Comprehensive analysis and comparison of proteins in salivary exosomes of climacteric and adolescent females.

Currently, it is difficult to extract exosomes with stable physicochemical properties from saliva. Furthermore, due to inadequate availability of basic data, the application of salivary exosomes as a diagnostic material is limited. In this study, we aimed to investigate an easier method for extraction of exosomes from whole saliva and compared proteins in salivary exosomes derived from subjects of two age groups. Salivary exosomes were extracted from nine females (56.7 ± 1.17 years old; climacteric or 19.9 ± 0.20 years old; adolescent) using commercial reagents and kits and detected using western blotting with anti-exosome marker antibodies. Exosome particle size and exosome-containing proteins were identified using NanoSight® and liquid chromatography with tandem mass spectrometry, respectively. In addition, an efficient method of exosome extraction from saliva using a reagent and without the use of an ultracentrifuge was shown. Our results showed a higher total protein content and larger particle size in climacteric exosomes than in adolescent exosomes. However, adolescent exosomes showed a larger variety of proteins (780 proteins) than the climacteric exosomes (573 proteins). Altogether, 893 proteins were identified in the salivary exosomes. Although viral process-, ribosome- and structural molecule-related proteins were higher in the adolescent exosomes, the levels of major salivary proteins such as immunoglobulins and amylase, were higher in the climacteric exosomes than in the adolescent exosomes. The data presented, which show the fundamental protein composition of salivary exosomes and the changes that occur with age, are beneficial in both diagnostic and biotechnological applications.

Properties of fucoidans beneficial to oral healthcare.

Fucoidans are sulfated polysaccharides that are found in marine algae and have many useful activities, including antitumor effects, promotion of apoptosis of cancer cells, and antiviral, anti-inflammatory, and antiallergic actions. In oral medicine, several case reports have shown that fucoidan-containing creams and tablets markedly improved recurrent aphthous stomatitis, symptomatic inflamed tongue, and recurrent oral herpes labialis. The aim of this study was to examine the properties of fucoidans for use in oral healthcare. The antimicrobial, anti-adhesion, endotoxin-neutralizing, and cyclooxygenase (COX)-1 and COX-2 inhibitory activities of fucoidans were examined. Four key results were obtained: fucoidans showed strong antimicrobial activity against Candida albicans, Streptococcus mutans, and Porphyromonas gingivalis; significantly inhibited the adhesion of S. mutans to bovine teeth and porcelain; were suggested to bind to and neutralize endotoxin (lipopolysaccharide) in an LAL assay; and showed COX-1 and/or COX-2 inhibitory activity. These results suggested that fucoidans may be useful in the field of oral healthcare.

Immunolocalization of enamel matrix protein-like proteins in the tooth enameloid of spotted gar, Lepisosteus oculatus, an actinopterygian bony fish.

Enameloid is a well-mineralized tissue covering the tooth surface in fish and it corresponds to the outer-most layer of dentin. It was reported that both dental epithelial cells and odontoblasts are involved in the formation of enameloid. Nevertheless, the localization and timing of secretion of ectodermal enamel matrix proteins in enameloid are unclear. In the present study, the enameloid matrix during the stages of enameloid formation in spotted gar, Lepisosteus oculatus, an actinopterygian, was examined mainly by transmission electron microscopy-based immunohistochemistry using an anti-mammalian amelogenin antibody and antiserum. Positive immunoreactivity with the antibody and antiserum was found in enameloid from the surface to the dentin-enameloid junction just before the formation of crystallites. This immunoreactivity disappeared rapidly before the full appearance of crystallites in the enameloid during the stage of mineralization. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures, which were probably derived from matrix vesicles in the previous stage. In inner dental epithelial cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the enamel matrix protein-like proteins originated from inner dental epithelial cells. Enamel matrix protein-like proteins in the enameloid matrix might be common to the enamel matrix protein-like proteins previously reported in the collar enamel of teeth and ganoine of ganoid scales, because they exhibited marked immunoreactivity with the same anti-mammalian amelogenin antibodies. It is likely that enamel matrix protein-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.

Potential synergistic effects of a mixture of mineral trioxide aggregate (MTA) cement and Bacillus subtilis in dental caries treatment.

Bacillus subtilis is nonpathogenic in humans and produces a number of useful substances and, therefore, this bacterium is used in probiotic therapy. There have been trials of B. subtilis for patients with periodontitis, but not for patients with caries. Similarly, mineral trioxide aggregate (MTA) cement has been widely used for endodontic treatment, but there are few reports of its use for caries. Therefore, examinations were performed regarding the benefits of addition of B. subtilis to MTA cement for treatment of dental caries. Indirect pulp capping with a mixture of MTA cement and B. subtilis spore powder is effective for avoiding pulpectomy or tooth extraction in such cases (personal communication). This study was planned to examine the scientific basis of this clinical finding, with examination of possible synergistic effects of MTA cement and B. subtilis. From these experiments, the following five results were obtained: (1) B. subtilis did not proliferate in liquid-culture media at pH ≥10. (2) B. subtilis proliferated when mixed with MTA cement. (3) There was no significant difference in proliferation of B. subtilis under aerobic and microaerobic conditions. (4) B. subtilis exhibited antibacterial effects on Staphylococcus aureus and Lactobacillus casei. (5) MTA cement exhibited antibacterial effects on S. aureus and Streptococcus mutans, but not on B. subtilis. These results support the hypothesis that a combination of B subtilis and MTA cement is likely to be clinically useful for treatment of dental caries.

Immunohistochemical and Western Blotting Analyses of Ganoine in the Ganoid Scales of Lepisosteus oculatus: an Actinopterygian Fish.

In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin.

ADP-ribose-acceptor hydrolase 2 ( Arh2 ) deficiency results in cardiac dysfunction, tumorigenesis, inflammation, and decreased survival.

ADP-ribosylation is a reversible reaction with ADP-ribosyltransferases catalyzing the forward reaction and ADP-ribose-acceptor hydrolases (ARHs) hydrolyzing the ADP-ribose acceptor bond. ARH2 is a member of the 39-kDa ARH family (ARH1-3), which is expressed in heart and skeletal muscle. ARH2 failed to exhibit any in vitro enzymatic activity. To determine its possible in vivo activities, Arh2 -knockout (KO) and - heterozygous (Het) mice were generated using CRISPR-Cas9. Arh2 -KO mice exhibited decreased cardiac contractility by MRI, echocardiography and dobutamine stress with cardiomegaly and abnormal motor function. Arh2 -Het mice showed results similar to those seen in Arh2 -KO mice except for cardiomegaly. Arh2 -KO and -Het mice and mouse embryonic fibroblasts (MEFs) developed spontaneous tumors and subcutaneous tumors in nude mice. We identified 13 mutations in Arh2 -Het MEFs and heterozygous tumors, corresponding to human ARH2 mutations in cancers obtained from COSMIC. Of interest, the L116R mutation in Arh2 gene plays a critical role in aggressive tumorigenesis in nude mice, corresponding to human ARH2 mutations in stomach adenocarcinoma. Both genders of Arh2 -KO and -Het mice showed increased unexpectedly deaths and decreased survival rate during a 24-month observation, caused by tumor, inflammation, non-inflammation (e.g., cardiomegaly, dental dysplasia), and congenital diseases. Thus, Arh2 plays a pivotal role in cardiac function, tumorigenesis, inflammation, and overall survival.

Can hypochlorous acid be a powerful sanitizer to replace alcohol for disinfection ? -Its bactericidal, degradation of the solutions under various storage condition, and steel rust effects.

Coronavirus disease pandemic has led to a shortage of disinfectant alcohol (DA; 76.9-81.4% ethanol solution) in Japan. This study investigated the availability of hypochlorous acid (HA) as an alternative disinfectant to DA in clinical environment. HA is a safe and economical disinfectant with a broad antibacterial spectrum and low risk of hand irritation, which is used worldwide. In this study, HA and sodium hypochlorite solution (SH) were degraded over time and the degradation was accelerated by temperature and lighting. Results of bactericidal effects of HA revealed that the realistic effective concentration should be 200 ppm. Compared with DA, the dental carbon steel burs were more severely rusted by SH and HA. These results indicated that high concentration of HA generated on-site could be an effective and safe disinfectant for use immediately after generation with consideration of object materials in clinical environment, as an alternative to DA.

Low temperature polytetrafluoroethylene (PTFE) coating improves the appearance of orthodontic wires without changing their mechanical properties.

A strong esthetic demand exists for white-colored rather than metallic-colored dental appliances. Polytetrafluoroethylene (PTFE), which is chemically stable with extremely low friction, is a suitable white-colored coating for dental appliances. In the conventional PTFE coating process, base materials are heated to approximately 400°C, which could change their mechanical properties. Examination of the PTFE-coating of stainless steel, nickel titanium (Ni-Ti), and ß-titanium (Ti-Mo and Ti-Nb) revealed that the conventional 380°C coating, but not the newly developed low temperature coating at 200°C, reduced elasticity and bendability, and changed the crystal structure, especially in Ni-Ti and Ti-Nb wires. PTFE-coating at 200°C resulted in less discoloration, microbial adhesion, and friction against brackets, and higher tolerance of wear than did the conventional 380°C coating. These results suggest that low temperature PTFE coating is an excellent method for improving the metallic appearance of orthodontic wires without changing their mechanical properties.

Polytetrafluoroethylene (PTFE): A resin material for possible use in dental prostheses and devices.

Polytetrafluoroethylene (PTFE) is chemically stable, non-toxic to humans, highly resistant to heat and chemicals, and has an extremely low coefficient of friction. Therefore, PTFE is used in medical applications. We focused on the physical properties of PTFE in relation to its application as a material for use in prostheses and dental devices/instruments. PTFE exhibited low wear (approximately 1/3 that of bovine tooth, and 1/2 that of type III gold alloy), low dynamic friction (approximately 1/5 that of the other specimens), low hardness (4.8HV1.0), low coloration, and low bacterial adhesion, compared to other specimens except porcelain in wear and coloration test (p<0.01). These results suggest that PTFE could have applications in some prostheses for provisional and/or permanent use, and dental instruments/devices by providing excellent impact absorption, high wear resistance for maintenance of occlusal vertical dimension and original function, and ease of cleaning.

Histochemical and immunohistochemical examination of odontoblasts (petroblasts) in petrodentine formation of lungfish.

OBJECTIVE: Petrodentine, the core of the lungfish tooth plate, is a well-mineralized tissue similar to mammalian enamel and analogous to enameloid in fish teeth. Petrodentine is formed solely by petroblasts, which are specialized odontoblasts, whereas enameloid is a composite tissue produced by both odontoblasts and dental epithelial cells. To clarify the details of petrodentine formation, petroblasts were investigated using histochemical and immunohistochemical techniques. METHODS: Extant lungfish (Lepidosiren paradoxa) were used in this study. Tooth plates during the stage of petrodentine formation were observed by means of histochemistry and immunohistochemistry. Commercial kits were used to detect enzyme activity. Correlative sections were immunostained using antibodies against selected peptides. Routine staining such as periodic acid-Schiff (PAS) reaction to identify glycogen and Elastica van Gieson staining for the detection of elastic fibers in histological sections were performed. In addition, conventional transmission electron microscopy was used for observing the fine structure. RESULTS: Petroblasts showed marked acid and alkaline phosphatase activities, and positive immunoreactivities against anti-nestin, anti-V-ATPase, and anti-Ca2+-ATPase, during the maturation stage, but in the matrix formation stage, reactions were much weaker than that of the maturation stage. During the maturation stage, petroblasts showed intense PAS reactivity, and glycogen particles were observed in petroblasts by transmission electron microscopy. Glucose transporter 1-immunoreactivity was observed in petroblasts in the matrix formation stage and the initial to mid part of the maturation stage. CONCLUSIONS: The results in this study suggested that petroblasts have two functional stages, matrix formation and maturation, and glycogen plays an important role in the modulation of petroblasts.

Intraoral electric potential via oral bacterial power generation -A novel mechanism of biofilm formation.

In the early stages of biofilm accumulation, the electric charge of the dental enamel and pellicle surfaces is known to be involved. We therefore investigated the relationship between oral hygiene and intraoral electric potential (IoP) in 45 male participants using a double-blind study. IoP, but not body surface electric potential, was loosely correlated with oral hygiene condition (Oral Hygiene Index; OHI). IoP was also loosely correlated with smartphone use; however, there was no significant correlation between smartphone use and OHI. IoP elevation might be caused by OHI elevation resulting from biofilm formation as an internal factor, with smartphone use as an external factor. This in vitro study revealed the generating capacity of Streptococcus mutans accompanied by biofilm accumulation using a microbial fuel cell. These results suggest that IoP elevation is caused by biofilm accumulation induced by power generation of oral bacteria, resulting in elevation of OHI.

A novel cysteine protease inhibitor with lectin activity from the epidermis of the Japanese eel Anguilla japonica.

A novel cysteine protease inhibitor (Eel-CPI-1) was isolated from the epidermis of the eel. Eel-CPI-1 was shown to bind strongly to both lactose- and carboxymethylated papain-affinity gels. Its molecular mass under reducing condition was determined to be 18 kDa by SDS-polyacrylamide gel electrophoresis but approximately 30.5 kDa under non-reducing-conditions. Eel-CPI-1 inhibited papain (K(i)=18 nM) and ficin (K(i)=120 nM) competitively. Combined with the data on amino acid and sequence analysis, Eel-CPI-1 is identical to the eel lectin, AJL-2. This is the first report describing a cysteine protease inhibitor with lectin activity.

Ontogenetic changes in neuropeptide Y-immunoreactive cerebrospinal fluid-contacting neurons in the hypothalamus of the cloudy dogfish, Scyliorhinus torazame (Elasmobranchii).

Ontogenetic changes in neuropeptide Y-immunoreactive (NPY-ir) cerebrospinal fluid (CSF)-contacting neurons in the dogfish hypothalamus were studied immunohistochemically. NPY-ir CSF-contacting neurons first appeared in the median infundibular floor of the embryo at the 34 mm stage. At the 40 mm stage, similar neurons were found also in the saccus vasculosus (SV). The number of these neurons increased during the 54-80 mm stages, and the cells in the infundibular floor extended their basal processes to the neuropil of the median eminence, whereas the cells in the SV sent their axonal fibers to the tractus sacci vasculosi. After hatching, NPY immunoreactivity in the ventral hypothalamus became less dense, and the labeled CSF-contacting neurons tended to be confined to the nucleus lateralis tuberis, similarly as in the adults. The occurrence of NPY-ir CSF-contacting neurons in the SV was transient during the embryonic periods.

Identification and characterization of a mammalian 39-kDa poly(ADP-ribose) glycohydrolase.

ADP-ribosylation is a post-translational modification resulting from transfer of the ADP-ribose moiety of NAD to protein. Mammalian cells contain mono-ADP-ribosyltransferases that catalyze the formation of ADP-ribose-(arginine) protein, which can be cleaved by a 39-kDa ADP-ribose-(arginine) protein hydrolase (ARH1), resulting in release of free ADP-ribose and regeneration of unmodified protein. Enzymes involved in poly(ADP-ribosylation) participate in several critical physiological processes, including DNA repair, cellular differentiation, and carcinogenesis. Multiple poly(ADP-ribose) polymerases have been identified in the human genome, but there is only one known poly(ADP-ribose) glycohydrolase (PARG), a 111-kDa protein that degrades the (ADP-ribose) polymer to ADP-ribose. We report here the identification of an ARH1-like protein, termed poly(ADP-ribose) hydrolase or ARH3, which exhibited PARG activity, generating ADP-ribose from poly-(ADP-ribose), but did not hydrolyze ADP-ribose-arginine, -cysteine, -diphthamide, or -asparagine bonds. The 39-kDa ARH3 shares amino acid sequence identity with both ARH1 and the catalytic domain of PARG. ARH3 activity, like that of ARH1, was enhanced by Mg(2+). Critical vicinal acidic amino acids in ARH3, identified by mutagenesis (Asp(77) and Asp(78)), are located in a region similar to that required for activity in ARH1 but different from the location of the critical vicinal glutamates in the PARG catalytic site. All findings are consistent with the conclusion that ARH3 has PARG activity but is structurally unrelated to PARG.

The 39-kDa poly(ADP-ribose) glycohydrolase ARH3 hydrolyzes O-acetyl-ADP-ribose, a product of the Sir2 family of acetyl-histone deacetylases.

The silent information regulator 2 (Sir2) family of NAD-dependent N-acetyl-protein deacetylases participates in the regulation of gene silencing, chromatin structure, and longevity. In the Sir2-catalyzed reaction, the acetyl moiety of N-acetyl-histone is transferred to the ADP-ribose of NAD, yielding O-acetyl-ADP-ribose and nicotinamide. We hypothesized that, if O-acetyl-ADP-ribose were an important signaling molecule, a specific hydrolase would cleave the (O-acetyl)-(ADP-ribose) linkage. We report here that the poly(ADP-ribose) glycohydrolase ARH3 hydrolyzed O-acetyl-ADP-ribose to produce ADP-ribose in a time- and Mg(2+)-dependent reaction and thus could participate in two signaling pathways. This O-acetyl-ADP-ribose hydrolase belongs to a family of three structurally related 39-kDa ADP-ribose-binding proteins (ARH1-ARH3). ARH1 was reported to hydrolyze ADP-ribosylarginine, whereas ARH3 degraded poly(ADP-ribose). ARH3-catalyzed generation of ADP-ribose from O-acetyl-ADP-ribose was significantly faster than from poly(ADP-ribose). Like the degradation of poly(ADP-ribose) by ARH3, hydrolysis of O-acetyl-ADP-ribose was abolished by replacement of the vicinal aspartates at positions 77 and 78 of ARH3 with asparagine. The rate of O-acetyl-ADP-ribose hydrolysis by recombinant ARH3 was 250-fold that observed with ARH1; ARH2 and poly(ADP-ribose) glycohydrolase were inactive. All data support the conclusion that the Sir2 reaction product O-acetyl-ADP-ribose is degraded by ARH3.

Ultrastructure of the agranular cells in the adenohypophysis of the South American lungfish, Lepidosiren paradoxa.

Light and electron microscopic examination demonstrated two types of non-endocrine agranular cells, cavity boundary cells and stellate cells, in the adenohypophysis of the South American lungfish, Lepidosiren paradoxa. The cavity boundary cells line the hypophyseal cleft and diverticulum and display few microvilli, occasional cilia, prominent junctional complexes, and many cytoplasmic microfilaments. The stellate cells are scattered in the glandular parenchyma and are devoid of microvilli and cilia. When adjacent, they are connected to one another by desmosomes. Pinocytotic vesicles or caveolae are frequently seen along the plasma membrane of the agranular cells adjoining the endocrine cells or abutting on the basement membrane. Possible roles of the agranular cells, physically and metabolically supportive functions, are discussed on the basis of their ultrastractural features.