Our investigation suggests that Epac, and not the canonical PKA, is the main effector involved in cAMP-mediated axon growth and guidance, and that the MAPK regulator, B-Raf, can be differentially regulated by both Epac and PKA.  Direct activation of Epac induces activation of B-Raf, whereas activation of PKA results in inhibition.  Moreover, in embryonic neurons, Epac-B-Raf signalling was found to be activated by the cAMP-dependent guidance molecule Netrin-1 (attractive to developing axons) and by cAMP activity at all levels tested, whereas in adult neurons, PKA signalling was activated by Netrin-1 (repulsive to adult neurons) and low levels of cAMP activity.  When cAMP was substantially elevated in adult neurons, Epac-B-Raf signalling was induced, and the response of adult axons to Netrin-1 was switched from repulsion to attraction, indicating B-Raf activity is consistent with chemoattractive axon turning.  Furthermore, B-Raf activity was found to localise towards the leading edge of a turning growth cone when visualised with fluorescent microscopy.  Neurons lacking B-Raf protein were unable to respond to cAMP-dependent guidance cues and postnatal neurons no longer responded to artificial cAMP elevation, which usually induces an increase in axon outgrowth.  Calcium influx into growth cones usually induced by axon guidance cues and cAMP activators, were also attenuated in neurons lacking B-Raf.  These data suggest B-Raf is a vital effector for cAMP-mediated axon growth and guidance.

No description available.

The function of CABP1, a novel cAMP-binding protein in Dictyostelium discoideum, has been investigated. Previously, it has been shown that the molecular weight of CABP1 varies in different strains and that this variation is correlated with differences in response to cAMP. Sequence analysis showed that compared to its counterpart in strain AX2, the gene encoding CABP1 of strain V12M2 is missing 27 bp. AX2 transformants expressing the capA gene of V12M2 exhibited no change in phenotype, suggesting that CABP1 is not involved in the features examined or that the V12M2 variant is recessive. Disruption of capA by homologous recombination was attempted with no success, suggesting that this gene might be essential. Proteins interacting with CABP1 have been identified by immunoprecipitation using specific antibodies, CABP1 was also found to copurify with annexin VII, a calcium-dependent phospholipid-binding protein, suggesting that CABP1 and annexin VII might interact.

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xxiv, 256 p.; also includes graphics (some col.). Includes bibliographical references (p. 228-256).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1971. / Typescript. Vita. Description based on print version record. Includes bibliographical references (leaves 92-107).

by Hing Kee Wong. / Title also in Chinese. / Thesis (M.Phil.)--Chinese University of Hong Kong. / Bibliography: leaves 67-74.

No description available.

published_or_final_version / Anatomy / Master / Master of Philosophy

Previous in vitro studies showed that exchange protein directly activated by cyclic AMP 1 (Epac1), which is a cAMP mediator, plays an important role in maintenance of endothelial barrier function. Diabetic retinopathy is characterized by impairment of retinal blood vessel integrity leading to breakdown of blood retinal barrier, retinal hypoxia, and neuronal damage. Here, we hypothesize that Epac1 regulates endothelial permeability and protects retina from the retinal damage associated with diabetes. To test such hypothesis, we first demonstrated that human retinal microvascular endothelial cells (HRMECs) exposed to high glucose concentration at 25 mM or 35 mM showed the decreased Epac1 expression level. Our preliminary data also showed that Epac1-downstream activator, Rap1, a member of Ras GTPase, was also altered by different glucose levels. In addition, retina from type 2 diabetic, db/db, mice also showed the decreased Epac1 expression compared to that of non-diabetic, db/m, mice. To further determine the role of Epac1 in diabetic retinopathy, we made use of Epac1-deficient mice. The pathogenesis of diabetic retinopathy share similar characteristics to that of ischemic retinopathy, such as neuronal cell death, glial reactivity, and glutamate toxicity. Therefore, we used our previous retinal ischemic model, i.e., transient middle cerebral artery occlusion (tMCAO). Firstly, we determined the retinal morphology of Epac1-/- mice under normal condition at 3wks. At 3 wks old, the Epac1-/- retinae showed a significantly decreased thickness of outer plexiform layer (OPL) with a trend of increase in inner nuclear layer (INL) thickness. Interestingly, there were obviously more glutamine synthetase (GS)-positive M?ller cells and protein kinase C (PKC)-α positive rod bipolar cells in INL. In addition, there were more IgG-positive blood vessels in OPL. To further determine whether these phenotypes will lead to more severe retinal damage, Epac1-/- mice were exposed to 2 hours of MCAO followed by 22 hours of reperfusion, which we have previously shown to induce retinal ischemia. There was no obvious difference in retinal thickness and expressions of glial fibrillary acidic protein (GFAP) and GS in the contralateral sides of Epac1+/+ and Epac1-/- retina after tMCAO suggesting that the Epac1-deficiency may be compensated by either protein kinase A (PKA) or Epac2. However, Epac2 level was not altered by Epac1-deficiency by Western blot analysis. The ipsilateral sides of the retina of Epac1+/+ and Epac1-/- after tMCAO also did not show obvious difference in swelling and cell death in inner retina, GFAP, glutamate, GS, nitrotyrosine (NT), and peroxiredoxin 6 (Prx6), suggesting that Epac1-deficiency may have been compensated by other cAMP mediators, such as Epac2. However, Epac2 expression in the ipsilateral side of Epac1+/+ and Epac1-/- retinae was not significantly different, although the activities of Epac and PKA were not determined. Taken together, the Epac1-deficient mice would serve as a useful model to determine the role of Epac1 in retinal development, and to determine the detail mechanisms of pathogenesis of diabetic and ischemic retinopathy. / published_or_final_version / Anatomy / Master / Master of Philosophy

The slime mould Dictyostelium discoideum contains a novel cAMP-binding protein, CABPl, that is composed of two subunits. Anti-CABPl monoclonal antibodies were used to isolate a cDNA from a $ lambda$gtll expression library. In hybrid selection experiments, this cDNA was found to be complementary to mRNAs encoding both CABPl subunits and also to share some homology with two polypeptides, p34 and p31, that copurify with CABPl. Further analysis showed that the two CABPl polypeptides are identical except for an additional 37 amino acids present in the larger polypeptide. Both polypeptides are encoded by a single gene and the production of the two CABPl mRNAs is accomplished by an unusual splicing event. The p34 and p31 polypeptides are smaller but highly homologous to the two CABPl polypeptides and are probably produced in a similar fashion. The carboxyl halves of the four polypeptides show a 50% similarity to two polypeptides of a tellurium anion resistance determinant encoded on a bacterial plasmid.