Detection of antigenic proteins in Porphyromonas gingivalis using two-dimensional electrophoresis and western blots

Using two-dimensional (2D) electrophoresis and western blot assay, we analyzed antigenic proteins in Porphyromonas gingivalis uniquely recognized by antibodies in sera of periodontitis subjects. Proteins in the total membrane fraction of P. gingivalis 381 were resolved into at least 70 protein spots by 2D electrophoresis. In the gel stained with silver, the substance around 47 kDa protein on the acidic side (at an isoelectric point of about 4.5) was stained as a smear. Antigenic substances were characterized using purified IgGs from sera of 16 adult periodontitis (AP), 19 rapidly progressive periodontitis (RPP) and 14 periodontally healthy volunteers. Western blots demonstrated that 75 kDa protein reacted with IgGs from 75% of AP patients (p>0.001), the antigenic substance around acidic 47 kDa protein reacted with IgGs from 81.3% of AP (p>0.01) and 68.4% of RPP patients (p>0.01) and the acidic 47 kDa protein reacted with 87.5% of AP (p>0.01) and 78.9% of RPP patients (p>0.01). The reaction frequency was significantly different from that of the healthy volunteers. Also 51 kDa and 41 kDa proteins reacted with 47 and 43 of 49 IgG samples, respectively. The substance around acidic 47 kDa protein reacted with mouse antiserum to P. gingivalis-LPS. After treatment with pronase or heat, the antigenic reactions disappeared not only from the proteins, but also from the area around the acidic 47 kDa protein. When the fraction was digested with lipase, the antigenic reaction of the area decreased. It appeared that the acidic 47 kDa protein and the LPS around it formed a complex, and that the LPS contained the lipid portion. These results suggested that the acidic 47 kDa protein can be classified among the lipid A-associated proteins, and that it is the major antigenic protein which is recognized by antibodies in sera of almost periodontal patients in P. gingivalis 381.     

Cadherin-related neuronal receptors in incisor development

Cadherins are cell adhesion molecules that are critical for tissue development. In this report, we identified members of the cadherin family cadherin-related neuronal receptors (CNRs) 1 and 5 expressed in rat incisors by the differential display method. Quantitative RT-PCR revealed that CNR1 mRNA is expressed in the secretory stage but reduced in the early-maturation stage, while CNR5 mRNA is expressed in both these stages. In situ hybridization showed that strong expression of CNR1 is strong in the secretory stage, but reduced in the early phase and diminished in the late phase of the early-maturation stage. CNR5 mRNA is expressed almost at the same levels in the secretory and in the early phase of the early-maturation stages but is absent in the late phase of the early-maturation stage. Both CNR1 and 5 mRNA are continuously expressed in odontoblasts. Immunohistology showed that CNR proteins are expressed in the secretory and early-maturation stages of ameloblasts, but no protein expression at the late-maturation stage was observed. CNR proteins were continuously expressed in odontoblasts. We found that recombinant CNR1 binds dental epithelial and mesenchymal cells through N-terminal domain EC1 in vitro. These results suggest that CNR1 and CNR5 may play an important role in enamel and dentin formation, probably through cell-cell and/or cell-matrix interactions.     

Evidence that experimentally induced sleep bruxism is a consequence of transient arousal

Spontaneous rhythmic masticatory muscle activity (RMMA) during sleep occurs more frequently following spontaneous transient micro-arousal in patients with sleep bruxism (SB) and normal controls. Here, we tested the hypothesis that an experimental arousal would be followed by an increase in RMMA. We identified RMMA on polygraphic recordings taken before and after sensory stimulation to induce experimental arousal in eight SB patients and eight matched normal subjects. The rate of experimental arousal and the level of resting electromyographic activity in masseter and suprahyoid muscles during sleep did not differ between the groups. In both, muscle tone and heart rate increased during the experimental arousal. Although post-arousal RMMA occurred in all SB patients, it was seen in only one normal subject. Moreover, tooth-grinding occurred during 71% of the evoked RMMA in SB patients. These results support the hypothesis that SB is an exaggerated form of oromotor activity associated with sleep micro-arousal.     

Development of casting investment preventing blackening of noble metal alloys Part 2. Application of developed investment for type 4 gold alloy

The objective of this study was to evaluate the efficacy of the developed investment for the prevention of blackening of a cast Type 4 gold and to analyze the oxides on its surface in relation to the blackening of the alloy. The experimental investments were prepared using a gypsum-bonded investment in which boron (B) or aluminum (Al) was added as a reducing agent. A Type 4 gold alloy was cast into the mold made of the prepared investment. The effect of the additives was evaluated from the color difference (deltaE*) between the as-cast surface and the polished surface of the cast specimen. B and Al were effective to prevent the blackening of a Type 4 gold alloy and the color of the as-cast surface approached that of the polished surface with increasing B and Al content. The prevention of the blackening of the gold alloy can be achieved by restraining the formation of CuO.     

Involvement of caspases in apoptotic cell death of murine macrophages infected with Actinobacillus actinomycetemcomitans

Infection of murine macrophages in vitro with periodontopathic bacterium Actinobacillus actinomycetemcomitans induces apoptotic cell death. In this study, we investigated the involvement of caspases in apoptotic cell death of A. actinomycetemcomitans-infected macrophages. Two peptide inhibitors of caspases, benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone (Z-DEVD-FMK), inhibited apoptotic cell death of murine macrophage cell line J774.1 infected with A. actinomycetemcomitans. During the process of apoptosis, interleukin-1beta (IL-1beta) was detected in the culture supernatants of J774.1 cells. IL-1beta secretion was blocked by the caspase-1 inhibitor, Z-VAD-FMK, indicating that caspase-1 is involved in not only the induction of apoptosis but also the IL-1beta secretion from A. actinomycetemcomitans-infected J774.1 cells. Immunoblot analysis revealed that the infection of A. actinomycetemcomitans to J774.1 cells induced the cleavage of retinoblastoma protein (Rb), suggesting that caspase-3 was activated by A. actinomycetemcomitans infection. The cytosol from A. actinomycetemcomitans-infected J774.1 cells induced Rb proteolysis in vitro, which was inhibited by the caspase-3 inhibitor, Z-DEVD-FMK. Furthermore, caspase-3-like activity was markedly increased in J774.1 cells infected with A.actinomycetemcomitans between 12 h and 24 h, which was subsequently inhibited by the addition of caspase-3 inhibitor, Z-DEVD-FMK. These findings indicate that caspase-3 induces apoptosis in J774.1 cells infected with A. actinomycetemcomitans. Taken together, these results suggest that caspase-1 and caspase-3 are involved in the induction of apoptosis in A. actinomycetemcomitans-infected macrophages.     

A comparison of mesial molar root canal preparations using two engine-driven instruments and the balanced-force technique

The purpose of this study was to compare the effects of two engine-driven, nickel-titanium instrument systems with hand files in the final shape of slight and moderately curved canals. A total of 72 mesial roots of extracted human mandibular molars were divided into three groups: ProFile .04 taper, Pow-R rotary systems, and Flex-R hand-filing technique. The roots were mounted and cross-sectioned at two different horizontal levels using a modified Bramante technique. Pre- and postinstrumented cross-sectional roots were imaged, recorded, and computer analyzed. Results showed that, at the middle third, in almost all groups, there was a tendency of cutting more toward the mesial side with only one exception: Pow-R cut more to the distal side (danger zone) (p < 0.02). At the apical third, Flex-R (p < 0.03) and ProFile (0.001) transported to the mesial side (danger zone) when the curvature increased. When the three techniques were compared analyzing each side and considering the two groups of curvature, at the middle third in the moderately curved-canal group, Flex-R cut statistically more than Pow-R toward the lingual side. The other comparisons showed no statistically significant difference. When the techniques were compared in relation with the degree of curvature, in the apical third, ProFile .04 cut statistically more toward the mesial side in the moderately curved canal group than in the slightly curved canal group. The other comparisons showed no statistically significant difference. Canal preparation time was shorter with hand instrumentation (p < .05) in a few instances.     

Two-dimensional changes in soft tissue profile following surgical mandibular advancement in Japanese retrognathic adults

The purpose of this study was to evaluate changes in the hard and soft tissues and to test a preliminary method for predicting the soft tissue profile changes in 14 Japanese retrognathic patients who underwent surgical mandibular advancement. A pair of pre- and postoperative lateral cephalograms was taken for each subject. A paired t test identified significant hard and soft tissue changes following surgery. A Pearson correlation analysis was used to identify soft tissue changes for hard tissue changes in the horizontal and vertical dimensions. Multiple regression models were generated to predict postoperative variables based on preoperative variables. Significant changes in the hard and soft tissues were found in the area inferior to the point Stomion in both the horizontal and vertical dimensions. The soft tissue changes demonstrated a significant correlation not only with the corresponding hard tissue, but also with noncorresponding anatomical points. Moreover, the tendency of changes in the soft tissue was different in the lower lip area from the chin area in both the horizontal and vertical dimensions. Significant R2 and higher contribution ratio were obtained from the multiple regression analysis for the chin area compared to the lower lip area.     

Histological study of lectin binding in regenerated rat junctional epithelium

We investigated the expression of carbohydrate residues in regenerated junctional epithelium (JE) cells histopathologically with lectin staining to clarify the mechanisms responsible for the changes in their expression in JE cells derived from residual oral epithelium. Curettage and root planing procedures were performed on the buccal gingival sulci of rat first lower molars, and JE and connective tissues were completely removed. The mandibles were resected after 12 h and 1, 2, 3, 4, 5, 7, 10 and 14 days, fixed with paraformaldehyde, decalcified with EDTA and embedded in paraffin. Serial sections were stained histochemically with four kinds of lectins (PNA, DBA, GS I, UEA I) to clarify the expression patterns of carbohydrate residues in regenerating epithelium. No binding of PNA or DBA was observed even when the regenerating epithelium was attached to the root surface, and they showed the same negative reactions as the basal cells of oral gingival epithelium (OGE). Positive reactions were, however, observed on the more stratified regenerating epithelium along the root surface. Positive reactions with GS I and negative reactions with UEA I were observed throughout the regeneration process, and these were the same as those observed in the basal cells of OGE. Therefore, we concluded that the basal cells and regenerated epithelium derived from OGE expressed the same carbohydrate residues. Futher-more, the expression of carbohydrate residues, one of the characteristics of JE, was related not only to the attachment to the tooth surface but also to changes of cell shape and cytoskeleton with stratification along the root surface.     

T2-weighted MRI for the assessment of joint effusion: comparative study of conventional spin-echo and fast spin-echo sequences

OBJECTIVE: This study aimed to evaluate the usefulness of suitable conventional spin-echo (CSE) and fast spin-echo (FSE) T2-weighted imaging parameters for the assessment of joint effusion in a phantom study and in a comparative study of CSE and FSE using clinical cases. STUDY DESIGN: In the phantom study, the signal ratios of water and oil signal fields were determined and studied comparatively. The shape and size of signals were evaluated separately. In the study of joint effusion images, 318 joints were evaluated. CSE T2-weighted imaging and FSE T2-weighted imaging were carried out, and a comparative assessment was performed. RESULTS: In both CSE and FSE imaging, the ratios of mean MRI signal values showed divergence as TR/TE values increased. The evaluation of joint effusion with FSE TR/TE 8000/120 msec was significantly better than that in all other groups (P <.01). CONCLUSION: The use of FSE requires investigation of TR/TE values. When a 0.5 T static field strength MRI apparatus is employed, TR/TE 8000/120 msec is recommended.     

Fluoride and magnesium concentrations in human dental calculus obtained from Japanese and Chinese patients

Calculus deposited on a total of 68 permanent teeth from patients 30-60 years old from Nagoya in Japan and Beijing in China was investigated. An abrasive microsampling method was used to examine the fluoride (F) and magnesium (Mg) distribution, using a fluoride ion-specific electrode and atomic absorption spectrophotometry, respectively. F concentrations decreased from the surface towards the interior of the calculus. Mg concentrations, however, gradually rose towards the innermost surface adjacent to the tooth. In all parts of the depth profiles, the average concentrations of both magnesium and fluoride were higher in the Japanese than in the Chinese calculus. Towards the inner surface of the calculus, F and Mg concentrations were also much higher in the Japanese than in the Chinese group. A greater intake of sea foods and greater use of fluoride dentifrices are possible reasons for the higher F and Mg concentrations in the Japanese individuals.