Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Patent
1997-10-02
1999-08-03
Elliott, George C.
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
435419, 4352523, 4352542, 4353201, 536 235, C12N 510, C12N 121, C12N 115, C12N 1512
Patent
active
059324775
ABSTRACT:
Isolated cDNA clones from human brain (frontal cortex) cDNA libraries that encode a unique subtype of the low K.sub.m, cAMP-specific phosphodiesterases (PDE IVs) are disclosed. Analysis of the distribution of hPDE IV.sub.B mRNA expression in various human tissues using a nonconserved fragment of the cDNA as a probe revealed a restricted pattern of expression, with an .about.4-kb MRNA detected in brain, heart, lung and skeletal muscle and not in placenta, liver, kidney or pancreas. Furthermore, an additional .about.5-kb hPDE IV.sub.B.sup.- related mRNA species was detected in brain tissue. Expression of hPDE IV.sub.B in a genetically-engineered PDE-deficient strain of the yeast Saccharomyces cerevisiae resulted in the overproduction of cAMP PDE activity which displayed the expected kinetic characteristics for a PDE IV: 1) low K.sub.m (4.3 .mu.M) for cAMP, 2) high K.sub.m (>3 mM) for cGMP, and 3) sensitivity to rolipram (K.sub.i =0.085 .mu.M), a selective inhibitor of binding data revealed curvilinear Scatchard plots, suggesting the presence of two non-interacting high affinity rolipram binding sites (K.sub.d =0.4 and 6 nM) or a negatively cooperative interaction among multiple binding sites. This novel enzyme is particularly useful for screening candidate compounds for their ability to serve as potential anti-depressant, antiasthmatic or anti-inflammatory agents.
REFERENCES:
Livi et al. Cloning and expression of cDNA for a human low-Km, rolipram-senistive cyclic AMP phosphodiesterase Mol. and Cell. Biol. vol. 10 2678-2686, 1990.
Shelton et al. Novel human rolopram-sensitive phosphodiesterase (PDE): cDNA cloning , expression and homology to rat family IV PDEs J. Cellular Biochem. Suppl. 16B 231, 1992.
Obernolte et al. Teh cDNA of a human lymphocyte cyclic-AMP phosohodiesterase (PDE IV) reveals a multigene family Gene vol. 129 239-247, 1993.
Sequence comparison Obernolte et al. (Accesion L12686) vs. SEQ ID No. 1 of 08/446386.
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Swinnen et al. Moledcular clonong of rat homologoues of the Drosophila melanogaster dunce cAMP phosphodiesterase: Evidence for a family of genes. Proc. Natl. Acad. Sci. USA vol. 86 pp. 5325-5329, 1989.
Livi George P.
McLaughlin Megan M.
Torphy Theodore J.
Brusca John S.
Elliott George C.
Gimmi Edward R.
Hecht Elizabeth T.
King William T.
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