Biomedical subjects

Hideto Watanabe

Publications and source records attributed to Hideto Watanabe.

23 records · Page 2Linked to original sources

PubMed2002

Molecular cloning and characterization of chondroitin polymerase from Escherichia coli strain K4.

Escherichia coli strain K4 produces the K4 antigen, a capsule polysaccharide consisting of a chondroitin backbone (GlcUA beta(1-3)-GalNAc beta(1-4))(n) to which beta-fructose is linked at position C-3 of the GlcUA residue. We molecularly cloned region 2 of the K4 capsular gene cluster essential for biosynthesis of the polysaccharide, and we further identified a gene encoding a bifunctional glycosyltransferase that polymerizes the chondroitin backbone. The enzyme, containing two conserved glycosyltransferase sites, showed 59 and 61% identity at the amino acid level to class 2 hyaluronan synthase and chondroitin synthase from Pasteurella multocida, respectively. The soluble enzyme expressed in a bacterial expression system transferred GalNAc and GlcUA residues alternately, and polymerized the chondroitin chain up to a molecular mass of 20 kDa when chondroitin sulfate hexasaccharide was used as an acceptor. The enzyme exhibited apparent K(m) values for UDP-GlcUA and UDP-GalNAc of 3.44 and 31.6 microm, respectively, and absolutely required acceptors of chondroitin sulfate polymers and oligosaccharides at least longer than a tetrasaccharide. In addition, chondroitin polymers and oligosaccharides and hyaluronan polymers and oligosaccharides served as acceptors for chondroitin polymerization, but dermatan sulfate and heparin did not. These results may lead to elucidation of the mechanism for chondroitin chain synthesis in both microorganisms and mammals.

Amino Acid Sequence↗

PubMed2002

Characterization of an anti-decorin monoclonal antibody, and its utility.

6B6 is a monoclonal antibody raised against a purified small dermatan sulfate proteoglycan from human ovarian fibroma capsule, has Although it been widely used as an anti-decorin monoclonal antibody, its epitope has not yet been characterized at the molecular level. Here, we show that 6B6 is specific to decorin. The antibody recognized human, mouse, and bovine decorin core protein, but not biglycan. Using recombinant decorin domains, we determined that the epitope lies within the region of amino acid residues 50-65, termed the cysteine cluster region. Cross-reactivity among species further narrowed it down to a primary sequence of residues 57-65. We also established the conditions for immunostaining. 6B6 stained both frozen and fixed sections. Whereas the glycosaminoglycan chain of decorin inhibited access of the antibody in immunoblotting, pretreatment of tissue sections with chondrotinase ABC did not affect the intensity of staining, suggesting that the glycosaminoglycan chain is integrated and the Cys cluster region oriented outside of the collagen fibrils in the tissue. When 6B6 was applied to enzyme-linked immunosorbent assay, a concentration as low as 0.5 microg/ml of decorin was detectable by either direct or sandwich ELISA. 6B6 is thus a sensitive and reliable antibody to study functions of decorin from various aspects.

Animals↗

PubMed2002

Establishment of a novel chondrocytic cell line N1511 derived from p53-null mice.

We established a clonal chondrocytic cell line N1511 derived from rib cartilage of a p53-null mouse. N1511 cells proliferated in polygonal shape and elicited differentiation at confluence when treated with combination of bone morphogenetic protein (BMP) 2 and insulin or parathyroid hormone (PTH) and dexamethasone. BMP-2/insulin-treated cells became refractile without forming cartilaginous nodules and reached terminal differentiation, became positive for alizarin red staining, and developed considerable ALP activity. In contrast, PTH/dexamethasone-treated cells formed Alcian blue-positive nodules but remained negative for alizarin red staining and ALP activity. Northern blot analysis revealed that BMP-2/insulin-treated cells sequentially expressed type II, IX, and X collagens, whereas PTH/dexamethasone-treated cells slowly expressed type II collagen and then type IX, and they did not exhibit type X collagen expression. These results show that BMP-2/insulin treatment induces full differentiation toward hypertrophy, whereas treatment with PTH/dexamethasone slows and limits differentiation. Recovery of p53 expression in N1511 cells by transient transfection inhibited cell proliferation, suggesting that cell proliferation could be regulated with p53 in this cell line. These results indicate that N1511 is the only cell line with known genetic mutation, which undergoes multiple steps of chondrocyte differentiation toward hypertrophy, and because proliferation could be regulated by expression of p53, N1511 could be an excellent model for studies of chondrogenesis, the function of p53, and genetic engineering of cartilage tissue.

Alkaline Phosphatase↗

PubMedDate not supplied

Chondrodysplasia of gene knockout mice for aggrecan and link protein.

The proteoglycan aggregate of the cartilage is composed of aggrecan, link protein, and hyaluronan and forms a unique gel-like moiety that provides resistance to compression in joints and a foundational cartilage structure critical for growth plate formation. Aggrecan, a large chondroitin sulfate proteoglycan, is one of the major structural macromolecules in cartilage and binds both hyaluronan and link protein through its N-terminal domain G1. Link protein, a small glycoprotein, is homologous to the G1 domain of aggrecan. Mouse cartilage matrix deficiency (cmd) is caused by a functional null mutation of the aggrecan gene and is characterized by perinatal lethal dwarfism and craniofacial abnormalities. Link protein knockout mice show chondrodysplasia similar to but milder than cmd mice, suggesting a supporting role of link protein for the aggregate structure. Analysis of these mice revealed that the proteoglycan aggregate plays an important role in cartilage development and maintenance of cartilage tissue and may provide a clue to the identification of human genetic disorders caused by mutations in these genes.

Aggrecans↗

PubMedDate not supplied

Ultrasound enhances transforming growth factor beta-mediated chondrocyte differentiation of human mesenchymal stem cells.

In clinical studies and animal models, low-intensity ultrasound (US) promotes fracture repair and increases mechanical strength. US also promotes cartilage healing by increasing glycosaminoglycan synthesis of chondrocytes. As mesenchymal stem cells (MSCs) have the ability to differentiate into chondrocytes, US may promote their differentiation. Here, we evaluated the effects of US on the differentiation of MSCs toward chondrocytes and cartilage matrix formation. When human MSCs cultured in pellets were treated with transforming growth factor beta (TGF-beta, 10 ng/mL), they differentiated into chondrocytes as assessed by alcian blue staining and immunostaining for aggrecan, but nontreated cell pellets did not. Furthermore, when low-intensity US was applied for 20 min every day to the TGF-beta-treated cell pellets, chondrocyte differentiation was enhanced. Biochemically, aggrecan deposition was increased by 2.9- and 8.7-fold by treatment with TGF-beta alone, and with both TGF-beta and US, respectively. In contrast, cell proliferation and total protein amount appeared unaffected by these treatments. These results indicate that low-intensity US enhances TGF-beta-mediated chondrocyte differentiation of MSCs in pellet culture and that application of US may facilitate larger preparations of chondrocytes and the formation of mature cartilage tissue.

Cartilage↗