Peroxynitric acid inhibits amyloid ß aggregation.

Peroxynitric acid (PNA), a reactive oxygen nitrogen species, has attracted attention in life science because of its unique properties such as high bacteriacidal activity. Since the bactericidal activity of PNA could be related to its reaction with amino acid residues, we speculate that PNA can be used for protein modifications. In this study, PNA was applied to inhibit aggregation of amyloid ß1-42 (Aß42), which is thought to cause Alzheimer's disease (AD). We demonstrated for the first time that PNA could inhibit the aggregation and cytotoxicity of Aß42. Since PNA could inhibit aggregation of other amyloidogenic proteins such as amylin and insulin, our study shed a light on a novel strategy for the prevention of various diseases caused by amyloids.

Limosilactobacillus (Lactobacillus) fermentum ALAL020, a Probiotic Candidate Bacterium, Produces a Cyclic Dipeptide That Suppresses the Periodontal Pathogens Porphyromonas gingivalis and Prevotella intermedia.

Periodontal disease develops as a result of oral microbiota in dysbiosis, followed by the growth of periodontal pathogens such as Porphyromonas gingivalis and Prevotella intermedia. In case of acute symptoms, antibacterial agents and disinfectants are administered, however the appearance of drug-resistant bacteria and allergies cause problems. In recent years, studies on the effects of probiotics have been conducted as an alternative therapy for periodontitis. However, the basic mechanism of the inhibitory effect of probiotic bacteria on periodontal disease has not been clearly elucidated. To clarify the antibacterial mechanism of probiotics against periodontal pathogens, we used Limosilactobacillus (Lactobacillus) fermentum ALAL020, which showed the strongest antibacterial activity against P. gingivalis and P. intermedia among 50 screened lactic acid bacteria strains. The antibacterial substances produced were identified and structurally analyzed. After neutralizing the MRS liquid culture supernatant of ALAL020 strain, the molecular weight (m/z) of the main antibacterial substance separated by gel filtration column chromatography and reverse phase HPLC was 226.131. This low molecular weight compound was analyzed by LC-MS and disclosed the composition formula C11H18O3N2, however the molecular structure remained unknown. Then, structural analysis by NMR revealed C11H18O3N2 as the cyclic dipeptide, "hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dion cyclo (Hyp-Leu) ". Based on the results of this analysis, cyclo (Hyp-Leu) was chemically synthesized and the antibacterial activity against P. gingivalis and P. intermedia was measured. The minimum inhibitory concentration (MIC) was 2.5 g/L and the minimum bactericidal concentration (MBC) was shown to be less than 5 g/L. In addition, an in vitro epithelial tissue irritation test at 10 g/L showed no tissue toxicity. So far there are no reports of this peptide being produced by probiotic bacteria. Furthermore, antibacterial activity of this cyclic dipeptide against periodontal disease bacteria has not been confirmed. The results of this study might lead to a comprehensive understanding of the antibacterial mechanism against periodontal disease bacteria in future, and are considered applicable for the prevention of periodontal disease.

Kinetics of Bacterial Inactivation by Peroxynitric Acid in the Presence of Organic Contaminants.

Low-temperature atmospheric-pressure plasma has been studied for disinfection purposes. When plasma is exposed to water, reactive oxygen and nitrogen species are generated and preserved in the water fraction (plasma-treated water [PTW]), which consequently exhibits bactericidal activity. At low temperatures, one of the bactericidal components of PTW is peroxynitric acid (PNA). Importantly, PNA can also be synthesized by chemical reaction, without exposure to plasma. In this study, we evaluated the bactericidal properties of PNA based on reaction kinetics in comparison with other disinfectants. The analysis, based on dose-dependent effects, showed that PNA exhibited about 1 and 10 times the bactericidal activity of hypochlorous acid (HOCl) and peracetic acid, respectively. In addition, we evaluated the influence of organic contaminants on the bactericidal effects of PNA and HOCl. The bactericidal potential of both disinfectants was reduced by bovine serum albumin (BSA); however, PNA showed about 30-times-higher resistance against BSA inhibition than HOCl. Analysis of the dose-dependent effects of PNA revealed that the inhibition of bactericidal effect was caused by its consumption. Further experiments using model substrates containing particular amino acid residues (Met, Cys, Lys, and Leu) suggested that the bacterial inactivation by PNA is less affected by BSA due to the low reactivity and narrow reactivity spectrum of PNA for amino acid residues. Overall, our results suggest that PNA has a great disinfection potential, especially in the presence of organic contaminants (e.g., on the surface of the human body and on medical instruments contaminated with biological fluids).IMPORTANCE A good disinfectant for the human body should have various properties, such as strong bactericidal activity, harmlessness to living tissues, and resistance against biological fluids (or other organic contaminants). Peroxynitric acid (PNA) showed a bactericidal effect that was several tens up to several hundred times higher per unit of molarity than that of sodium hypochlorite and peracetic acid, which are used as general disinfectants for medical equipment. Moreover, the high resistance of PNA to organic load was confirmed, indicating that PNA will inactivate bacteria effectively even on contaminated surfaces, such as used medical devices or the human body surface. Therefore, we propose that PNA can be used as a strong disinfectant for the human body.

Kinetics Analysis of the Reactions between Peroxynitric Acid and Amino Acids.

Plasma disinfection using low-temperature atmospheric pressure plasma is widely studied in many applications, and the use of plasma-treated water (PTW) for disinfection is being developed by many researchers. Exposing plasma to water supplies and preserves reactive oxygen and nitrogen species (RONS) in the water, and this PTW exhibits bactericidal activity. In our previous study, it was revealed that peroxynitric acid (O2NOOH, PNA) was the dominant bactericidal component in PTW. PNA can be easily synthesized without plasma treatment, and the physicochemical properties of PNA have been well-analyzed. As the application of PNA in fields related to medicine and biology has not been reported, a basic study on the behavior of PNA is required. In this study, the bactericidal activity of PNA and its reactivities with 20 naturally occurring amino acids were evaluated to understand its reaction mechanism with biomolecules. Interestingly, PNA exhibited 10-6 times lower reactivities with amino acids when compared with hypochlorous acid and other RONS, although its bactericidal activity was 310 times higher than that of sodium hypochlorite. In addition, the reactivity of PNA with methionine was over 100 times higher than that with other amino acids, indicating that the reactions of PNA with amino acids are highly specific. No other oxidants have been reported to react selectively with only methionine. As methionine is involved in specific activities in cells, the unique reaction profile of PNA was examined in the context of biological systems.

High microbicidal effect of peroxynitric acid on biofilm-infected dentin in a root carious tooth model and verification of tissue safety.

OBJECTIVES: Root-caries, which frequently occurs in elderly people, is more difficult to treat than caries in a tooth crown, especially in filling restorations. To overcome this difficulty, it is essential to find a strategy for sufficiently sterilizing the infected dentin; however, techniques for sterilizing carious pathogens inside the biofilm, called dental plaque, have not yet been established. Recently, dental applications of plasma sterilization technology have attracted attention. The mechanism of plasma sterilization became clear, and revealed that peroxynitric acid (PNA) is an effective sterilization substance. Highly concentrated PNA solutions can be chemically synthesized in large quantities without using plasma technology. We thought that the application of PNA solution could be a novel treatment for root caries, and examined the microbicidal effect and safety of PNA. METHODS: A sterilization experiment was performed using an extracted tooth model infected with Streptococcus mutans. Subsequently, a biofilm of S. mutans and Candida albicans was formed on a plate or a dentin slice, and sterilization experiments were performed in comparison with chlorhexidine. Furthermore, a toxicity test of PNA was performed using an epithelial tissue model. RESULTS: In the infection model, sterilization was achieved with a 22 mM PNA solution in only 10 s. In the biofilm model, a 22 mM PNA solution showed a higher microbicidal effect than 2.0% chlorhexidine. In the toxicity test, 2.0% chlorhexidine was toxic, but a 220 mM PNA solution showed no toxicity. CONCLUSIONS: PNA is an unprecedented disinfectant that has high microbicidal activity on biofilm and is safe for tissues.

Investigation of a novel sterilization method for biofilms formed on titanium surfaces.

The development of effective methods to disinfect biofilms on dental materials is medically important. This study evaluated the bactericidal effects of peroxynitric acid (HOONO2; PNA) on biofilms formed on titanium surfaces. Streptococcus gordonii was cultured on either machined or rough titanium discs that were then used to evaluate the bactericidal effects of seven reagents, i.e., normal saline, benzalkonium chloride disinfectant solution, chlorhexidine digluconate solution, three concentration types of PNA, and inactivated PNA. Using low concentration of PNA, the bacterial count based on a CFU assay reached an undetectable level within 10 s; this bactericidal effect was the strongest observed for the seven tested reagents. Thus, PNA may be more useful than other disinfectants for sterilizing biofilms on titanium surfaces that have been contaminated with bacteria.

A Proposal of Remedies for Oral Diseases Caused by Candida: A Mini Review.

An opportunistic pathogen, Candida is not only related to oral problems such as oral candidiasis and denture stomatitis, but also to systemic diseases such as aspiration pneumonia and fungemia. The carriage rate of Candida species in the oral cavity of individuals wearing dentures and with removable orthodontic appliances, has increased. Moreover, it is one of the causal pathogens in refractory infected root canals because of its resistance to antifungal drugs in root canal therapy and poses a great challenge during the treatment of patients. This problem has led to the search for alternative strategies for the treatment and management of C. albicans infections. In this mini review, recent preventive strategies against Candida infection in the oral mucosa with natural product-derived antifungal molecules were discussed. Inhibitory strategies by introducing competitive naturally-derived antifungal peptide molecules with Candida adhesion molecules were specifically introduced. In addition, novel sterilization methods for Candida-infected root canals and tooth structures in the oral cavity were considered, with focused attention on the activities of reactive oxygen species. The possibility of application of these novel strategies in clinical treatments and daily life was also proposed.

Plasma-treated water eliminates Streptococcus mutans in infected dentin model.

Non-mechanical procedures for removing caries-infected dentin are warranted in dentistry. We previously demonstrated the marked sterilization effect for direct irradiation of low-temperature plasma using dentin model infected with Streptococcus mutans. However it requires 180 s of intraoral plasma irradiation to eliminate bacteria. We alternatively investigated whether plasma-treated water (PTW), i.e., pure water exposed to plasma in an atmosphere, has a same bactericidal activity with the plasma irradiation. In the infected dentin model, the viable S. mutans counts recovered by bur at depth of 0.8-2.4 mm from the cavity floor were 104-106 CFU/round bur. After PTW application for only 10 s, the count was significantly decreased to below the detection limit (2.5 CFU/round bur) or 3.0±5.0 CFU/round bur. Since the bactericidal activity of PTW is rapidly deactivated at body temperature (37°C), PTW is likely to be biocompatible and holds significant potential for non-mechanical procedures for removing caries-infected dentin.

Ion-exchange chromatographic analysis of peroxynitric acid.

Ion-exchange chromatographic analysis of peroxynitric acid (O2NOOH) was performed by combining an acidic eluate with an UV-vis detector and immersing the separation column in an ice-water bath. The decomposition behavior of peroxynitric acid in the solution was also studied using this system. The fraction for the peroxynitric acid peak was collected. Ion-exchange chromatographic analysis of this fraction, after standing at room temperature for 24h, showed that the decomposition products were mainly nitrate ions with a very small amount of nitrous acid. The peroxynitric acid peak area correlated perfectly with the total amount of decomposition products. The ion-exchange chromatographic isolation allowed us to evaluate the molar extinction coefficient of peroxynitric acid precisely in a wider wavelength range than previous reports. The value decreases monotonically from 1729±26M(-1)cm(-1) at 200nm to 12.0±0.5M(-1)cm(-1) at 290nm.

Cytotoxic activity of Bacillus thuringiensis Cry proteins on mammalian cells transfected with cadherin-like Cry receptor gene of Bombyx mori (silkworm).

Cry1Aa, an insecticidal protein produced by Bacillus thuringiensis, has been shown to bind to cadherin-like protein, BtR175, in Bombyx mori (silkworm) midgut. We previously reported three variant alleles of BtR175 (BtR175a, b and c). When transiently expressed in COS7 cells, all the three BtR175 variants bound to Cry1Aa. We stably expressed BtR175b in HEK293 cells. These BtR175b-expressing cells swelled and died in the presence of activated Cry1Aa in a dose- and time-dependent manner, showing that BtR175b itself can impart Cry1Aa-susceptibility to mammalian cells. These cells were more susceptible to Cry1Aa than to Cry1Ab and Cry1Ac. Since dispersed B. mori midgut cells were reported to be highly susceptible to Cry1Ac, this result suggested that other Cry1Ac-specific receptor(s) were simultaneously working with BtR175 in the midgut cells. Advantages are also discussed of applying these transfected mammalian cells to toxicity assays of mutant Cry proteins.