Inhibitory effects of a novel cationic dodecapeptide [CL(14-25)] derived from cyanate lyase of rice on endotoxic activities of LPSs from Escherichia coli and periodontopathic Aggregatibacter actinomycetemcomitans.

OBJECTIVE: CL(14-25), a dodecapeptide of cyanate lyase from rice, is a novel cationic α-helical antimicrobial peptide. In this study, we examined inhibitory ability of CL(14-25) against endotoxic activities of lipopolysaccharides (LPSs) from Escherichia coli and periodontal pathogenic Aggregatibacter actinomycetemcomitans. METHODS: Endotoxin-neutralizing activity of CL(14-25) was evaluated by inhibition to induction of cytokine and nitric oxide in human aortic endothelial cells (HAECs) and RAW264 mouse macrophage cells, respectively. Protective effect of CL(14-25) was determined in mice against lethal toxicity of LPS. RESULTS: IL-6 in HAECs was induced by stimulation with LPS preparations of A. actinomycetemcomitans and E. coli tested in this study, and addition of CL(14-25) to the medium caused inhibition of their induction in a dose-dependent manner. CL(14-25) inhibited NO induction in RAW264 cells by a smooth type LPS of E. coli O55:B5 and an Rc type LPS of E. coli J5 as well as lipid A of E. coli R515 in a dose-dependent manner. Simultaneous injection of E. coli O55:B5 LPS and CL(14-25) in BALB/c mice resulted in prevention of lethal toxicity of the former. The results of a Limulus amebocyte lysate assay and surface plasmon resonance analysis of interaction between CL(14-25) and E. coli LPS or lipid A showed that CL(14-25) specifically binds to a lipid A moiety of LPS. CONCLUSION: The results of present study suggest that CL(14-25) has a potential to be used as a nutraceutical agent for periodontal therapy.

Effect of a mouthrinse containing rice peptide CL(14-25) on early dental plaque regrowth: a randomized crossover pilot study.

BACKGROUND: We aimed to evaluate clinically the effect of mouthrinse containing a rice peptide on early dental plaque regrowth. METHODS: The study was designed as a double-masked, two-group crossover randomized pilot trial, involving 10 periodontally healthy volunteers. After receiving a professional tooth cleaning at baseline, over the next 3 days each participant refrained from all oral hygiene measures and had two daily rinses with 20 ml of the test mouthrinse containing 0.4 % rice peptide CL(14-25) or placebo rinse. At the end of each experimental period, plaque score was assessed using the modified Volpe's method, and the participants filled out a questionnaire. Each participant underwent a 7-day washout period followed by a second allocation. The plaque score was the primary outcome of the study and participant perception was the secondary outcome. RESULTS: No adverse effects were observed in the participants during the study. Clinically, the mean plaque score of the examined teeth was significantly lower in the test group (2.44 ± 0.74, CI: 1.91-2.96) than the placebo group (2.65 ± 0.63, CI: 2.20-3.10) (P < 0.05). When analyzed according to the type of teeth, a significantly lower score of the premolars/molars was observed in the test group (2.39 ± 0.68, CI: 2.08-2.71) than that in the placebo group (2.66 ± 0.58, CI: 2.39-2.93) (P < 0.05). CONCLUSIONS: The mouthrinse containing 0.4 % rice peptide CL(14-25) was effective in reducing the early regrowth of dental plaque. However, clinical relevance of this efficacy needs to be validated in a future large-scale study. TRIAL REGISTRATION: UMIN Clinical Trials Registry (UMIN-CTR) R000014000. Date of formal registration: November 1, 2013.

Prevalence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in Japanese patients with generalized chronic and aggressive periodontitis.

This study aimed to investigate the prevalence and levels of major periodontal pathogens, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in subgingival plaque samples of a group of Japanese patients with aggressive periodontitis (AgP) and chronic periodontitis (CP). A total of 40 patients with clinical diagnosis of AgP or CP and 10 periodontally healthy volunteers were subjected to clinical and microbiological analysis. Subgingival plaque samples were analyzed for A. actinomycetemcomitans, P. gingivalis and T. forsythia with a real-time polymerase chain reaction (PCR) technique. The prevalence of P. gingivalis and T. forsythia was relatively high in patients with periodontitis: over 60% of AgP or CP patients harbored these pathogens whereas they were not detected in the subgingival plaque samples from periodontally healthy individuals. P. gingivalis and T. forsythia were relatively frequently detected together in AgP and CP patients. No significant differences in the prevalence or level of the 3 pathogens were found between periodontitis groups. The proportion of T. forsythia was approximately 4-fold higher in CP group than in AgP group (P = 0.02). In periodontitis patients, a significant positive correlation was found between periodontal parameters (probing depth and clinical attachment level) and the numbers of total bacteria, P. gingivalis and T. forsythia. No distinct pattern of the subgingival profile of these pathogens was discerned between the two disease entities, except for the difference in the proportion of T. forsythia. The red complex bacteria, P. gingivalis and T. forsythia were highly prevalent in this population of Japanese AgP and CP patients, collaborating their roles in periodontitis.

Effect of eel galectin AJL-1 on periodontopathic bacterial biofilm formation and their lipopolysaccharide-mediated inflammatory cytokine induction.

Porphyromonas gingivalis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans, infectious pathogenic bacteria found in oral biofilm, cause periodontal disease. The inhibitory effect of AJL-1, a galectin present in the skin mucus of the Japanese eel Anguilla japonica, on biofilm formation by each of these strains was investigated by staining adherent bacteria on culture plates with crystal violet. An ATP bioluminescence assay was used to determine whether inhibition of biofilm formation was due to the bactericidal activity of AJL-1. The effect of AJL-1 on cytokine induction in human umbilical vascular endothelial cells (HUVECs) by lipopolysaccharide (LPS) isolated from A. actinomycetemcomitans was also investigated by enzyme-linked immunosorbent assay (ELISA). AJL-1 significantly inhibited biofilm formation by A. actinomycetemcomitans strains Y4, ATCC 29523 and ATCC 29524 but not by any strain of P. gingivalis or P. intermedia, and showed no bactericidal activity against A. actinomycetemcomitans strains. AJL-1 markedly suppressed interleukin (IL)-6 and IL-8 induction in HUVECs by LPS from A. actinomycetemcomitans strains Y4 and ATCC 29523. These observations indicate that AJL-1 is an effective inhibitor of biofilm formation by A. actinomycetemcomitans as well as of inflammatory cytokine induction in HUVECs by LPS. These finding indicate that AJL-1 may be of therapeutic value in A. actinomycetemcomitans-associated periodontal diseases.

Genotoxicity of 3,6-dinitrobenzo[e]pyrene, a novel mutagen in ambient air and surface soil, in mammalian cells in vitro and in vivo.

3,6-Dinitrobenzo[e]pyrene (3,6-DNBeP), newly identified in airborne particles and surface soil, is a potent mutagen in Salmonella typhimurium. The present study investigated the genotoxic potency of 3,6-DNBeP in vitro and in vivo using mammalian cell strains (Chinese hamster CHL/IU and human HepG2) and ICR mice, respectively. In the hprt gene mutation assay using HepG2 cells, the spontaneous mutant frequency was 61.1 per 10(5) clonable cells, which increased to 229 per 10(5) clonable cells after treatment with 1.0 microg/ml (3 microM) 3,6-DNBeP. Notably, in HepG2 cells with increased N-acetyltransferase 2 activity, the mutant frequency increased to 648 per 10(5) clonable cells by treatment of 1.0 microg/ml (3 microM) 3,6-DNBeP. The sister chromatid exchange frequency increased approximately three times the control level in HepG2 cells treated with 3,6-DNBeP at a concentration of 1.0 microg/ml (3 microM). In HepG2 and CHL/IU cells, the frequency of the cells with micronuclei was 0.9 and 1.2%, and the frequencies increased to 2.3 and 7.6% after 1.0 microg/ml (3 microM) 3,6-DNBeP-treatment, respectively. The H2AX phosphorylation level increased 8-fold compared with the background level with 1.0 microg/ml (3 microM) 3,6-DNBeP-treatment in HepG2 cells. Moreover, the comet assay showed that 3,6-DNBeP produced DNA damage in the cells of liver, kidney, lung and bone marrow in ICR mice 3 h after intraperitoneal injection at 40 mg/kg (0.12 mmol/kg) body weight. These data indicate that 3,6-DNBeP is genotoxic to mammalian cells in vitro and in vivo.