Takamune Hino

Differential Effects of Various Growth Factors and Cytokines on the Syntheses of DNA, Type I Collagen, Laminin, Fibronectin, Osteonectin/Secreted Protein, Acidic and Rich in Cysteine (SPARC), and Alkaline Phosphatase by Human Pulp Cells in Culture

The purpose of this study is to differentiate roles of several growth factors and cytokines in proliferation and differentiation of pulp cells during development and repair. In human pulp cell cultures, laminin and type I collagen levels per cell remained almost constant during the whole culture period (22 days). On the other hand, secreted protein, acidic and rich in cysteine (SPARC/osteonectin) and alkaline phosphatase (ALPase) levels markedly increased after the cultures reached confluence. Laminin and type I collagen, as well as fibronectin, stimulated the spreading of pulp cells within 1 h. Adding transforming growth factor‐β (TGF‐β) decreased laminin and ALPase levels, whereas it increased SPARC and fibronectin levels 3‐ to 10‐fold. Western and Northern blots showed that TGF‐β enhanced SPARC synthesis at the protein and mRNA levels. Basic fibroblast growth factor (bFGF) decreased type I collagen, laminin, SPARC, and ALPase levels without changing the fibronectin level. Platelet‐derived growth factor (PDGF) selectively decreased laminin, SPARC, and ALPase levels. Epidermal growth factor (EGF) also decreased SPARC and ALPase levels. Tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) decreased type I collagen and laminin levels, and abolished SPARC and ALPase syntheses. Of these peptides, bFGF and PDGF showed the greatest stimulation of [3H]thymidine incorporation into DNA. TGF‐β, EGF, and TNF‐α had less effect on DNA synthesis, whereas IL‐1β inhibited DNA synthesis. These findings demonstrated that TGF‐β, bFGF, EGF, PDGF, TNF‐α, and IL‐1β have characteristically different patterns of actions on DNA, laminin, type I collagen, fibronectin, ALPase, and SPARC syntheses by pulp cells. J. Cell. Physiol. 174:194–205, 1998.

The green tea polyphenol (−)-epigallocatechin gallate precipitates salivary proteins including alpha-amylase: biochemical implications for oral health

Green tea is a popular drink throughout the world, and it contains various components, including the green tea polyphenol (-)-epigallocatechin gallate (EGCG). Tea interacts with saliva upon entering the mouth, so the interaction between saliva and EGCG interested us, especially with respect to EGCG-protein binding. SDS-PAGE revealed that several salivary proteins were precipitated after adding EGCG to saliva. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting indicated that the major proteins precipitated by EGCG were alpha-amylase, S100, and cystatins. Surface plasmon resonance revealed that EGCG bound to alpha-amylase at dissociation constant (K(d)) = 2.74 × 10(-6) M, suggesting that EGCG interacts with salivary proteins with a relatively strong affinity. In addition, EGCG inhibited the activity of alpha-amylase by non-competitive inhibition, indicating that EGCG is effective at inhibiting the formation of fermentable carbohydrates involved in caries formation. Interestingly, alpha-amylase reduced the antimicrobial activity of EGCG against the periodontal bacterium Aggregatibacter actinomycetemcomitans. Therefore, we considered that EGCG-salivary protein interactions might have both protective and detrimental effects with respect to oral health.

Neodymium-doped Yttrium-Aluminium-Garnet Laser Irradiation Abolishes the Increase in Interleukin-6 Levels Caused by Peptidoglycan through the p38 Mitogen-activated Protein Kinase Pathway in Human Pulp Cells

The anti-inflammatory effects of low-power laser irradiation have previously been reported. However, how the laser irradiation regulates the expression of inflammatory cytokines remains unknown. In the present study, to elucidate the mechanism behind the anti-inflammatory effect, we examined the effects of low-power neodymium-doped yttrium-aluminium-garnet (Nd:YAG) laser irradiation on interleukin (IL)-6 expression in human pulp (HP) cells stimulated by peptidoglycan (PGN) and focused on intracellular signaling pathways. Low-power Nd:YAG laser irradiation obviated the PGN-induced increase in IL-6 levels in HP cells. A p38 mitogen-activated protein kinase inhibitor, SB203580, also inhibited the increase in IL-6 messenger RNA levels. PGN stimulated the activity of phosphorylated p38 in HP cells. Low-power laser irradiation inhibited the activity. Thus, suppression of the phosphorylated p38 activity by low-power laser irradiation in HP cells culminates in inhibition of the increase in IL-6 induced by PGN, suggesting that low-power laser irradiation regulates intracellular signaling molecule activities to exert its anti-inflammatory effect.

Proliferative ability and alkaline phosphatase activity with in vivo cellular aging in human pulp cells.

Little is known about the effect of aging on characteristic functions of pulp cells. When damaged pulp is recovered and mineralized tissue is formed to protect remaining pulp tissue, the general responses of pulp tissue after adequate stimuli (pulp cell proliferation and activation of alkaline phosphatase [ALPase]) are thought to be essential. In this study, we compared proliferative ability and ALPase activity between cultures of human pulp (HP) cells obtained from young and aged donors. The in vitro proliferative lifespan of HP cells from young donors was longer than HP cells from aged donors. Growth rates and ALPase activity of HP cells decreased with increasing donor age. These findings suggest that impaired repair of pulp and dentin in aged patients is partly due to a decrease in the proliferative ability and ALPase activity in aged pulp cells.

Purification of Staphylococcal Exfoliative Toxin by High Pressure Liquid Chromatography

Exfoliative toxin (ET) isolated from a clinical strain of Staphylococcus aureus was purified to homogeneity, using a 3-step HPLC system. NH2-terminal 20 amino residues of purified ET was found to be identical with ETA of S. aureus TA (7), S. aureus TC16 (9) and S. aureus ZM (10), but stability of purified ET was completely different from that of ETA. This purification system gave a high yield of pure ET, which exhibited higher purity than specimens purified by more complicated and time-consuming procedures. It is useful for small-scale purification for the comparative study of ET and easy to scale up for preparative purification.

Cellular immuno-competence of infected root canal contents in pathogenesis of periapical lesions

The soluble fractions of infected root canal contents (IRCC) were collected from about 300 human extracted teeth and examined for the presence of mononuclear cell (MNC) chemotaxis and cellular immunocompetence. IRCC showed remarkable chemotactic activity for polymorphonuclear leukocytes but a weak activity for MNC. However, generation of intrinsic MNC chemotaxis and induction of cellular immunity were confirmed in rats given repeated injections of IRCC.