Michiko Kiyama-Kishikawa

Effect of low-level laser irradiation on osteoglycin gene expression in osteoblasts

AbstractMany studies have attempted to elucidate the mechanism of the biostimulatory effects of low-level laser irradiation (LLLI), but the molecular basis of these effects remains obscure. We investigated the stimulatory effect of LLLI on bone formation during the early proliferation stage of cultured osteoblastic cells. A mouse calvaria-derived osteoblastic cell line, MC3T3-E1, was utilised to perform a cDNA microarray hybridisation to identify genes that induced expression by LLLI at the early stage. Among those genes that showed at least a twofold increased expression, the osteoglycin/mimecan gene was upregulated 2.3-fold at 2 h after LLLI. Osteoglycin is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix which was previously called the osteoinductive factor. SLRP are abundantly contained in the bone matrix, cartilage cells and connective tissues, and are thought to regulate cell proliferation, differentiation and adhesion in close association with collagen and many other growth factors. We investigated the time-related expression of this gene by LLLI using a reverse transcription polymerase chain reaction (RT-PCR) method, and more precisely with a real-time PCR method, and found increases of 1.5–2-fold at 2–4 h after LLLI compared with the non-irradiated controls. These results suggest that the increased expression of the osteoglycin gene by LLLI in the early proliferation stage of cultured osteoblastic cells may play an important role in the stimulation of bone formation in concert with matrix proteins and growth factors.

Further development of an electroosmotic medium pump system for preparative disk gel electrophoresis

A simple and practical 6.8-cm-diameter (36.30-cm(2) cross-sectional-area) preparative disk gel electrophoresis device, based on the design of M. Hayakawa et al. (Anal. Biochem. 288 (2001) 168), in which the elution buffer is driven by an electroosmotic buffer flow through the membrane into the elution chamber from the anode chamber was constructed. We have found that the dialysis membranes employed provide suitable flow rates for the elution buffer, similar to those of an earlier 3.6-cm-diameter device, resulting in the prevention of excess eluate dilution. The efficiency of this device was demonstrated by the fractionation of a bovine serum albumin (BSA) Cohn V fraction into monomer, dimer, and oligomer components using nondenaturing polyacrylamide gel electrophoresis (native-PAGE). The maximum protein concentration of the eluate achieved was 133 mg/ml of BSA monomer, which required a dilution of the eluate for subsequent analytical PAGE performance. As a practical example, the two-dimensional fractionation of soluble dipeptidyl peptidase IV (sDPP IV) from 50 ml fetal bovine serum (3.20 g protein) per gel is presented. The sDPP IV enzyme protein was recovered in a relatively short time, utilizing a 6.5% T native-PAGE and subsequential sodium dodecyl sulfate-PAGE system. This device enhances the possibility of continuous electrophoretic fractionation of complex protein mixtures on a preparative scale.

Hemin-binding protein 35 (HBP35) plays an important role in bacteria–mammalian cells interactions in Porphyromonas gingivalis

Hemin-binding protein 35 (HBP35) may be an essential protein for bacterial survival in evasion from environmental stress in Porphyromonas gingivalis. The anti-recombinant HBP35 antibody inhibits P. gingivalis hemagglutination. This study considered the role of this protein for hemagglutination and adherence to host cells using the HBP35-deficient mutant (MD774) derived from P. gingivalis FDC381. FDC381 had strong hemagglutination activity, whereas MD774 had no activity. Anti-130-kDa hemagglutinin antibody, mAb-Pg-vc, reacted mainly with the 43- and 49-kDa molecules in the membrane fraction. However, no proteins reacted in the MD774. The hemolytic activity in MD774 was much lower than that in FDC381. Anti-recombinant HBP35 antibody strongly inhibited the P. gingivalis FDC381 adherence to epithelial cells. In addition, MD774 exhibited a significant decrease in the adherence. The hydrophobicity of MD774 was equal to 19.4% of that of FDC381. SDS-PAGE profiling of the membrane fractions of both strains showed very different profiles. Taken together, these findings suggest that HBP35 plays a role, not only in hemin-binding, but also in multiple P. gingivalis binding to erythrocytes, and host epithelial gingival cells. In addition, this protein may directly and/or indirectly affect the virulence of this organism.

Inhibition of hemagglutinating activity by monoclonal antibody against Porphyromonas gingivalis 40-kDa outer membrane protein.

Periodontitis is a chronic inflammatory disease of periodontal tissues that results in alveolar bone loss, and Porphyromonas gingivalis, which has a high hemagglutinating activity, has been implicated as an important pathogen in the development of periodontitis. This bacterium has a high hemagglutinating activity. We previously succeeded in gene cloning the 40-kDa outer membrane protein (OMP) from P. gingivalis 381. Although recombinant (r) 40-kDa OMP itself did not show hemagglutinating activity, its polymeric form, constructed with a cross-linking reagent, significantly expressed that activity. Furthermore, an affinity-purified antibody against r40-kDa OMP inhibited the hemagglutinating activity of P. gingivalis vesicles. In the present study, in order to clarify the pathological role of 40-kDa OMP and develop passive immunotherapy, we examined the inhibitory effect of monoclonal antibodies (MAbs) against r40-kDa OMP on the hemagglutinating activity of P. gingivalis vesicles. The MAbs reacted with r40-kDa OMP, the outer membrane fraction, vesicles, and P. gingivalis cell extracts, and significantly inhibited the hemagglutinating activities of the polymeric r40-kDa OMP as well as of P. gingivalis vesicles. These findings suggest that MAbs against 40-kDa OMP may be useful for the development of passive immunotherapy and for assessing treatment for periodontal diseases caused by P. gingivalis infection.