Thesis (M. Phil.)--University of Hong Kong, 2006. / Also available online.

M.Sc. (Biochemistry) / The culture of plant anther tissue in vitro, is an important tool which could be used, when perfectedr in the conventional breeding of crop plants. Using this technique it may be possible to generate bomozygotIc lines in one or two generations in contrast to the seven or eight generations required by conventional breeding programs. Physiological data on the behavior of cultured anther tissue suggests that the genotype is the most Important factor Influencing this factor with respect to wheat barley and most other crop plants. Optimal culture conditions with respect to culture medium and environmental factors have already been established. It would appear that a critical factor, present in the pollen cells of anther tissue, governs the variation and differentiation of the embrionic pollen in the formation of haploid plants. Many important systems of cellular signalling and therefore cellular regulation are mediated by protein kinases and phosphatases. An examination of protein kinase activity in normal and anther tissue may yield information about the process of differentiation. Identification of key kinase activities may provide plant breeders with a means of selecting more responsive genotypes for the use In the anther culture technique In VItro. In this manuscript a caparison is made between protein kinase activities in crude extracts of meristematic...

No description available.

It is generally held that adenosine 3',5'-cyclic monophosphate (cAMP) mediates smooth muscle relaxation by the activation of cAMP-dependent protein kinase (PKA). This hypothesis was tested in two intact smooth muscle preparations, the rat vas deferens and the bovine coronary artery, using exogenously applied cAMP and cAMP analogs. After 30 minutes of incubation, N⁶,2'-0-dibutyryl-cAMP (dBu-cAMP) (1 - 100 μM) inhibited phenylephrine (PE)-induced tension generation in the rat vas deferens in a dose-dependent manner. This analog (10 μM) also activated the soluble fraction of PKA but did not activate the particulate fraction kinase. In contrast, 8-bromo-cAMP (8Br-cAMP) (10 -100 μM) did not have any significant effect on inhibition of PE-induced tension after 30 minutes of incubation but, at a concentration of 10 μM, significantly activated both the soluble and particulate fractions of PKA. The time course of activation of soluble PKA activation by 8Br-cAMP (10 μM) demonstrated that the kinase was significantly activated only after 30 minutes of exposure to the analog. In the bovine coronary artery, cAMP (10 - 100 μM) relaxed potassium-depolarized helical strips and significantly activated soluble PKA in a dose-dependent manner. dBu-cAMP (10 - 100 μM) affected neither tension nor soluble PKA activity. 8Br-cAMP (10 - 100 μM) did not affect the coronary artery tension but did activate soluble PKA. Both smooth muscle preparations were homogenized with charcoal prior to the determination of PKA activity in order to minimize artifactual assay results. As a further precaution, extracellularly associated cAMP and analogs were also washed from bovine coronary artery strips after the incubation period. These controls allowed for a valid assessment of PKA activity in the cyclic nucleotide-treated tissues. The results of the tension and kinase studies demonstrate a lack of correlation between activation of PKA and inhibition of rat vas deferens contraction or relaxation of bovine coronary artery. This does not support the hypothesis that the kinase is responsible for cAMP-induced relaxation of vascular and non-vascular smooth muscle. While the mechanism by which exogenous cAMP and specific analogs induce relaxation in some smooth muscle preparations remains unclear, it can be suggested that PKA activation is not necessarily required for the final functional effect. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

A description of the nature of the transition state structure for phosphoryl transfer in the cAPK reaction requires a measurement of the primary 180 isotope effect at the serine hydroxyl acceptor. Since it is difficult to obtain primary 180 isotope effect directly, the 15N/1 4N ratio of the a-amine of the C-terminal glycine in the peptide Leu Arg-Lys-Ala-Ser-Leu-Gly (when serine is phosphorylated) was used to represent on the phosphorylation at serine. 15N Glycine, ' 4N-Glycine and 180 serine were synthesized and used to synthesize two peptides, one containing 1 80-serine/' 5 N glycine and second 1 60-serine/1 4N-glycine. Methods were developed for hydrolyzing the peptides and quantitatively isolating glycine. Partitioning results suggest that catalytic rate was slow compare to substrate dissociation. The 180 primary isotope effect will be determined in the near future using the method developed herein.

The kinetic mechanism of the cAMP-dependent protein kinase has been determined to be random in the direction of MgADP phosphorylation by using initial velocity studies in the absence and presence of the product, phospho-Serpeptide (Leu-Arg-Arg-Ala-Ser[P]-Leu-Gly) , and dead-end inhibitors. In contrast to the kinetic parameters obtained in the direction of Serpeptide phosphorylation, the only kinetic parameters affected by Mg^2+ are the dissociation constants for E:phospho-Serpeptide and E:MgADP, which are decreased by about 4-fold. The dead-end analog MgAMPCP binds with an affinity equal to that of MgADP in contrast to MgAMPPCP, which binds weaker than MgATP. The ratio of the maximum velocities in the forward and reverse reactions is about 200, and the Haldane relationship gives a K-eq of (7.2 ± 2) x 10^2. The latter can be compared to the K-eq obtained by direct measurement of reactant concentrations (2.2 ± 0.4) x 10^3 and 31-P NMR (1 ± 0.5) x 10^3. Data for the pH dependence of kinetic parameters and inhibitor dissociation constants for the cAMP dependent protein kinase are consistent with a mechanism in which reactants selectively bind to an enzyme with the catalytic base unprotonated and an enzyme group required protonated for Ser-peptide binding. Preferentially MgATP binds fully ionized and requires an enzyme residue (probably lysine) to be protonated. The maximum velocity and V/K-MgATP are pH independent. The V/K for Serpeptide is bell-shaped with estimated pK values of 6.2 and 8.5. The dependence of 1/K-i for Leu-Arg-Arg-Ala-Ala-Leu-Gly is also bell-shaped, giving pK values identical with those obtained for V/K-Serpeptide, while the K-i for MgAMPPCP increases from a constant value of 650 μM above pH 8 to a constant value of 4 mM below pH 5.5. The K-i for uncomplexed Mg^2+ obtained from the Mg^2+ dependence of V and V/K-MgATP is apparently pH independent.

Myocardial ischemia and ischemia/reperfusion activate several protein kinase pathways. Protein kinase activation potentially regulates the onset of myocardial cell injury and the reduction of this injury by ischemic and pharmacologic preconditioning. The primary protein kinase pathways that are potentially activated by myocardial ischemia/reperfusion include: the MAP kinases, ERK 1/2, JNK 1/2, p38 MAPKα/β; the cell survival kinase, Akt; and the sodium-hydrogen exchanger (NHE) kinase, p90RSK. The literature does not support a role for ischemia/reperfusion in the activation of the tyrosine kinases, Src and Lck, or the translocation and activation of PKC. This review will detail the role of these protein kinases in the onset of myocardial cell death by necrosis and apoptosis and the reduction of this injury by preconditioning.

The regulation and maintenance of normal cell movements and shape play a vital role in the normal development and health of every living thing. The characterization of 6 zebrafish Zipper Interacting Protein Kinase homolog has helped to better understand how changes in cell cytoskeletal elements can lead to changes in cell shape and movement. Zebrafish are ideal model organisms for studying ZIPK because it has been previously shown that zebrafish ZIPK has closer sequence homology to human ZIPK than rodent ZIPK, and because zebrafish embryos are ideal for studying cell shape and movement in vivo. Using Whole Mount In Situ Hybridization we found that the zebrafish ZIPK is expressed during all stages of embryonic development, but most importantly during gastrulation when cell motility and changes in cell shape can best be studied. To determine where zebrafish ZIPK is expressed at the sub-cellular level, GFP-ZIPK and Flag-ZIPK clones were created and used for transfecting into Hek293T cells and Hela Cells. From these transfections, cell counterstaining and confocal microscopy we found that ZIPK is expressed ubiquitously throughout the cell, although mainly cytoplasmic. To study the effects on cell shape various ZIPK mutants were created through site-directed mutagenesis. These mutants were made to study the effects of the kinase domain of the protein, or other functional domains within the protein. From these studies it was shown that ZIPK does affect cell shape through changes in the actomyosin cytoskeleton resulting in aberrant cytoskeletal structures. Finally, we have also shown through phosphorylation assays that ZIPK phosphorylates and thus regulates MYPT-1, a scaffolding protein of the myosin protein phosphate complex and directly phosphorylates myosin light chain, both of which play a role in changes in cell shape and movement.

No description available.

No description available.