Satoshi Ikawa

Chemical modification of amino acids by atmospheric-pressure cold plasma in aqueous solution

Plasma medicine is an attractive new research area, but the principles of plasma modification of biomolecules in aqueous solution remain elusive. In this study, we investigated the chemical effects of atmospheric-pressure cold plasma on 20 naturally occurring amino acids in aqueous solution. High-resolution mass spectrometry revealed that chemical modifications of 14 amino acids were observed after plasma treatment: (i) hydroxylation and nitration of aromatic rings in tyrosine, phenylalanine and tryptophan; (ii) sulfonation and disulfide linkage formation of thiol groups in cysteine; (iii) sulfoxidation of methionine and (iv) amidation and ring-opening of five-membered rings in histidine and proline. A competitive reaction experiment using 20 amino acids demonstrated that sulfur-containing and aromatic amino acids were preferentially decreased by the plasma treatment. These data provide fundamental information for elucidating the mechanism of protein inactivation for biomedical plasma applications.

Free radicals induced in aqueous solution by non-contact atmospheric-pressure cold plasma

To understand plasma-induced chemical processing in liquids, we investigated the formation of free radicals in aqueous solution exposed to different types of non-contact atmospheric-pressure helium plasma using the spin-trapping technique. Both hydroxyl radical (OH·) and superoxide anion radical (O2−·) adducts were observed when neutral oxygen gas was additionally supplied to the plasma. In particular, O2−· can be dominantly induced in the solution via oxygen flow into the afterglow gas of helium plasma. This type of plasma treatment can potentially be used in medical applications to control infectious diseases, because the O2−· is crucial for sterilization of liquids via atmospheric-pressure plasma.

Molecular mechanism of plasma sterilization in solution with the reduced pH method: importance of permeation of HOO radicals into the cell membrane

Sterilization of certain infected areas of the human body surface is necessary for dental and surgical therapies. Because the blood is filled with body fluid, sterilization in solution is essential. In vitro solution sterilization has been successively carried out using a combination of low-temperature atmospheric-pressure plasma and the reduced pH method, where the solution is sufficiently acidic. Here, we show the molecular mechanism of such plasma sterilization in solution based on microbiology. Three kinds of bacteria were inactivated by plasma treatment under various pH conditions. The theoretical and experimental models revealed that the sterilization was characterized by the concentration of hydroperoxy radicals (HOO·), which were dependent on the pH value. Bacterial inactivation rates were proportional to the HOO· concentrations calculated by the theoretical model. To evaluate the penetration of radicals into the cell membrane, a bacterial model using dye-included micelles was used. Decolouration rates of the model were also in proportion with the calculated HOO· concentrations. These results indicate that the key species for plasma sterilization were hydroperoxy radicals. More importantly, the high permeation of hydroperoxy radicals into the cell membrane plays a key role for efficient bactericidal inactivation using the reduced pH method.

Physicochemical properties of bactericidal plasma-treated water

Plasma-treated water (PTW), i.e. distilled water (DW) exposed to low-temperature atmospheric pressure helium plasma, exhibited strong bactericidal activity against Escherichia coli in suspension even within a few minutes of preparation. This effect was enhanced under acidic conditions. The bactericidal activity of PTW was attenuated according to first-order kinetics and the half-life was highly temperature dependent. The electron spin resonance (ESR) signal of an adduct of the superoxide anion radical () was detected in an aqueous solution using a spin-trapping reagent mixed with PTW, and adding superoxide dismutase to the PTW resulted in a loss of the bactericidal activity and weakening of the ESR adduct signal of in the spin-trapping. These results suggest that plays an important role in imparting bactericidal activity to PTW. Moreover, molecular nitrogen was required both in the ambient gas and in the DW used to prepare the PTW. We, therefore, suggest that the reactive molecule in PTW with bactericidal effects is not a free reactive oxygen species but nitrogen atom(s)-containing molecules that release , such as peroxynitrous acid (ONOOH) or peroxynitric acid (O2NOOH). Considering the activation energy for degradation of these species, we conclude that peroxynitric acid stored in PTW induces the bactericidal effect.

Diagnosis of superoxide anion radical induced in liquids by atmospheric-pressure plasma using superoxide dismutase

To confirm the formation of the superoxide anion radical (O2−) in liquids by atmospheric-pressure plasma, we investigated plasma-induced radical species in water using the electron spin resonance (ESR) spin-trapping technique combined with two proteins: superoxide dismutase (SOD), which has enzymatic activity to quench the superoxide anion radical, and bovine serum albumin (BSA), which does not have this enzymatic activity. Different setups of contact and non-contact atmospheric-pressure helium plasma were tested with an additional supply of oxygen gas. For each setup of plasma, no superoxide anion adduct ESR signal was observed in the aqueous solution with SOD, whereas the ESR signal appears in the samples with BSA and without any additive proteins. This means that a superoxide anion radical in the solution is sufficiently quenched by SOD before the formation of the spin adduct. The superoxide anion radical is actually induced in an aqueous solution by atmospheric-pressure plasma when ambient gases contain oxygen.

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.

Evaluation of fatty acid oxidation by reactive oxygen species induced in liquids using atmospheric-pressure nonthermal plasma jets

We investigated fatty acid oxidation by atmospheric-pressure nonthermal helium plasma using linoleic acid, an unsaturated fatty acid, together with evaluating active species induced in liquids. If the ambient gas contains oxygen, direct plasma such as plasma jets coming into contact with the liquid surface supplies various active species, such as singlet oxygen, ozone, and superoxide anion radicals, to the liquid. The direct plasma easily oxidizes linoleic acid, indicating that fatty acid oxidation will occur in the direct plasma. In contrast, afterglow flow, where the plasma is terminated in a glass tube and does not touch the surface of the liquid sample, supplies mainly superoxide anion radicals. The fact that there was no clear observation of linoleic acid oxidation using the afterglow reveals that it may not affect lipids, even in an atmosphere containing oxygen. The afterglow flow can potentially be used for the sterilization of aqueous solutions using the reduced pH method, in medical and dental applications, because it provides bactericidal activity in the aqueous solution despite containing a smaller amount of active species.

Determination of the Antibacterial Constituents Produced by Lactobacilliagainst a Periodontal Pathogen: Sodium Lactate and a Low MolecularWeight Substance

Background and objectives: Probiotics are living bacteria which can improve the balance of microbiota. There are many recent studies on the effects of probiotics, including oral health promotion and the prevention of oral diseases. However, the mechanisms that underlie the activity of probiotic bacteria against periodontal pathogens have not been clearly elucidated. The purpose of this study was to examine the effects of lactic acid bacteria as probiotics in the prevention and treatment of periodontal disease. Material and Methods: The growth inhibitory effects of the culture supernatants of 50 strains of lactobacilli on the periodontal pathogen Porphyromonas gingivalis ATCC33277 were examined. To obtain a substance with antibacterial properties under neutral pH conditions, each culture supernatant was neutralized and purified by gel filtration column chromatography and reverse-phase HPLC. The molecular weight of purified substances was analyzed with LC-MS. Results: The results showed that two strains of Lactobacillus plantarum 122 (derived from the oral cavity) and L. fermentum ALAL020 (derived from fermented soy milk food products) had strong growth inhibition effects. The major antibacterial substance produced by L. plantarum 122 was thought to be sodium lactate. On the other hand, the molecular weight of the major antibacterial substance produced by L. fermentum ALAL020 we purified was 226.131 Da. An LC-MS analysis revealed that it had the following composition: C11H18O3N2. Conclusion: The antibacterial substance of L. plantarum 122 against P. gingivalis was sodium lactate, and that of L. fermentum ALAL020 we purified was a novel low molecular substance. This antibacterial substance has a possibility for using periodontal disease prevention.

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.