Salivary glands synthesize and secrete cyclic AMP-reactive proteins (cARP)-regulatory (R) subunits of cyclic AMP (cAMP)-dependent protein kinase. The R subunits are generally intracellular eukaryotic cARP which regulate protein phosphorylation by formation or dissociation of the holoenzyme. Recent animal studies have shown that cARP (mainly the type Il isoform) are present in saliva and in other body fluids and may have separate extracellular function. The purpose of this study is to prepare and test specific monoclonal antibodies (mAb) using cARP purified from human saliva as antigen (Ag). Whole saliva and parotid fluid were collected from a population of healthy volunteers without diagnosed disease or oral pathology. Salivary cARP activity was measured using photoaffinity labeling with an azido analog of AMP, separating proteins by electrophoresis and identifying cARP by autoradiography. Densitometric analyses of the radioactive bands from saliva of 12 male and 11 female volunteers showed that each sample of parotid and whole saliva contained cARP (in a ratio of about 10:1) with the relative mobility of RII compared to standards, providing a sufficient quantity of Ag for immunization (approximately 0.1 to 1.0 ug/ml). Isolation of cARP will be by affinity chromatography using cAMP-Sepharose which binds only R subunits. The cARP will be eluted, collected, checked for purity by photoaffinity labeling and electrophoresis, concentrated and used in immunizations. Antibodies to human salivary cARP will be raised in mice to produce antibody secreting cells for fusion to form hybridomas which secrete MAb. Limiting dilution clonal selection and characterization of MAb will be carried out using ELlSA, immunodiffusion. Western blotting and immunocytochemistry to ensure that the optimal testing methods hake been selected. As in other tissues, cARP in oral epithelium is likely to mediate cellular signal processing and secretory cARP in saliva may be used as an index of systemic, hormone- dependent, or drug-induced disorders. The MAb will be used to determine baseline values of cARP in human saliva of a population of normal subjects and to immunocytochemically localize cAPK subunits in glandular epithelium of rat parotid, exorbital lacrimal and pancreatic gland tissue as well as in ameloblasts involved in secretion of rat incisor enamel. Additionally, these Ab will be used for the intracellular localization and quantitation of cARP in oral tissues (archival samples of human tissue) and in saliva of patients with diagnosed periodontitis. Our preliminary findings showed unique, individual cARP pleiomorphy. These reagents may also be commercially developed for application in clinical or forensic science.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Small Research Grants (R03)
Project #
1R03DE010105-01A3
Application #
2131071
Study Section
NIDCR Special Grants Review Committee (DSR)
Project Start
1994-04-01
Project End
1996-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Dentistry
Type
Schools of Dentistry
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
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Mednieks, Maija I; Szczepanski, Andrew; Clark, Brett et al. (2009) Protein expression in salivary glands of rats with streptozotocin diabetes. Int J Exp Pathol 90:412-22
Mednieks, Maija; Lin, Marcus; Hand, Arthur R (2008) Immunocytochemical analysis of cyclic AMP receptor proteins in the developing rat parotid gland. Arch Oral Biol 53:429-36
Piludu, M; Mednieks, M I; Hand, A R (2002) Cyclic AMP-receptor proteins in human salivary glands. Eur J Morphol 40:219-25
Szczepanski, A; Mednieks, M I; Hand, A R (1998) Expression and distribution of parotid secretory proteins in experimental diabetes. Eur J Morphol 36 Suppl:240-6
Mednieks, M I; Hand, A R; Grindeland, R E (1998) Cyclic AMP-receptor responses to hypergravity. Aviat Space Environ Med 69:A28-36
Mednieks, M I; Merheb, N N; Eisenmann, D R et al. (1998) Cyclic AMP-receptor proteins in the enamel matrix. Connect Tissue Res 38:287-94;discussion 295-303
Zaki, A E; Hand, A R; Mednieks, M I et al. (1996) Quantitative immunocytochemistry of Ca(2+)-Mg2+ ATPase in ameloblasts associated with enamel secretion and maturation in the rat incisor. Adv Dent Res 10:245-51