Biomedical subjects

Gary Z Morris

Publications and source records attributed to Gary Z Morris.

4 recordsLinked to original sources

PubMed2004

Characterization of the cAMP-dependent protein kinase catalytic subunit Cgamma expressed and purified from sf9 cells.

The Cgamma and Calpha subunits of the cAMP-dependent protein kinase (PKA) contain 350 amino acids that are highly homologous (83% amino acid sequence), with 91% homology within the catalytic domain (a.a. 40-300). Unlike Cgamma, the Calpha subunit has been readily purified and characterized as a recombinant protein in vitro, in intact cells, and in vivo. This report describes for the first time the expression, purification, and characterization of Cgamma. The expression of active Cgamma was eukaryote-specific, from mammalian and insect cells, but not bacteria. Active recombinant Cgamma was optimally expressed and purified to homogeneity from Sf9 cells with a 273-fold increase in specific activity and a 21% recovery after sequential CM-Sepharose and Sephacryl S-300 chromatography. The specific activity of pure Cgamma was 0.31 and 0.81 U/mg with kemptide and histone as substrates, respectively. Physical characterization showed Cgamma had a lower apparent molecular weight and Stokes radii than Calpha, suggesting differences in tertiary structures. Steady-state kinetics demonstrated that like Calpha and Cbeta, Cgamma phosphorylates substrates requiring basic amino acids at P-3 and P-2. However, Cgamma generally exhibited a lower Km and Vmax than Calpha for peptide substrates tested. Cgamma also exhibited a distinct pseudosubstrate specificity showing inhibition by homogeneous preparations of RIalpha and RIIalpha-subunits, but not by pure recombinant protein kinase inhibitors PKIalpha and PKIbeta, PKA-specific inhibitors. These studies suggest that Cgamma and Calpha exhibit differences in structure and function in vitro, supporting the hypothesis that functionally different C-subunit isozymes could diversify and/or fine-tune cAMP signal transduction downstream of PKA activation.

Amino Acid Sequence↗

PubMed2002

Resveratrol induces apoptosis in LNCaP cells and requires hydroxyl groups to decrease viability in LNCaP and DU 145 cells.

BACKGROUND: This study was conducted to determine the effects of resveratrol on prostate cancer cell viability through apoptosis induction and the significance of the three hydroxyl groups on resveratrol to the measured effect. METHODS: Hormone-sensitive LNCaP cells and hormone-insensitive DU 145 cells were treated with resveratrol, tri-methoxy-resveratrol, or diethylstilbestrol (DES; the positive control for toxicity and apoptosis). Cell viability was determined by using an MTS assay. Apoptosis was determined by the appearance of apoptotic morphology, annexin V-FITC-positive intact cells, and caspase activation. RESULTS: Resveratrol and DES decreased viability in LNCaP cells, but only resveratrol-treated cells expressed apoptotic morphology, annexin V-FITC-positive cells, and caspase activation. Tri-methoxy-resveratrol had no effect on DU 145 cell-viability and was less toxic to LNCaP cells than resveratrol. CONCLUSION: Resveratrol was toxic to cells regardless of whether the cells were hormone-responsive or -unresponsive. This finding suggests that the cell's hormone responsive status is not an important determinant of the response to resveratrol. Furthermore, the hydroxyl-groups on resveratrol are required for cell toxicity. Finally resveratrol but not DES induced caspase-mediated apoptosis.

Antineoplastic Agents, Hormonal↗

PubMed2002

Differential transcriptional regulation by the alpha- and gamma-catalytic subunit isoforms of cAMP-dependent protein kinase.

The C gamma and C alpha isoforms of the cAMP-dependent protein kinase (PKA) share 83% identity including all critical catalytic and substrate-binding residues defined to date. Compared to C alpha, C gamma has a different substrate specificity and a selective pseudosubstrate specificity, exhibiting inhibition by regulatory subunits, but not by the protein kinase inhibitor. In these studies, C gamma-mediated gene transcription regulation was compared with that of C alpha in four cell lines using transient transfection/dual luciferase assays. As compared to C gamma, C alpha more efficiently activated a cAMP-response element (CRE)-regulated fragment of the human alpha-glycoprotein hormone promoter which was coupled to a firefly luciferase reporter gene (pGH alpha-fluc). This occurred in Cos7, Y1, and Kin8 adrenal cells by 23-, 6.5-, and 1.4-fold, respectively. In contrast, C gamma, but not C alpha, activated the Sp1RE-regulated herpes simplex virus thymidine kinase promoter which was coupled to a Renilla luciferase reporter (pTK-rluc). In Sp1-deficient Sf9 cells, pGH alpha-fluc expression was maintained for both isoforms, but cotransfection with an Sp1 expression plasmid was necessary and sufficient for activation of pTK-rluc expression by C gamma. In all cell lines, cotransfection with a PDK1 expression plasmid enhanced the transcriptional activation of both C alpha and C gamma (1.5- to 3-fold), while a catalytically inactive PDK1 mutant (PDK.KD) did not. These results suggest that both C alpha and C gamma can activate CRE-responsive genes; however, C alpha does so with better efficiency than C gamma. In contrast to C alpha, C gamma activates transcription of genes containing pTK-like Sp1RE sites. Activation of different C subunit isoforms can provide a means to diversify cAMP-mediated transcription, possibly affecting cell phenotype.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2002

Dietary phytoestrogens and their synthetic structural analogues as calcium channel blockers in human platelets.

Phytoestrogens have been shown to inhibit platelet activation by blocking platelet calcium channels. This study examined the effect of several synthetic derivatives of trans-resveratrol, genistein, and daidzein on platelet free intracellular calcium ([Ca2+]i) elevation in thrombin-activated platelets and the possible mechanisms of this inhibitory effect. Studies were conducted on fresh human platelets from healthy volunteers. The fluorescent dye fura-2 was used to monitor [Ca2+]i in platelets. At 10 microM-resveratrol, triacetyl-trans-resveratrol, and trimethoxy-trans-resveratrol produced, respectively, 57 +/- 4%, 40 +/- 4%, and 21 +/- 1% inhibition; genistein, acetylgenistein, and dihydrogenistein produced 51 +/- 10%, 26 +/- 7%, and 16 +/- 2% inhibition, respectively; daidzein and diacetyldaidzein produced 56 +/- 5% and 45 +/- 10% inhibition of thrombin-induced [Ca2+]i elevation. The inhibitory effect was immediate and appeared to directly affect the calcium influx channels. Phytoestrogen action on [Ca2+]i did not cause alteration in nitric oxide signaling. Tyrosine phosphorylation was not involved in the inhibition of [Ca2+]i elevation by phytoestrogens, because the percent inhibition produced by the tyrosine kinase inhibitor genistein and its inactive analogue daidzein on thrombin-induced and thapsigargin-induced [Ca2+]i elevation was not significantly different for either compound at any concentration tested. Structure-activity relationship studies on this limited set of compounds reveal the requirements for the stilbene pharmacophore for the calcium-blocking activity.

Adult↗