Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes.

Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.

Preparation of monoclonal antibodies to the avian progesterone receptor.

In an effort to obtain additional probes for analysis of the avian progesterone receptor, this receptor was isolated and used to prepare several monoclonal antibodies. Progesterone receptor purified from oviduct cytosol by chromatography on deoxycorticosterone-Sepharose and heparin-agarose was used as the immunizing antigen. Twenty-nine hybridoma cultures which tested positive in an enzyme-linked immunosorbent assay against the receptor preparation were subcloned resulting in establishment of 12 stable cell lines. Of these, 5 produced antibodies capable of complexing receptor-bound progesterone from cytosol as measured by adsorption of receptor-antibody complexes onto antimouse immunoglobulin G-agarose. Each was used to generate ascites and the purified antibodies were designated alpha PR 6, 11, 13, 16, and 22. In addition to precipitating receptor-bound progesterone from cytosol, the antibodies were also effective in increasing the sedimentation velocity of progesterone receptor centrifuged on glycerol gradients, and in recognizing receptor proteins that were resolved by denaturing gel electrophoresis and transferred to nitrocellulose (Western blots). Immunoisolation of receptor was also demonstrated using receptor labeled covalently with the synthetic progestin, R5020. The antibodies were specific for progesterone receptor and did not cross-react with estrogen receptor from the oviduct or glucocorticoid receptor from chick liver. Two antibodies, alpha PR 6 and alpha PR 22, also recognized some mammalian forms of the progesterone receptor. Both antibodies reacted with progesterone receptor from the rabbit uterus and alpha PR 6 recognized human progesterone receptor. Four of the antibodies recognized both A and B forms of the avian receptor while alpha PR6 was specific for the B form.

Development of monoclonal antibodies against the human sodium iodide symporter: immunohistochemical characterization of this protein in thyroid cells.

The thyroid sodium-iodide symporter (NIS) is responsible for iodide concentrating ability within thyroid follicular cells. We sought to develop monoclonal antibodies against human NIS (hNIS) for use as reagents in structure-function studies of the protein, as well as potential tools in the assessment of NIS expression in benign and malignant thyroid tissues. Synthetic peptides corresponding to the second ExMD and to the carboxy-terminal ExMD of hNIS were produced and utilized as antigens to develop monoclonal antibodies, which were tested by Western blotting using membranes prepared from COS-7 cells transiently transfected with a pcDNA3 plasmid containing the gene for the full-length hNIS, or a control vector. Western blotting showed a major band with molecular weight (MW) of approximately 97 kDa and several minor bands with MW of approximately 160 kDa, 68 kDa, 30 kDa, and 15 kDa, all specific for hNIS-transfected cells. Immunohistochemistry was performed in various types of thyroid tissues and nonthyroidal tissues, using the monoclonal antibodies. Strong immunostaining was observed in Graves' tissue, intermediate staining in papillary and follicular thyroid cancer, and no staining in Hürthle cell cancer or in nonthyroidal tissue. The staining was specific for the follicular epithelium in each of the tissues and was most intense in the basolateral portion of the cell membrane. Overall, our observations indicate that the monoclonal antibodies are specific for hNIS and will be invaluable reagents for investigating the role of NIS in thyroid disease.

Determination of tolerance to self E alpha peptides by clonal elimination of H-2E reactive T cells and antigen presentation by H-2A molecules.

A series of three synthetic peptides spanning H-2E alpha k chain residues (90-110), (110-130), and (130-150) were synthesized and purified. Mice representative of H-2E- (B6, B10, B10.M, B10.Q, B10.S) and H-2E+ (B10.D2, B10.K, B10.RIII) were immunized with individual peptides and lymph node cells challenged in vitro. Both B6 and B10 mice respond to in vitro challenge to peptides (90-110) (cpm 20,000), (110-130) (cpm 40,000), and (130-150) (cpm 60,000). In contrast all H-2E+ haplotypes were unresponsive to all three peptides (cpms < 10,000). Furthermore, B10 mice could be rendered hyporesponsive to E alpha k peptide challenge following expression of an E alpha k transgene or mating to an H-2E+ strain. The H-2Ad,k,f,q,s alleles were associated with reduced peptide recognition. Furthermore, alteration of the H-2A beta chain in bm12 mutant mice resulted in impaired responses to all three peptides. Immunization with synthetic peptides comprising major histocompatibility molecules may yield insights into mechanisms of self-tolerance.

Monoclonal antibodies against the human sodium iodide symporter: utility for immunocytochemistry of thyroid cancer.

The recent cloning of the thyroidal protein that is responsible for iodide transport, the sodium iodide symporter (hNIS), has made possible studies designed to characterize its structure, function and expression in thyroidal tissues. Using a mannose binding protein (MBP)-hNIS fusion protein as antigen, we have developed mouse monoclonal antibodies against hNIS to utilize as tools in such studies. Twenty-four clones were initially recovered which recognized the MBP-hNIS fusion protein, but only two of them were specific for hNIS while the others recognized MBP alone. Both antibodies were found to be immunoglobulin G (IgG) 1kappa (kappa). The specificity of antibodies was tested by Western blotting using membranes prepared from COS-7 cells transiently transfected with the pcDNA3 plasmid containing the full-length hNIS cDNA, or cells transfected with the pcDNA3 vector. A major band with a molecular weight (MW) of approximately 97 kDa, and several minor bands with MW of approximately 160 kDa, approximately 68 kDa, approximately 30 kDa and approximately 15 kDa, were detected specifically in the hNIS-transfected cells. After enzymatic deglycosylation, the major band was present at 68 kDa, as expected based upon the amino acid sequence of hNIS. Immunohistochemistry was performed with several different types of thyroid tissue and non-thyroidal tissues, using the monoclonal antibodies. Strong immunostaining was observed in Graves' tissue, with intermediate staining in papillary and follicular thyroid cancers and an absence of staining in Hürthle cell cancer. The staining was specific for the follicular epithelium and was concentrated in the basolateral portion of the cell membrane. These monoclonal hNIS antibodies should prove useful in the characterization of NIS expression in benign and malignant thyroid tissue and in studies characterizing its structure and function.

Genetic control of the immune response to Trichinella spiralis: recognition of muscle larval antigens.

Host antibody recognition of muscle larval (ML) antigens of Trichinella spiralis was examined. Monoclonal antibodies (MoAbs) to known host protective ML antigens have been produced in order to aid this examination. Eleven strains of mice with independent MHC haplotypes and seventeen T. spiralis infected human patients were all found to recognize the same three major antigens as the monoclonal antibodies; i.e., of mw 41, 46 and 55 kD. However all serum samples tested also recognized further ML antigens and this recognition varied with the individual or strain. This variation in antigen recognition also applied to the MoAb. Mutual inhibition studies demonstrated that even where the MoAb apparently recognized the same antigens, specific epitope recognition was disparate. Hence some of the major antigens recognized by hosts of T. spiralis, regardless of whether vaccinated or infected, correspond with antigens which have considerable host protective properties. There also appear to be a number of epitopes upon these antigens and the biological implications of this are discussed.

Hormone-dependent processing of the avian progesterone receptor.

Avian progesterone receptor exists as two forms, A and B, with molecular weights of 79,000 and 110,000 daltons, respectively. The origin and significance of these two forms is an area of active investigation and debate. Monoclonal antibodies produced against these two forms were used to examine receptor stability in cytosol and changes in the receptor forms induced by hormone binding. The lability of hormone binding at elevated temperatures is well documented. Analysis by Western blotting showed the receptor was stable in freshly prepared oviduct cytosol for 2 hr at 37 degrees C, while hormone binding was lost within 30 min. However, loss of receptor through degradation was seen when cytosol was prepared from frozen tissue or when homogenization was excessive. Progesterone was injected into diethylstilbestrol-stimulated chicks to examine, in vivo, effects of hormone treatment on receptor forms in the cytosol and nuclear fractions. Progesterone treatment caused a time- and dose-dependent conversion of the A receptor to a form (A') with a slower electrophoretic mobility. The cytosolic progesterone receptor was divided equally between the B and A forms, while the nuclear receptor was predominantly A'. The amount of nuclear receptor was consistently less than cytosolic receptor. Receptor phosphorylation was analyzed by incubating tissue minces with [32P]orthophosphate with or without progesterone followed by immune isolation of receptor forms. Progesterone treatment caused a time-dependent increase in cytosol receptor phosphorylation which was evident after 5 min of treatment. This phosphorylation was observed with both the A and B receptor forms. The results indicate that receptor phosphorylation is a very early event during progesterone action.