The differential expression of mgl mRNA by Porphyromonas gingivalis affects the production of methyl mercaptan.

OBJECTIVE: A large number of individuals have halitosis. The total amount of volatile sulfur compounds, which are the main cause of halitosis, has been correlated with periodontitis following bacterial infection. In this study, Porphyromonas gingivalis (Pg), a major periodontopathogenic bacterium, was isolated from patients with halitosis by the amplification of 16S rRNA, and the ability of isolated Pg to produce methyl mercaptan (CH3 SH) was determined to clarify the relationship between halitosis and Pg infection. MATERIALS AND METHODS: CH3 SH concentrations were measured in patients using Oral Chroma. The production of CH3 SH by Pg standard and clinical strains was also measured in vitro. Real-time PCR was performed to compare the expression of mgl mRNA (which encoded l-methionine-a-deamino-g-mercaptomethane-lyase) among the Pg strains. The production of CH3 SH and the expression of mgl mRNA were also determined to assess the effects of oriental medicine. RESULTS: The production of CH3 SH and the expression of mgl mRNA strongly correlated with each other in the presence of l-methionine. The expression of mgl mRNA by Pg W83 was strongly inhibited by magnoliaceae. CONCLUSION: The production of CH3 SH was correlated with the expression of mgl. Furthermore, the oriental medicine, magnoliaceae, may represent a potential treatment for halitosis.

HTHQ (1-O-hexyl-2,3,5-trimethylhydroquinone), an anti-lipid-peroxidative compound: its chemical and biochemical characterizations.

Recently, it has become apparent that reactive oxygen species (ROS) play many important roles in biological systems. For example, relationships between many diseases, such as cancer, cardiac infarction and arteriosclerosis, and ROS have been found. It is also well known that anti-oxidative agents scavenge ROS in biological systems, which in turn prevents ROS-related diseases. In our previous efforts to develop effective anti-oxidative compounds, we found that 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), which is a hydroquinone monoalkyl ether, is a potent anti-oxidative agent. Here, the scavenging activities of HTHQ against ROS, such as superoxide anion radicals, hydroxyl radicals, t-butyl peroxyl radicals and singlet oxygens, were examined by the ESR (electron spin resonance)-spin trapping method. Among ROS, HTHQ scavenged t-butyl peroxyl radicals most effectively (IC50=0.31+/-0.04 mM), showing approximately twice the activity of a well-known lipophilic anti-oxidant, D,L-alpha-tocopherol (IC50=0.67+/-0.06 mM), as measured by IC50 values defined as the 50% inhibition concentration of the generated ROS. In addition, a relatively stable ESR spectrum of free radicals due to HTHQ was observed during the reaction of HTHQ and t-butyl peroxyl radicals, indicating a direct reaction of HTHQ and t-butyl peroxyl radicals. The free radicals due to HTHQ were more stable than those derived from D,L-alpha-tocopherol under the same conditions examined. On the basis of these results, we evaluated anti-lipid-peroxidative activity of HTHQ in three systems involving micelles, liposomes and rat liver microsomes. HTHQ exhibited a similar anti-oxidative activity to that of D,L-alpha-tocopherol against lipid peroxidation in linolate micelles initiated by addition of Fe2+. On the other hand, HTHQ exhibited approximately 4.8-fold higher anti-lipid-peroxidation activity than that of D,L-alpha-tocopherol against the peroxidation in phosphatidylcholine liposomes initiated by addition of Fe2+. Furthermore, HTHQ scavenged the lipid peroxides at a rate approximately 150 times higher than that of D,L-alpha-tocopherol against Fe3+ -ADP-induced lipid peroxidation in rat liver microsomes, indicating that the anti-lipid-peroxidation activity of HTHQ might be substantially elevated in biological systems in comparison with that of D,L-alpha-tocopherol. Based on these results, we suggest that HTHQ reacts directly with peroxyl radicals, such as t-butyl peroxyl radicals and peroxides of linolate micelles, liposomes and microsomes, by scavenging them to form stable free radicals. The resulting free radicals are presumed to be reduced by several reducing mechanisms in biological systems similarly to those of D,L-alpha-tocopherol, and then the lipid-peroxidation reactions will be terminated. In conclusion, HTHQ was found to be a potent anti-lipid-peroxidative compound and its antioxidation activity to be extremely elevated in biological systems, such as that of liver microsomes via the generation of stable free radicals. We propose that HTHQ is a potent anti-oxidative agent for use in future treatments for lipid-peroxide relevant diseases.

Insolubilization of apatite-collagen composites by UV irradiation.

The physico-chemical properties and biocompatibility of insolubilized apatite-collagen composites were examined. A carbonate-containing hydroxyapatite with chemical composition and crystallinity similar to that of bone was synthesized at pH 7.4 and 60 degrees C. The apatite powder was mixed with collagen solution, whose antigenicity had been removed by enzymatic treatment and formed into apatite-collagen pellets. After insolubilization by UV irradiation for 4 h, the composites showed remarkably reduced disintegration and showed good biocompatibility when implanted into rat abdomen.

Pulmonary delivery of insulin with nebulized DL-lactide/glycolide copolymer (PLGA) nanospheres to prolong hypoglycemic effect.

Insulin loaded PLGA nanospheres having weight mean diameters of 400 nm were prepared by the modified emulsion solvent diffusion method in water. The nanosphere recovery and the drug recovery in the nanospheres were 74.8%+/-4.71 and 46.8%+/-7.01, respectively. Eighty five percent of the drug was released from the nanospheres at the initial burst, followed by prolonged releasing of the remaining drug for a few hours in saline at 37 degrees C. The aqueous dispersions (6 mg/ml) of PLGA nanospheres were nebulized by a sieve type ultrasonic nebulizer to discrete droplets of 5 approximately 7 microm in mean diameters, 75% of which were successfully delivered into the alveolar fraction in a cascade impactor inhaled at 28.3 l/min. The nebulized PLGA nanospheres were administered via a spacer by using a constant volume respirator into the trachea of the fasted guinea pig for 20 min. After the administration of 3.9 I.U./kg insulin with the PLGA nanospheres, the blood glucose level was reduced significantly and the hypoglycemia was prolonged over 48 h, compared to the nebulized aqueous solution of insulin as a reference (6 h). This result could be attributed to the sustained releasing of insulin from the nanospheres deposited widely on to the whole of lung.

Properties of a peptide containing DL-lactide/glycolide copolymer nanospheres prepared by novel emulsion solvent diffusion methods.

Novel emulsion solvent diffusion methods in water or oil were developed to prepare the peptide (TRH and elcatonin)-loaded PLGA nanospheres, via the coprecipitation of drug and polymer in the emulsion droplets induced by the diffusion of solvent. The PLGA nanospheres prepared by the emulsion solvent diffusion method in water were unimodally dispersed particles with an average diameter of about 250 nm, whereas those prepared by the emulsion solvent diffusion method in oil and the phase separation method exhibited bimodal distribution with average diameter of about 700 and 800 nm, respectively. The content and recovery of the peptide and nanospheres prepared by the emulsion solvent diffusion method in oil were superior to those of nanospheres prepared using other methods. The drug release properties from nanospheres depended strongly on their preparation processes. The nanospheres prepared by the emulsion solvent diffusion method in oil, released the drug continuously over 14 days, the rate of which was determined by the diffusion of drug in the rigid matrix structure of the nanosphere. The drug release behavior of nanospheres prepared by the phase separation method exhibited a triphasic release pattern with an initial burst and an induction period followed by the diffusion of drug through the walls of the reservoir type nanospheres, which were produced via the phase separation of polymer deposited around the emulsion droplets of the drug.

Prolonged circulation time of doxorubicin-loaded liposomes coated with a modified polyvinyl alcohol after intravenous injection in rats.

The purpose of this study was to evaluate the functions of a modified polyvinyl alcohol (PVA-R), which has a hydrophobic moiety, as a coating material for liposomes to be loaded with the anticancer drug, doxorubicin. The size controlled liposomes (egg phosphatidylcholine: cholesterol=1:1 molar ratio) were prepared by the hydration method followed by extrusion. Drug encapsulation and surface modification with polymers (PVA and PVA-R) were carried out simultaneously using a modified pH gradient method. The existence of a thick polymer layer on the surface of the liposomes was confirmed by an increase in particle size and the amount of polymer on the liposomal surface, especially for the PVA-R-coated liposomes. The effects of polymer coating on the behavior of the liposomes in vivo were evaluated by measuring the circulation time and biodistribution of the drug after i.v. administration of the liposomal drug in rats. The PVA-R-coated liposomes showed a more prolonged circulating time for the drug with less uptake by the reticuloendothelial system after i.v. administration in rats, compared with non-coated liposomes. These results confirm that polymer possessing a hydrophobic anchor at its end, like PVA-R, is a suitable material for modifying the surface of doxorubicin-loaded liposomes to improve their stability in the circulating blood.

In vitro drug release behavior of D,L-lactide/glycolide copolymer (PLGA) nanospheres with nafarelin acetate prepared by a novel spontaneous emulsification solvent diffusion method.

Nanospheres with D,L-lactide/glycolide copolymer (PLGA) were prepared as a biodegradable and biocompatible polymeric carrier for peptide drugs by a novel spontaneous emulsification solvent diffusion method. Nafarelin acetate (NA), a luteinizing hormone-releasing hormone analogue, was employed as a model peptide drug to investigate the encapsulation efficiency. The drug and PLGA, dissolved in an acetone-dichloromethane-water mixture, were poured into an aqueous solution of polyvinyl alcohol under moderate stirring at room temperature. Spontaneous emulsification arising from a rapid diffusion of acetone from the organic to the aqueous phase enables preparation of PLGA submicron spheres 200-300 nm in size. The entrapment of NA in nanospheres was improved by blending low molecular weight (Mw = 4500) PLGA with higher molecular weight PLGA due to the synergistic effect of the rapid deposition of PLGA and the ionic interaction between NA and PLGA. By coadmixing a small amount of negatively charged phospholipids such as dipalmitoyl phosphatidylglycerol or dicetyl phosphate, the leakage of water-soluble NA was further prevented. The NA encapsulated in PLGA nanospheres was more stable than native NA in acidic medium (pH = 1.2). The drug-release behavior from nanospheres suspended in the disintegration test solution no. 1 (Japanese Pharmacopeia XII) exhibited a biphasic pattern. It was found tht the initial burst of release might be due to the degradation of the PLGA chain, as monitored by gel permeation chromatography. At a later stage, the drug was released more slowly, the rate of which was determined by the diffusion of the drug in the porous matrix structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Hollow microspheres for use as a floating controlled drug delivery system in the stomach.

Hollow microspheres (microballoons), loaded with drug in their outer polymer shells, were prepared by a novel emulsion-solvent diffusion method. The ethanol:dichloromethane solution of drug (tranilast or ibuprofen) and an enteric acrylic polymer were poured into an agitated aqueous solution of polyvinyl alcohol that was thermally controlled at 40 degrees C. The gas phase generated in the dispersed polymer droplet by the evaporation of dichloromethane formed an internal cavity in the microsphere of the polymer with the drug. The drugs incorporated in the solidified shell of the polymer were found to be partially or completely amorphous. The flowability and packability of the resultant microballoons were much improved compared with the raw crystals of drug. The microballoons floated continuously over the surface of acidic dissolution media containing surfactant for greater than 12 h in vitro. The drug release behavior of the microballoons was characterized as an enteric property, and drug release rates were drastically reduced depending on the polymer concentration at pH 6.8.

Clinical periodontal findings and microflora profiles in children with chronic neutropenia under supervised oral hygiene.

BACKGROUND: This is the first known case report that used a polymerase chain reaction (PCR)-based method to help identify the oral microflora in patients with chronic neutropenia. In this study, we report clinical periodontal findings and microflora profiles of 2 children, 1 with severe congenital neutropenia (SCN, Kostmann type) and 1 with cyclic neutropenia (CN). METHODS: The SCN patient had severe gingivitis, whereas the patient with CN had mild gingivitis in the gingival margins. Monthly oral cleaning instruction and review were performed without subsequent periodontal therapy. Oral hygiene conditions remained satisfactory and visible plaque was scarce, despite the persistence of mild gingivitis. Under supervised oral hygiene, we examined the presence of periodontal pathogens from patient plaque samples. RESULTS: By a PCR-based method, Prevotella nigrescens, Bacteroides forsythus, Campylobacter rectus, and Capnocytophaga gingivalis were detected in the SCN patient and P. intermedia, C. rectus, C. gingivalis, and C. sputigena in the CN patient, suggesting the existence of periodontal pathogens. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, and C. ochracea were not found in either patient. CONCLUSIONS: Use of 1% povidone iodine solution and local antibiotic application under supervised oral hygiene were helpful to improve gingival conditions in patients with chronic neutropenia.

Application of a local drug delivery system to periodontal therapy: I. Development of collagen preparations with immobilized tetracycline.

For the purpose of applying a local drug delivery system to periodontal therapy, atelocollagen preparations with immobilized tetracycline (TC) were prepared by modifying the form of the collagen, the concentration of the immobilized TC, and the time of the cross-link process with glutaraldehyde. The course of the TC release from the collagen preparations into an aqueous solution was determined in relation to time. The preparations were also inserted into periodontal pockets, and the amount of TC remaining in the pocket was determined daily. The results obtained were as follows: 1) The degree of drug release could be controlled to some extent by adjusting the TC concentration and the time of the cross-link process; and 2) an amount of TC exceeding the effective dose in the gingival crevicular fluid was present in the periodontal pocket even 10 days after the insertion of TC fixed in the cross-linked processed collagen film in the periodontal pockets.

Cellular immuno-competence of infected root canal contents in pathogenesis of periapical lesions.

The soluble fractions of infected root canal contents (IRCC) were collected from about 300 human extracted teeth and examined for the presence of mononuclear cell (MNC) chemotaxis and cellular immunocompetence. IRCC showed remarkable chemotactic activity for polymorphonuclear leukocytes but a weak activity for MNC. However, generation of intrinsic MNC chemotaxis and induction of cellular immunity were confirmed in rats given repeated injections of IRCC.

[Aerosolization of lactide/glycolide copolymer (PLGA) nanospheres for pulmonary delivery of peptide-drugs].

Lactide/glycolide copolymer (PLGA) nanospheres with nafarelin acetate (NA) used as a model peptide-drug have been developed by the emulsion-phase separation method in an oil phase as reported previously. In the present study, the aerosolization of nanospheres in dry and wet dispersion methods was investigated in vitro to evaluate their applications to pulmonary delivery of peptide-drug. The size distribution of aerosolized nanosphere dispersions and pulmonary deposition behaviors of inhaled particles were investigated using a lazer diffraction light-scattering particle sizer and an artificial model lung (Cascade Impactor: CI), respectively. It was found that the freeze-drying of nanospheres with a hydrophilic surface active agent effectively improved the inhalation behavior. Further, the mixing of nanospheres with lactose led to the reduction of their adhesion to an inhalation device (Spinhaler). It was supposed that the mists of nanosphere-suspension generated from a jet nebulizer might be inhaled into the lower bronchus and alveolus, because their respirable fraction (RF) value, i.e., the deposition percentage on stage 2 to 6 of CI, defined as a pulmonary inhalation index, reached a maximum of over 50%.

[Evaluation of mica crystal glass ceramic crown marginal fitness].

The purpose of this study is to investigate the influence of three marginal shape of abutment teeth (Flat, Moderate and Steep) on the fitness of castable glass ceramics (DICOR) crown clinically. The following results were obtained: 1. The change of width of three marginal shapes showed the same tendency on each firing procedure. 2. The vertical change of three marginal shapes showed the most constant on Flat and decreased on Steep. 3. On the marginal edge, the Labiopalatal gaps were larger than Mesiodistal gaps. 4. On Labiopalatal section, the gaps of Steep were larger than those of Flat and Moderate especially as cast. 5. The gaps Flat were almost same on Labial, Palatal, Mesial and Distal parts.

Assessment of the plasma/serum IgG test to screen for periodontitis.

Chronic periodontitis is a silent infectious disease prevalent worldwide and affects lifestyle-related diseases. Therefore, efficient screening of patients is essential for general health. This study was performed to evaluate prospectively the diagnostic utility of a blood IgG antibody titer test against periodontal pathogens. Oral examination was performed, and IgG titers against periodontal pathogens were measured by ELISA in 1,387 individuals. The cut-off value of the IgG titer was determined in receiver operating characteristic curve analysis, and changes in periodontal clinical parameters and IgG titers by periodontal treatment were evaluated. The relationships between IgG titers and severity of periodontitis were analyzed. The best cut-off value of IgG titer against Porphyromonas gingivalis for screening periodontitis was 1.682. Both clinical parameters and IgG titers decreased significantly under periodontal treatment. IgG titers of periodontitis patients were significantly higher than those of healthy controls, especially in those with sites of probing pocket depth over 4 mm. Multiplied cut-off values were useful to select patients with severe periodontitis. A blood IgG antibody titer test for Porphyromonas gingivalis is useful to screen hitherto chronic periodontitis patients.

[A mechanical study on new ceramic crowns and bridges for clinical use].

By the recent development of new ceramics, i.e. castable glass ceramics and high strength porcelain, the clinical use of all-ceramic bridges as well as all-ceramic crowns have been expected. The purpose of this study was to evaluate the mechanical properties of new ceramics and to analyze the stress distributions in new ceramic crowns and bridges. The Young's modulus, flexural strength and diametral tensile strength of four types of new ceramics (DICOR, BIORAM-C, OPTEC, and HI-CERAM) were measured, and the fracture loads of new ceramic anterior crowns and bridges on the metal abutments were evaluated. Three dimensional finite element analyses of new ceramic anterior crowns and bridges were also carried out to investigate the effects of various mechanical factors; locations of loading point, types of ceramics, thickness of crowns, luting materials, core materials, and designs of fixed joints. In each experiment, the loading forces were applied at 45 degree to the tooth axis. The results were summarized as follow; 1) DICOR showed the highest flexural strength. HI-CERAM hard core porcelain showed the highest Young's modulus and tensile strength. 2) HI-CERAM crowns showed the highest fracture load among the new ceramic crowns. DICOR bridges were significantly stronger than BIORAM-C bridges. The stress analyses of the experimental cases indicated that the fractures of crowns and bridges occurred by the concentration of tensile stresses. 3) By the load at the incisal edge, the highest tensile stresses were caused in the crown. In the crown with 0.5 mm thickness at axial wall, high tensile stresses were observed at more wide regions of palatal side than in the crown with 0.75 mm or 1 mm thickness. However, in the case with an enamel layer remained on the surface of the abutment tooth, the stresses were reduced in spite of the crown thickness. When the abutment tooth was restored with a metal post and core, the stresses of the crown decreased in comparison with the natural abutment tooth. 4) In case of bridges, high tensile stresses concentrated at the fixed joints under any loading point. The stresses tended to rise slightly according to the increase of the Young's modulus of bridges. The aluminous core material which had high Young's modulus was effective for the reduction of the stresses at the surface of the bridge. Those tensile stresses were reduced remarkably by increasing the thickness of the fixed joints toward the labial and vertical side.(ABSTRACT TRUNCATED AT 400 WORDS)

[Disintegration of visible light-cured composite resins caused by long-term water immersion].

The purpose of this study is to clarify a cause of disintegration of composite resins by long-term immersion in distilled water. Three kinds of visible light-cured composite resins (Heliosit, Plurafil Super and Visio Dispers) and one conventional composite resin (Clearfil F II) were prepared as the specimens with a 20 mm diameter and 1 mm thickness. These specimens were immersed in distilled water at 37 +/- 1 degree C for 3 years. These specimens were analysed and observed by a comprehensive multi analyzer and scanning electron microscope. The other hand residues in distilled water were analysed by infrared (IR) and nuclear magnetic resonance (NMR) spectrometers. The surface layer of all four composite resins showed signs of disintegration. The composite resins with abundant dissolved substances had disintegrated markedly, and such disintegration occurred deep inside the specimens. In IR and 1H-NMR spectra of dissolved substances, two visible light-cured composite resins (Heliosit and Plurafil Super) could be detected unreacted monomers, but one visible light-cured composite resin (Visio Dispers) and one conventional composite resin (Clearfil F II) could not be detected them. In 1H-NMR spectra of dissolved substances of all four composite resins, new signals not composed originally were observed. The progress of disintegration were demonstrated clearly. The dissolved substances were shown as the disintegrated substance between resin matrixs and silane coupling agents. It is suggested that the disintegration of composite resins by long-term water immersion is derived from hydrolysis.

Epithelial regeneration in collagen-coated and uncoated patch grafts implanted into dog tracheas.

The paper compares ultrastructural regeneration of the tracheal epithelium on patch grafts coated or not coated with collagen. A longitudinal patch window 2 x 1 cm (four cartilaginous rings in cephalocaudal extent) was made on the ventral wall of the cervical trachea in dogs, and replaced by a polypropylene Malex mesh graft. Grafts coated with collagen allowed normal connective tissue ingrowth and subsequent epithelial spreading. From the cut edge, flat, stratified, poorly differentiated cells migrated and spread on the newly formed stroma. Six weeks after the operation, the prosthesis was thoroughly covered by pseudostratified ciliated columnar epithelium with a newly formed basal lamina. On uncoated grafts, in contrast, some regenerated ciliated cells formed compound cilia or irregular microvilli and dilated endoplasmic reticulum. At 6 weeks, no normal basal cells differentiated, although some bizarre flat cells with extremely extended cytoplasm were located in the basal area of the epithelium. The basal lamina was thick and discontinuous. The underlying stroma included abnormally elongated fibroblasts with condensed cytoplasm and curved, randomly dispersed collagen fibrils. These ultrastructural results indicate that (1) regenerated epithelial cells were derived from poorly differentiated cells; (2) a plastic implant may lead to abnormal regeneration in the connective tissues and epithelium; and (3) collagen coating of the graft may allow fibroblasts to produce normal connective tissue substances.

Effects of beta-carotene and alpha-tocopherol on rumen bacteria in the utilization of long-chain fatty acids and cellulose.

Addition of safflower oil to a growth medium depressed the growth of mixed rumen bacteria above 200 mg/L and did not significantly increase bacteria, even at lower concentrations. However, when 10 mg/L of beta-carotene were added to 50 to 100 mg/L of safflower oil, bacterial growth was significantly increased. When more than 200 mg/L of safflower oil were present, beta-carotene markedly restored the growth capacity. alpha-Tocopherol was more effective than beta-carotene, although it inhibited growth at high concentrations. The combination of beta-carotene and alpha-tocopherol (each 5 mg/L) exerted partially additive effects. beta-Carotene plus alpha-tocopherol enhanced bacterial cell yield in the presence of safflower oil, caprate, stearate, or linoleate, suggesting that beta-carotene and alpha-tocopherol increase the utilization of fatty acids. beta-Carotene plus alpha-tocopherol also stimulated cellulose digestion in the presence of 100 mg/L of safflower oil, evidently through the increased growth of cellulolytic bacteria.