No description available.

No description available.

No description available.

Recent work on Drosophila limb development has indicated that short-range interactions between distinctly specified populations of cells (compartments) establish organizing centers at compartment boundaries. These organizing centers direct pattern formation and growth in the developing limbs. In the Drosophila wing imaginal disc, there are at least two such organizing centers, located at the anterior/posterior (A/P) and dorsal/ventral (D/V) compartment boundaries. The genetic hierarchies which establish these organizers are starting to be understood, and it also appears that the key mediators of some of the organizers have been identified. Thus Decapentaplegic (Dpp, a secreted signalling molecule of the TGF-B family) is the mediator of the A/P organizer, while Wingless (DWnt-l, a secreted molecule of the Wnt family) is a key mediator of the D/V organizer. In this thesis, several aspects of Wingless function in the wing imaginal disc are examined. Two regulatory mutations, spadeflag (spdfg) and Sternopleural (Sp), that affect Wingless expression in the wing imaginal disc are characterized. The analysis of the mutation spdfg, together with other data, identifies a role of Wingless as a localized mitogen in the developing wing hinge, and also indicates that cells in different regions of the wing disc respond very differently to the Wingless signal. The mutations spdfg and Sp are also among the tools used to examine the position of Wingless in the genetic hierarchy that establishes and mediates the activity of the D/V organizer. These experiments extend the evidence suggesting that Wingless mediates both short-range and long-range effects of the D/V organizer. Wg does so by controlling the expression domains of different target genes, including the acheate-scute genes, Distal-less and vestigial. Finally, the mechanism by which Wingless mediates the activity of the D/V organizer is examined. The results obtained suggest that Wingless functions as a long-range morphogen.

Hepatocyte growth factor (HGF) is a multifunctional mitogen, stimulating cell proliferation, motility, angiogenesis and morphogenesis via activating its receptor, c-Met tyrosine kinase. Overexpression of HGF and c-Met has been shown as a characteristic of cancer transformation and metastasis. Inhibition of HGF/c-Met signaling may abrogate the malignant and metastatic states of cancer cells and offer a useful therapeutic approach for treating cancers. Thus with the aim of creating inhibitors to HGF-cMet signaling, we constructed plasmids containing truncated N-terminus of HGF, NK1, NK2, NK3 and NK4, respectively, and transfected into MDA MB 435S cells by electroporation. After selection with antibiotics, stable transfectants were obtained. Proliferation assay showed that the truncated NKs significantly inhibited the growth of the cells. Moreover, wound healing assay showed that migration of the NK-transfected cells were also significantly inhibited in comparison with GFP-transfected and nontransfected cells. These results therefore suggest that truncated NKs may be good inhibitors to HGF/c-Met signaling in the proliferation and migration of human breast cancer cells in vitro. In the future, the in vivo animal model is needed to be carried out to further clarify the clinical values of truncated NKs for application to cancer therapy.

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Chu Kit Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 142-168). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.iv / Abbreviations --- p.v / Publications Based on Work in this thesis --- p.viii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- G protein-coupled receptors --- p.1 / Chapter 1.1.1 --- Introduction --- p.1 / Chapter 1.1.2 --- Heterotrimeric G proteins --- p.3 / Chapter 1.1.3 --- Second messenger systems --- p.4 / Chapter 1.1.4 --- Mechanism of GPCR activation --- p.6 / Chapter 1.2 --- Prostacyclin and its receptors --- p.9 / Chapter 1.2.1 --- General properties of prostacyclin --- p.9 / Chapter 1.2.1.1 --- Synthesis of prostacyclin --- p.9 / Chapter 1.2.1.2 --- Prostacyclin analogues --- p.10 / Chapter 1.2.2 --- Characterization of IP-receptors --- p.12 / Chapter 1.2.2.1 --- Distribution of IP-receptors --- p.12 / Chapter 1.2.2.2 --- Cloning of IP-receptors --- p.14 / Chapter 1.2.2.3 --- Structure of IP-receptors --- p.15 / Chapter 1.2.3 --- Coupling of IP-receptors to G proteins --- p.16 / Chapter 1.2.3.1 --- Interaction with Gs --- p.16 / Chapter 1.2.3.2 --- Interaction with Gq --- p.17 / Chapter 1.2.3.3 --- Interaction with Gi --- p.18 / Chapter 1.2.3.4 --- Interaction with PPARs --- p.20 / Chapter 1.2.4 --- Role of prostacyclin in mitogenesis/anti-mitogenesis --- p.20 / Chapter 1.3 --- Signal transduction network of MAPK family --- p.27 / Chapter 1.3.1 --- MAPK modules in mammalian cells --- p.29 / Chapter 1.3.1.1 --- Extracellular regulated kinase (ERK) cascade --- p.30 / Chapter 1.3.1.2 --- Stress-activated protein kinase (JNK and p38) cascades --- p.33 / Chapter 1.3.2 --- Activation ofERKl/2 through GPCRs --- p.35 / Chapter Chapter 2 --- Materials and solutions --- p.53 / Chapter 2.1 --- Materials --- p.53 / Chapter 2.2 --- "Culture media, buffer and solutions" --- p.58 / Chapter 2.2.1 --- Culture media --- p.58 / Chapter 2.2.2 --- Buffers --- p.59 / Chapter 2.2.3 --- Solutions --- p.62 / Chapter Chapter 3 --- Methods --- p.65 / Chapter 3.1 --- Maintenance of cell lines --- p.65 / Chapter 3.1.1 --- Chinese Hamster ovary (CHO) cells --- p.65 / Chapter 3.1.2 --- Human neuroblastoma (SK-N-SH) cells --- p.66 / Chapter 3.1.3 --- Rat/mouse neuroblastoma/glioma hybrid (NG108-15) cells --- p.66 / Chapter 3.2 --- Transient transfection of mammalian cells --- p.67 / Chapter 3.3 --- Measurement of ERK activity --- p.68 / Chapter 3.3.1 --- PathDetect® Elkl trans-Reporting System --- p.68 / Chapter 3.3.1.1 --- Introduction --- p.68 / Chapter 3.3.1.2 --- β-galactosidase assay --- p.72 / Chapter 3.3.1.3 --- Transient transfection of cells --- p.72 / Chapter 3.3.1.4 --- Cell assay --- p.73 / Chapter 3.3.1.5 --- Luciferase assay --- p.74 / Chapter 3.3.1.6 --- Micro β-gal assay --- p.74 / Chapter 3.3.1.7 --- Data analysis --- p.75 / Chapter 3.3.2 --- Western Blotting --- p.79 / Chapter 3.3.2.1 --- Introduction --- p.79 / Chapter 3.3.2.2 --- Transient transfection of cells --- p.79 / Chapter 3.3.2.3 --- Cell assay --- p.79 / Chapter 3.3.2.4 --- Protein electrophoresis and transfer --- p.80 / Chapter 3.3.2.5 --- Immunodetection --- p.80 / Chapter 3.4.1 --- Measurement of adenylyl cyclase activity --- p.83 / Chapter 3.4.1 --- wyo-[3H]-inositol labelling method --- p.83 / Chapter 3.4.1.1 --- Preparation of columns --- p.83 / Chapter 3.4.1.2 --- Incubation of cells --- p.84 / Chapter 3.4.1.3 --- Measurement of [3H]-cyclic AMP production --- p.84 / Chapter 3.4.1.4 --- Data analysis --- p.85 / Chapter 3.5 --- Measurement of phospholipase C activity --- p.85 / Chapter 3.5.1 --- wyo-[3H]-inositol labelling method --- p.85 / Chapter 3.5.1.1 --- Preparation of columns --- p.86 / Chapter 3.5.1.2 --- Incubation of cells --- p.86 / Chapter 3.5.1.3 --- Measurement of [3H]-inositol phosphate production --- p.87 / Chapter 3.5.1.4 --- Data analysis --- p.88 / Chapter Chapter 4 --- Results --- p.89 / Chapter 4.1 --- Validation of PathDetect® Elkl Trans-Reporting System --- p.89 / Chapter 4.1.1 --- Introduction --- p.89 / Chapter 4.1.2 --- Internal control --- p.89 / Chapter 4.1.3 --- Response to cicaprost and ATP --- p.91 / Chapter 4.1.4 --- Normalisation of ERK1/2 activity with transfection efficiency --- p.92 / Chapter 4.1.5 --- Cicaprost response in CHO cells in the absence of mIP- receptor --- p.93 / Chapter 4.1.6 --- Normalised luciferase activity reflecting ERK1/2 activation --- p.93 / Chapter 4.1.7 --- Conclusion --- p.95 / Chapter 4.2 --- Characterization of IP-receptors --- p.101 / Chapter 4.2.1 --- IP-receptor activation of adenylyl cyclase and phospholipase C --- p.101 / Chapter 4.2.2 --- IP-receptor activation ofERKl/2 in mIP-CHO cells --- p.102 / Chapter 4.2.2.1 --- PathDetect System --- p.102 / Chapter 4.2.2.2 --- Western Blotting --- p.103 / Chapter 4.2.2.3 --- Conclusion --- p.104 / Chapter 4.2.3 --- Role of the Gs-mediated pathway in cicaprost-stimulated ERK1/2 activation --- p.104 / Chapter 4.2.3.1 --- Role of cyclic AMP --- p.105 / Chapter 4.2.3.2 --- Role of protein kinase A --- p.106 / Chapter 4.2.4 --- Role of the Gq-mediated pathway in cicaprost-stimulated ERK1/2 activation --- p.106 / Chapter 4.2.4.1 --- Role of IP3 --- p.107 / Chapter 4.2.4.2 --- Role of protein kinase C --- p.108 / Chapter 4.2.4.3 --- Conclusion --- p.108 / Chapter 4.2.5 --- IP-receptor activation of ERKl/2 in hIP-CHO cells --- p.109 / Chapter 4.2.5.1 --- Activation ofERKl/2 in hIP-CHO cells --- p.109 / Chapter 4.2.5.2 --- Role of the Gq-mediated pathway in cicaprost- stimulated ERK 1/2 activation --- p.110 / Chapter 4.2.5.3 --- Role of the Gs-mediated pathway in cicaprost- stimulated ERK 1/2 activation --- p.111 / Chapter 4.2.5.4 --- Conclusions --- p.113 / Chapter 4.2.6 --- IP-receptor activation of ERX1/2 in neuroblastoma cells --- p.114 / Chapter 4.2.6.1 --- Rat/mouse neuroblastoma/glioma (NG108-15) cells --- p.114 / Chapter 4.2.6.2 --- Human neuroblastoma (SK-N-SH) cells --- p.115 / Chapter Chapter 5 --- General Discussion and Conclusions --- p.137 / References --- p.142

Background: Regulation of vascular tone is complex. Various complementary signaling pathways causing contraction and relaxation of vascular smooth muscle take place to ensure proper blood flow within the vasculature. Mitogen activated protein kinase (MAPK) signaling cascade is observed to be one of the many signaling pathways that regulate vascular tone. Aim: This study examines the role of the following MAPK: mitogen-activated extracellular-regulated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and p38 MAPK in the regulation of relaxation in the endothelium and smooth muscle. Method: Isometric tension of isolated porcine coronary artery rings were measured with organ chamber setup. The effects of MEK inhibitor, PD98059 (30 μM), ERK inhibitor, U0126 (10 μM) and p38 MAPK inhibitor, SB203580 (10 μM), on relaxations induced by bradykinin (a vasodilating peptide), SKA-31 [an activator of small and intermediate conductance calcium-activated potassium channels (SKCa and IKCa,, respectively)], Deta NONOate (a nitric oxide donor) and forskolin (an adenylate cyclase activator) were examined in arteries with and without endothelium, contracted with an thromboxane A2 analog, U46619 (300 nM – 1 μM). In some experiments, rings were also incubated with the following pharmacological inhibitors, indomethacin (cyclooxygenase inhibitor, 10 μM), L-NAME (nitric oxide synthase inhibitor, 300 μM), TRAM34 (IKCa blocker, 1 μM), and UCL1684 (SKCa blocker, 1 μM), alone or in combination. Results: 1. Bradykinin-induced relaxation was potentiated by MEK and ERK inhibition but not by p38 MAPK inhibition. 2. SKA-31-induced relaxation was potentiated by MEK and p38 MAPK inhibition but not by ERK inhibition. 3. Deta NONOate-induced relaxation was potentiated by MEK, p38 MAPK inhibition, but not by ERK inhibition except in the presence of indomethacin, TRAM-34 plus UCL1684. 4. Forskolin-induced relaxation was potentiated by MEK and p38 MAPK inhibition, but not by ERK inhibition. Discussion: MAPK plays a role in regulating the vascular tone in both the endothelium and smooth muscle of porcine coronary arteries. MEK appears to have an inhibitory action on relaxation that is downstream of the generation of endothelium-derived nitric oxide, activation of IKCa and SKCa and activation of adenylate cyclase. ERK are unlikely to be the downstream target of MEK for inhibiting relaxation, in view of the lack of effects of its inhibitor on endothelium-derived hyperpolarizing factor (EDHF)-mediated and endothelium-independent relaxations. The involvement of ERK in relaxation pathways in the endothelium appears to be complicated, since U0126 caused opposing effects (inhibition and potentiation) on bradykinin-induced relaxation in the presence of indomethacin without and with L-NAME or TRAM-34 plus UCL1684. As inhibition of p38 MAPK results in potentiation of relaxations to all relaxing agents tested except bradykinin, this MAPK may have opposing action in the endothelium and smooth muscle; endothelial p38 MAPK may facilitate relaxation while smooth muscle p38 MAPK attenuates it. In conclusion, this study provided additional information on the influences of MEK, ERK and p38 MAPK on relaxation; this knowledge may contribute to the understanding of the mechanisms underlying the development of vascular disorders. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

Plants possess integrated signalling networks that mediate the responses to various environmental conditions. Mitogen-activated protein kinases (MAPKs) constitute a highly conserved family of enzymes in eukaryotes, and in plants MAPK-based signal transduction modules regulate a large number of physiological processes, including responses to environmental stresses and phytohormones. Regulated dephosphorylation of active MAPKs is a key component of the control of MAPK signalling cascades, and in mammals, members of the MAPK phosphatase (MKP) sub-class of dual-specificity tyrosine phosphatases have been recognized as key players for inactivating MAPKs. Five MKP homologues are found in Arabidopsis thaliana, but only limited information is available concerning their properties and biological roles. Based on initial data derived from my reverse genetics and protein interaction studies of these five potential MKPs, as well as gene function information in the literature, I chose to focus on two putative Arabidopsis MKPs, AtMKP2 and Indole-3-Butyric Acid-response 5 (IBR5). By using a combination of genetic and biochemical studies, I established that the previously uncharacterized MKP designated AtMKP2, participates in the regulation of cellular homeostasis in ozone-challenged tissue, and can influence the activation state of two MAPKs, MPK3 and MPK6. AtMKP2-suppressed plants displayed significantly prolonged MPK3 and MPK6 activation during ozone treatment, and recombinant AtMKP2 was able to dephosphorylate both phospho-MPK3 and phospho-MPK6 in vitro, providing direct evidence that AtMKP2 may target these oxidant-activated MAPKs. A mutation in IBR5, one of the five potential AtMKPs, was previously reported to confer reduced sensitivity to auxin and ABA in Arabidopsis. My protein interaction studies demonstrated that IBR5 and MPK12 are physically coupled and that the C-terminus of MPK12 is essential for its interaction with IBR5. In vitro dephosphorylation assays indicated that recombinant phosphoMPK12 is efficiently dephosphorylated by IBR5. In transgenic plants with reduced expression of the MPK12 gene, root growth is hypersensitive to exogenous auxins, consistent with the lower auxin sensitivity reported for ibr5 mutants. Taken together, my data demonstrate for the first time that both AtMKP2 and IBR5 are bona fide Arabidopsis MAPK phosphatases and that they serve as important regulators of oxidative stress and auxin signalling, respectively, in Arabidopsis.