To investigate the pathogenicity, genomic pattern, and o-like hemolysin of Staphylococcus lugdunensis (S. lugdunensis) in acute oral infection, S. lugdunensis was isolated from patients with an acute oral infection and from healthy persons. Antibiotic susceptibility, in vitro cellular toxicity, in vivo virulence, and hemolytic activity were tested, and plasmid DNA and restriction pattern of whole genomic DNA were analyzed to characterize the staphylococci. The dot blot and Southern blot hybridization analysis of staphylococcal DNA were performed with o-hemolysin gene probe. The isolation ratio of S. lugdunensis in the patients was higher than that in the healthy persons. S. lugdunensis from the patients with an acute oral infection showed resistance to penicillin, ampicillin, methicillin, cephalothin, and clindamycin. In the analysis of plasmid, there was a clear band about 6.5 kb in three strains of S. lugdunensis isolated from the patients with infection. S. lugdunensis in the patients had cellular toxicity in vitro and virulence in vivo. All strains of S. lugdunensis had o-like hemolysin activity against rabbit erythrocytes. Four of the six strains of S. lugdunensis gave synergistic hemolysis with Staphylococcus aureus (S. aureus) on sheep blood agar plates. In the analysis of genomic pattern, four strains of S. lugdunensis that gave synergistic hemolysis with S. aureus showed a similar genetic pattern with HindIII enzyme digests. In dot blot analysis, all strains of S. lugdunensis showed a positive reaction with the probe of 5-hemolysin gene in S. aureus. In Southern blot analysis, a 7.3 kb HindIII fragment was observed in DNA of S. lugdunensis that gave synergistic hemolysis with S. aureus, and a 2.5 kb band was observed in HindIII digests of S. aureus in the patients. These results suggest that S. lugdunensis may be an important pathogen in an acute oral infection and the 7.3 kb HindIII fragment from S. lugdunensis DNA may contain o-like hemolysin gene.
Agar ; Ampicillin ; Blotting, Southern ; Cephalothin ; Clindamycin ; DNA ; Erythrocytes ; Hemolysis ; Humans ; Methicillin ; Penicillins ; Plasmids ; Sheep ; Staphylococcus aureus ; Staphylococcus lugdunensis* ; Staphylococcus* ; Virulence

Streptococcus mutans is one of the important bacteria that forms dental biofilm and cause dental caries. Virulence genes in S. mutans can be classified into the genes involved in bacterial adhesion, extracellular polysaccharide formation, biofilm formation, sugar uptake and metabolism, acid tolerance, and regulation. The genes involved in bacterial adhesion are gbps (gbpA, gbpB, and gbpC) and spaP. The gbp genes encode glucan-binding protein (GBP) A, GBP B, and GBP C. The spaP gene encodes cell surface antigen, SpaP. The genes involved in extracellular polysaccharide formation are gtfs (gtfB, gtfC, and gtfD) and ftf, which encode glycosyltransferase (GTF) B, GTF C, and GTF D and fructosyltransferase, respectively. The genes involved in biofilm formation are smu630, relA, and comDE. The smu630 gene is important for biofilm formation. The relA and comDE genes contribute to quorum-sensing and biofilm formation. The genes involved in sugar uptake and metabolism are eno, ldh, and relA. The eno gene encodes bacterial enolase, which catalyzes the formation of phosphoenolpyruvate. The ldh gene encodes lactic acid dehydrogenase. The relA gene contributes to the regulation of the glucose phosphotransferase system. The genes related to acid tolerance are atpD, aguD, brpA, and relA. The atpD gene encodes F1F0-ATPase, a proton pump that discharges H⁺ from within the bacterium to the outside. The aguD gene encodes agmatine deiminase system and produces alkali to overcome acid stress. The genes involved in regulation are vicR, brpA, and relA.
Agmatine ; Alkalies ; Antigens, Surface ; Bacteria ; Bacterial Adhesion ; Biofilms ; Dental Caries ; Glucose ; Lactic Acid ; Metabolism ; Oxidoreductases ; Phosphoenolpyruvate ; Phosphopyruvate Hydratase ; Proton Pumps ; Streptococcus mutans ; Streptococcus ; Virulence

Recently, many natural medicines, which have advantage of less side effects and possibility of long-term use have been studied for their capacity of anti-bacterial, anti-inflammatory and regenerative potential of periodontal tissues. Olibanum has the effects to hemostasis, analgesic and anti-inflammatory, and it also has been traditionally used as a drug for the treatment of bone disease in oriental medicine. The purpose of the present study was to investigate the effects of Olibanum extracts on the activity and differentiation of MC3T3-E1 cells, alkaline phosphatase(ALP) synthesis, formation of bone nodules and expression of type I collagen of MC3T3-E1 cells. To examine the cellular activity, MC3T3-E1 cells were cultured with alpha-MEM(control) and each concentration of Olibanum for 2 days and 4 days. To compare the ALP synthesis, MC3T3-E1 cells were cultured with alpha-MEM(negative control), dexamethasone(positive control), and each concentration of Olibanum for 2 days and 4 days. To compare the bone nodule formation, MC3T3-E1 cells were cultured for 21 days, and to compare the type I collagen expression, MC3T3-E1 cells were cultured for 4 days. The cellular activity of MC3T3-E1 cells treated with 1 microgram/ml of Olibanum extracts was significantly increased at 4-day(p<0.05) to control. The activity of ALP in MC3T3-E1 cells treated with 1 microgram/ml Olibanum extracts was significantly increased at 4-day(p<0.05). All the experimental groups showed much more bone nodule formation than control groups. The group treated with 1 microgram/ml of Olibanum extracts was the highest bone nodule formation, and showed much more type I collagen expression than negative control. These results indicate that Olibanum extracts may be considered effective in the activity and differentiation of MC3T3-E1 cells.
Bone Diseases ; Boswellia* ; Collagen Type I ; Hemostasis ; Medicine, East Asian Traditional

The present study was performed to evaluate the clinical effects following local application of 30% minocycline strip(polycaprolactone), 2% minocycline gel(hydro-carbon gel) and 12% minocycline strip(polylactide, Minodent) to augment scaling and root planing in patients with chronic adult periodontitis. Forty teeth with periodontitis were enrolled in the study anddistributed into 4 groups including control group. All patients performed standardized oral hygiene instructions and mechanical debridement at the beginning of the study and then each local delivery drugs were inserted into periodontal pocket in each groups. Examinations regarding plaque index(PI), papillary bleeding index (PBI), probing pocket depth (PPD) were carried out at 0, 2, 4 weeks. All experimental groups showed statistically significant differences between baseline and 2 and 4 weeks in every clinical indices. Especially, 30%minocycline strip and Minodent group showed a significant improvement in PBI at 2 weeks and in PPD at 2 and 4 weeks. In conclusion, highly bio-resorbable Minodent delivered subgingivally as an adjunct to scaling and root planing induces better clinical effects for periodontal health than 2% minocycline gel and control group.
Adult ; Male ; Female ; Humans

Cyclosporin A is a cyclic polypeptide produced by the metabolism of fungi. It is widely used at present as immunosuppressive treatment following organ transplants. It is also used to deal with autoimmune diseases such as rheumatoid arthritis or type II diabetes. Gingival hyperplasia is one of the most frequent side-effects associated with the prescription of Cyclosporin A. The mechanisms involved in Cyclosporin A induced gingival hyperplasia are not yet clear. In vitro Cyclosporin A promotes proliferation of gingival fibroblasts, that Cyclosporin A act as a mitogen. Its action is based on mitosis of gingival fibroblasts regulated by cell cycle regulatory proteins. It was the purpose of the present study to examine the effects of Cyclosporin A on human gingival fibroblasts by means of biological and biochemical criteria. In this present study, we examined change of cell proliferation, cell activity, cell viability and cell cycle progression after application of Cyclosporin A. We also examined expression of cell cycle regulatory proteins by western blot analysis. Human gingival fibroblasts were cultured for 48 hours with application of Cyclosporin A at concentrations of 0.01, 0.1, 1, and 10 ng/ml. Cyclosporin A(1 ng/ml) significantly increased the cell activity of gingival fibroblast. Proliferation and viability of gingival fibroblasts were also increased in group treated with 1 ng/ml of Cyclosporin A compared to control group. In the cell cycle analysis, S phase was increased and G1 phase was decreased in the group treated with 1 ng/ml of Cyclosporin A. Cyclosporin A increased the expression of cdk4 and inhibited the expression of pRB and p21. These results suggest that 1 ng/ml of Cyclosporin A may increase the cell cycle progression of human gingival fibroblasts, and its mechanisms may increase the expression of cdk4 and decrease the expression of pRB and p21.
Arthritis, Rheumatoid ; Autoimmune Diseases ; Blotting, Western ; Cell Cycle Proteins ; Cell Cycle* ; Cell Proliferation ; Cell Survival ; Cyclosporine* ; Fibroblasts* ; Fungi ; G1 Phase ; Gingival Hyperplasia ; Humans* ; Metabolism ; Mitosis ; Prescriptions ; S Phase ; Transplants

Normal gingival fibroblasts functioning is fundamental for the maintenance of periodontal connective tissue as well as wound healing. Nicotine have been found to affect DNA synthesis and cell proliferation, which appear to depend on the type of cells. This in vitro study was done to determine the effects of nicotine, a major component of tobacco, on cell proliferation, viability, activity, cell cycle distribution, and expression of cell cycle regulatory proteins in human gingival fibroblasts. Nicotine has been tested for 2 days or 4 days in 5 different concentrations; 0.1 microgram/ml; 1 microgram/ml; 10 microgram/ml; 100 microgram/ml; 1000 microgram/ml. To assess cell proliferation and viability, viable and non-viable cells were counted by hemocytometer; to evaluate cellular activity, MTT assay was employed; to analyze cell cycle distribution, fluorescent propidium iodide-DNA complex were measured using fluorocytometer; to determine the expression of cell cycle regulatory proteins, western blot analysis was performed. After 2 days and 4 days incubation respectively, at concentrations of 1 microgram/ml - 1000 microgram/ml, nicotine significantly inhibited proliferation comparing to non-supplemented controls. The cell viability was significantly decreased after 2 days and 4 days at concentrations of 1 microgram/ml - 1000 microgram/ml and at 10 microgram/ml - 1000 microgram/ml respectively. After 2 days and 4 days, the cellular activity was significantly decreased at concentrations of 10 microgram/ml - 1000 microgram/ml. Treatment with 100 microgram/ml nicotine for 48 hours caused an increase in the proportion of G1-phase cells (from 46.41% to 53.46%) and a decrease in the proportion of S-phase cells (from 17.80% to 14.27%). The levels of cyclin D1 and CDK 4 proteins in nicotine-treated fibroblasts were lower than that of controls, whereas the levels of p16 and pRB were higher than that of controls. These results suggest that the decrease of cell proliferation and lengthened Gap phases (G1) by nicotine may due to the increased expression of p16 and pRB as well as decreased expression of cyclin D1 and CDK 4 in human gingival fibroblasts.
Blotting, Western ; Cell Cycle Proteins* ; Cell Cycle* ; Cell Proliferation ; Cell Survival ; Connective Tissue ; Cyclin D ; Cyclin D1 ; DNA ; Fibroblasts* ; Humans* ; Nicotine* ; Propidium ; Tobacco ; Wound Healing

Retinoic acid plays an important role in the regulation of cell growth and differentiation. In our present study, we evaluated the effects of all-trans retinoic acid (RA) on cell proliferation and on the cell cycle regulation of human gingival fibroblasts (HGFs). Cell proliferation was assessed using the MTT assay. Cell cycle analysis was performed by flow cytometry, and cell cycle regulatory proteins were determined by western blot. Cell proliferation was increased in the presence of a 0.1 nM to 1 microM RA dose range, and maximal growth stimulation was observed in cells exposed to 1 nM of RA. Exposure of HGFs to 1 nM of RA resulted in an augmented cell cycle progression. To elucidate the molecular mechanisms underlying cell cycle regulation by RA, we measured the intracellular levels of major cell cycle regulatory proteins. The levels of cyclin E and cyclin-dependent kinase (CDK) 2 were found to be increased in HGFs following 1 nM of RA treatment. However, the levels of cyclin D, CDK 4, and CDK 6 were unchanged under these conditions. Also after exposure to 1 nM of RA, the protein levels of p21WAF1/CIP1 and p16INK4A were decreased in HGFs compared with the control group, but the levels of p53 and pRb were similar between treated and untreated cells. These results suggest that RA increases cell proliferation and cell cycle progression in HGFs via increased cellular levels of cyclin E and CDK 2, and decreased cellular levels of p21WAF1/CIP1 and p16INK4A.
Blotting, Western ; Cell Cycle ; Cell Cycle Proteins ; Cell Proliferation ; Cyclin D ; Cyclin E ; Cyclins ; Fibroblasts ; Flow Cytometry ; Humans ; Phosphotransferases ; Tretinoin

Gingival fibroblasts are major cellular component of gingiva. However, the molecular mechanisms of senescence of human gingival fibroblasts are unknown. Human fibroblasts undergo replicative senescence in vitro after a limited number of population doublings. A reduced rate of proliferation is a prominent phenomenon observed in senescent fibroblasts. This phenomenon is happened with cell cycle arrest that was controled by cell cycle regulatory proteins. The purpose of present study was to investigate the effect of replicative senescence on cell cycle progression and to find out its molecular mechanisms in human gingival fibroblasts. Replicative senescence of gingival fibroblasts were induced by subsequent cultures that were repeated up to 18 passage. In the present study, I examined change of cell proliferation, cell activity, cell viability and cell cycle progression during the replicative process. Also, I examined expression of cell cycle regulatory proteins which was estimated by western blot analysis. Cell proliferation, cell activity and cell viability of gingival fibroblasts were notably decreased with increase of population doubling level(PDL). S phase was decreased and G1 phase was increased with increase of PDL. Western blot analysis showed that levels of p16, p21 and p53 of senescent gingival fibroblasts(PDL41, PDL58) were higher than young fibroblasts(PDL27) and cdk4 were lower than young fibroblasts(PDL27). In conclusion, these results suggest that proliferative function of human gingival fibroblasts may be decreased by replicative senescence and its molecular mechanisms may be activatied with p16, p21, p53 and pRB, and repressed wtih cdk4.
Aging ; Blotting, Western ; Cell Aging* ; Cell Cycle Checkpoints ; Cell Cycle Proteins ; Cell Cycle* ; Cell Proliferation ; Cell Survival ; Fibroblasts* ; G1 Phase ; Gingiva ; Humans* ; S Phase

Ampicillin ; Anti-Bacterial Agents ; Berberine ; Biological Products ; Child ; Fibroblasts ; Humans ; Mouth ; Staphylococcus aureus ; Staphylococcus

Hyaluronic acid (HA) is an almost essential component of extracellular matrices. Early in embryogenesis mesenchymal cells migrate, proliferate and differentiate, in part, because of the influence of HA. Since the features of embryogenesis are revisited during wound repair, including bone fracture repair, this study was initiated to evaluate whether HA has an effect on calcification and bone formation in an in vitro system of osteogenesis. Mouse calvaria Pre-osteoblast (MC3T3-E1) cells were cultured in alpha-MEM medium with microorganism-derivative hyaluronic acid that was produced by Strep. zooepidemicus which of molecular weight was 3 million units. The dosages were categorized in each 0.5, 1.0 and 2.0 mg/ml concentration experimental groups. After 2 and 4 days cultures in expeirmental and control groups, the tendency of cell proliferation, MTT assay, protein synthesis ability, collagen synthesis and alkaline phosphatase activity were analysed and bone nodule formation capacity were measured with Alizarin Red S stain after 29 days cultures. The cell proliferation was increased in time, especially the group of 0.5 and 1.0 mg/ml concentration of HA were showed prominent cell proliferation. After 2 and 4 days culture, experimental groups in general were greater cell activity in MTT assay. The protein synthesis was increased in all experimental groups compared to control group, especially most prominent in 1.0 mg/ml concentration group. The collagen synthesis capacity were increased in HA experimental groups, especially prominent in 1.0 mg/ml group and the activity of alkaline phosphatase were increased, especially also prominent in 1.0 mg/ml group, compared to control group. Above these, the activity of mouse carvarial pre-osteoblast cells was showed greater bone osteogenesis activity in all applied HA experimental group, especially group of 1.0 mg/ml concentration of HA.
Alkaline Phosphatase ; Animals ; Cell Proliferation ; Collagen ; Embryonic Development ; Extracellular Matrix ; Female ; Fractures, Bone ; Hyaluronic Acid* ; Mice* ; Molecular Weight ; Osteogenesis* ; Pregnancy ; Skull* ; Wounds and Injuries