Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-03-03
2002-08-20
Gambel, Phillip (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007940, C436S504000, C436S501000
Reexamination Certificate
active
06436636
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the detection and treatment of heart failure based on the down-regulatory activity of the AUF1 or an AUF1-related polypeptide toward human &bgr;-adrenergic receptors.
BACKGROUND OF THE INVENTION
The condition of heart failure is associated with heightened activity of the adrenergic nervous system (Bristow et al.,
Circulation
82:(Suppl.I) 12-25 (1990)), the severity of failure correlating with increases in circulating and cardiac concentrations of the catecholamine, norepinephrine (Cohn et al.,
N. Engl. J. Med.
311:819-823 (1984)). As a consequence of this increased “adrenergic drive” the cardiac &bgr;-adrenergic receptor (&bgr;-AR)/G-protein/adenylyl cyclase pathway can become markedly desensitized. One major component of the desensitization is selective down-regulation of the dominant adrenergic receptor subtype within the human myocardium, the &bgr;
1
-AR (Bristow et al.,
Circulation
82:(Suppl.I) 12-25 (1990); Bristow et al.,
Circ. Res.
59:297-309 (1986); Brodde et al.,
J. Cardiovasc. Pharmacol.
8:1235-1242 (1986); Bristow et al.,
J. Clin. Invest.
89:803-815 (1992)). Recently, it has been demonstrated that the observed decrease in &bgr;
1
-adrenergic receptors in failing human heart is closely associated with a corresponding down-regulation of &bgr;
1
-AR mRNA (Bristow et al.,
J. Clin. Invest.
92:2737-2745 (1993); Ungerer et al.,
Circulation
87:454-463 (1993)).
Experiments performed using hamster DDT1-MF2 smooth muscle cells (Hadcock et al.,
J. Biol. Chem.
264:19928-19933 (1989)) suggest that down-regulation of the endogenously expressed &bgr;
2
-AR mRNA does not appear to be caused by a decrease in the rate of transcription; rather, it appears that agonist exposure decreases the half-life of &bgr;-AR mRNA from approximately 12 to 5 h (Hadcock et al.,
J. Biol. Chem.
264:19928-19933 (1989)). This regulatory mechanism has been demonstrated previously to be important for numerous mRNAs encoding proto-oncogenes, lymphokines and cytokines. For these gene products regulation of mRNA stability has also been associated with the interaction of the mRNA with a family of cytosolic proteins (Mr 30,000-40,000) that often bind to A+U-rich elements (ARE) commonly within the 3′ untranslated region (3′UTR) of the mRNA. This interaction induces mRNA degradation by mechanisms poorly understood. However, for some mRNAs including those containing AREs (Savant-Bhonsale and Cleveland,
Genes Dev.
6:1927-1939 (1992); Winstall et al.,
Mol. Cell. Biol.
15:3796-3804 (1995)), the degradation of mRNA may be associated with the process of translation. The cytosolic A+U-rich mRNA binding proteins are in general considered to be distinct from other mRNA binding proteins such as the heterogeneous nuclear ribonucleoproteins (hnRNPs) (Dreyfuss,
Annu. Rev. Cell Biol.
2:459-498 (1986); McCarthy and Kollmus,
TIBS
20:191-197 (1995)), however, the role of hnRNP A1 and C proteins as cytoplasmic factors regulating mRNA stability is currently undergoing reassessment (Gorlach et al.,
EMBO J.
11:3289-3295 (1992); Hamilton et al.,
J. Biol. Chem.
268:8881-8887 (1993)).
From previous studies (Port et al.,
J. Biol. Chem.
267:24103-24108 (1992); Huang et al.,
J. Biol. Chem.
268:25769-25775 (1993); Tholanikunnel et al.,
J. Biol. Chem.
270:12787-12793 (1995)) using cytosolic extracts produced from DDT1-MF2 hamster smooth muscle cells, the properties of a &bgr;-AR mRNA-binding polypeptide (&bgr;-ARB), which binds to hamster &bgr;
2
-adrenergic and human &bgr;
1
-adrenergic receptor mRNAs, have undergone preliminary characterization. Binding of &bgr;-ARB to mRNA was determined to involve regions of the 3′UTR of the hamster &bgr;
2
-AR mRNA containing an ARE (Port et al.,
J. Biol. Chem.
267:24103-24108 (1992); Huang et al.,
J. Biol. Chem.
268:25769-25775 (1993)). In addition, agonist stimulation of the &bgr;-AR pathway or protein kinase A (PK-A) activation by a cAMP analogue resulted in significant up-regulation (3-4 fold) of &bgr;-ARB as detected by UV-crosslinking. Conversely, treatment of DDT1-MF2 cells with dexamethasone, which up-regulates &bgr;
2
-AR mRNA, down-regulated &bgr;-ARB by ~50%. Therefore, agents that regulate hamster &bgr;
2
-AR mRNA stability and abundance appear to affect reciprocally the abundance of &bgr;-ARB. Among the family of G-protein coupled receptors, the mRNAs of the hamster &bgr;
2
-AR, the human &bgr;
1
- and &bgr;
2
-AR, and the thrombin receptor have all been demonstrated to interact with &bgr;-ARB (Port et al.,
J. Biol. Chem.
267:24103-24108 (1992); Huang et al.,
J. Biol. Chem.
268:25769-25775 (1993); Tholanikunnel et al.,
J. Biol. Chem.
270:12787-12793 (1995)). To date, the identity of &bgr;-ARB has remained unresolved.
The cytoplasmic RNA-binding polypeptide, AUF1 (A+U-rich element RNA-binding/degradation Factor) (Zhang et al.,
Mol. Cell. Biol
13:7652-7665 (1993)), has recently been cloned and characterized. AUF1 binds to the 3′UTRs of several highly regulated mRNAs including c-myc, granulocyte/macrophage colony-stimulating factor (GM-CSF), and c-fos. Further, there is evidence of “cause and effect” between AUF1 and regulation of mRNA stability in that partially purified AUF1 can selectively accelerate the degradation of c-myc mRNA in an in vitro mRNA decay system (Brewer,
Mol. Cell. Biol.
11:2460-2466 (1991)).
SUMMARY OF THE INVENTION
The present invention involves a method of detecting in a biological sample the amount of the AUF1 or an AUF1-related polypeptide for indicating heart failure. This involves generating an antibody to the products of the AUF1 or an AUF1-related polypeptide. Then contacting the antibody with the biological sample and detecting the amount of immune complex formation as an indication of the amount of the polypeptide in the biological sample. A higher amount of immune complex formulation, than found in a normal cell, indicates that heart failure is present. The higher than normal amount of the AUF1 or an AUF1-related polypeptide is a result of a high level of the AUF1 or an AUF1-related gene. This higher than normal amount of AUF1 or AUF1-related polypeptide is involved in the compensatory response to heart failure and/or involved in the worsening the condition of heart failure. This detection procedure would preferably involve contacting a biological sample from a human patient with an antibody (e.g., monoclonal antibody) which specifically reacts with the polypeptide. Then determining the amount of immune complex formation present in the biological sample. A higher amount of immune complex formation, than found in a normal cell, indicates that heart failure is present. The biological sample can be obtained from human ventricular myocardium, serum, and blood cells.
Another method for determining heart failure is the use of probes that bind to the RNA of the AUF1 or an AUF1-related gene. This involves hybridizing an RNA extracted from a biological sample of a human patient with a probe specific for the gene. Then determining the degree of hybridization to the mRNA as an indication of the amount of the gene in the biological sample. A higher degree of hybridization, than found in a normal cell, indicates that heart failure is present.
Another aspect of the invention involves a method for detecting a pharmacological that inhibits the down-regulatory activity of the AUF1 or an AUF1-related polypeptide for limiting the expression of &bgr;-adrenergic receptor genes by decreasing the binding affinity of the polypeptide for the &bgr;-adrenergic receptor gene. This involves mixing the polypeptide, a radiolabeled ARE-RNA of the &bgr;-adrenergic gene, and the pharmacological. The resulting mixture is filtered through a membrane such as nitrocellulose. Pharmacologicals that inhibits the down-regulatory activity of the polypeptide do so by decreasing the binding affinity of the polypeptide for the ARE-RNA. Since the polypeptide binds to the membrane, the radiolabeled ARE-RNA complexed with the polypeptide will adhere by means of the
Brewer Gary
Port J. David
Dann Dorfman Herrell and Skillman
Gambel Phillip
Wake Forest University
LandOfFree
Methods for identifying pharmacologicals for the treatment... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods for identifying pharmacologicals for the treatment..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for identifying pharmacologicals for the treatment... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2965044