ABCA1 is believed to affect macrophage inflammatory responses, but the mechanism by which ABCA1 may impact cytokine secretion in macrophages has yet to be fully defined. We observed that the induction of ABCA1 expression in three different cell lines, namely BHK, RAW 264.7 macrophages, and primary bone marrow derived macrophages (BMDMs), results in a significant increase in phosphorylated CREB, a known protein kinase A (PKA) substrate. In RAW macrophages, induction of ABCA1 expression by the LXR-agonist T0901317 is correlated with a decrease in LPS-stimulated secretion of proinflammatory cytokines IL-6 and TNF-α. Additionally, the secretion of anti-inflammatory cytokine IL-10 was increased upon ABCA1 induction. A similar trend was observed in BMDMS: ABCA1-expressing BMDMs released less TNF-α and more IL-10 compared to ABCA1-knockout BMDMs. We speculated that the inflammation modulating effects of ABCA1 in macrophages could be a result of PKA activation. Indeed, we found that the LXR-induced ABCA1 phenotype can be mimicked by cAMP in macrophages. 8-bromo-cAMP, a PKA activator, dose-dependently suppressed inflammatory cytokine secretion while promoting IL-10 release in the absence of ABCA1 expression. Finally, we found that the T0901317-induced ABCA1 expression is correlated with higher expression levels of MKP-1, a downstream target of PKA known to suppress inflammatory responses. Together, our results suggest that ABCA1 expression may activate PKA and CREB and that such activation may contribute to the inflammatory modulating effects of ABCA1.

Traumatic neuronal injury inevitably causes plasmalemmal damage, and sometimes leads to axonal severance. For any eukaryotic cell to survive following traumatic injury, the plasmalemma must be repaired (sealed). Plasmalemmal sealing occurs via a Ca²⁺-dependent accumulation of vesicles or other membranous structures that form a plug at the damage site. Using uniquely identified and damaged rat hippocampal B104 cells that extend neurites with axonal properties, or rat sciatic nerves, plasmalemmal sealing is assessed by exclusion of an extracellular dye from each damaged B104 cell, or sciatic nerves ex vivo. B104 cells with neurites transected nearer (<50 [micrometres]) to the soma seal at a lower frequency and slower rate compared to cells with neurites transected farther (>50 [micrometres]) from the soma. Sealing in B104 cells is enhanced by 1) increased [cAMP], 2) increased PKA activity, 3) increased Epac activity, 4) H₂O₂ and 5) Poly-ethylene glycol (PEG). Sealing is decreased by 1) PKA inhibition, 2), Botulinum toxins A, B, E, 3) Tetanus toxin 4), NEM, 5) Brefeldin A, 6) nPKC inhibition, 7) DTT, 8) Melatonin and 9) Methylene Blue. Substances (NEM, Bref A, PKI, db-cAMP, PEG) that affect plasmalemmal sealing in B104 cells in vitro have similar effects on plasmalemmal sealing in rat sciatic nerves ex vivo. Based on data from co-application of enhancers and inhibitors of sealing, I propose a plasmalemmal sealing model having four partly redundant, parallel pathways mediated by 1) PKA, 2) Epac, 3) cytosolic oxidation and 4) nPKCs. The identification and confirmation of these pathways may provide novel clinical targets for repairing and/or recovery from traumatic injury. The fusogenic compound PEG rapidly repairs axonal continuity of severed axons, potentially by rejoining severed proximal and distal axons. PEG-fusion is influenced by plasmalemmal sealing, since unsealed axons are easier to PEG fuse. I demonstrate that PEG restores morphological continuity, and improves behavioral recovery following crush-severance to sciatic nerves in rats in vivo. Co-application of Mel or MB prior to PEG application further improves PEG fusion (as measured by electrophysiology) and behavioral recovery following crush-severance in vivo. These PEG data may provide novel clinical techniques for rapidly repairing axonal severance. / text

In nature, non-covalent interactions are as important and dynamic as they are elusive. As such, the study of non-covalent interactions both in vivo and in vitro has proven to be challenging. Given the potential benefits of elucidating protein-protein, ligand-receptor, and other biologically relevant interactions, the development of methodologies for the study of non-covalent interactions is an attractive goal.Biologically encoded protein and peptide libraries that connect the genotypic information with the expressed phenotype have emerged in recent years as powerful methods for studying non-covalent interactions. One of the quintessential platforms for the creation of such libraries is phage display. In phage display, the connection between genetic information and the corresponding protein allows for the iterative isolation and amplification of library members that possess a desired function. Hence, an in vitro selection can be used to isolate epitopes that bind to desired targets or display specific attributes.We have sought to develop novel phage display methodologies that have the potential to expand the scope of this in vitro selection platform. Specifically, we developed a method for the non-covalent attachment of a small molecule ligand to a cyclic peptide library. This system localizes the phage display library to the ligand binding site, thus allowing for the translation of the selected cyclic peptides to a covalently tethered bivalent inhibitor.The first class of biological molecules that we chose to target with our methodology is the biologically and therapeutically important class of enzymes called protein kinases. In the first demonstration of this strategy, we were able to isolate cyclic peptide ligands for the model kinase PKA (cAMP-dependent protein kinase), which were subsequently translated to a bivalent inhibitor. This inhibitor showed both increased affinity and selectivity for PKA in relation to other protein kinases.In a separate project, we sought to develop a method for the isolation of small molecule-responsive mutants of a well-characterized protein scaffold from a phage display library. During these investigations, we discovered interesting homologous single-point mutations of the protein that resulted in large spherical oligomers that may mimic species relevant to the study of protein misfolding diseases such as Alzheimer's.

Studies have shown that long-term opioid agonist (such as morphine) treatment produces antinociceptive tolerance and increased pain sensitivity (hyperalgesia and/or allodynia), limiting the clinical efficacy of morphine. Prolonged opiate administration also upregulates spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) levels and enhances evoked CGRP release in the dorsal horn of rats. It was suggested that augmented spinal pain neurotransmission may contribute to paradoxical pain sensitization and antinociceptive tolerance. The cellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter are not fully clarified.Sustained morphine treatment was shown to augment the concentrations of inflammatory mediators, such as PGE2 in the spinal cord. Studies have shown that PGE2 stimulates cAMP formation and CGRP release by activation of Gs protein-coupled prostaglandin receptor types in primary sensory neurons. Interestingly, it was found earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). It is well demonstrated that cAMP activates cAMP-dependent protein kinase (PKA), which plays an important role in the modulation of presynaptic neurotransmitter release. Therefore, in this study, we investigate the physiological role of Raf-1 mediated AC superactivation and subsequent PKA activation in A. sustained morphine-mediated augmentation of basal or evoked pain neurotransmitter release in vitro, in cultured primary sensory neurons, and B. in vivo, in sustained morphine mediated paradoxical pain sensitization and antinociceptive tolerance in rats.Our data demonstrates that A. sustained morphine treatment augments both basal and capsaicin-evoked CGRP release from isolated primary sensory neurons in a PKA- and Raf-1- dependent manner. B. sustained morphine treatment- augments of PGE2-evoked CGRP release from these cells. C. selective knockdown of spinal PKA or Raf-1 protein levels by intrathecal PKA- or Raf-1-specific siRNA pretreatment completely attenuates sustained morphine-mediated thermal hyperalgesia, tactile allodynia and greatly reduces antinociceptive tolerance in rats.In conclusion, we suggest that Raf-1-mediated AC superactivation may have a crucial trigger role in sustained morphine-mediated compensatory adaptations in the nervous system. Thus, we expect that pharmacological attenuation of Raf-1-mediated AC superactivation may improve the clinical treatment of chronic and neuropathic pain.

Vascular endothelial cells (VECs) play an essential role in regulating the passage of macromolecules and cells between the blood stream and underlying tissues. The second messenger 3’, 5’ cyclic adenosine monophosphate (cAMP) regulates numerous events in VECs, including permeability. Since human VECs express several distinct cAMP-hydrolyzing phosphodiesterases (PDEs), and these are the only enzymes that catalyze the inactivation of cAMP, we investigated if selective pharmacological inhibition of PDEs could impact VEC permeability. Interestingly, we found that PDE4 inhibitors decreased human aortic VEC (HAEC) permeability and PDE4 and PDE3 inhibitors decreased human microvascular VEC (HMVEC) permeability. Consistent with a role for both protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in regulating VEC permeability, selective activators of these enzymes significantly decreased permeability. Since neither PDE4 nor PDE3 inhibitors significantly increased cAMP in these cells, our data are consistent with the idea that PDE inhibition causes small localized increases in “pools” of cAMP that regulate permeability. In order to test if PDE4 enzymes could act locally on pools of cAMP that regulated permeability, we selectively isolated the adherens junctional protein VE-cadherin from confluent monolayers of HAECs or HMVECs, and immunoblotted these isolates for cAMPeffectors and PDEs. Briefly, we found that each PKA-II, EPAC1, and a PDE4D variant, but not PDE3 enzymes, each could be isolated in VE-cadherin-based complexes from these cells. These novel findings identify PKA-II, EPAC1, and PDE4D as members of VE-cadherin-based signaling complexes in human VECs and are consistent with the idea that localized cAMP-signaling regulates permeability in these cells. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2007-09-14 15:52:20.216

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ABCA1 is believed to affect macrophage inflammatory responses, but the mechanism by which ABCA1 may impact cytokine secretion in macrophages has yet to be fully defined. We observed that the induction of ABCA1 expression in three different cell lines, namely BHK, RAW 264.7 macrophages, and primary bone marrow derived macrophages (BMDMs), results in a significant increase in phosphorylated CREB, a known protein kinase A (PKA) substrate. In RAW macrophages, induction of ABCA1 expression by the LXR-agonist T0901317 is correlated with a decrease in LPS-stimulated secretion of proinflammatory cytokines IL-6 and TNF-α. Additionally, the secretion of anti-inflammatory cytokine IL-10 was increased upon ABCA1 induction. A similar trend was observed in BMDMS: ABCA1-expressing BMDMs released less TNF-α and more IL-10 compared to ABCA1-knockout BMDMs. We speculated that the inflammation modulating effects of ABCA1 in macrophages could be a result of PKA activation. Indeed, we found that the LXR-induced ABCA1 phenotype can be mimicked by cAMP in macrophages. 8-bromo-cAMP, a PKA activator, dose-dependently suppressed inflammatory cytokine secretion while promoting IL-10 release in the absence of ABCA1 expression. Finally, we found that the T0901317-induced ABCA1 expression is correlated with higher expression levels of MKP-1, a downstream target of PKA known to suppress inflammatory responses. Together, our results suggest that ABCA1 expression may activate PKA and CREB and that such activation may contribute to the inflammatory modulating effects of ABCA1.

Made available in DSpace on 2014-06-12T15:54:51Z (GMT). No. of bitstreams: 2 arquivo607_1.pdf: 1573923 bytes, checksum: 64f355c17fab0797315fdf979c778510 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Faculdade Intergrada do Recife / Este estudo investigou os efeitos agudos do extrato aquoso do chá verde (GTE) e dos seus constituintes polifenóis (-)-epigalocatecina-3-galato (EGCG) e (-)-epicatecina (EC) sobre a produção de testosterona basal e estimulada, em células de Leydig de ratos in vitro. Células de Leydig purificadas foram incubadas por 3 horas com GTE, EGCG ou EC e com o precursor da testosterona androstenediona, na presença ou ausência de ativadores da proteína quinase A (PKA) e da proteína quinase C (PKC). O GTE e a EGCG, mas não a EC, inibiram ambas as produções de testosterona, basal e quinaseestimuladas. Células pré-tratadas por 15 minutos com GTE ou EGCG e recuperadas por 1 hora foram submetidas a tratamento com gonadotrofina coriônica humana (hCG), hormônio liberador de gonadotrofinas (LHRH), 22OHColesterol ou androstenediona. Nestas condições o efeito inibitório do GTE/EGCG em suas maiores concentrações utilizadas (69,2 e 100 &#956;g/mL, respectivamente) sob a produção de testosterona estimulada por hCG/LHRH ou 22OHColesterol se manteve, enquanto que a produção de testosterona estimulada pela androstenediona retornou para os níveis do controle, indicando que o efeito inibitório sob a função da enzima 17&#946;-hidroxidesidrogenase (17&#946;-HSD) foi reversível. Nestas mesmas condições de pré-tratamento, porém utilizando menores concentrações de GTE/EGCG (13,8 e 20 &#956;g/mL, respectivamente) observou-se que o efeito inibitório destes polifenóis sobre a produção de testosterona estimulada pelo 22OHColesterol foi revertida e até excedeu os níveis do controle, indicando que o efeito inibitório dos polifenóis sob a função da enzima de clivagem da cadeia lateral (P450scc) em mitocôndrias foi reversível. Conclui-se que os efeitos inibitórios do GTE podem ser explicados, pelo menos em parte, pela ação da EGCG, seu principal componente, e que a presença do grupo galato em sua estrutura parece ser importante para sua alta eficácia na inibição da síntese de testosterona. Os mecanismos envolvidos nos efeitos do GTE e da EGCG são provavelmente diversos e envolvem a inibição das cascatas de sinalização da PKA/PKC, assim como a inibição da função das enzimas P450scc e 17&#946;-HSD

Syftet med detta examensarbete var att undersöka högskolestudenters erfarenheter av övergången från studier inom kemi på gymnasial nivå till högskolenivå för att se om det finns ett glapp i undervisningen. Undersökningen bestod av dels en enkätundersökning och dels svaren från tentamensfrågor av samma karaktär som enkätfrågorna. Resultatet visar att studenterna anser att det är en skillnad mellan att läsa kemi på gymnasial nivå och på högskolenivå och att den största skillnaden ligger i ett förändrat arbetssätt. Av studenternas svar på enkätfrågorna och tentamen kan slutsatsen dras att studenterna känner till termer och begrepp, men har svårt att använda dem för att förklara kemiska fenomen.

Efficient directed migration requires tight regulation of chemoattractant signal transduction pathways in both space and time, but the mechanisms involved in such regulation are not well understood. Here, we investigated the role of protein kinase A (PKA) in controlling signaling of the chemoattractant cAMP in Dictyostelium discoideum. We found that cells lacking PKA display severe chemotaxis defects, including impaired directional sensing. Although PKA is an important regulator of developmental gene expression, including the cAMP receptor cAR1, our studies using exogenously expressed cAR1 in cells lacking PKA, cells lacking adenylyl cyclase A (ACA) and cells treated with the PKA-selective pharmacological inhibitor H89, suggest that PKA controls chemoattractant signal transduction, in part, through the regulation of RasG, Rap1 and TORC2. As these pathways control the ACA-mediated production of intracellular cAMP, they lie upstream of PKA in this chemoattractant signaling network. Consequently, we propose that the PKA-mediated regulation of the upstream RasG, Rap1 and TORC2 signaling pathways is part of a negative feedback mechanism controlling chemoattractant signal transduction during Dictyostelium chemotaxis.