Toshio Kawase

Impairment of spatial memory and changes in astroglial responsiveness following loss of molar teeth in aged SAMP8 mice

In order to evaluate the mechanism(s) responsible for senile impairment of cognitive function as a result of reduced mastication, the effects of the loss of the molar teeth (molarless condition) on the hippocampal expression of glial fibrous acidic protein (GFAP) and on spatial memory in young adult and aged SAMP8 mice were studied using immunohistochemical and behavioral techniques. Aged molarless mice showed a significantly reduced learning ability in a water maze test compared with age-matched control mice, while there was no difference between control and molarless young adult mice. Immunohistochemical analysis showed that the molarless condition enhanced the age-dependent increase in the density and hypertrophy of GFAP-labeled astrocytes in the CA1 region of the hippocampus. These effects increased the longer the molarless condition persisted. When the extracellular K+ concentration ([K+]o) was increased from 4 to 40 mM for hippocampal slices in vitro, the mean increase in the membrane potential was about 57 mV for fine, delicate astrocytes, the most frequently observed type of GFAP-positive cell in the young adult mice, and about 44 mV for the hypertrophic astrocytes of aged mice. However, there was no significant difference in resting membrane potential between these cell types. The data suggest that an impairment of spatial memory and changes in astroglial responsiveness occur following the loss of molar teeth in aged SAMP8 mice.

Expression of Cementum-derived Attachment Protein in Bovine Tooth Germ During Cementogenesis

Cementum-derived attachment protein (CAP) is a 56 kDa collagenous protein that promotes attachment of mesenchymal cells. Previous studies have shown that the presence of CAP is restricted to cementum in adult human tissues. In this study, we report generation of a monoclonal antibody against CAP and its use for the investigation of CAP in developing bovine tooth germs. Mice were immunized with CAP purified from bovine cementum, and a monoclonal antibody, 3G9, was produced. Immunohistochemical staining of bovine tooth germ at root forming stage using 3G9 antibody showed that the tissue distribution of CAP expression was limited to cementum matrix and cementoblasts during cementogenesis. Alveolar bone did not stain with the 3G9 antibody, whereas anti-type I collagen stained positively. CAP was purified from bovine tooth germs with immunoaffinity purification using the 3G9 antibody. Examination of the immunoaffinity-purified fraction showed that CAP existed in tooth germ as a 65 kDa protein. The protein was susceptible to bacterial collagenase. To investigate the possible biological function of CAP during cementogenesis, we isolated dental follicle cells from the bovine tooth germ, and showed that they adhered to surfaces containing CAP. These data demonstrate that CAP is expressed by bovine cementoblasts as a 65 kDa protein and that the CAP may have a function in cementogenesis.

Evidence for involvement of dysfunctional teeth in the senile process in the hippocampus of SAMP8 mice

In order to evaluate the involvement of dysfunctional teeth in age-related deficits in hippocampal function, we examined the effect of removal of molar teeth (molarless condition) on neuronal degeneration and glial fibrous acidic protein (GFAP) expression in the hippocampus and on learning ability in a water maze test in young, middle-aged, and aged accelerated senescence-prone mice (SAMP8). The molarless condition enhanced an age-dependent decrease in both learning ability and the number of neurons in the hippocampal CA1 subfield and the age-dependent increase in the number and hypertrophy of GFAP-labeled astrocytes in the same subfield. These observations suggest that the molarless condition may be involved in the senile process in the hippocampus in SAMP8 mice.

Effect of ipriflavone and estrogen on the differentiation and proliferation of osteogenic cells

SummaryThe effect of ipriflavone (IP) on the proliferation and differentiation of rat osteoblast-like (ROB) cells and human periodontal ligament fibroblasts (HPLF) was studied in the presence and absence of estrogen. ROB cells were isolated from newborn rat calvaria by sequential collagenase digestion and HPLF from the outgrowth of human periodontal ligament in culture. The alkaline phosphatase (ALP) activity, employed as a marker of bone cell differentiation, was significantly enhanced by IP in both cell types; however, the concentration at which IP had a maximal effect was lower in ROB cells than in HPLF (10−10 versus 10−7 M, respectively). Cell proliferation judged by DNA content was either constant (ROB cells) or slightly increased (HPLF) by IP up to 10−10 M, and decreased significantly above that concentration. In addition, the dose-dependent effect of estrogen on the growth and differentiation of each cell type in the presence and absence of IP was also tested. At the concentrations of IP which showed maximum effects in the induction of ALP, 10−10 M for ROB cells and 10−7 M for HPLF, IP inhibited DNA increase in an estrogen-independent manner. Estradiol (10−10-10−9 M) itself increased the growth rate of both cell types significantly in a dose-dependent manner. Regardless of the concentrations of estradiol tested, ALP activities of both ROB cells and HPLF were elevated by the addition of IP. The ratio of ALP in the presence and absence of IP was similar over the range of estradiol concentrations tested. Thus, we conclude that IP modulates osteogenic cell differentiation of both ROB cells and HPLF and the effect is estrogen independent.

Stimulation of Motility of Human Renal Cell Carcinoma by SPARC/Osteonectin/BM-40 Associated with Type IV Collagen

SPARC is known to be important in development and tissue remodelling. Here, we examined the effects of SPARC (secreted protein, acidic and rich in cysteine; osteonectin) derived from a rat osteosarcoma cell line on migration of renal cell carcinoma (RCC) by a Boyden chamber assay. YCR RCC cells migrated through type IV collagen-coated filters without stimuli (basal level). SPARC in the lower compartment stimulated chemotactic activity to 120% of the basal level, whereas premixing of YCR with purified SPARC before inoculation reduced their migration to 72% of the basal level. Furthermore, SPARC mixed with type IV collagen more efficiently stimulated their migration in a concentration-dependent manner (up to 170% of the basal level). This suggests that SPARC bound to type IV collagen plays a role in tumor invasion.

In situ Phosphorylation of Bone and Dentin Proteins by the Casein Kinase II-like Enzyme

Our previous studies suggested the possibility of extracellular phosphorylation of matrix phosphoproteins into more phosphorylated forms by mature odontoblasts and osteocytes (Mikuni-Takagi et al., 1995; Satoyoshi et al., 1995). To elucidate such phosphorylation of bone and dentin proteins, we developed a histochemical method using frozen sections to determine the sites of enzymatic processing by the casein kinase II-like enzyme. It was observed that proteins in bone, dentin, and predentin are phosphorylated by the endogenous enzyme when the tissue slices were incubated with [γ-32P] GTP, suggesting that there are both substrates and the enzyme in these matrices. In vivo, phosphate donors, ATP and GTP, may be supplied through dentinal canals and osteocyte canaliculi. Immunohistochemical analysis of frozen sections showed that the extremely intense staining of phosphoserine residues by anti-phosphoserine antibodies appeared in dentin only after demineralization of the tissue samples. It implies that these phosphoserine residues become bound to mineral as soon as the phosphorylation is completed, thereby being inaccessible to the antibodies without demineralization. The data support our notion that the extracellular phosphorylation of dentin/bone proteins, regulated by the developmental stages of bone and dentin cells, occurs prior to matrix mineralization.

Identification of osteoblast-specific monoclonal antibodies

SummaryA series of four antibodies against rat osteoblasts have been produced using the hybridoma technique. After bone cells isolated from newborn rat calvariae by a sequential digestion procedure were cultured for 3 days, the cells were trypsinized and further maintained in rotation cultures overnight. Out of the cultured bone cells alkaline phosphatase-positive cells were sorted by flow cytometry and used as immunogens. The clones secreting the antibodies were selected on the basis of the abilities of these antibodies to bind to the bone cells but not to fibroblasts from neonatal rat head skins, in an enzyme-linked immunosorbent assay. Clones of two hybridomas, designated AOB-1 and AOB-2, were used to characterize the antigenic determinant(s) in osteogenic cells. The antibody showed the reactivity with isolated alkaline phosphatase-positive cells, osteogenic tissue cells in newborn rat calvaria, and mandibula, but not with the cells in head skin, lung, kidney, liver, or stomach as determined by immunofluorescence study. Western blot analysis has identified the antigenic determinants possessing apparent molecular weights of 210,000, 110,000, 65,000, 58,000, 40,000, 36,000, 32,000, 28,000, 25,000, 17,000, and 15,000 of osteoblast-rich monolayer cultured cells. According to the cell surface detection with biotin-avidin protein blotting technique, these fractions appear to be present as components of the cell surface of the osteoblast.

Calcium in osteoblast-enriched bone cells

The exchangeability, location, and amount of the total calcium in bone cells were studied in relation to their osteoblastic activity. Cells were isolated from neonatal rat calvariae by sequential collagenase digestion and incubated with 45Ca2+ before or after various treatments. 45Ca2+, 40Ca2+, and DNA were determined on each cell sample. Long-term experiments were performed on cultured cells. The cells closest to the forming bone had the highest alkaline phosphatase activity and the highest Ca2+ content (i.e., 17 mm Ca2+/l cell water, as compared to soft tissue cells, which have 2-3 mm Ca2+/l (Borle 1981). About 50% of this Ca2+ exchanges readily with 45Ca2+ and appears to be located almost entirely in the cell membrane. Thirty-five percent exchanges only slowly with a t1/2 of 27 hours, and is located within the cell, principally in the mitochondria as seen in pyroantimonate fixed cells (Neuman et al. 1985). In spite of its slow exchange-ability, this Ca2+ fraction can be mobilized or augmented rapidly when Ca2+ supply is reduced or increased in vitro or in vivo. 1,25(OH)2D3 given in vivo increased this intracellular Ca2+ when the Ca2+ supply was low and released it when the Ca2+ supply was high. About 15% of the Ca2+ in these cells was nonexchangeable. These results suggest that osteoblasts do process more Ca2+ when closer to the mineralization site. Whether this Ca2+ is en route from blood to bone or from bone to blood will require further study.

Age-associated changes in the dopamine synthesis as determined by GTP cyclohydrolase I inhibitor in the brain of senescence-accelerated mouse-prone inbred strains (SAMP8)

Our objective in this study was to elucidate the mechanism underlying the decrease in dopamine (DA) levels in the brain with ageing We administered 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I to senescence-accelerated mouse-prones (SAMP8), to inhibit DA and serotonin syntheses, and following immunohistochemical staining, analyzed the immunoreactive intensities (IR-Is) for DA in the nigrostriatal dopaminergic neurons by microphotometry. The DA-IR-Is in the substantia nigra pars compacta and neostriatum of young mice (2 months old) reached a minimal value 3 h after DAHP administration and returned to the control value 12 h after the administration. However, in aged mice (10 months old), the minimal value was reached 6 h after the administration and the value remained at approximately 70 and 80% of the control value at 24 and 72 h, respectively, after DAHP administration. The results suggest that DA turnover is lower in aged mice than in young mice.

EXTRACELLULAR PROCESSING OF BONE AND DENTIN PROTEINS IN MATRIX MINERALIZATION

There are two steps in the process of matrix-mediated bone and dentin mineralization. First, as in other soft tissues, osteoblasts/odontoblasts synthesize collagenous matrices and second, mineral deposits in these matrices at a location distant from the cells that synthesized the matrices. We suggest a sequence of events that lead the matrix to mineralization: the phosphoproteins of bone and dentin are posttranslationally processed by limited proteolysis, then they are extracellularly processed into a more phosphorylated species that, we believe, facilitates mineralization. Our in situ phosphorylation experiments done with [gamma-32P] GTP suggest the existence of extracellular phosphorylation by a casein kinase II (CKII)-like enzyme, the enzyme known to phosphorylate most of the phosphate residues in dentin phosphophoryn and bone sialoproteins (osteopontin and BSP II).