No abstract

A DNA fragment conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae was isolated from a library of yeast genomic DNA. Its nucleotide sequence revealed the presence of a single open reading frame (ORF; 1326 bp) having the potential to encode a protein of 442 amino acid residues (molecular mass of 48.3 kDa). A frameshift mutation introduced within the ORF abolished resistance to heavy metal ions, indicating the ORF is required for resistance. Therefore, we termed it the ZRC1 (zinc resistance conferring) gene. The deduced amino acid sequence of the gene product predicts a rather hydrophobic protein with six possible membrane-spanning regions. While multiple copies of the ZRC1 gene enable yeast cells to grow in the presence of 40 mM Zn2+, a level at which wild-type cells cannot survive, the disruption of the chromosomal ZRC1 locus, though not a lethal event, makes cells more sensitive to zinc ions than are wild-type cells.

We attempted to determine natural agonists of GPR39 in fetal bovine serum (FBS). FBS was conditioned to extract peptides and fractionated by two types of HPLC. The activity of each fraction was monitored by intracellular calcium mobilization. Then the purified active ingredient was analyzed by inductively coupled plasma mass spectrometry. In this fashion, Zn2+ ion was identified as an agonist of GPR39, though no peptidergic molecules were found. The calcium-mobilizing activity of Zn2+ was not abolished by pertussis toxin but was by a phospholipase C (PLC) inhibitor, U73122, indicating that the activity of GPR39 is mediated through the Gqalpha -PLC pathway. In addition, Zn2+ also activated mouse and rat GPR39, showing that the function of GPR39 as a Zn2+ receptor is conserved across species. This study is the first exploration of GPR39 agonists in FBS and indicates that GPR39 functions as a Gq-coupled Zn2+-sensing receptor.

Cartilage is unique among tissues of mesenchymal origin in that it is resistant to vascular invasion due to an intrinsic angiogenic inhibitor. During endochondral bone formation, however, calcified cartilage formed in the center of the cartilaginous bone rudiment allows vascular invasion, which initiates the replacement of cartilage by bone. The transition of cartilage from the angioresistant to the angiogenic status thus plays a key role in bone formation. However, the molecular basis of this phenotypic transition of cartilage has been obscure. We report here purification of an endothelial cell growth inhibitor from a guanidine extract of bovine epiphyseal cartilage. The N-terminal amino acid sequence indicated that the inhibitor was identical to chondromodulin I (ChM-I), a cartilage-specific growthmodulating factor. Purified ChM-I inhibited DNA synthesis and proliferation of vascular endothelial cells as well as tube morphogenesis in vitro. Expression of ChM-I cDNA in COS7 cells indicated that mature ChM-I molecules were secreted from the cells after post-translational modifications and cleavage from the transmembrane precursor at the predicted processing signal. Recombinant ChM-I stimulated DNA synthesis and proteoglycan synthesis of cultured growth plate chondrocytes, but inhibited tube morphogenesis of endothelial cells. In situ hybridization and immunohistochemical studies indicated that ChM-I is specifically expressed in the avascular zone of cartilage in developing bone, but not present in calcifying cartilage. These results suggest a regulatory role of ChM-I in vascular invasion during endochondral bone formation.

During endochondral bone formation, cartilage cells show increased matrix synthesis and rapid proliferation. We found that cartilage matrix contains at least two types of heparin binding growth-promoting components. One, with a higher affinity to heparin, was identified as chondromodulin I (Hiraki, Y., Tanaka, H., Inoue, H., Kondo, J., Kamizono, A., and Suzuki, F. (1991) Biochem. Biophys. Res. Commun. 175, 871-977). In this study, we isolated a novel growth-promoting component, chondromodulin II, which has a lower heparin affinity, from the dissociative extracts of fetal bovine epiphyseal cartilage. Chondromodulin II stimulated the proteoglycan synthesis in rabbit cultured growth plate chondrocytes, an expression of the differentiated phenotype of chondrocytes. It also stimulated DNA synthesis in chondrocytes in both the absence and the presence of fibroblast growth factor-2. The apparent molecular mass of chondromodulin II on SDS-polyacrylamide gel electrophoresis was 16 kDa. Its complete amino acid sequence was determined by overlapping sequences of the peptides released by endopeptidase digestion and CNBr cleavage. Chondromodulin II consists of 133 amino acids (calculated M r ‫؍‬ 14,548). The sequence was unique but homologous to the repeats 1 and 2 of the deduced amino acid sequence of the chicken mim-1 gene, which is specifically transactivated by the v-Myb oncogene product in promyelocytes. We also found a minor component with a higher heparin affinity, chondromodulin III, in cartilage extracts. Chondromodulin III stimulated DNA synthesis in chondrocytes in vitro, and its N-terminal sequence was identical with ribosomal protein L31 lacking the N-terminal three amino acids. These findings suggest that the growth and differentiation of chondrocytes are regulated by multiple components in the cartilage matrix.The growth of cartilage plays a key role in endochondral bone formation during embryonic development and during the longitudinal growth of bone. Fibroblast growth factor (FGF) 1 exhibits pleiotropic effects depending on the target tissue. Cartilage is a major source of FGF (1), although growth factors of the FGF family are also widely distributed in the body (2). Recent DNA analysis has revealed point mutations in the FGF receptor 3 gene in achondroplasia (3,4). In individuals with this disorder, the growth cartilage of the long bones undergoes minimal proliferation. Thus, FGF signaling is considered to be important for the support of cartilage growth. FGF-2 is the most potent mitogen for chondrocytes (5) and stabilizes the phenotypic expression of these cells (6). As we have reported previously, some proteinaceous components in cartilage synergistically stimulated DNA synthesis and the growth of cultured chondrocytes in vitro as well as stimulating proteoglycan synthesis in chondrocytes (6, 7). These findings suggest that FGF, in combination with some unique growth-promoting component(s) in cartilage, may act on chondrocytes.In the course of the initial screening of these active components in the extrac...