Masae Goseki

Expression of bone morphogenetic protein genes in the human dental pulp cells

Dental pulp has a potential to induce ectopic bone formation, but little is known about its mechanism. We thought that bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-beta) superfamily, are involved in the osteoinductive activity of dental pulp. In order to prove this assumption, we constructed a cDNA library from primary culture cells of human dental pulp (HDP cells), and screened the library with previously cloned cDNAs for mouse BMP-2 and -6 as probes. Three distinct cDNA clones encoding human BMP-2, -4 and -6 were isolated. By Northern blot analysis, specific transcripts of the genes of those BMPs were detected in the HDP cells. It was concluded that the BMPs were expressed in a certain population of dental pulp cells and might play some roles in ectopic bone formation by dental pulp.

Properties of Alkaline Phosphatase of the Human Dental Pulp

Enzymatic and immunological properties of alkaline phosphatase [ALP; orthophosphoric monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1.] in the human dental pulp were investigated. In inhibition and thermal inactivation studies, dental pulp ALP showed properties of universal-type ALP (kidney/bone/liver type). Dental pulp ALP cross-reacted with polyclonal and monoclonal antibodies against purified swine-kidney ALP, and with monoclonal antibody against ALP of human osteoblast-like cells in the same manner as ALPs of human bone and kidney. The sodium dodecyl sulfate-gel electrophoretic pattern showed a 140, 000-Mr native protein band. These data suggest that dental pulp ALP can be classified as a universal-type ALP having antigenic determinants common to ALP of the kidney and bone.

Partial breakdown of glycated alkaline phosphatases mediated by reactive oxygen species.

The lower levels of serum alkaline phosphatase (AP) activity found in patients with diabetes mellitus apparently originate from the selective disappearance or decrease in bone AP activity in the circulation. Hence, we investigated in vitro the effect of glycation on the activities of five AP isozymes. Aseptic incubation with 25 mmol/L of D-glucose and APs rapidly reduced bone and placental AP activities before those of liver, kidney and intestinal enzymes. The resulting bone and placental AP molecules were clearly glycated, according to the result of aminophenylboronic acid affinity chromatography. Furthermore, Western blotting analysis revealed that the placental AP molecule was fragmented, and its partial cleavage took place at Ala154 on the AP molecule. Since glycation of serum proteins causes the generation of reactive oxygen species, the effects of reactive oxygen species on placental AP activity were assayed, and the results indicated that hydroxyl radicals might be a major factor for the specific inactivation of AP activities. The reduction in AP activity by incubation with glucose in vitro was reversed by the further addition of catalase. Furthermore, ferrous ion with hydrogen peroxide, which generates hydroxyl radicals, had an inhibitory effect on AP activities. These findings suggest that the reduced AP activity in diabetic patients might result from partial cleavage of the bone AP molecule by reactive oxygen species induced by glycation.

Purification and subunit structure of alkaline phosphatase from bovine enamel organ.

A highly-purified alkaline-phosphatase preparation having a specific activity of 703 U/mg protein was obtained from bovine enamel organ by a series of procedures: butanol extraction, isoelectric and acetone precipitation, ion-exchange, concanavalin A affinity and gel-filtration chromatography. The purified enzyme showed the same properties as kidney-type isozyme and contained carbohydrate moieties which react with concanavalin A. Analysis by polyacrylamide gel electrophoresis showed that the purified enzyme split into half the size of the native molecule (160,000 in mol. wt) after being heated in sodium dodecyl sulphate (SDS) solution and the subunit had no catalytic activity. The results indicate that the enzyme is a dimeric glycoprotein comprised of two identical subunits, each having a molecular weight of 80,000.