linking of monocyte CD40. Inhibitors of tyrosine kinase activity blocked both Tm(A) and anti-CD40-mediated rescue of monocytes from apoptosis, suggesting a primary role of a tyrosine kinase signaling pathway in the events controlling monocyte longevity.
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NF-κ Activation Is Required for the Development of Cardiac Hypertrophy in VivoLi, Yuehua, Ha, Tuanzhu, Gao, Xiang, Kelley, Jim, Williams, David L., Browder, I. William, Kao, Race L., Li, Chuanfu 01 October 2004 (has links)
In the present study, we examined whether NF-κB activation is required for cardiac hypertrophy in vivo. Cardiac hypertrophy in rats was induced by aortic banding for 1, 3, and 5 days and 1-6 wk, and age-matched sham-operated rats served as controls. In a separate group of rats, an IκB-α dominant negative mutant (IκB-αM), a superrepressor of NF-κB activation, or pyrrolidinedithiocarbamate (PDTC), an antioxidant that can inhibit NF-κB activation, was administered to aortic-banded rats for 3 wk. The heart weight-to-body weight ratio was significantly increased at 5 days after aortic banding, peaked at 4 wk, and remained elevated at 6 wk compared with age-matched sham controls. Atrial natriuretic peptide and brain natriuretic peptide mRNA expressions were significantly increased after 1 wk of aortic banding, reached a maximum between 2 and 3 wk, and remained increased at 6 wk compared with age-matched sham controls. NF-κB activity was significantly increased at 1 day, reached a peak at 3 wk, and remained elevated at 6 wk, and IKK-β activity was significantly increased at 1 day, peaked at 5 days, and then decreased but remained elevated at 6 wk after aortic banding compared with age-matched sham controls. Inhibiting NF-κB activation in vivo by cardiac transfection of IκB-αM or by PDTC treatment significantly attenuated the development of cardiac hypertrophy in vivo with a concomitant decrease in NF-κB activity. Our results suggest that NF-κB activation is required for the development of cardiac hypertrophy in vivo and that NF-κB could be an important target for inhibiting the development of cardiac hypertrophy in vivo.
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Analyses of alternative cell signal transduction pathwaysGong, Yunchen, 1965- January 2004 (has links)
No description available.
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Study on the effect of Leishmania donovani infection on signal transduction in macrophagesDescoteaux, Albert January 1991 (has links)
No description available.
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Phosphotyrosine-mediated signal transduction pathways essential for RET/PTC-1-induced tumor formation /Buckwalter, Tara Lynne Furminger January 2000 (has links)
No description available.
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Molecular and genetic dissection of sugar signal transduction pathway in Arabidopsis thalianaKang, Shin Gene 29 September 2004 (has links)
No description available.
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Mechanism and consequence of P75 SignalingHarrington, Anthony W. 11 March 2005 (has links)
No description available.
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