Pathogenicity of exopolysaccharide-producing Actinomyces oris isolated from an apical abscess lesion.

AIM: To demonstrate a capacity for producing exopolysaccharides (EPSs) and an ability to form biofilm on abiotic materials of Actinomyces oris strain K20. METHODOLOGY: The productivity of EPSs and the ability to form biofilm of strain K20 were evaluated by measuring viscosity of spent culture media and by scanning electron microscopy (SEM) and the biofilm assay on microtitre plates, respectively. High-performance liquid chromatography was used to determine the chemical composition of the viscous materials. To examine the role of the viscous materials attributable to the pathogenicity in this organism, the ability of strain K20 to induce abscess formation was compared in mice to that of ATCC 27044. RESULTS: The viscosity of the spent culture media of K20 was significantly higher than that of ATCC 27044. Strain K20 showed dense meshwork structures around the cells and formed biofilms on microtitre plates, whereas ATCC 27044 did not. Chemical analysis of the viscous materials revealed that they were mainly composed of neutral sugars with mannose constituting 77.5% of the polysaccharides. Strain K20 induced persistent abscesses in mice lasting at least 5 days at a concentration of 10(8) cells mL(-1), whereas abscesses induced by ATCC 27044 healed and disappeared or decreased in size at day 5. CONCLUSIONS: Strain K20 produced EPSs, mainly consisting of mannose, and formed biofilms. This phenotype might play an important role for A. oris to express virulence through the progression of apical periodontitis.

Analysis of proteins in human gingival crevicular fluid by mass spectrometry.

BACKGROUND AND OBJECTIVE: Gingival crevicular fluid is a bodily fluid transuded from periodontal tissues into the gingival crevice and periodontal pocket, and contains many species of components. Proteins in gingival crevicular fluid have been studied as markers for periodontal diseases. Mass spectrometric analysis is used for the analyses of proteins, lipids, saccharides and metals, and expected as an approach for disease diagnosis. For better analysis of the protein components in gingival crevicular fluid, we investigated proteins in gingival crevicular fluid samples from the healthy gingival crevice and periodontal pocket using mass spectrometry. MATERIAL AND METHODS: Gingival crevicular fluid samples were collected from subjects who gave their informed consent and were periodontally healthy or had diseased pockets. These samples were electrophoretically separated, and each fraction on the gels was analysed by nano liquid chromatography coupled with tandem mass spectrometry. Antimicrobial peptides detected in gingival crevicular fluid were confirmed by western blotting. RESULTS: One hundred and four proteins were detected in gingival crevicular fluid samples from both healthy sites and sites of periodontitis; 64 proteins were contained only in gingival crevicular fluid from healthy sites and 63 proteins were observed only in gingival crevicular fluid from periodontitis sites. These proteins were blood-, cytoskeleton-, immunity-, inflammation- and lipid-related proteins and enzymes. Some proteins, including ceruloplasmin, glycogen phosphorylase, glutathione S-transferase, phosphoglycerate mutase, psoriasin, S100A11 and resistin, were identified for the first time in gingival crevicular fluid. Antimicrobial peptides, such as lactoferrin, α1-antitrypsin, lipocalin, S100A7, S100A8, S100A9 and cathelicidin, were observed by mass spectrometry and western blotting. CONCLUSION: Multiple protein components in gingival crevicular fluid were analysed at the same time using mass spectrometry, and this approach may be useful for the diagnosis of periodontal diseases.

Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity.

Orexins (hypocretins) are a pair of neuropeptides implicated in energy homeostasis and arousal. Recent reports suggest that loss of orexin-containing neurons occurs in human patients with narcolepsy. We generated transgenic mice in which orexin-containing neurons are ablated by orexinergic-specific expression of a truncated Machado-Joseph disease gene product (ataxin-3) with an expanded polyglutamine stretch. These mice showed a phenotype strikingly similar to human narcolepsy, including behavioral arrests, premature entry into rapid eye movement (REM) sleep, poorly consolidated sleep patterns, and a late-onset obesity, despite eating less than nontransgenic littermates. These results provide evidence that orexin-containing neurons play important roles in regulating vigilance states and energy homeostasis. Orexin/ataxin-3 mice provide a valuable model for studying the pathophysiology and treatment of narcolepsy.

Structure, tissue distribution, and pharmacological characterization of Xenopus orexins.

We isolated the Xenopus gene encoding prepro-orexin to predict the structures of orexins in submammalian chordates. Putative mature Xenopus orexin-A and -B are highly similar to each mammalian counterpart. Especially, the C-terminal 10 residues were highly conserved among these species and isopeptides. Immunohistochemical examination of Xenopus brain revealed that orexin-containing neurons were highly specifically localized in the ventral hypothalamic nucleus. A rich network of immunoreactive fibers was found in various regions of the Xenopus brain. The distribution was similar to that of mammalian orexins. Xenopus orexin-A and -B specifically bind and activate human orexin receptors expressed in Chinese hamster ovary cells. Of interest, Xenopus orexin-B had several-fold higher affinity to human OX2R compared with human orexins. These results suggest that Xenopus orexin-B might be a useful pharmacological tool as an OX2R selective high-affinity agonist.

Orexin-induced hyperlocomotion and stereotypy are mediated by the dopaminergic system.

We demonstrated involvement of the ventral tegmental area (VTA) dopaminergic system in orexin-induced hyperlocomotion and stereotypy in rats. In double-label immunohistochemical study of rat brain, we found that tyrosine hydroxylase (TH)-immunoreactive cells in the VTA received innervation from orexin immunoreactive-fibers. Orexin-A induced an increase in [Ca(2+)](i) in isolated A10 dopamine neurons in a dose-dependent manner. In behavioral studies, we found that orexin-A induced hyperlocomotion, stereotypy and grooming behavior when administered centrally in rats, and these effects were abolished by dopamine D(2) (haloperidol and sulpiride) or D(1) (SCH23390) antagonists. These results suggest that the orexin-induced hyperlocomotion, stereotypy and grooming behavior are mediated by the dopaminergic system and this pathway might be involved in orexin-induced emotional responses.

Orexins/hypocretins regulate drinking behaviour.

Orexin/hypocretins are recently identified neuropeptides which regulate feeding behaviour. We found orexins increased water intake when administrated intracerebroventricularly to rats. The effect of orexin-A was more potent as compared with orexin-B, suggesting the possible involvement of OX(1) receptor. The efficacy of orexin-A was almost comparable with that of angiotensin II, and the effect lasted more than 3 h. Prepro-orexin mRNA level was up-regulated when rats were deprived of water. Orexin-immunoreactive varicose axons were observed in the subfornical organ and area postrema, regions implicated in drinking behaviour. These observations suggest a physiological role for orexin as mediators that regulate drinking behaviour.

Distribution of orexin neurons in the adult rat brain.

Orexin (ORX)-A and -B are recently identified neuropeptides, which are specifically localized in neurons within and around the lateral hypothalamic area (LHA) and dorsomedial hypothalamic nucleus (DMH), the regions classically implicated in feeding behavior. Here, we report a further study of the distribution of ORX-containing neurons in the adult rat brain to provide a general overview of the ORX neuronal system. Immunohistochemical study using anti-ORX antiserum showed ORX-immunoreactive (ir) neurons specifically localized within the hypothalamus, including the perifornical nucleus, LHA, DMH, and posterior hypothalamic area. ORX-ir axons and their varicose terminals showed a widespread distribution throughout the adult rat brain. ORX-ir nerve terminals were observed throughout the hypothalamus, including the arcuate nucleus and paraventricular hypothalamic nucleus, regions implicated in the regulation of feeding behavior. We also observed strong staining of ORX-ir varicose terminals in areas outside the hypothalamus, including the cerebral cortex, medial groups of the thalamus, circumventricular organs (subfornical organ and area postrema), limbic system (hippocampus, amygdala, and indusium griseum), and brain stem (locus coeruleus and raphe nuclei). These results indicate that the ORX system provides a link between the hypothalamus and other brain regions, and that ORX-containing LHA and DMH neurons play important roles in integrating the complex physiology underlying feeding behavior.

Orexin-induced food intake involves neuropeptide Y pathway.

Orexins (orexin-A and -B) are recently identified neuropeptides, which are thought to be implicated in the regulation of feeding behavior. We used a NPY-Y1 receptor specific antagonist, BIBO3304, to examine whether NPY is involved in orexin-induced feeding behavior. Intracerebroventricular administration of orexin-A (10 nmol) induced food intake in rats (food intake for 3 h; vehicle 0.3+/-0.2 g vs. orexin-A 10 nmol, 4.0+/-0.5 g, n=4). Orexin-induced feeding behavior was partially inhibited by prior administration of BIBO3304 (3 h food intake: orexin-A 10 nmol, 4.0+/-0.5 g vs. BIBO3304 (60 microgram) + orexin-A 10 nmol, 2.2+/-0.2 g, n=4). A low dose of BIBO3304 (30 microgram) did not show a significant inhibitory effect. BIBO3457, an inactive enantiomer, used as a negative control, did not show any inhibitory effect on orexin-A-induced feeding behavior. Fos expression was observed in NPY-containing neurons in the arcuate nucleus 1 h after orexin-A (10 nmol) was administered intracerebroventricularly (control 0.3+/-0.08%, orexin-A 10.2+/-0.8%, n=5 rats/group). These observations suggest that NPY is involved in orexin-induced feeding behavior. However, BIBO3304 did not completely abolish the effect of orexin-A. These results suggest that orexin-A elicits feeding behavior partially via the NPY pathway. The NPY system could be the one of downstream pathways by which orexin-A induces feeding behavior. Another pathway may also be involved in orexin-A-induced feeding behavior, because BIBO3304 did not completely abolish orexin-A-induced feeding behavior.

Neurons containing orexin in the lateral hypothalamic area of the adult rat brain are activated by insulin-induced acute hypoglycemia.

Orexin-A and -B (also known as hypocretin-1 and -2) are neuropeptides which stimulate food intake when administered intracerebroventricularly. Orexins are specifically localized in neurons within and around the lateral hypothalamic area (LHA). Previous electrophysiological studies have demonstrated that some neurons in the LHA are activated by hypoglycemia, and are therefore termed 'glucose-sensitive neurons'. In the present study, we examined whether orexin-containing neurons are activated in the hypoglycemic states, using Fos-like immunoreactivity (FLI) as a marker of neuronal activation. We observed that FLI was induced in the LHA by acute insulin treatment. Double staining with anti-Fos and anti-orexin antibodies revealed that up to 33% of the orexin-containing neurons in the LHA also expressed FLI under the hypoglycemic condition. These results suggest that some populations of neurons which contain orexins are activated under hypoglycemic conditions.

Deformation of the distal femur: a contribution towards the pathogenesis of osteochondrosis dissecans in the knee joint.

Osteochondrosis dissecans (OD) is a process of subchondral bone necrosis occurring predominantly in young individuals at specific sites. The aetiology of this disease remains controversial with mechanical processes due to trauma and/or ischaemic factors being proposed. This study aims at explaining the aetiology of OD in the knee joint as a result of the particular deformation of the condyles. A finite element analysis of the distal third of the femur was performed. A three-dimensional model was developed based on computed tomography scans of a normal femur, consisting of cortical bone, cancellous bone and articular cartilage. This model was subjected to physiological loads at 0, 30, 60 and 90 degrees of knee flexion. A complex deformation was found within each condyle as well as between the two condyles. Both medial and lateral condyles are deformed in the medio-lateral direction and at the same time compressed between the patella and the tibia in the antero-posterior direction. This effect is highest at 60 degrees of knee flexion. In both planes, the medial condyle is distorted more than the lateral one. Strain concentration in the subchondral bone facing the patella varies with flexion, especially for angles exceeding 60 degrees. The deformation of the femur in the predominant locus of OD in the medial condyle exceeds that of the lateral condyle considerably. The analysis shows that repeated vigorous exercise including extreme knee flexion may produce rapidly changing strains which in turn could ultimately be responsible for local subchondral bone collapse.

The trophic effects of long chain triglycerides on the atrophic ileal mucosa of rats.

Rats with atrophic intestinal mucosa due to enteral nutrition supplied by an elemental diet (ED) for 4 weeks or more, received a fat-enriched ED containing 10% long chain triglycerides (10% FED) orally. The atrophic ileal mucosa became trophic 4 weeks after administration of the 10% FED. Ornithine decarboxylase activity in the ileal mucosa increased 3 days after the administration of 10% FED. Rats with atrophic intestinal mucosa that had undergone a 70% proximal jejunoileectomy, received an oral ED containing 4% long chain triglycerides (4% FED). In the jejunoileectomized rats, marked proliferation of the remaining ileum was observed irrespective of diet, when compared with the transected control group. In the transected group, the 4% FED had trophic effects on the ileum, but in the jejunoileectomized group, the 4% FED had no significant trophic effect on the remaining ileum. In conclusion, long chain triglycerides had mild trophic effects on ileal mucosa and were effective in the treatment of atrophic intestinal mucosa. However, the trophic effects of fat were apparently masked by the marked proliferation of the ileal mucosa following jejunoileectomy.

Behaviour of pathogenic E. coli and Salmonella enteritidis in small domestic sewage treatment apparatus ("Johkasou").

"Johkasou" is a small sewage treatment apparatus commonly used in Japan which can effectively treat domestic wastewater in places where a public sewage system is difficult to supply. The behaviour of enterohaemorrhagic E. coli O157 and Salmonella enteritidis in a "Johkasou" was studied. Their reduction rates depended significantly on the water temperature in the "Johkasou" with minimal decrease in numbers at 10 degrees C within 48 h. The reduction rates increased at 20 degrees C and 30 degrees C where 4 log reduction could be expected. The reduction rates were influenced by the BOD of the solutions that contained the pathogens with the lower the BOD the higher the reduction rate. The reduction rates were about the same between both pathogens. The result showed that it was necessary to disinfect the effluent as some pathogens can pass through the apparatus when some users of the apparatus excrete pathogens.

Influence of water on the transverse strength of posterior composite resins.

In an attempt to evaluate the influence of water on the mechanical properties of composite resin, the transverse strengths of 15 commercially available composites were determined in water at 37 degrees C and compared with the strength measured in air after storage in water for periods of 1 day to 3 months. Some of the composites, including two which contained a blend filler, showed remarkable reduction of transverse strength when measured in air, according to the procedures specified in ISO 4049, in comparison with transverse strength measured in water. The change of water content in the material can predominantly affect the mechanical behavior of composite resin.

Polymerization shrinkage and contraction force of composite resin restorative inserted with "Megafiller".

This study quantified the contraction force and polymerization shrinkage of composite resins with/without beta-Quartz Glass Ceramic Inserts (BQCI) as "Megafiller". The materials used for the determination included a chemically cured composite and five light-cured composites. The system for measuring contraction force consisted of a transparent teflon tube for preparing the specimen, a small load cell, a dynamic strain gauge and a pen-recorder. After the composite was packed into the teflon mold, a BQCI (Type R3) was inserted through the opening and the specimen was cured. Linear polymerization shrinkage of the composites was measured every 10 seconds from the start of mixing or irradiation to 90 minutes by the mercury bath method. Three pieces each of BQCI (Type T3) were inserted in each specimen. The results suggested that BQCI was markedly effective in reducing polymerization shrinkage, but was not always effective in reducing the contraction force during polymerization.

[A case of icteric hepatoma which succeeded internal fistulation after radiotherapy].

A case of icteric hepatoma was treated by TAE, arterial chemoinfusion and photoirradiation to the hepatic hilum. The patient was successfully discharged after radiotherapy, which was most effective for internal fistulation of obstructed bile duct and decreasing the size of portal tumor thrombus.

[CT in staging gastric cancer].

CT pictures of 26 cases of advanced gastric cancer were reviewed. All the cases of gastric cancer were staged surgically and also histologically. 1) Gastric cancer was enhanced intensely. 2) About the diagnosis of the extension of the tumor, there were many problems to be solved in the future, but the result of final staging was satisfactory. 3) With further advent of technology and methodology in the field of CT, CT will play an important role in staging diagnosis of gastric cancer.

[Ultrasonography of the extremities: nontumor lesions].

Ultrasonographic imagings of non-tumor disorders in extremities were reviewed and discussed. All cases underwent surgery. In our retrospective evaluation, US was found to be effective in cases of tendon injuries, articular osteochondritis, tenosynovitis and the displacement of chondral navicula in infantile club foot, etc.

[CT in gallbladder cancer; especially the extension of the tumor].

An efficacy of CT in the preoperative staging diagnosis of gallbladder cancer was evaluated. CT findings were recorded following general rules for surgical and pathological studies on cancer of biliary tract proposed by Japanese society of biliary surgery. Serosal invasion (S factor), direct hepatic invasion (H inf factor), lymph node metastasis (N factor), bile duct invasion (B factor), duodenal invasion (Du factor) and finally preoperative staging diagnosis were included in this study. Conclusion is as follows. 1) False positive results were predominating in S and H inf factor, sensitivity was 76.0% and 50.0% respectively. 2) In B factor, the result was poor, the sensitivity 44.4%. 3) In N factor, CT was effective, the sensitivity 77.6%. 4) Finally it was concluded that CT can play an important role in staging diagnosis of gallbladder cancer, the sensitivity was 93.1%.

[CT of pulmonary eosinophilic granuloma].

CT findings of four cases of pulmonary eosinophilic Granuloma were discussed. In three cases, multiple nodular shadows or multiple cavitary lesions were observed in upper and middle lung field predominant. However, in one patient who had history of recurrent pneumothorax, CT revealed a honeycomb appearance and some bullous change as well. These findings seemed to represent a far advanced state of the disease. X-ray CT is excellent in demonstrating such pathological changes, and is indispensable in the diagnosis and follow up study of pulmonary eosinophilic granuloma.