Increases in cyclic AMP (cAMP) are pro-apoptotic in numerous cell types, but the mechanisms of cAMP-promoted apoptosis are poorly defined. We have used murine S49 T-lymphoma cells as a model to provide insight into these mechanisms. Increases in cAMP in wild-type (WT) S49 cells were first noted to kill these cells in the 1970 s, but only in recent years, it was shown that this occurs by the intrinsic (mitochondria-dependent) apoptotic pathway. The apoptotic response does not occur in protein kinase A-null (kin-) clonal variants of WT S49 cells and thus is mediated by protein kinase A (PKA). A second S49 clonal variant, cAMP-Deathless (D-), has PKA activity but lacks cAMP-promoted apoptosis. Apoptosis in WT S49 cells occurs many hours after cAMP/PKA-promoted G1 cell cycle arrest and involves increased expression of Bim, a pro-apoptotic member of the Bcl-2 (B-cell lymphoma-2) family. This increase in Bim expression does not occur in kin- or D- S49 cells and knockdown of Bim blunts cAMP-mediated apoptosis in WT cells. Cytotoxic T lymphocyte antigen-2 also appears to contribute to cAMP/PKA-promoted apoptosis of S49 cells. Based on time-dependent differences in gene expression between WT, D- and kin- S49 cells following incubation with 8-(4-chlorophenylthio)-cAMP, additional genes and proteins are likely involved in this apoptosis. Studies with S49 cells should reveal further insight regarding the mechanisms of cAMP/PKA-promoted cell death, including the identification of proteins that are targets to enhance (e. g., in cancer) or inhibit (e. g., cardiac failure) apoptosis in response to hormones, neurotransmitters, and drugs.

Tyrosine hydroxylase (TH), the rate‐limiting enzyme in catecholamine biosynthesis, has a common repeat polymorphism, (TCAT)n. We asked whether variation at TH influenced the autonomic stress responses in a human twin study design. Autonomic traits displayed substantial heritability (h2), up to h2=56.8±7.5% (p<0.0001) for norepinephrine secretion, and h2=61±6% (p<0.001) for heart rate. Common (TCAT)n alleles, particularly (TCAT)6 and (TCAT)10i, influenced such traits (including catecholamine secretion, as well as basal and post‐stress heart rate) in allele copy number dose‐dependent fashion, though in directionally opposite ways, indicating functional allelic heterogeneity. Multivariate ANOVA documented genetic pleiotropy: joint effects of the (TCAT)10i allele to influence both biochemical (norepinephrine) and physiological (heart rate) traits. (TCAT)6 allele frequencies were lower in normotensive twins at genetic risk of hypertension, consistent with an effect to protect against later development of hypertension, and suggesting that the traits predicted by these variants in still‐normotensive subjects are early, heritable, “intermediate phenotypes” in the pathogenetic scheme for later development of sustained hypertension. We conclude that common allelic variation within the TH locus exerts a heritable effect on autonomic control of the circulation, and that such variation may have implications in later development of cardiovascular disease. Clinical Pharmacology & Therapeutics (2005) 77, P6–P6; doi:

Poster SessionsC786 peak wavelength of gold, and processed with HKL2000. Phasing, model building and refinement were conducted using PHENIX, REFMAC5, COOT and other programs of CCP4. Electron densities for the catalytic domain of the enzyme were clearly obtained, but those for the linker and PHB-binding domains were not, in spite that there is remaining space for these domains to be packed in the crystal lattice.The overall structure of the catalytic domain represented an α/β hydrolase fold which is often observed for the large superfamily of esterases including lipases and carboxypeptidases. A cervice was formed on the surface of the protein, at the bottom of which are located catalytic triad residues Ser-166, Asp-241 and His-300. The structure was compared with that of a fungal single-domain depolymerase with circular permuted polypeptide connectivity [1]. These two enzymes differ in the product composition. The main product is dimer of R-3-hydroxybutyrate for the bacterial enzyme, whereas it is monomer for the fungal enzyme. Structures of loop regions around the active site were different between the two enzymes. This may differentiate the mode of interactions with monomer units of the substrate polymer for these enzymes, which may explain the difference in the product composition.[1] T.