A basic protein was isolated from aqueous extracts of barley seeds. It contains 91 amino acid residues including 8 half-cystines, but no phenylalanine or tryptophan; the calculated molecular weight is 9,694. Amino acid composition and N-terminal sequence show that the protein corresponds to the probable amylase/protease inhibitor (PAPI) encoded by a formerly described cDNA. The protein is 50% homologous with an a-amylase inhibitor I-2 from seeds oflndian finger millet and it shows a distant homology with two Bowman Birk type protease inhibitors. However, the barley protein does not inhibit any of the enzymes tested. They include a-amylases, a 13-amylase. [3-glucanases. serine proteases, a sulfhydryl protease, aspartate proteases, and serine carboxypeptidases. Nevertheless. difference UV-spectroscopy indicated that the protein interacts with porcine pancreatic a-amylase. NMR-spectroscopy revealed that it possesses tertiary structure.
The acetamidomethyl (Acrn) group attached at the thiol function of cysteine was found to be cleaved quantitatively by treatment with silver trifluoromethanesulphonate in trifluoroacetic acid, this reagent being more effective than silver trifluoroacetate and cleaved the S-benzamidomethyl and the S-p-methoxybenzyl groups as well; this new S-deprotecting reagent has been used in the syntheses of oxytocin and chicken calcitonin.
Experiments have been carried out to determine the mechanisms involved in the formation of osteoclast-like cells from spleen cells in mice. Osteoclasts were defined as tartrate-resistant acid phosphatase-positive multinucleated cells (TRACP-positive MNCs) in which specific calcitonin receptors were identified by autoradiography with labeled salmon calcitonin. Furthermore, cultures rich in these cells produced resorption pits when grown on dentine slices. Several clonal cell lines were obtained from fetal mouse calvariae and screened for their ability to induce TRACP-positive MNCs in response to 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3] in co-cultures with spleen cells. A cell line, KS-4, was identified with the greatest potency in inducing osteoclast-like cell formation in co-culture with spleen cells. The capacity of KS-4 cells to produce this effect was much greater than that of two bone marrow-derived stromal cell lines (MC3T3-G2/PA6 and ST2 cells), which we have previously shown to be effective in this system but to require treatment with dexamethasone in addition to 1 alpha, 25(OH)2D3 (Udagawa et al.: Endocrinology 125:1805-1813, 1989). Parathyroid hormone (PTH) increased cAMP production in KS-4 cells, and PTH and interleukin-1 alpha also induced TRACP-positive MNCs in co-cultures with spleen cells. Contact between living KS-4 and spleen cells was necessary for osteoclast formation to take place, since this did not occur when the two populations were separated by a membrane filter, or when the KS-4 cells were killed by fixation. Separate cultures of either spleen cells or KS-4 cells formed no TRACP-positive MNCs. KS-4 cells synthesized predominantly type I collagen, formed bone nodules without added of beta-glycerophosphate in a long-term culture, and expressed increasing alkaline phosphatase activity after confluence in culture. These results indicate that the KS-4 cells have properties consistent with progression toward the osteoblast phenotype and represent a single cell line with the ability to promote osteoclast formation by a contact-requiring process.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.