Yasuko Shibata

INHIBITION OF PROSTAGLANDIN E2 AND INTERLEUKIN 1-BETA PRODUCTION BY LOW POWER LASER IRRADIATION IN STRETCHED HUMAN PERIODONTAL LIGAMENT CELLS

It is well-known that orthodontic treatment usually causes some discomfort and pain to the patients. Recently, it has been reported that low-power laser irradiation is effective in reducing the pain accompanying tooth movement. However, the mechanism of such pain relief cannot be elucidated. Since high levels of prostaglandin (PG) E2 and interleukin (IL)-1β are found in the periodontal ligament (PDL) during tooth movement, and both factors are involved in the induction of pain, the effects of low-power laser irradiation on PGE2 and IL-1β production in stretched human PDL cells were studied in vitro. The PDL cells, derived from healthy premolars extracted for orthodontic treatment, were utilized for experiments. Cells were seeded in flexible-bottomed culture plates, and the bottom of each plate was elongated (18% increase) under vacuum at 6 cycles per min for 1, 3, or 5 days. The stretched cells were irradiated with a Ga-Al-As low-power diode laser (60 mW) once a day for 3, 6, or 10 min (from 10.8 to 36.0 J) for 1, 3, or 5 days. PGE2 and IL-1β levels in the medium were measured by radioimmunoassay. In response to mechanical stretching, human PDL cells showed a marked elevation in PGE2 production in a time-dependent manner. IL-1β production was also elevated, but this remained constant. The increase in PGE2 production was significantly inhibited by laser irradiation in a dose-dependent manner. The increase in IL-1β production was also significantly inhibited by laser irradiation, although the inhibition was only partial. Its inhibitory effects on PGE2 and IL-1β production suggest that laser irradiation may have great therapeutic benefits in bringing about relief of the pain that accompanies orthodontic treatment.

Effects of reactive oxygen species (ROS) on antioxidant system and osteoblastic differentiation in MC3T3-E1 cells

Oxidative stress regulates cellular functions in multiple pathological conditions, including bone formation by osteoblastic cells. However, little is known about the cellular mechanisms responsible for the effects of oxidative stress on osteoblast functions in senescence. To clarify the inhibitory effects of oxidative stress on osteoblastic mineralization, we examined the relationship between the antioxidant system and bone formation in MC3T3‐E1 cells. After a single exposure to H2O2 within range of a non‐toxic concentration for cells, the mineralization level was diminished half. Under the same conditions, gene expression of the transcription factor Nrf2, which regulates antioxidant enzymes, was up‐regulated. In addition, gene expression for the osteogenic markers Runx2, ALP, and BSP was lower than that in non‐treated cells, whereas expression of the osteocalcin gene was up‐regulated following H2O2 exposure. These results suggest that reduced mineralization by MC3T3‐E1 cells after H2O2 exposure is the result of an up‐regulated antioxidant system and altered osteogenic gene expression. IUBMB Life, 59: 27‐33, 2007

Effect of different magnitudes of tension force on prostaglandin E2 production by human periodontal ligament cells

Periodontal ligament (PDL) cells are known to produce prostaglandin E2 (PGE2) in response to mechanical stress. However, the rate of PGE2 production from PDL cells in response to different magnitudes of tension forces has not been examined. This study, therefore, was undertaken to determine the effect of different magnitudes of tension forces on PGE2 production and inositol trisphosphate (IP3) levels in PDL cells in vitro. Human PDL cells were cultured on flexible-bottomed plates and placed on a Flexercell strain unit. Cells were flexed at six cycles (5-s strain, 5-s relaxation) at six steps of tension force (9, 12, 15, 18, 21, 24% increase in surface area) for 5 days. PGE2 production and IP3 levels were determined by radioimmunoassay. There was a 6- and 25-fold increase in the rate of PGE2 production by cells exposed to low (9%) and high (24%) tension forces, respectively, and these increases were tension force-dependent. Tension force also induced increases in the intracellular levels of IP3 that did not seem to be directly related to the production of PGE2. The different rates of PGE2 production by PDL cells in response to different magnitudes of mechanical stress may be of importance in PDL and alveolar bone metabolism.

Effects of Different Magnitudes of Tension-force on Alkaline Phosphatase Activity in Periodontal Ligament Cells

Alkaline phosphatase (ALP) activity is involved in the process of calcification in various mineralizing tissues, and it is found at much higher levels in the periodontal ligament (PDL) than in other connective tissues. Since the PDL lies between hard tissues and functions as a cushion mitigating mechanical stress, such as occlusal and orthodontic forces, this stress may modulate ALP activity in PDL cells, which themselves may affect adjacent alveolar bone metabolism. The objective of this study was to determine the level of ALP activity and the gene expression of liver/bone/kidney (L/B/K) ALP in human PDL fibroblasts in response to cyclic tension-forces. Human PDL cells were cultured on flexible-bottomed plates and placed on a Flexercell Strain Unit. Cells were flexed at 6 cycles/min (5 sec strain, 5 sec relaxation) at 6 levels of tension-force (9%, 12%, 15%, 18%, 21%, and 24% increase in surface area) for 5 days. There was no significant difference in cell proliferation between the cells subjected to the tension-force and the controls. There was a 10% and 42% decrease, respectively, in the ALP activity in PDL cells exposed to low (9%) and high (24%) tension-forces, and these decreases were dependent on the magnitude of the tension-force. The finding of inhibited ALP activity in response to tension-force was consistent with the observation that L/B/K ALP mRNA levels were decreased in response to cyclic tension-force. These results suggest that tension-force may affect PDL metabolism, depending on the functional role of ALP.

A 35-kDa co-aggregation factor is a hemin binding protein in Porphyromonas gingivalis.

It has been known that Porphyromonas gingivalis has an obligate requirement for hemin or selected heme- or Fe-containing compounds for its growth. In addition, the influence of hemin on the expression of several putative virulence factors produced by this bacterium has also been recently documented; however, the mechanisms involved in hemin uptake are poorly defined. We succeeded in cloning the gene coding for the 35-kDa protein, which was specifically expressed in P. gingivalis and seemed to confer colonizing activities. Recently, we have constructed the P. gingivalis 381 mutant defective in the 35-kDa protein by insertion mutagenesis. The beige mutant exhibited little co-aggregation and the virulence was also decreased. Based on these results and homology search analysis, we focused on assessing the hemin bindings and found the heme regulatory motif (HRM) as a hemin direct binding site. The 35-kDa protein did possess the binding ability of selected protoporphyrins involving the hemin. These results demonstrated that 35-kDa protein is one of the hemin binding proteins in P. gingivalis and suggested that hemin binding ability of 35-kDa protein is important for the expression of virulence in P. gingivalis.

Stimulation by interleukin-1 of interleukin-6 production by human periodontal ligament cells

Interleukin-1(IL-1), a cytokine present in the gingiva and crevicular fluid of patients with periodontitis and in the periodontal ligament (PDL) of experimentally moved teeth, has multiple biological activities, including the ability to elicit bone resorption. Interleukin-6, also found in the gingiva of patients with periodontitis, may induce osteoclastic bone resorption through an effect on osteoclastogenesis. Here IL-6 production and its gene expression in response to recombinant IL-1 beta were examined in primary cultures of PDL cells. IL-1 beta stimulated IL-6 production by these cells in a dose- and time-dependent manner; this increase in IL-6 production was much higher than that in human gingival fibroblasts. In situ hybridization, using a synthetic oligonucleotide DNA probe of the IL-6 gene, revealed that most PDL cells expressed IL-6 mRNA in response to IL-1 beta treatment. The finding that IL-6 is produced by PDL cells and is regulated by IL-1 beta has revealed a potentially important mechanism for controlling alveolar bone resorption.

Roles of Arg- and Lys-gingipains in coaggregation of Porphyromonas gingivalis: Identification of its responsible molecules in translation products of rgpA, kgp, and hagA genes

Abstract Arg- (Rgp) and Lys-gingipains (Kgp) are two individual cysteine proteinases produced by Porphyromonas gingivalis, an oral anaerobic bacterium, and are implicated as major virulence factors in a wide range of pathologies of adult periodontitis. Coaggregation of this bacterium with other oral bacteria is an initial and critical step in infectious processes, yet the factors and mechanisms responsible for this process remain elusive. Here we show that the initial translation products of the rgpA, kgp and hemagglutinin hagA genes are responsible for coaggregation of P. gingivalis and that the proteolytic activity of Rgp and Kgp is indispensable in this process. The rgpA rgpB kgp- and rgpA kgp hagA-deficient triple mutants exhibited no coaggregation activity with Actinomyces viscosus, whereas the kgp-null and rgpA rgpB-deficient double mutants significantly retained this activity. Consistently, the combined action of Rgp- and Kgp-specific inhibitors strongly inhibited the coaggregation activity of the bacterium, although single use of Rgp- or Kgp-specific inhibitor significantly retained this activity. We also demonstrate that the 47- and 43-kDa proteins produced from the translation products of the rgpA, kgp, and hagA genes by proteolytic activity of both Rgp and Kgp are responsible for the coaggregation of P. gingivalis.

In vitro senescence enhances IL-6 production in human gingival fibroblasts induced by lipopolysaccharide from Campylobacter rectus

The production of interleukin-6 (IL-6) in human gingival fibroblasts (Gin cells) is increased by lipopolysaccharide (LPS) from Campylobacter rectus (C. rectus), which is associated with adult periodontitis; however, the age-related changes in the susceptibility of Gin cells to C. rectus LPS remain unclear. We examined the influence of in vitro senescence on C. rectus LPS-stimulated IL-6 production in Gin cells. LPS was prepared from C. rectus ATCC 33238 using hot phenol-water. The Gin cells were established from healthy gingival tissue removed from three patients, aged 10-12 years. The cells were cultured until confluence then stimulated with LPS (0.01, 0.1, 1.0 and 10.0 micrograms/ml). Levels of IL-6 released in the medium were measured after incubation for 3, 6, 9, 12, and 24 h. In both young (5-6 population doublings) and senescent (17-20 population doublings) cells, LPS stimulated IL-6 production in a dose- and time-dependent manner. In response to 0.01-10.0 micrograms/ml of LPS, IL-6 production in the senescent cells was higher than that in the young cells. Using cells from each of the three donors, we found that this phenomenon of higher LPS-stimulated IL-6 production in senescent cells was reproducible. The greater capacity of the senescent cells to synthesize IL-6 in response to LPS was a higher production of mRNA for IL-6. This increase of IL-6 production induced by C. rectus LPS in senescent Gin cells could help to explain the increased susceptibility to periodontal diseases shown by aged individuals.

Production of a single-chain variable fraction capable of inhibiting the Streptococcus mutans glucosyltransferase in Bacillus brevis: construction of a chimeric shuttle plasmid secreting its gene product

Periodontitis and dental caries are common oral diseases, in these days, and the passive immunization is one of the most effective approaches for prevention. For this purpose, we have constructed mouse and human monoclonal antibodies to inhibit the Porphyromonas gingivalis-associated hemagglutination and coaggregation. In addition, an artificial antibody, single-chain variable fraction, or scFv, which also inhibited the hemagglutination, was constructed. Specifically for dental caries, mouse and human monoclonal antibodies that inhibited the glucosyltransferase (GTF) activity, responsible for biofilm formation, were also constructed. The advantage of scFv over the native antibody is that the former molecule does not induce possible side-effects due to Fc, such as autoimmune disease, because it consists only of variable regions originating from both heavy and light chains. To increase the abilities of the antibody preparations, we attempted to construct an additional scFv using Bacillus brevis, a secretion-proficient gram-positive bacterium, as a host cell. An scFv protein possessing the same biological activity as that of the parental antibody was successfully secreted from a B. brevis transformant following the construction of a chimeric shuttle plasmid, which was accomplished by employing a new heterodimer system.

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.