Inhibitory effects of Oenothera biennis (evening primrose) seed extract on Streptococcus mutans and S. mutans-induced dental caries in rats.

BACKGROUND: Oenothera biennis (evening primrose) seed extract (OBSE) is known to contain polyphenols, which may possess antioxidant activities. Polyphenols extracted from several plants are reported to exhibit cariostatic activities by inhibiting mutans streptococcus growth and glucosyltransferase activities. The purpose of the present study was to examine the inhibitory effects of OBSE on the development of dental caries, both in vitro and in vivo. METHODS: OBSE was investigated for its inhibitory effects on cellular aggregation, hydrophobicity, sucrose-dependent adherence and insoluble glucan synthesis. Furthermore, biofilm formation was examined in the presence of OBSE, using confocal microscopic imaging. An animal experiment was also performed to examine the in vivo effects. RESULTS: OBSE induced a strong aggregation of Streptococcus mutans MT8148 cells, while cell surface hydrophobicity was decreased by approximately 90% at a concentration of 0.25 mg/ml. The sucrose-dependent adherence of the MT8148 cells was also reduced by addition of OBSE, with a reduction rate of 73% seen at a concentration of 1.00 mg/ml. Additionally, confocal microscopic observations revealed the biofilm development phase to be remarkably changed in the presence of OBSE. Furthermore, insoluble glucan synthesis was significantly reduced when OBSE was present at concentrations greater than 0.03 mg/ml. In an animal experiment, the caries scores in rats given OBSE (0.05 mg/ml in drinking water) were significantly lower than those in rats given water without OBSE. CONCLUSION: Our results indicate that OBSE has inhibitory activity on dental caries.

Characterization of aortic aneurysms in cardiovascular disease patients harboring Porphyromonas gingivalis.

OBJECTIVE: Porphyromonas gingivalis was recently shown to cause intimal hyperplasia in a mouse model by a novel cholesterol-independent mechanism, suggesting to be a pathogen-specific feature of cardiovascular diseases. The aim of this study was to characterize the clinical and histopathological features of aortic aneurysms in cardiovascular disease patients harboring oral P. gingivalis. SUBJECT AND METHODS: Aortic aneurysm specimens were collected from 76 Japanese patients who underwent surgery, of whom dental plaque specimens were also collected from 31 patients. Bacterial DNA was extracted from each specimen to detect P. gingivalis by polymerase chain reaction. Histopathological analyses of the aortic aneurysm specimens, including immunohistochemical staining for embryonic myosin heavy chain isoform (SMemb) and S100 calcium-binding protein A9 (S100A9), were also performed. RESULTS: The number of aneurysms occurring in the distal aorta was significantly higher in subjects positive for P. gingivalis in dental plaque compared with those who were negative. The expressions of S100A9 and SMemb were also significantly greater in the subjects positive for P. gingivalis in dental plaque. On the other hand, there were no significant differences in adipocellular accumulation between the groups. CONCLUSIONS: These results suggest that aortic aneurysms in patients harboring oral P. gingivalis have greater expression of S100A9 and proliferative smooth muscle cells, which was different from the present patients without oral P. gingivalis.

Molecular interactions of alanine-rich and proline-rich regions of cell surface protein antigen c in Streptococcus mutans.

INTRODUCTION: Streptococcus mutans has been implicated as a primary causative agent of dental caries in humans, and its cell surface protein antigen c (PAc) is known to be associated with sucrose-independent adhesion to tooth surfaces. PAc is composed of several domains, including an N-terminal signal sequence, an alanine-rich repeat region (A-region), a proline-rich repeat region (P-region), and an anchor region. METHODS: To investigate the functions of each domain, an A-region-deficient mutant strain of S. mutans was constructed, and recombinant PAc and A- and P-region proteins were also constructed. The interactions of each domain with the recombinant proteins were analyzed using surface plasmon resonance spectroscopy with a biomolecular interaction analyzing system. RESULTS: The A-region-deficient mutant strain showed the lowest levels of adherence to saliva-coated hydroxyapatite. Furthermore, findings in an immunoblot assay indicated that the A-region protein reacted strongly with proline-rich proteins in saliva, while the recombinant P-region protein interacted more quickly with PAc than the recombinant A-region protein. CONCLUSION: These results suggest that the A-region has a strong relationship with adhesion to tooth surfaces, while the P-region has a high affinity for PAc.

Distribution of Porphyromonas gingivalis fimA genotypes in cardiovascular specimens from Japanese patients.

INTRODUCTION: Porphyromonas gingivalis, a major periodontal pathogen, is gaining increasing attention for its possible association with cardiovascular diseases. Its fimbriae are classified into six genotypes (types I-V and Ib) based on the diversity of the fimA genes encoding the fimbrial subunits. In this study, fimA genotypic distribution was analyzed in P. gingivalis-infected cardiovascular specimens. METHODS: A total of 112 heart valves and 80 atheromatous plaque specimens were collected from patients undergoing cardiovascular surgery, as well as 56 dental plaque specimens. Bacterial DNA was extracted from each, and polymerase chain reaction analysis was carried out with a P. gingivalis-specific set of primers. P. gingivalis-positive specimens were further analyzed to discriminate the fimA genotype using polymerase chain reaction with fimA type-specific primer sets. RESULTS: P. gingivalis was detected in 10.4% of the cardiovascular specimens and 50.0% of the dental plaque samples. In the latter, type II was most frequently detected (35.7%), followed by types I (28.6%) and IV (21.4%), while types IV and II were detected with considerable frequencies of 45.0% and 30.0%, respectively, in the cardiovascular specimens. In contrast, the occurrence of type I was limited (5.0%) in the cardiovascular specimens. CONCLUSION: These results suggest that specific fimA genotypic clones, which are reportedly associated with periodontitis, are also frequently harbored in cardiovascular specimens, indicating the possible involvement of type II and IV clones in the initiation and progression of cardiovascular diseases.

Heterogenic virulence and related factors among clinical isolates of Porphyromonas gingivalis with type II fimbriae.

BACKGROUND/AIMS: Porphyromonas gingivalis is a periodontal pathogen whose fimbriae are classified into six genotypes (types I-V and Ib) based on the diversity of the fimA genes encoding the fimbrial subunits. Accumulated evidence suggests that P. gingivalis strains with type II fimbriae are more virulent as compared to those with other types. However, it is unknown if strong virulence is uniformly conserved among clones with type II fimbriae. In the present study, we compared infectious inflammatory changes in clinical isolates of P. gingivalis with type II fimbriae using a mouse abscess model to examine their pathogenic heterogeneity and heterogeneity-related factors. METHODS: Suspensions of nine different clinical isolates with type II fimbriae were subcutaneously injected into female BALB/c mice and inflammatory parameters, such as serum sialic acid concentration, were compared. RESULTS: Many of the type II fimbrial isolates caused severe inflammation in the mice, though some were less causative, as was the control strain ATCC 33277 (type I fimbria strain). These results showed that pathogenic heterogeneity exists among P. gingivalis clones with type II fimbriae. Further, the heterogeneity-related factors of P. gingivalis strains were analyzed and the pathogenic potentials showed positive relationships to gingipain activities and invasive efficiency but not to hydrophobicity or autoaggregation. In addition, invasive efficiency was related to the activities of gingipains that were extracellularly secreted. CONCLUSION: These results suggest that pathogenic heterogeneity has relationships with the invasive and proteolytic activities of P. gingivalis clones with type II fimbriae.

Detection and serotype distribution of Actinobacillus actinomycetemcomitans in cardiovascular specimens from Japanese patients.

Actinobacillus actinomycetemcomitans, an important pathogen in periodontitis, has also been detected in cardiovascular tissues. Sixty heart valves were collected during valve replacement surgery from 60 patients (one from each), 10 were from patients with infective endocarditis (IE group) and 50 were from patients with other valvular diseases (non-IE group). In addition, 46 samples of aneurysmal tissue were taken from 46 patients with a thoracic or abdominal aneurysm (Aneurysm group, one from each). Dental plaque samples were taken from 54 of the patients, 31 in the IE and non-IE groups and 23 in the aneurysm group. First, the distribution of A. actinomycetemcomitans in all specimens was analysed using a polymerase chain reaction method, which resulted in a positive reaction in 33 (31.1%) of the cardiovascular specimens and 25 (46.3%) of the dental plaque samples. Next, using serotype-specific sets of primers, the serotype distribution of A. actinomycetemcomitans in the cardiovascular specimens and dental plaque samples was found to be significantly different compared to dental plaque samples from Japanese subjects reported previously.

Antibacterial activity of polyphenol components in oolong tea extract against Streptococcus mutans.

The purpose of the present study was to determine the antibacterial activity of oolong tea extract on oral streptococci, including Streptococcus mutans and Streptococcus sobrinus, and to identify the response to its components. Antibacterial activity was found when the extract was added to S. mutans cells in chemically defined medium but not in complex broth media. Further, pretreatment with bovine serum albumin reduced the antibacterial activity. The extract showed antibacterial activity against all of the oral streptococci examined, with the highest activity against S. mutans MT8148R. This activity was found to originate from a monomeric polyphenol-rich fraction, and it was stronger than that of pure polyphenols. Moreover, some combinations of monomeric polyphenols showed the highest level of antibacterial activity. These results suggest that the antibacterial activity of oolong tea extract is caused by a synergistic effect of monomeric polyphenols, which can easily bind to proteins.

Molecular analysis of the inhibitory effects of oolong tea polyphenols on glucan-binding domain of recombinant glucosyltransferases from Streptococcus mutans MT8148.

An oolong tea polyphenol (OTF6) has been shown to possess a strong anti-glucosyltransferase (GTF) activity and inhibit experimental dental caries in rats infected with mutans streptococci. The effects of OTF6 on the functional domains of GTFs of Streptococcus mutans, an N-terminal catalytic domain (CAT), and a C-terminal glucan-binding domain (GBD), were examined. The maximum velocity of glucan synthesis by recombinant GTFB (rGTFB) and GTFD (rGTFD) became significantly slower in the presence of OTF6, however, Km values remained stable when compared in their absence. These results suggest that OTF6 reduces glucan synthesis by non-competitively inhibiting the GBD of S. mutans GTFB and GTFD. Further, the recombinant proteins of CAT (rCAT) and GBD (rGBD) were expressed using Escherichia coli, and purified by affinity column chromatography. rGBD but not rCAT was found to possess dextran-binding activity, which was shown to be inhibited by OTF6. These results indicate that OTF6, a polymeric polyphenol specific for oolong tea is able to reduce glucan synthesis by inhibiting the GBD of S. mutans GTFB.

Identification of cariostatic substances in the cacao bean husk: their anti-glucosyltransferase and antibacterial activities.

The cacao bean husk has been shown to possess two types of cariostatic substances, one showing anti-glucosyltransferase (GTF) activity and the other antibacterial activity, and to inhibit experimental dental caries in rats infected with mutans streptococci. In the present study, chromatographic purification revealed high-molecular-weight polyphenolic compounds and unsaturated fatty acids as active components. The former, which showed strong anti-GTF activity, were polymeric epicatechins with C-4beta and C-8 intermolecular bonds estimated to be 4636 in molecular weight in an acetylated form. The latter, which showed bactericidal activity against Streptococcus mutans, were determined to be oleic and linoleic acids, and demonstrated a high level of activity at a concentration of 30 microgram/mL. The cariostatic activity of the cacao bean husk is likely caused by these biologically active constituents.

Radicular cyst associated with a primary molar following pulp therapy: a case report.

A radicular cyst arising from the primary second molar and causing displacement of the permanent successor to the lower border of the mandible, with accompanying buccal expansion, was examined clinically and radiographically. Extraction of the primary molar and extirpation of the cyst led to uneventful healing. The primary molar had received pulp treatment with therapeutic agents approximately 1.5 years prior to the patient's first visit. The relationship between pulp treatment and rapid growth of the radicular cyst is discussed.

Tooth eruption in a patient with craniometaphyseal dysplasia: case report.

Craniometaphyseal dysplasia (CMD) is a very rare genetic disorder of bone remodeling caused by osteoclast dysfunction. The clinical and radiographical features of oral findings are presented in a sporadic case of CMD in a child (age 10 years, 7 months). An intraoral examination showed severe malocclusions, including anterior crossbite and deep bite. Furthermore, a radiographic examination showed increased radiopacity of the maxilla and mandibular bones due to hyperostosis and sclerosis of the jaw. There was no root resorption of the canines or molars in the primary dentition, although root formation of the permanent teeth was proceeding. Dental age was calculated to be approximately 1 year, 4 months younger than his chronological age. The eruption speed of the permanent lateral incisors after the gingival emergence was shown to be within normal values, and we discuss whether the canines and premolars in the permanent dentition could erupt or not.

Cloning and characterization of the human ameloblastin gene.

We isolated the full-length human ameloblastin (AMBN) cDNA clone using reverse transcription-polymerase chain reaction (RT-PCR) methods. Sequence analysis of the AMBN cDNA revealed an open reading frame of 1341bp encoding a 447-amino-acid protein. Comparison with pig, cattle, rat, and mouse AMBN sequences showed a high amino acid sequence similarity and led to the identification of a novel 78bp (26 amino acids) insert resulting from internal sequence duplication. By DNA analysis of a human genomic clones, the AMBN gene was shown to consist of 13 exons and a novel 78bp segment, which proved to comprise two small exons. Human ameloblastomas express AMBN transcripts that contain some mutations.

Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription.

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration.

Calbindin D28k-like immunoreactivity during the formation of the enamel-free area in the rat molar teeth.

Previous studies have demonstrated the presence of calbindin D28k in the ameloblasts derived from the inner enamel epithelium. The occlusal surfaces of the rodent molars partly lack the enamel covering, which is referred to as enamel-free area (EFA). In the present study, we compared the immunohistochemical localization of calbindin D28k-like immunoreactivity (CB-LI) in the cells at the EFA (EFA cells) and ameloblasts of the rat molar teeth at the light microscopic level. CB-LI was strong in the ameloblasts of the presecretory through the protective stages, while it was faint at the late secretory to transitional stages. However, some mature ameloblasts lacked the immunoreactivity. On the other hand, the majority of EFA cells showed distinct polarization and elongation that were absent in few cells at the early stage of EFA formation. At all stages, the EFA cells adjacent to the ameloblasts showed CB-LI, however, some cells adjacent to the mature ameloblasts lacked the reaction. Intensive CB-LI was demonstrated in EFA cells at the reduced enamel epithelium. These immunohistochemical findings suggest EFA cells have cytochemical properties similar to those of ameloblasts.

Simultaneous occurrence of unusual odontodysplasia and oligodontia in the permanent dentition: report of a case.

Odontodysplasia is an uncommon clinicopathological condition with a variety of expressions. Although it is generally recognized as a localized disorder of dental tissue, its aetiology has not yet been well explained. In the present case, odontodysplasia with oligodontia in the permanent dentition is reported. The patient was in good health with normal stature and no other physical abnormalities. His parents and siblings were dentally and medically normal. The primary teeth appeared to be normal except for the primary second molars, where the enamel was malformed. However, the permanent incisors that had erupted into the oral cavity showed rough and hypoplastic enamel. An orthopantomogram showed 17 congenitally missing permanent teeth and malformation of the other 11 permanent teeth and tooth-germs. Because these findings were caused by developmental disturbances of both the mesodermal and ectodermal dental components, we diagnosed the present case as odontodysplasia accompanied by oligodontia in the permanent dentition.

Inhibitory effects of oolong tea extract on caries-inducing properties of mutans streptococci.

The inhibitory effects of oolong tea extract (OTE) on the caries-inducing properties of mutans streptococci were examined in vitro. OTE reduced the rate of acid production by mutans streptococci accompanied with the retardation of growth rate of mutans streptococci, while the action by chromatographically isolated oolong tea polyphenol (OTF6) was weak. On the other hand, both oolong tea products decreased cell surface hydrophobicity of almost all the oral streptococci examined in the present study, and also induced cellular aggregation of Streptococcus mutans, Streptococcus oralis, Streptococcus sanguis or Streptococcus gordonii. In these reactions, OTF6 showed a more pronounced activity than OTE. Furthermore, the oolong tea products inhibited the adherence of mutans streptococci to saliva-coated hydroxyapatite. These results suggest that OTF6 may inhibit bacterial adherence to the tooth surfaces by reducing the hydrophobicity of mutans streptococci, and OTE may inhibit caries-inducing activity of mutans streptococci by reducing the rate of acid production.

Comparison of the cariostatic effects between regimens to administer oolong tea polyphenols in SPF rats.

The cariostatic effect of oolong tea polyphenols administered according to several regimens was examined in specific pathogen-free (SPF) Sprague-Dawley rats given both a diet containing 20% sucrose and infected with S. sobrinus 6715. The crude preparation (OTE) of oolong tea polyphenols showed the most prominent effect on caries reduction in SPF rats when OTE was administered in the drinking water beginning 1 day prior to the inoculation of S. sobrinus 6715, when compared with chromatographically isolated polyphenol fractions (OTF1 and OTF6) of OTE. Reduction in caries development was found even when OTE was given 1 day after inoculation of the organism. OTE was shown to significantly inhibit dental caries in rats at the concentrations of either more than 5 microg/ml in drinking water or more than 10 microg/g in diet. OTF1 and OTF6 also showed significant inhibition of caries induction, with the minimum inhibitory concentration of OTF6 being 50 microg/ml in drinking water and the minimum inhibitory concentration of OTF1 being 100 microg/g in diet. These results indicate that cariostatic activity of OTE was effective even after the establishment of S. sobrinus in the oral cavity and was more effective in drinking water than in diet. Furthermore, OTE may contain some anticaries substances that affect the virulence of S. sobrinus other than glucosyltransferases.

[Two cases of primary mediastinal germ cell tumor].

We have experienced two patients with primary mediastinal germ cell tumor. The 1st case was a 26-year-old male with high AFP levels. The tumor was completely removed with en-bloc resection of the left upper lobe and postoperative combination chemotherapy was effective. He is free of disease for ten years after the surgery. The 2nd case was a 25-year-old male with normal AFP and HCG level. After 2 courses of combined chemotherapy, the residual tumor was resected. Viable tumor cells were not presented in the resected tumor. He is free of disease for 3 months after surgery.

Peroxidase-catalyzed generation of catechin oligomers that inhibit glucosyltransferase from Streptococcus sobrinus.

Oolong tea extract (OTE) and the purified polymeric polyphenols from OTE have been found to inhibit glucosyltransferase (GTase) of mutans streptococci. In view of the partial fermentation characteristic of oolong tea, we describe here an in vitro model reaction system to produce partially fermented products of D-(+)-catechin or green tea extract (GTE) using horseradish peroxidase. A dimeric catechin molecule was identified as dehydro-dicatechin A by instrumental analyses. The molecular size of some oligomeric catechins was estimated by the elution profile with HPLC. These catechin oligomers markedly inhibited GTase from Streptococcus sobrinus 6715. As the degree of polymerization of catechin or GTE increased, GTase was inhibited more effectively. These results suggest that polymeric polyphenols found in OTE are synthesized by partial fermentation due to oxidases/peroxidases present in tea leaves.

The prevalence of developmental anomalies of teeth and their association with tooth size in the primary and permanent dentitions of 1650 Japanese children.

The prevalence of microdontia, macrodontia, peg-shaped tooth, Carabelli's tubercle, protostylid, paramolar tubercle, central tubercle and palatal accessory cusp were examined in Japanese children. This study included 905 children with primary dentitions (mean age 4 years 7 months) and 745 high-school students with permanent dentitions (mean age 16 years 8 months). Microdontia, macrodontia, Carabelli's tubercle, protostylid and paramolar tubercle were more frequent in the primary dentition, whereas peg-shaped tooth, central tubercle and palatal accessory cusp were more frequent in the permanent dentition. The association between the presence of developmental anomalies and the size of the remaining teeth was significant in permanent dentitions. Both the literature and this study indicate that developmental anomalies of tooth number, size and morphology should be studied as a group rather than as isolates.