Inhibitory effects of sword bean extract on alveolar bone resorption induced in rats by Porphyromonas gingivalis infection.

BACKGROUND: The domesticated legume, Canavalia gladiata (commonly called the sword bean), is known to contain canavanine. The fruit is used in Chinese and Japanese herbal medicine for treating the discharge of pus, but its pharmacological mechanisms are still unclear. OBJECTIVES: This study examined the effect of sword bean extract (SBE) on (i) oral bacteria and human oral epithelial cells in vitro, and (ii) the initiation and progression of experimental Porphyromonas gingivalis-induced alveolar bone resorption in rats. MATERIAL AND METHODS: A high-performance liquid chromatography/ultraviolet method was applied to quantitate canavanine in SBE. By assessing oral bacterial growth, we estimated the minimum inhibitory concentration and minimum bactericidal concentration of SBE, canavanine, chlorhexidine gluconate (CHX) solution. The cytotoxicity of SBE, canavanine, CHX, leupeptin and cystatin for KB cells was determined using a trypan blue assay. The effects of SBE, canavanine, leupeptin and cystatin on Arg-gingipain (Rgp) and Lys-gingipain (Kgp) were evaluated by colorimetric assay using synthetic substrates. To examine its effects on P. gingivalis-associated periodontal tissue breakdown, SBE was orally administered to P. gingivalis-infected rats. RESULT: Sword bean extract contained 6.4% canavanine. SBE and canavanine inhibited the growth of P. gingivalis and Fusobacterium nucleatum. The cytotoxicity of SBE, canavanine and cystatin on KB cells was significantly lower than that of CHX. Inhibition of Rgp with SBE was comparable to that with leupeptin, a known Rgp inhibitor, and inhibition of Kgp with SBE was significantly higher than that with leupeptin at 500 µg/mL ( p < 0.05). P. gingivalis-induced alveolar bone resorption was significantly suppressed by administration of SBE, with bone levels remaining comparable to non-infected animals ( p < 0.05). CONCLUSION: The present study suggests that SBE might be effective against P. gingivalis-associated alveolar bone resorption.

Anatomical location of various condylar points for jaw movement analysis in Japanese women.

The aim of this study was to determine anatomical locations of the hinge axis point, kinematic axis point and reference point for the palpated lateral condylar pole on lateral cephalograms. Subjects comprised 18 Japanese women selected according to following criteria: normal occlusion; and absence of signs and symptoms of stomatognathic function. Jaw movement and the condylar reference points noted earlier were recorded three-dimensionally with six degrees of freedom, and kinematic axis point and hinge axis point were determined using an optoelectronic jaw-tracking system. Lateral cephalograms were used to determine anatomical locations of the three points in the condyle. Mean location of hinge axis point was 12.9 mm anterior of the porion and 5.3 mm inferior to the Frankfort horizontal plane, the kinematic axis point was situated in 12.8 mm anterior and 0.1 mm inferior, and the reference point for the palpated lateral condylar pole was situated 10.7 mm anterior and 0.8 mm inferior, respectively. The kinematic axis point was located outside the condyle in the majority of subjects. The reference point for the palpated lateral pole offers a useful indicator in the analysis of condylar movements.

Identification of oral species of the genus Veillonella by polymerase chain reaction.

INTRODUCTION: Members of the genus Veillonella cannot be reliably distinguished by their biochemical characteristics and phenotypic features. Moreover, DNA-DNA hybridization and sequence analyses of the 16S ribosomal RNA gene including random fragment length polymorphism analysis, are complex and time-consuming procedures that are not well-suited to identifying oral species of Veillonella: Veillonella atypica, Veillonella denticariosi, Veillonella dispar, Veillonella parvula, and Veillonella rogosae. METHODS: In this study, five forward primers and a reverse primer were designed for polymerase chain reaction (PCR) according to the partial sequences of the rpoB genes of these oral Veillonella species. RESULTS: The forward primers were species-specific for these five Veillonella species, and could produce specific amplicons when used together with reverse primer and individual DNA templates of these species in PCR. These primer pairs were also found to discriminate between the respective species, and the Veillonella strains isolated from human oral cavities were successfully assigned to one of the five oral species of the genus Veillonella based on their specific products by PCR. CONCLUSION: A simple two-step PCR procedure using the five sets of primer pairs developed in the present study is a rapid and reliable method for the identification of the recognized oral Veillonella species.

Reconstituting telomerase activity using the telomerase catalytic subunit prevents the telomere shorting and replicative senescence in human osteoblasts.

The rate of bone formation is largely determined by the number of osteoblasts, which in turn is determined by the rate of replication of progenitors and the life span of mature cells, reflecting the timing of death by apoptosis. However, the exact age-dependent changes of the cellular activity, replicative potential, and life span of osteoblasts have not been investigated to date. Here, we present evidence that the cellular activity, telomere lengths, and replicative life span of osteoblastic cells obtained from juxta-articular bone marrow gradually decrease with the advance of donor age. Recently, telomerase reverse transcriptase (hTERT) has been identified as a human telomerase catalytic subunit. We transfected the gene encoding hTERT into telomerase-negative human osteoblastic cells from donors and osteoblastic cell strain NHOst 54881 cells and showed that expression of hTERT induces telomerase activity in these osteoblastic cells. In contrast to telomerase-negative control cells, which exhibited telomere shortening and senescence after 10-15 population doublings, telomerase-expressing osteoblastic cells had elongated telomere lengths and showed continued alkaline phosphatase activity and procollagen I C-terminal propeptide (PICP) secretion for more than 30 population doublings. These results indicate that osteoblasts with forced expression of hTERT may be used in cell-based therapies such as ex vivo gene therapy, tissue engineering, and transplantation of osteoblasts to correct bone loss or osteopenia in age-related osteoporotic diseases.

Up-regulation of glial fibrillary acidic protein in the retina of primate eyes with experimental glaucoma.

OBJECTIVE: To identify molecular mechanisms of retinal responses to intraocular pressure elevation in primate experimental glaucoma. METHODS: An experimental glaucoma model was created by repeated laser trabeculophotocoagulation. After the preparation of complementary DNAs from extracted total RNAs in the retinas, polymerase chain reaction (PCR) experiments were performed for the following screening target genes: beta-tubulin beta 2 and beta 5 and glial fibrillary acidic protein (GFAP). To investigate the amplified sequences derived from the PCR experiments, sequencing, subcloning, and Southern blot analysis of PCR products were performed. In addition, an immunohistochemical analysis was performed in an attempt to show the distribution of the target gene products in the retinas. RESULTS: A series of PCR experiments suggested up-regulation of gene expression for GFAP but not for beta-tubulins. Sequencing of the PCR products and results of the Southern blot analysis showed that the amplified sequences were derived mainly from the target gene, GFAP, and that increased expression of GFAP was found despite the severity of glaucoma. Immunohistochemical studies also demonstrated increased expression of GFAP proteins in Müller cells and astrocytes in the retinas of primate eyes with experimental glaucoma. CONCLUSIONS: Our study showed up-regulation of GFAP at gene and protein levels, which suggests that glial components in the retina may contribute to the pathologic processes induced by elevated intraocular pressure.

Multilocational hepatocyte transplantation for treatment of congenital ascorbic acid deficiency rats.

We attempted multilocational hepatocyte transplantation (HCTx) including hepatocyte-bearing polyurethane foam (PUF) to treat congenitally ascorbic acid (AsA) biosynthetic enzyme-deficient (ODS-od/od) rats. Hepatocytes isolated from the liver of congeneic rats were transplanted into the portal vein (Pv), spleen (Sp), omentum (Om), and mesentery (Ms). Hepatocyte-bearing PUF was transplanted into the Om and Ms. Experimental groups were divided into four groups (group I; Pv + Sp, group II; Pv + Sp + Om + Ms, group III; Pv + Sp + hepatocyte-bearing PUF, group IV; control). The average serum AsA level of the surviving rats in group II and III was significantly higher than that in group I 3 mo after HCTx. Histological examination showed small foci of surviving hepatocytes in the Om and Ms tissues and in the connective tissue in the PUF. ODS-od/od rats survived for a long time by multilocational HCTx.

Developmental expression of cytochrome P450S within intrasplenically transplanted fetal hepatocytes.

Fetal hepatocytes were harvested at day 20 of gestation from spontaneously hypertensive rats (SHR) and then transplanted into recipient adult SHR spleens. Morphological examination of the recipient spleens revealed that, after 4 and 10 wk, large masses of hepatocytes were present in the red pulp with apparent cord-like structures. Larger batches of hepatocytes were observed in the spleens at 10 wk after than at 4 wk after transplantation. Of major significance was the fact that hepatocyte transplanted spleens were able to express several families of cytochrome P450 (cyto P450) proteins 2-10 wk after transplantation. Immunochemical determinations revealed that cytos P450 IA1, P450 IIB1, P450 p, P450 HLp, and P450 LA omega could be detected without any prior induction. All were intensely expressed 6 wk after transplantation; however, P450 IA1 and P450 IIB1 did not appear to be expressed by 2 wk after transplantation. Although cytos P450 p and P450 HLp did not appear to be expressed by 10 wk after transplantation, they were induced with dexamethasone at that time. Cyto P450 LA omega and peroxisomal acyl CoA oxidase were expressed 6 wk after transplantation in a 70% hepatectomized host. These results demonstrate that fetal hepatocytes can be successfully transplanted into the spleens of recipients and that the fetal hepatocytes appear to grow and develop cyto P450 metabolizing systems.

Corticosteroid treatment induces chondrocyte apoptosis in an experimental arthritis model and in chondrocyte cultures.

OBJECT: In order to examine the mechanisms involved in steroid-induced arthropathy after intra-articular corticosteroid injection, a histological examination was performed in vivo using severe combined immunodeficiency (SCID) mice that were implanted with human articular cartilage into the back (SCID/hu model). In addition, the effect of corticosteroids on chondrocyte apoptosis was evaluated in vitro using cultured human chondrocytes. METHOD: Human articular cartilage was obtained during knee surgery and implanted subcutaneously into the backs of SCID mice. One month later, weekly injections of corticosteroid (hydrocortisone acatate: 1 mg/0.2 ml, triamcinolone acetonide: 0.2 mg/0.2 ml, dexamethasone acetate: 0.1 mg/0.2 ml) in the subcutaneous cavity around the grafted cartilage in SCID mice were initiated. After six weeks of treatment, the grafted cartilage pieces were removed from the SCID mice and examined histologically. Chondrocyte apoptosis after corticosteroid treatment was also investigated using cultured human chondrocytes. RESULT: In the corticosteroid treated, grafted articular cartilage, apoptotic chondrocytes were apparent in the superficial and middle layers of cartilage. But a reduced intensity of Safranin O staining was not remarkable. In the cultured chondrocytes, apoptotic changes were also observed after corticosteroid treatment. CONCLUSION: Corticosteroid treatment induces chondrocyte apoptosis and it may be important to understand the steroid-induced arthropathy.

Structural studies of the antigenic polysaccharide of Eubacterium saburreum, strain T17.

The antigenic polysaccharide produced by Eubacterium saburreum, strain T17, is a homoglycan composed of D-glycero-D-galacto-heptose (Hep) residues having the pentasaccharide repeating-unit----6)-[alpha-Hepf-(1----4)] -beta-Hepp-(1----6)-[alpha-Hepf-(1----2), alpha-Hepf-(1----4)]-beta-Hepp-(1----. The polysaccharide does not contain O-acetyl group.

Isolation and identification of 3,6-dideoxy-3-(L-glyceroyl-amino)-D-glucose. a constituent of the antigenic polysaccharide of Eubacterium saburreum, strain V5.

3,6-Dideoxy-3-(L-glyceroylamino)-D-glucose has been identified, for the first time, as a sugar component of the antigenic polysaccharide of Eubacterium saburreum strain V5, principally by n.m.r. and mass spectrometry.

Structural studies of the antigenic polysaccharide of Eubacterium saburreum, strain T27.

The antigenic polysaccharide produced by Eubacterium saburreum, strain T27, is a homoglycan composed of D-glycero-D-galacto-heptose (Hep) residues having a nonasaccharide repeating-unit with the structure (leads to 6)-[alpha-Hepf-(1 leads to 4)]-beta-Hepp-(1) leads to (3)6)-[alpha-Hepf-(1 leads to 2), alpha-Hepf-(1 leads to 4)]-beta-Hepp-(1 leads to. The polysaccharide contains acetyl groups linked to O-2 (except to the 2,4,6-linked heptopyranosyl residue), O-3 and O-7 of part of both heptopyranosyl and heptofuranosyl residues. The assignment of an acetyl group at O-3 of part of the terminal heptofuranosyl and 4,6-linked heptopyranosyl groups is tentative.

Structural studies of the antigenic polysaccharide of Eubacterium saburreum, strain T19.

The antigenic polysaccharide produced by Eubacterium saburreum, strain T19, contains unusual sugars, including D-glycero-D-galacto-heptose (Hep) and D-fucose (D-Fuc). A repeating unit of the polysaccharide is composed of a linear chain of D-glycero-D-galacto-heptopyranosyl tetrasaccharide as its backbone structure, i.e., -[-->6)-beta-Hep p-(1-->3)-beta-Hep p-(1-]2-->, and a D-fucofuranosyl disaccharide as a branched group, i.e., alpha-D-Fuc f-(1-->2)-alpha-D-Fuc f-(1-->, which is linked to O-4 of one (1--6)-linked D-glycero-D-galacto-heptopyranosyl residue. The polysaccharide also contains O-acetyl groups.

[Retinal toxicity of intravitreally injected steroids on the rabbit eye].

The retinal toxicity of intravitreally injected steroids, fluorometholone and tetrahydrocortisol, was examined. The concentration of the steroids was 20 mg/ml, and 0.05 ml of each solution were injected into the rabbit vitreous cavity. An equal volume of saline solution was injected as a control. In addition to ophthalmoscopy, intraocular pressure was measured and electroretinography, light microscopy and electron microscopy were performed. The results showed no remarkable changes in all eyes. Fluorometholone and tetrahydrocortisol seem to have neither toxicity to the retina nor adverse effect of elevating intraocular pressure when intravitreally injected with this amount.

Asaccharolytic anaerobic gram-negative coccobacilli (AAGNC) isolated from infected root canals and periodontal pockets.

BACKGROUND/AIMS: Culture-difficult bacteria, including asaccharolytic anaerobic gram-negative coccobacilli (AAGNC), may constitute a predominant group of organisms in oral sites. This study aimed to characterize phylogenetically 10 AAGNC isolated from endodontic lesions and periodontal pockets. METHODS: 16S rDNA sequence and G + C content were determined. Strains sharing more than 98% sequence similarities and similar G + C content were considered the same bacterial species. RESULTS: One isolate resembled Dialister pneumosintes (the type species of the genus Dialister) with 35 mol% G + C content and 97% sequence similarity. Of eight isolates having 45-47 mol% G + C content, seven were identified as D. invisus and one resembled Dialister invisus with 97% sequence similarity. However the 16S rDNA sequence similarities with D. pneumosintes were relatively low, indicating the strains may belong to a new genus. The last isolate revealed 35 mol% G + C content, but had higher 16S rDNA sequence similarity with D. invisus than with D. pneumosintes. CONCLUSION: The group of oral AAGNC isolates need to be reclassified.

Immunological specificity of oral Eubacterium species.

Antigens of Eubacterium species including E. alactolyticum, E. brachy, E. nodatum, E. saburreum, E. timidum, E. yurii subsp. yurii and E. yurii subsp. margaretiae, which have been isolated frequently from periodontal pockets and associated with periodontal diseases, were extracted by ultrasonication from whole bacterial cells. Antigens were also prepared from E. aerofaciens, E. lentum and E. rectale, which have been found in intestinal tracts and infected abscesses in human oral cavities. The antigens of the oral Eubacterium species were compared with antigens from E. limosum, the type species of the genus Eubacterium, by using SDS-PAGE and Western immunoblot assays. SDS-PAGE gels stained with Coomassie brilliant blue indicated that no major peptide bands were common among the Eubacterium species examined. The protein profile patterns were distinctly different from each other. Western immunoblotting reactions with rabbit antisera showed that the Eubacterium species could be clearly distinguished serologically, and that the species-specific antigens were peptide components of ultrasonic extracts from the whole bacterial cells. The present study demonstrates that these Eubacterium species show great heterogeneity in their peptide components and immunological reactions, which may be useful for identification of the Eubacterium species from human oral specimens.

DNA-DNA relatedness and phylogenetic positions of Slackia exigua, Slackia heliotrinireducens, Eggerthella lenta, and other related bacteria.

Recently, two asaccharolytic Eubacterium species, Eubacterium exiguum and Eubacterium lentum, and Peptostreptococcus heliotrinreducens have been reclassified as Slackia exigua, Eggerthella lenta and Slackia heliotrinireducens in the novel genera on the basis of 16S rDNA sequence analysis. But DNA-DNA relatedness among these species and other related bacteria have not been reported yet. DNA-DNA relatedness is the standard arbiter and the recommended method for the designation and evaluation of new species, particularly closely related ones. In the present study, DNA-DNA hybridization studies were performed on S. exigua, S. heliotrinireducens and E. lenta together with the other bacterial species in the related genera. The phylogenetic relationships of these species were also investigated by comparison analysis of 16S rDNA sequence data. In the DNA-DNA hybridization studies, S. exigua showed a DNA homology level of 33% to S. heliotrinireducens and 11% to E. lenta. DNA-DNA homology between S. heliotrinireducens and E. lenta was 10%. But these three species showed very low homology (less than 5%) to the related asaccharolytic species such as Eubacterium and Mogibacterium. In conclusion, the DNA-DNA relatedness data together with the evolutionary data in the present paper further support the reclassification of Eubacterium exiguum, Peptostreptococcus heliotrinreducens and Eubacterium lentum as Slackia exigua, Slackia heliotrinireducens and Eggerthella lenta, respectively.

Immunochemical and structural characterization of the antigenic polysaccharide from Eubacterium saburreum T18.

An antigenic surface polysaccharide produced by Eubacterium saburreum strain T18, isolated from human dental plaque, was purified from formamide extract of whole cells. Methylation analysis, Smith degradation, optical rotation data and nuclear magnetic resonance spectra demonstrated that the purified antigen was a homopolysaccharide composed of D-glycero-D-galacto-heptose (Hep.) residues. The structure of the repeating unit in the polysaccharide was: -[----6)-[alpha-Hep.furanosyl-(1----4)]-beta-Hep.pyranosyl- (1----6)-[alpha-Hep.furanosyl-(1----2), alpha-Hep.furanosyl-(1----4)]-beta- Hep.pyranosyl-(1-)4----6)-beta-Hep.pyranosyl-(1----. No heptose residues were acetylated. Immunodiffusion reactions in agar gel suggested that the immunodeterminant of the antigenic polysaccharide was D-glycero-D-galacto-heptofuranosyl residues as branched nonreducing terminals.