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This study determined the influence of intravenous (i.v.) oncolytic vaccinia virus mpJX-594 (mpJX) on antitumor activity of anti-programmed death receptor-1 antibody (aPD1) in functional and metastatic pancreatic neuroendocrine tumors (PanNETs). One i.v. dose of mpJX, engineered for mice with the same plasmid design as clinical virus Pexa-Vec, was administered alone or with repeated dosing of aPD1 (mpJX+aPD1) to two contrasting genetic models of PanNET: one developing benign insulin-secreting tumors (RIP1-Tag2;C57BL/6J mice) and the other developing liver metastases (RIP1-Tag2;AB6F1 mice). Experiments revealed that aPD1 had synergistic actions with mpJX on CD8+ T cell and natural killer (NK) cell influx, apoptosis, and suppression of proliferation in PanNETs. After mpJX+aPD1, the 53-fold increase in apoptosis (5 days) and 85% reduction in proliferation (20 days) exceeded the sum of mpJX and aPD1 given separately. mpJX+aPD1 also stabilized blood insulin and glucose in mice with functional PanNETs, regressed liver metastases in mice with aggressive PanNETs, and prolonged survival of both. The findings revealed that mpJX+aPD1 converted “cold” PanNETs into immunogenic tumors with widespread cytotoxic T cell influx, tumor cell killing, and suppression of proliferation. Reduction of tumor insulin secretion from functional PanNETs prolonged survival, and anti-metastatic actions on aggressive PanNETs reduced the metastatic burden to less than before treatment. The findings support the efficacy of the vaccinia virus with aPD1 for functional and metastatic PanNETs., Graphical abstract, Vaccinia virus mpJX-594 administered intravenously to mice with pancreatic neuroendocrine tumors increases the antitumor response of an anti-PD1 antibody by turning immunologically “cold” tumors into “hot” tumors. Synergistic actions of the treatment combination amplify cytotoxic T cell influx and lead to smaller, less functional primary tumors and to the regression of metastases.

ATP6AP2, also known as (pro)renin receptor, has been shown to be expressed in several tissues including pancreatic β cells. Whereas ATP6AP2 plays an important role in regulating insulin secretion in mouse pancreatic β cells, the expression profiles and roles of ATP6AP2 in human pancreatic endocrine cells and neuroendocrine tumor cells remain unclear. Here in this study, we investigated the expression profiles of ATP6AP2 in pancreatic endocrine cells, and found that ATP6AP2 is robustly expressed in pancreatic insulinoma cells as well as in normal β cells. Although ATP6AP2 was also expressed in low-grade neuroendocrine tumors, it was not or faintly detected in intermediate- and high-grade neuroendocrine tumors. Knockdown experiments of the Atp6ap2 gene in rat insulinoma-derived INS-1 cells demonstrated decreased cell viability accompanied by a significant increase in apoptotic cells. Taken together, these findings suggest that ATP6AP2 plays a role in maintaining cellular homeostasis in insulinoma cells, which could lead to possible therapeutic approaches for endocrine tumors.

The anti-bone resorptive drugs denosumab, an anti-human-RANKL antibody, and zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, have recently been applied for treatment of pediatric patients with bone diseases, though details regarding their effects in growing children have yet to be fully elucidated. In the present study, we administered these anti-resorptive drugs to mice from the age of 1 week and continued once-weekly injections for a total of 7 times. Mice that received the anti-RANKL antibody displayed normal growth and tooth eruption, though osteopetrotic bone volume gain in long and alveolar bones was noted, while there were nearly no osteoclasts and a normal of number osteoblasts observed. In contrast, ZOL significantly delayed body growth, tooth root formation, and tooth eruption, with increased osteoclast and decreased osteoblast numbers. These findings suggest regulation of tooth eruption via osteoblast differentiation by some types of anti-resorptive drugs.

Aims/Introduction Non‐alcoholic fatty liver disease is frequently associated with type 2 diabetes, and constitutes an important risk factor for the development of hepatic fibrosis and hepatocellular carcinoma. Because there remains no effective drug therapy for non‐alcoholic fatty liver disease associated with type 2 diabetes, we evaluated the efficacy of sodium–glucose cotransporter 2 inhibitor. Methods and Materials In the present pilot, prospective, non‐randomized, open‐label, single‐arm study, we evaluated the effect of 100 mg canagliflozin administered once daily for 12 months on serological markers, body composition measured by bioelectrical impedance analysis method and hepatic fat fraction measured by magnetic resonance imaging in type 2 diabetes patients with non‐alcoholic fatty liver disease. Results Canagliflozin significantly reduced body and fat mass, and induced a slight decrease in lean body or muscle mass that did not reach significance at 6 and 12 months. Reductions in fat mass in each body segment (trunk, arms and legs) were evident, whereas those in lean body mass were not. The hepatic fat fraction was reduced from a baseline of 17.6 ± 7.5% to 12.0 ± 4.6% after 6 months and 12.1 ± 6.1% after 12 months (P

Objectives Nectins are immunoglobulin-like cell–cell adhesion molecules and are of four types, namely, nectin-1, nectin-2, nectin-3, and nectin-4. Cleft lip/palate-ectodermal dysplasia is caused by a mutation in the nectin-1 gene locus. However, nectin-1-deficient (KO) mice only show mild tooth defects. This study determined the intracellular localization of nectins in mouse mandibular incisors to identify their heterophilic interactions during amelogenesis. Methods Nectin localization was determined by performing immunohistochemical analysis with confocal microscopy. Nectin gene expression was determined by performing quantitative reverse transcription-PCR. Phenotypes of nectin-2 - KO mice were examined by performing micro-computed tomography, histological analysis, and organ culture. Results We found that mRNA levels of nectin-1 and nectin-3 genes were higher than those of nectin-2 and nectin-4 genes in mouse enamel organs. Nectin-2 and nectin-4 were strongly expressed at the apical adherens junctions of secretory-stage ameloblasts, whereas nectin-1 and nectin-3 were distributed between the basal adherens junction of maturation-stage ameloblasts and the stratum intermedium and papillary layers. Nectin-2-KO mice showed normal mandibular tooth shape, incisor surface pigmentation, and histology. Moreover, organ cultures of the tooth organs of nectin-2-KO mice proceeded normally. Conclusion These results indicate that nectins show graded and overlapping distribution in mouse incisors.

Aim Gingivitis commonly progresses to periodontitis in permanent dentition but rarely in deciduous teeth. Little is known about the biochemical differences between gingiva of deciduous and permanent teeth. Here we compared the protein profiles of gingival crevicular fluids (GCF) from the gingiva of deciduous and permanent teeth. Materials and Methods Forty children with mixed dentition (Hellman's dental age IIIA) were selected and GCF samples were collected from deciduous cuspids and central incisors in the maxilla. Pairs of GCF samples were labelled using isobaric tags to permit quantitative comparison of protein abundance in the samples using liquid chromatography-electron spray ionization-tandem mass spectrometry. Results Sixty-two proteins were upregulated in deciduous teeth GCF and 54 in permanent teeth GCF. In particular, neutrophil-derived proteins, including myeloperoxidase and lactoferrin, were repeatedly higher in deciduous teeth GCF than in permanent teeth GCF. These differences were verified using ELISA (P < 0.01). In contrast, immunoglobulin components were upregulated in permanent teeth GCF. Conclusions Neutrophil-related proteins were enriched in deciduous teeth GCF and immunoglobulins in permanent teeth GCF. This suggests that neutrophil accumulation plays a protective role in innate immunity against bacterial infection in gingival tissue of deciduous teeth. This article is protected by copyright. All rights reserved.

Histamine receptors are densely expressed in the mesencephalic trigeminal nucleus (MesV) and trigeminal motor nucleus. However, little is known about the functional roles of neuronal histamine in controlling oral-motor activity. Thus, using the whole-cell recording technique in brainstem slice preparations from Wistar rats aged between postnatal days 7 and 13, we investigated the effects of histamine on the MesV neurons innervating the masseter muscle spindles and masseter motoneurons (MMNs) that form a reflex arc for the jaw-closing reflex. Bath application of histamine (100 μM) induced membrane depolarization in both MesV neurons and MMNs in the presence of tetrodotoxin, whereas histamine decreased and increased the input resistance in MesV neurons and MMNs, respectively. The effects of histamine on MesV neurons and MMNs were mimicked by an H1 receptor agonist, 2-pyridylethylamine (100 μM). The effects of an H2 receptor agonist, dimaprit (100 μM), on MesV neurons were inconsistent, whereas MMNs were depolarized without changes in the input resistance. An H3 receptor agonist, immethridine (100 μM), also depolarized both MesV neurons and MMNs without changing the input resistance. Histamine reduced the peak amplitude of postsynaptic currents (PSCs) in MMNs evoked by stimulation of the trigeminal motor nerve (5N), which was mimicked by 2-pyridylethylamine but not by dimaprit or immethridine. Moreover, 2-pyridylethylamine increased the failure rate of PSCs evoked by minimal stimulation and the paired-pulse ratio. These results suggest that histaminergic inputs to MesV neurons through H1 receptors are involved in the suppression of the jaw-closing reflex although histamine depolarizes MesV neurons and/or MMNs.

Background/Purpose Periapical bone resorption is mainly induced by inflammation resulting from an infected root canal. However, chemical and physical stimuli (e.g., traumatic occlusions and excessive orthodontic force) can also cause periapical bone resorption. Here, we report the induction of a periapical bone resorption model without infection in rats. Methods Wire ligature was applied between the upper first and second molars for 2 weeks. At 2 weeks and 4 weeks after removal of the wire, the maxillary bones were processed for micro-computed tomography and histological analyses. Results The micro-computed tomography analysis showed that periapical bone resorption had occurred and that the bone volume had significantly decreased between Week 2 and Week 4 after wire removal. Histological examination showed a normal apical periodontal ligament. No accumulation of inflammatory cells and no abscess formation were detected in the periapical region. Meanwhile, many tartrate-resistant acid phosphatase-positive osteoclasts were detected in the periapical region of non-wire-ligated rats in which the dental pulp had been exposed; however, active osteoclasts were still located at the alveolar bone in rats even at 4 weeks after wire removal. Conclusion These results indicate that induction of periapical bone resorption occurred in the absence of infection and that a bone resorption system different from the infection-associated bone resorption one accounted for the changes.

Objective Respiratory disease occurs frequently in children. The nasal cavity, trachea, and oral cavity are considered the primary routes of infection. We aimed to investigate the types of microbial pathogens present in the oral cavities of children with respiratory disease. In addition, we compared the detection rates of different pathogens between the oral cavity and the nasopharynx according to Hellman dental age. Materials and methods We included 32 children who were hospitalized for respiratory disease and classified them according to Hellman dental age. Furthermore, 32 control children were included as controls. Specimens were collected using two palate swabs: one taken at hospital admission and the other at discharge. The culture results of the samples were compared with those of the nasopharyngeal cultures. Results The following six bacterial strains were detected in both the palate and the nasopharynx: α-Streptococcus , Corynebacterium , Haemophilus influenzae , Haemophilus parainfluenzae , methicillin-resistant Staphylococcus aureus (MRSA), and Neisseria . In the comparison of the detection rates, Neisseria were significantly more frequently detected in the palate. MRSA was detected at a significantly higher rate in the nasopharynx. When the detection rates of different bacteria were compared according to Hellman dental age, significant differences were observed in the detection rates of α-Streptococcus in stage IA and Neisseria in stages IC and IIA. Discussion We noted that the bacterial load of specific bacteria in the palate increased with Hellman dental age. Furthermore, the increase in bacterial load in the oral cavity at stage IA, when teeth have not yet erupted, suggests the need for oral management.

Although it is known that osteoclasts are multinucleated cells that are responsible for bone resorption, the mechanism by which their size is regulated is unclear. We previously reported that an actin-rich superstructure, termed the zipper-like structure, specifically appears during the fusion of large osteoclast-like cells (OCLs). Actin cytoskeleton reorganization in osteoclasts is regulated by a signaling network that includes the macrophage colony-stimulating factor (M-CSF) receptor, a proto-oncogene, Src, and small GTPases. Here, we examined the role of actin reorganization in the multinucleation of OCLs differentiated from RAW 264.7 cells using various pharmacological agents. Jasplakinolide, which stabilizes actin stress fibers, induced the development of small OCLs, and the Src inhibitor SU6656 and the dynamin inhibitor dynasore impaired the maintenance of the podosome belt and the zipper-like structure. These inhibitors decreased the formation of large OCLs but increased the number of small OCLs. M-CSF is known to stimulate osteoclast fusion. M-CSF signaling via Src up-regulated Rac1 activity but down-regulated Rho activity. Rac1 and Rho localized to the center of the zipper-like structure. Rho activator II promoted the formation of small OCLs, whereas the Rho inhibitor Y27632 promoted the generation of large OCLs. These results suggest that the status of the actin cytoskeleton signaling network determines the size of OCLs during cell fusion.

Objective The optimal polymerization of resin-based luting cements plays a critical role in the long-term clinical success of dental prostheses and indirect restorations. This study investigated a mutual action between the conformational changes and mechanical properties of a dimethacrylate resin-based luting cement with and without pre-application of the acidic functional monomer 10-methacryloxydecyl dihydrogen phosphate. Methods Degree of conversion in the luting cement was measured using conventional infrared spectrophotometry. Mechanical properties of the luting cements were also evaluated by quasi-static and dynamic nanoindentation tests. Results The results of infrared spectrophotometry and nanoindentation testing were proportional in samples without functional monomer pretreatment. When considerable residual monomer remains within the final products, the mechanical properties of the resin-based luting cements could possibly be impaired. Although the apparent degree of conversion increased with a mixture of functional monomer, a reduction in the cross-linking polymer network may have resulted in the highest viscoelastic creep behavior of the luting cement. The time-dependent behaviors found in the nanoindentation tests likely resulted from linear polymerization chains of the functional monomer. Significance The application of an acidic functional monomer may affect the viscosity of resin-based luting cements. Quasi-static or dynamic nanoindentation is a useful tool for assessing the polymerization qualities of resin composites.

A 52-year-old man was scheduled for the repair of inguinal hernia recurrence. When he was 48 years of age, he received a heart transplantation due to severe heart failure resulting from ischemic heart disease. When he was 50 years old, he suffered from inguinal hernia, and it was repaired under spinal anesthesia. During this surgery, he experienced pain because of the inadequate effect of anesthesia, but his blood pressure and heart rate were stable. We suspected that this was because of denervation of the heart. On hernia repair for inguinal hernia recurrence, general anesthesia was chosen, induced with midazolam, rocuronium, and fentanyl and maintained with sevoflurane, rocuronium, fentanyl, and remifentanil. The blood pressure was mostly stable during anesthesia, but we noted an increase in the heart rate when the trachea was intubated and extubated and when surgical incision started. This phenomenon may indicate reinnervation of the transplanted heart. We could safely manage anesthesia without invasive monitoring because the transplanted heart functioned favorably and surgery was minimally invasive.

Hereditary sensory and autonomic neuropathy (HSAN) is a rare peripheral nerve disorder associated with sensory dysfunction (pain, touch, and pressure) and various degrees of autonomic dysfunction. We administered general anesthesia for a 54-year-old woman with HSAN type II undergoing amputation of the left hallux. She had reduced sensation for pain, pressure, and temperature since birth and frequently injured her hands and legs. Before the operation, she did not report pain in the hallux. Only propofol was given for anesthesia without use of analgesia. Intraoperatively, her vital signs were stable. To evaluate the sympathetic nervous response to surgical stimulation, we measured the plasma catecholamine levels before tracheal intubation and just before and during surgery. Plasma catecholamine levels were normal at all time points, indicating no sympathetic responses to surgical stimulation. This case suggests that anesthesia for HSAN II patients can be safely managed with propofol alone.

Dental fear in children can be a psychological barrier for maintenance of oral health, thus it is important to objectively evaluate the responses to stress stimuli produced during dental treatments. In this study, we analyzed the stress responses of 15 preschool (3- to 6-year) and 22 school-aged (6- to 12-year) children to dental air turbine noise by measuring salivary levels of chromogranin A (CgA), a marker of psychological stress. Salivary CgA was significantly increased during exposure to the noise in the school-aged subjects, especially in those who had previously experienced tooth excavation, while there was no significant difference in CgA levels before and during noise exposure in those without such experience. In the preschool children, the noise did not induce increased secretion of CgA even in those with previous excavation experience. In addition, the levels of dental fear were assessed using a dental subscale of children's fear survey schedule (CFSS-DS), while reactions during dental care were scored according to the Frankl Behavior Rating scale. In the school-aged children, change in salivary CgA level was not correlated with CFSS-DS ( r =0), while there was a weak negative correlation with Frankl score ( r =−10.202). Our results indicate that air turbine noise causes psychological stress in school-aged children, especially in those who have previously received dental treatments.

It is important to know racial differences in the characteristics of the dental arch form from the view point not only of dental interests but also of anthropology. We got an opportunity to investigate the status of oral condition of children living in the Changchum-city, Jilin Province in China. The size and the form of the deciduous dental arch were measured using plaster casts prepared from the impressions taken at the time of oral examination. The same measurement was conducted using the dental plaster cast of the Japanese children and the size and shape of the dental arch were compared between the Japanese and the Chinese children. The subjects were the Chinese children who had the Hellman IIA dentition lining in Changchum-city. The average age of the Chinese children was 9 years and 4 months and that of the Japanese children was 4 years and 1 month. The measurement results showed that the Chinese children were bigger than Japanese children in every measurement items. As for the dental arch form, Japanese children had a U-shaped arch form with wider width in the front part of the dental arch. On the other hand, the Chinese children had a half-circle-shape arch form that the front arch width was not so wide but that of the back was wide.

The aims of this study were to investigate the condition of dental caries in 1–4 years old Japanese children with cleft lip and/or palate, and also to examine the relationships between age, location of the cleft, oral health behavior of cleft children and the incidence of dental caries. The study was carried out through a dental examination of a sample of children and a questionnaire to their parents which included questions about infant feeding practices, frequency of consumption of specific drinks and children's frequency of tooth brushing. A sample of 116, 1–4 years old Japanese children (58 boys and 58 girls) with oral clefts referred to the Department of Pediatric Dentistry Clinic, Showa University, after receiving surgical treatment, participated in this study. Results indicated that the prevalence of caries in cleft children was related to patient age, location of clefts and oral health behavior. A higher level of dental caries was recognized in children with cleft lip and alveolus/palate than in those with cleft lip alone. Incidence of caries increased with patient age. Children who had been fed in a determined time were less affected than those who had been fed at will. Incidence of caries was also high in the children who ingested drinks with sugar than in those who did not. The results of this study suggest that it is important to improve the oral health behavior of cleft children to minimize the risk of caries. Moreover, children with clefts and their parents should as early as possible undergo a preventive program to ensure preservation of primary dentition.

Previous studies using a sortase-deficient mutant (SrtA − mutant-) of Streptococcus mutans have demonstrated that the sortase (SrtA) catalyzes cell wall anchoring of the surface protein antigen PAc, a dextranase and a glucan-binding protein C. In this study, cell wall anchoring of a wall-associated protein antigen A (WapA) and an exo- β -D-fructosidase (FruA) in S. mutans was examined by Western blot analysis with a specific antiserum. In the SrtA − mutant, FruA and WapA were not bound to the cell wall but were secreted into the culture supernatant. In contrast, in the wild type, both proteins were associated with the cell wall of S. mutans . Biological properties of the SrtA − mutant were examined by determination of fructan fermentation and adherence to a smooth surface. Both the SrtA − mutant and the wild type retained the ability to ferment levan. In addition, adherence to a smooth surface of the SrtA − mutant was as extensive as that of the wild-type 109c when sucrose was present. However, in the absence of sucrose, the adhesion of the SrtA − mutant remarkably decreased as compared with that of the wild type. These results suggest that SrtA catalyzes anchoring of WapA and FruA to the cell wall in S. mutans and that surface proteins anchored by SrtA are involved in the initial adhesion of S. mutans to smooth surface. In addition, it was shown that both cell wall-anchored and extracellular FruA are related to the degradation of extracellular fructan as a nutrient source.

To evaluate the ease of manipulation and durability of 11 commercially available silicone-based resilient denture liners, extrusion force, hardness, weight change, and bond strength were determined. Extrusion force from the cartridge of each material ranged from 0.25 to 1.26 MPa at an extrusion rate of 1 cm/min. Durometer hardness, after set materials were stored in distilled water at 37 degrees C for one day, ranged from A5.9 to A47.7, and after four weeks their values increased by 4.0 to 275%. Bond strength ranged from 1.01 to 2.88 MPa after set materials were stored in distilled water at 37 degrees C for one day, but decreased to 0.59 to 1.99 MPa after 10,000 thermal cycles. These results suggested that except for one material, the rest of the evaluated materials exhibited good handling properties--for example, mixing and spreading of material can be done easily. However, some materials exhibited inadequate durability for clinical service, because hardness increased during storage and/or bond strength decreased after thermal cycling.

The purpose of the present study was to evaluate the efficacy of an experimental self-etching dentin primer composed of TEGMA and phenyl-P using primary and young permanent teeth. The efficacy of the self-etching dentin primer was evaluated by measuring the wall-to-wall polymerization contraction gap width and the shear bond strength to the flat dentin surface.The contraction gap formation was prevented completely in the specimens primed with the 35 vol% TEGMA and 20% phenyl-P for 30 sec.

The aim of this study was to investigate the setting characteristics of seven silicone-based resilient denture liners during setting. Maximum extrusion force--at the flow rate of 1 cm/min--ranged from 0.183 to 1.26 MPa. Working time and setting time ranged from 2.0 to 5.0 minutes and 1.9 to 6.8 minutes respectively. Consistency as described in JIS T6519-2000 was 32.4 to 46.5 mm. As normal load of 1 kgf was applied to a specimen held between two glass plates after mixing, the time at which film thickness reached up to 1 mm was 4.0 to 13.0 minutes at 23 degrees C and 2.2 to 5.5 minutes at 37 degrees C. These results suggested that the reaction rate of materials used in this study differed from one brand to another. Against this backdrop of differing reaction rates amongst brands, it is therefore vital that a denture liner material's setting characteristics be carefully considered whenever it is used in clinical dentistry to prevent any undesirable or unexpected results and outcome.

The purposes of this study were to investigate cavity surfaces morphologically, and compare microleakage at cavities prepared by Er:YAG laser after composite resin restoration versus conventional mechanical treatment in human primary teeth in vitro.There have been few reports on microleakage at cavities prepared by Er:YAG laser irradiation.A total of 30 cavities (class V) in human primary teeth were used. Half of the cavities were prepared by an Er:YAG laser system at 300 mJ pulse energy and 4 Hz, and the other half were prepared with a high-speed diamond bur. Five cavities from each group were investigated by scanning electron microscopy (SEM) and histopathological examination. Remaining cavities were filled with a composite resin without an acid-etching technique and then subjected to microleakage test in 0.6% rhodamine B solution under thermocycling.Microleakage (score: 2.45 +/- 1.07) at cavities prepared by laser was significantly less than that by bur (score: 1.30 +/- 0.95; p0.05). SEM observation showed that, compared with the relatively flat appearance of cavities prepared by bur, cavity margins prepared by laser were irregular but there was almost no smear layer at the cavity walls.It can be concluded that cavity surfaces prepared by Er:YAG laser are irregular, but microleakage at cavities prepared by the laser after filling with composite resin is better than that by mechanical bur using the dye penetration method.

The purpose of the present study was to investigate the efficacy of dentin adhesives and to examine acid resistance in primary and permanent teeth. The efficacy of dentin adhesives was evaluated by SEM observation and by measuring the wall-to-wall polymerization contraction gap and dentin hardness before and after conditioning. The detailed mechanism of dentin bonding was the same in both primary and permanent teeth.

Although it is known that osteoclasts are multinucleated cells that are responsible for bone resorption, the mechanism by which their size is regulated is unclear. We previously reported that an actin-rich superstructure, termed the zipper-like structure, specifically appears during the fusion of large osteoclast-like cells (OCLs). Actin cytoskeleton reorganization in osteoclasts is regulated by a signaling network that includes the macrophage colony-stimulating factor (M-CSF) receptor, a proto-oncogene, Src, and small GTPases. Here, we examined the role of actin reorganization in the multinucleation of OCLs differentiated from RAW 264.7 cells using various pharmacological agents. Jasplakinolide, which stabilizes actin stress fibers, induced the development of small OCLs, and the Src inhibitor SU6656 and the dynamin inhibitor dynasore impaired the maintenance of the podosome belt and the zipper-like structure. These inhibitors decreased the formation of large OCLs but increased the number of small OCLs. M-CSF is known to stimulate osteoclast fusion. M-CSF signaling via Src up-regulated Rac1 activity but down-regulated Rho activity. Rac1 and Rho localized to the center of the zipper-like structure. Rho activator II promoted the formation of small OCLs, whereas the Rho inhibitor Y27632 promoted the generation of large OCLs. These results suggest that the status of the actin cytoskeleton signaling network determines the size of OCLs during cell fusion.

Fusarium oxysporum, a soilborne phytopathogenic fungus, produces endo polygalacturonase (PG) which has been suggested to be related to disease development. A polyclonal antibody (PAb) APG1 was prepared against purified PG from F. oxysporum f. sp. lycopersici race 2. By direct tissue-blotted immunobinding assay (DT-IBA) with the antibody, PG production by the pathogen in the stem of the host plant was confirmed. Degenerate primers were designed based on partial amino acid residue information for the PG protein from the fungus: part of the PG-encoding gene was obtained by PCR. Using TAIL-PCR, the complete gene encoding PG was cloned and sequenced. The structural gene comprises 1318 bp coding for 371 amino acids with a putative signal peptide of 22 amino acids, and the open reading frame is interrupted by four introns of 47, 51, 50, and 54bp. The deduced amino acid sequence of the mature protein showed 82.9, 29.0, 27.6, 14.2, and 26.9% homology with those of F. moniliforme PG, Cochliobolus carbonum PGNI, Aspergillus niger PG, A. oryzae PG, and Sclerotinia sclerotiorum PGI, respectively.

Fructanase enzymes hydrolyze the β-2,6 and β-2,1 linkages of levan and inulin fructans, respectively. We analyzed the influence of fructan on the growth of Prevotella intermedia. The growth of P. intermedia was enhanced by addition of inulin, implying that P. intermedia could also use inulin. Based on this finding, we identified and analyzed the genes encoding a putative fructanase (FruA), sugar transporter (FruB), and fructokinase (FruK) in the genome of strain ATCC25611. Transcript analysis by RT-PCR showed that the fruABK genes were co-transcribed as a single mRNA and semi-quantitative analysis confirmed that the fruA gene was induced in response to fructose and inulin. Recombinant FruA and FruK were purified and characterized biochemically. FruA strongly hydrolyzed inulin, with slight degradation of levan via an exo-type mechanism, revealing that FruA is an exo-β-d-fructanase. FruK converted fructose to fructose-6-phosphate in the presence of ATP, confirming that FruK is an ATP-dependent fructokinase. These results suggest that P. intermedia can utilize fructan as a carbon source for growth, and that the fructanase, sugar transporter, and fructokinase proteins we identified are involved in this fructan utilization.

The aim of this study was to investigate the relationships between eruption of deciduous teeth and eating habits determined by health examinations of infants.We verified eruption of deciduous teeth based on observations of 455 fourteen-month-old infants at health examinations in a ward of Tokyo, and performed a questionnaire survey involving their mothers regarding the hardness of infants' meals and their eating habits. We examined 420 infants excluding 17 whose births were 'pre-term delivery (born at or before 36 weeks)' and 18 whose questionnaire had excessive omissions.The percentage of infants who began a weaning diet at 5 to 6 months of age was 81.4%, and 71.2% of mothers considered their infant's age in months before starting a weaning diet. We divided the children into three stages: those not showing full eruption of the eight front deciduous teeth (stage I, 27.4%); those with full eruptions of the eight front deciduous teeth excluding the first primary molars (stage II, 61.9%); those with full eruptions of the first primary molars (stage III, 10.7%). Most mothers cooked meals considering the hardness of the gingival gums (stage I; 53.5%, stage II; 54.4%, stage III; 40.0%). The percentage of mothers who cooked meals considering the hardness of the primary molars was 14.0 and 15.1% in stages I and II, respectively. In addition, the percentage who cooked meals while considering the hardness in relation to adult meals was 7.0, 9.7, and 24.4% in stages I, II, and III, respectively. Moreover, the percentage considering the salt-taste in relation to adult meals was 13.2, 17.3, and 22.2% in stages I, II, and III, respectively.In the present study, we obtained valuable data showing that the timing deciduous teeth eruption varies among individuals. These results suggested that nutritional education on the appropriate quality of meals for infants based on their state of deciduous teeth eruption is necessary.

Dental materials that fluoresce affect the reading of the laser fluorescence device DIAGNOdent. Tetracycline hydrochloride (TCH) shows fluorescence and is retained in teeth. The aim of this study was to evaluate the effects of TCH on the DIAGNOdent reading. Filter-paper discs that contained various amounts of TCH were prepared (0.16–10 mg per disc). One-day-old newborn rats were subcutaneously injected with TCH for 29 days, and their mandibles were then removed. The DIAGNOdent values (D-V) of the discs and first molars of the rats were measured before and after they were subjected to ultraviolet irradiation (UV). The D-V of discs containing TCH increased depending on the amount of TCH. The D-Vs of discs with lower amounts of TCH (0.16–1.25 mg) were approximately 10–15, and these values increased to 30–40 under UV. In addition, the D-Vs of molars after UV were twofold greater than those before UV. These results suggest that TCH might affect the readings obtained by DIAGNOdent.

To detect the presence of Porphyromonas gingivalis (Pg) and Actinobacillus actinomycetemcomitans (Aa) using polymerase chain reaction (PCR) in the oral plaque samples from children and investigate the relationship between bacteria and clinical parameters.A total of 151 children aged 7 to 12 years were selected from Changchun Ziqiang primary school. The supragingival plaque sample was collected from the mesiobuccal and labial surfaces of the right maxillary central incisor and the right maxillary first molar. Extracted DNA from plaque samples was used for PCR analysis. Intraoral examination, probing depth (PD) and bleeding on probing (BOP) were performed and recorded.The detection rate for Pg was 27.6% and Aa 54.3% in supragingival plaque. The detection rates for Pg in molars were much higher than those in incisors (P0.01). The detection rate of Pg was positively related to BOP+ and PD. The detection rate for Pg increased gradually with aging, and the detection rate for Aa was highest in the group aged 11 to 12 and the detection rates for Pg and Aa were higher in the gingiva with BOP+ than that with BOP- (P0.05). The detection rates for Pg increased remarkably with BOP+ and especially when PD was greater than 4 mm.Detection rates of putative periodontal pathogens from healthy children of 7 to 12 years of age were high. The detection rates for Pg in molars were much higher than those in incisors,and the presence of Pg and Aa in supragingival plaque was related to periodontal parameters.

The tissue-nonspecific alkaline phosphatase (TNSALP) gene in four unrelated patients with hypophosphatasia was analyzed using polymerase chain reaction–single strand conformation polymorphism and the direct sequencing method. Of the participating patients, one had childhood-type and three had perinatal-type disease. All carried a deletion of T at cDNA number 1559, which causes a frameshift downstream from codon L503, as a heterozygote. In the childhood-type patient, an F310L mutation was detected in the opposite allele. Similarly, a perinatal-type patient carried a V365I mutation in the opposite allele. Mutations in the opposite alleles were not detected in the other two patients with perinatal-type disease. In addition, although both parents carried the deletion as a heterozygote in two families with childhood-type and perinatal-type disease, patients from those families were not homozygous for the deletion. Several single-nucleotide polymorphisms (SNPs) were also detected, which were shown to be useful for haplotype analysis. Allele frequency of the deletion among Japanese patients was 36% (10 of 28 alleles) but none occurred in Caucasian patients. These findings indicate that regardless of clinical type, deletion in the TNSALP gene occurs frequently among Japanese patients. Furthermore, haplotype analysis using SNPs suggested that the deletion might have derived from more than a single founder.

The xenoestrogenic substance bisphenol-A is widely used as a synthetic precursor of resin monomers, such as bisphenol-A diglycidyl methacrylate. Reports describing the release of bisphenol-A from polymerized resin into saliva have aroused considerable concern regarding exposure to xenoestrogen by dental treatment. The purpose of the present study was to demonstrate a reliable methodology of detecting the trace amounts of bisphenol-A in dental materials. Bisphenol-A was separable from bisphenol-A diglycidyl methacrylate, which is often employed as the principal dimethacrylate monomer, by selective extraction with a Sep-Pak C18 cartridge. Using this extraction method in combination with a gas-chromatography mass-spectrometry, we have obtained evidence that all unpolymerized materials used in this study were contaminated with bisphenol-A. Quantitative analysis using a deuterium-labeled compound as an internal standard revealed bisphenol-A contents in commercial dental materials ranging from < 1 microgram/g material to about 20 micrograms/g material. The polymerized dental materials released up to 91.4 ng bisphenol-A/g material into phosphate buffered saline during 24-h incubation. These results indicate that bisphenol-A can be released from dental materials, however the leachable amount would be less than 1/1000 of the reported dose (2 micrograms/kg body weight/day) required for xenoestrogenisity in vivo.

Qualitative and quantitative analyses were conducted on four kinds of resin-modified glass ionomer (RMGI) cement liquids, LC, LC II, LC III (hereinafter referred to as LCs) and VM, using HPLC and laser Raman spectroscopic methods. HPLC revealed that among the RMGI liquids LCs contain 31-32% HEMA (2-Hydroxyethyl methacrylate), and VM contains 18% of the same. The composition of RMGI cement liquids varied significantly between manufacturers. In Raman spectroscopic analyses, the spectra of liquids of various ratios of polyacrylic acid and HEMA were measured, and calculations were made on the peak intensity ratios of C=C stretch vibration to C=O stretch vibration, common in both HEMA and polyacrylic acid. The composition ratio of polycarboxylic acid to HEMA of commercial glass ionomer cements was assessed by the regression curve generated by a combination of peak intensity ratios and composition ratios. In addition, Raman spectroscopy was able to identity the differences in form of the methacryloyloxy group.