Hiroshige Sasahara

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.

ROLE OF PORPHYROMONAS GINGIVALIS 40-kDa OUTER MEMBRANE PROTEIN IN THE AGGREGATION OF P. GINGIVALIS VESICLES AND ACTINOMYCES VISCOSUS

Porphyromonas gingivalis, an important pathogen in periodontitis, produces extracellular vesicles that aggregate with Actinomyces viscosus cells. A 40-kDa outer membrane protein (OMP)-coding gene from P. gingivalis was cloned and the protein was found to be localized in these vesicles. The recombinant 40-kDa OMP did not show aggregation activity. However, affinity-purified antibody against the recombinant protein significantly inhibited aggregation of P. gingivalis vesicles with A. viscosus cells. The antibody also inhibited cellular coaggregation of several strains of P. gingivalis with A. viscosus cells, but not with other periodontal pathogens. Moreover, aggregation of A. viscosus cells with P. gingivalis vesicles was inhibited in a dose-dependent manner by pre-treatment of the A. viscosus cells with the recombinant protein. These findings suggest that the 40-kDa OMP may be an important aggregation factor of P. gingivalis.

Gene cloning of Porphyromonas gingivalis specific antigens recognized by serum of adult periodontitis patient

1. Porphyromonas gingivalis is believed an important pathogen of adult periodontitis. A gene library of P. gingivalis 381 was constructed in lambda phage vector L47.1. The library was probed with serum obtained from patients of severe adult periodontitis. Two clones, lambda MDBG101 and lambda MDBG103 which were expressed, 200 and 160 kDa respectively, were selected and further studied. 2. The expressed antigens in these two clones were also reacted with rabbit antiserum against whole cells, capsular fraction and cell surface fraction of P. gingivalis. 3. Genes coding protein antigens in lambda MDBG101 and lambda MDBG103 were subcloned into high-copy-number plasmid vector pACYC184 and subclones obtained were designated as MD101 and MD103. Recombinant plasmids, pMD101 and pMD103, differed in their restriction endonuclease digestion. 4. Immunodiffusion analysis showed that cloned proteins from MD101 and MD103 reacted with antiserum against P. gingivalis but did not react with antiserum against Prevotella intermedia, Prevotella loescheii and Prevotella asaccharolyticus. 5. These data suggest that P. gingivalis species-specific antigens has been successfully cloned and expressed in Escherichia coli. Since these cloned specific antigens were recognized by adult periodontitis patient sera, the recombinant antigen will be useful material for the development of serodiagnosis of P. gingivalis infection in adults periodontitis.

Caffeine Enhances the Expression of the Angiotensin II Type 2 Receptor mRNA in BeWo Cell Culture and in the Rat Placenta

Although chronic caffeine exposure during pregnancy has been shown to affect fetal growth, adverse effects of caffeine on embryogenesis are not only well understood, but also controversial. We have used gene chip technology in an attempt to identify to what extent, if any, caffeine could possibly alter gene expressions in the cytotrophoblast-like cell line BeWo. Few down-regulated genes were found; most of the genes were up-regulated, suggesting that chronic caffeine exposure during the gestational period could exert certain influences on embryogenesis. The highest up-regulated gene expression of BeWo cells by caffeine was angiotensin II type 2 (AT(2)) receptor gene. We focused the genes of the renin-angiotensin system (RAS), angiotensin II type 1 (AT(1)) and AT(2)receptors and angiotensin I converting enzyme, for study on caffeines responsive gene expression in BeWo cells and in the placentae of pregnant rats that were fed a diet supplemented with caffeine (2 mg/100 g body weight) during gestation, and analysed the gene expressions using RT-PCR and LightCycler system. A significantly increased AT(2)receptor gene expression and a slight decreased AT(1)receptor gene expression demonstrated the caffeines effect to the placental RAS.

Effects of maternal caffeine with zinc intake during gestation and lactation on bone development in newborn rats

On day 9 of gestation, pregnant dams were randomly divided into 3 groups. Dams of group 1 were fed a 20% protein diet as a control. Dams of group 2 were fed a 20% protein diet supplemented with caffeine. Dams of group 3 were fed a 20% protein diet supplemented with caffeine and zinc. The amount of caffeine added to the maternal diet was 2 mg/100 g body weight; the amount of zinc was 0.6 g/kg of diet. At birth, pups were mixed within each group, and 8 randomly selected pups from each group were assigned to each dam of the respective group and were continuously fed the same diet. On day 15, the pups were killed and cranial bones, mandibles and femurs removed. The bones were measured, and the mineral content of the mandibles and femurs was determined. Although there were no differences in the dimensions of the cranial bones among the groups, the measurements and mineral content of the mandibles and femurs were consistently affected by the caffeine in the diet. On the other hand, supplementation of the caffeine-added diets with zinc led to greatly improved bone development, reaching values up to or beyond control levels. Thus zinc supplementation of a caffeine diet given to the dams during gestation and lactation can favourably influence the otherwise impaired bone development of their offspring.

Genome science-based gene expression monitoring in osteoblasts altered by low-level laser irradiation

Abstract Although the biostimulatory effects of low-level laser irradiation (LLLI) have been reported, the molecular basis of the mechanisms are not fully elucidated. To elucidate the mechanism of bone regeneration by low-level laser irradiation (LLLI), gene expressions altered in osteoblast by LLLI were identified using the subtractive gene cloning and cDNA microarray technologies. Many gene expressions in osteoblast were altered by LLLI. Many genes whose expression levels were altered by LLLI were shown, including ATP synthesis, DNA replication, signal transduction, enzymes, structural proteins, ESTs and other unknown function genes. These findings suggest that LLLI may demonstrate biostimulatory effects through the expression of many genes, and genome science-based gene expression monitoring will provide unprecedented access to elucidate the mechanism of bone regeneration stimulated by LLLI.

Characterization of Rat Monoclonal Antibodies against Human β-Defensin-2

Defensins are a family of cationic antimicrobial peptides that participate in host defense. Human β-defensin (hBD)-2 has a potent bactericidal activity against a wide spectrum of microorganisms. Because human gingival epithelium is constantly exposed to a variety of microbial challenges, it is considered that hBD-2 has an important role in the protective mechanisms against oral bacterial infection. However, little is known about the production of hBD-2 in tissues of the oral cavity. Six rat monoclonal antibodies (MAbs) raised against chemically synthesized hBD-2 have been characterized. Rat MAbs were specific for the conformational epitopes on hBD-2, but not to hBD-1. To identify the epitope on hBD-2, a series of six overlapping peptides covering the hBD-2 whole sequence were synthesized and the immunoreactivities of six MAbs were examined. The FCPRRYK domain in hBD-2 was recognized by all six MAbs and suggested to be an epitope region. By immunocytochemistry, hBD-2 was localized focally in the epidermis ...

Effect of linear polarized light near-infrared irradiation on chemokines production in synovial cells from human temporomandibular joint

Abstract Immediate effects of linear polarized light near-infrared irradiation (LPLI), such as anti-inflammation and pain relief from temporomandibular joint (TMJ) disorders, have been reported. However, the molecular-based mechanisms are not yet elucidated. Synovial cells are believed to play pathological roles in the development and continuation of inflammation. Chemokines, such as interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1, have been found in synovial fluid from patients with TMJ synovitis as well as IL-1β. The purpose of this study was to examine the effect of LPLI on chemokines production stimulated in human synovial (HTS) cells treated with IL-1β. The cells were isolated from TMJ synovial tissues and cultured using an outgrowth method. The confluent-stage cells were treated with IL-1β and LPLI was applied. The amounts of chemokines in conditioned medium were measured by ELISA kit. LPLI significantly reduced the production of IL-8 and MCP-1. These findings suggest that LPLI may have an anti-inflammatory effect on TMJ disorders through the reduction of chemokine production.