Capsaicin receptor expression in the rat temporomandibular joint

Experimentally, temporomandibular joint (TMJ) nerve units respond to capsaicin, which is used clinically to treat TMJ pain. However, the existence of capsaicin receptors in the TMJ has not previously been clearly demonstrated. Immunohistochemical analysis has revealed the presence of transient receptor potential vanilloid subtype 1 (TRPV1) expression in the nerves and synovial lining cells of the TMJ. TRPV1-immunoreactive nerves are distributed in the synovial membrane of the joint capsule and provide branches to the joint compartment. The disc periphery is supplied by TRPV1 nerves that are mostly associated with small arterioles, and occasional nerves penetrate to the synovial lining layer. Double immunofluorescence has shown that many TRPV1-immunoreactive nerves are labeled with neuropeptide calcitonin gene-related peptide, whereas few are labeled with IB4-lectin. The results provide evidence for the presence of TRPV1 in both nerves and synovial lining cells, which might thus be involved in the mechanism of nociception and inflammation in the TMJ. This study was supported by a grant-in-aid for Scientific Research (C), no. 15591938, from the Japanese Ministry of Education, Science, Sports, Culture, and Technology (to M.A.K.).     

Bacteriocin Protein BacL1 of Enterococcus faecalis Is a Peptidoglycan d-Isoglutamyl-l-lysine Endopeptidase

Enterococcus faecalis strains are commensal bacteria in humans and other animals, and they are also the causative agent of opportunistic infectious diseases. Bacteriocin 41 (Bac41) is produced by certain E. faecalis clinical isolates, and it is active against other E. faecalis strains. Our genetic analyses demonstrated that the extracellular products of the bacL₁ and bacA genes, which are encoded in the Bac41 operon, coordinately express the bacteriocin activity against E. faecalis. In this study, we investigated the molecular functions of the BacL₁ and BacA proteins. Immunoblotting and N-terminal amino acid sequence analysis revealed that BacL₁ and BacA are secreted without any processing. The coincidental treatment with the recombinant BacL₁ and BacA showed complete bacteriocin activity against E. faecalis, but neither BacL₁ nor BacA protein alone showed the bacteriocin activity. Interestingly, BacL₁ alone demonstrated substantial degrading activity against the cell wall fraction of E. faecalis in the absence of BacA. Furthermore, MALDI-TOF MS analysis revealed that BacL₁ has a peptidoglycan d-isoglutamyl-l-lysine endopeptidase activity via a NlpC/P60 homology domain. These results collectively suggest that BacL₁ serves as a peptidoglycan hydrolase and, when BacA is present, results in the lysis of viable E. faecalis cells.     

Purification and Properties of Wall-bound α-Glucosidase from Suspension-cultured Rice Cells

Wall-bound α-glucosidase (EC 3.2.1.20) has been solubilized from suspension-cultured rice cells with Sumyzyme C and Pectolyase Y-23 and isolated by a procedure including fractionation with ammonium sulfate, Sephadex G-100 column chromatography, CM-cellulose column chroma-tography, Sephadex G-200 column chromatography, and preparative disc gel electrophoresis. The molecular weight of the enzyme was 64,000. The enzyme readily hydrolyzed maltose, maltotriose, and amylose, but hydrolyzed isomaltose and soluble starch more slowly. The Michaelis constant for maltose of the enzyme was estimated to be 0.272 mm. The enzyme produced panose as the main α- glucosyltransferred product from maltose.     

Polyphenol oxidase from wheat bran is a serpin

Polyphenol oxidase (PPO; EC 1.10.3.2) was isolated from wheat bran by a procedure that included ammonium sulfate fractionation, batch adsorption by DEAE-cellulofine, CM-cellulofine column chromatography, DEAE-cellulofine column chromatography, preparative isoelectric focusing, adsorption on the membrane of a Vivapure Q Maxi H spin column, and heat treatment. These procedures led to 150-fold purification with 4.2% recovery. The PPO was homogeneous by SDS/PAGE. The relative molecular weight of the PPO was estimated to be 37,000 based on its mobility in SDS/PAGE. The isoelectric point of the PPO was 4.4. The K(m) values of the PPO for caffeic acid, chlorogenic acid, pyrocatechol, 4-methyl catechol and l-DOPA as substrates were 0.077, 0.198, 1.176, 1.667 and 4.545 mM. The PPO was strongly inhibited by tropolone. The K(i) value for tropolone is 2.2 x 10(-7) M. The sequence of the 15 N-terminal amino-acid residues was determined to be ATDVRLSIAHQTRFA, which was identical to those of serpin from Triticum aestivum and protein Z from Hordeum vulgare. The PPO strongly inhibited the activity of trypsin, which is an enzyme of serine proteases; 50% inhibition was observed with 1.5 x 10(-7) M PPO. The K(i) value for PPO is 2.3 x 10(-8) M. The wheat bran PPO should be a very important protein for protecting wheat against disease, virus, insect and herbivore damages by both the activities of PPO and protease inhibitor.     

TRPV2 expression in rat oral mucosa

The oral mucosa is a highly specialised, stratified epithelium that confers protection from infection and physical, chemical and thermal stimuli. The non-keratinised junctional epithelium surrounds each tooth like a collar and is easily attacked by foreign substances from the oral sulcus. We found that TRPV2, a temperature-gated channel, is highly expressed in junctional epithelial cells, but not in oral sulcular epithelial cells or oral epithelial cells. Dual or triple immunolabelling with immunocompetent cell markers also revealed TRPV2 expression in Langerhans cells and in dendritic cells and macrophages. Electron microscopy disclosed TRPV2 immunoreactivity in the unmyelinated and thinly myelinated axons within the connective tissue underlying the epithelium. TRPV2 labelling was also observed in venule endothelial cells. The electron-dense immunoreaction in junctional epithelial cells, macrophages and neural axons occurred on the plasma membrane, on invaginations of the plasma membrane and in vesicular structures. Because TRPV2 has been shown to respond to temperature, hypotonicity and mechanical stimuli, gingival cells expressing TRPV2 may act as sensor cells, detecting changes in the physical and chemical environment, and may play a role in subsequent defence mechanisms.     

Pharmacologic stem cell based intervention as a new approach to osteoporosis treatment in rodents

Background

Osteoporosis is the most prevalent skeletal disorder, characterized by a low bone mineral density (BMD) and bone structural deterioration, leading to bone fragility fractures. Accelerated bone resorption by osteoclasts has been established as a principal mechanism in osteoporosis. However, recent experimental evidences suggest that inappropriate apoptosis of osteoblasts/osteocytes accounts for, at least in part, the imbalance in bone remodeling as occurs in osteoporosis. The aim of this study is to examine whether aspirin, which has been reported as an effective drug improving bone mineral density in human epidemiology studies, regulates the balance between bone resorption and bone formation at stem cell levels.

Methods and Findings

We found that T cell-mediated bone marrow mesenchymal stem cell (BMMSC) impairment plays a crucial role in ovariectomized-induced osteoporosis. Ex vivo mechanistic studies revealed that T cell-mediated BMMSC impairment was mainly attributed to the apoptosis of BMMSCs via the Fas/Fas ligand pathway. To explore potential of using pharmacologic stem cell based intervention as an approach for osteoporosis treatment, we selected ovariectomy (OVX)-induced ostoeporosis mouse model to examine feasibility and mechanism of aspirin-mediated therapy for osteoporosis. We found that aspirin can inhibit T cell activation and Fas ligand induced BMMSC apoptosis in vitro. Further, we revealed that aspirin increases osteogenesis of BMMSCs by aiming at telomerase activity and inhibits osteoclast activity in OVX mice, leading to ameliorating bone density.

Conclusion

Our findings have revealed a novel osteoporosis mechanism in which activated T cells induce BMMSC apoptosis via Fas/Fas ligand pathway and suggested that pharmacologic stem cell based intervention by aspirin may be a new alternative in osteoporosis treatment including activated osteoblasts and inhibited osteoclasts.     

Glycolytic enzyme Pgk1 is strongly expressed in the developing tooth germ of the mouse lower first molar

This study examined detailed in situ expression patterns and possible functional roles of phosphoglycerate kinase 1 (Pgk1) gene in the developing tooth germ of the mouse lower first molar. The strong expression of Pgk1 mRNA was seen in the odontogenic epithelial cells and surrounding mesenchymal cells of the tooth germ from embryonic day 10.5 (E10.5) to E18.0. Western blotting analysis demonstrated that Pgk1 protein formed 84-kDa protein complex in these embryonic organs. The results of immunoprecipitation-western blotting also suggested this complex to be formed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover, the immunofluorescence expression of those proteins was shown to overlap each other in the tooth germ at E15.0. A strong immunofluorescence expression of both Pgk1 and GAPDH also corresponded to the in situ expression of those mRNAs. These results suggested that Pgk1 plays some functional roles in the development of tooth germ and other embryonic organs by forming protein complex with GAPDH.     

Differential changes in cell wall matrix polysaccharides and glycoside-hydrolyzing enzymes in developing wheat seedlings differing in drought tolerance

The growth kinetics and variations in cell wall matrix polysaccharides and glycoside hydrolases during seedling development of the drought-tolerant wheat cultivar (cv. Hong Mang Mai) were compared with the drought-sensitive cultivar (cv. Shirasagikomugi). After 15d of culture in water at 22 degrees C under constant irradiance of 98mumolm(-2)s(-1), the length of the coleoptile and leaf sheath of Hong Mang Mai seedlings was 1.7 times longer than those of Shirasagikomugi seedlings. In the cell walls isolated from coleoptiles and leaf sheaths of the seedling of the two cultivars, the contents of arabinose, xylose, and glucose changed during development. The cell walls were fractionated progressively with 50mM CDTA, 50mM Na(2)CO(3), 1M KOH and 4M KOH, and sugar composition was determined. The amount of CDTA-soluble fraction from the Hong Mang Mai cell walls was 2.4-fold higher than that from the Shirasagikomugi cell walls at 6d of culture, and a considerable decrease was observed during development. The ratio of arabinose to xylose in 1M KOH-soluble fraction from the two cultivars decreased. The amount of 4M KOH-soluble fraction from the Shirasagikomugi cell walls was affected much more than those of the Hong Mang Mai cell walls. Many glycoside hydrolase activities were detected in the protein fractions from coleoptiles and leaf sheaths of the two cultivars, and the activities of licheninase, 1,3-1,4-beta-glucanase, and 1,3-beta-glucanase in the LiCl-soluble protein fraction increased drastically during development of the Shirasagikomugi seedlings. These findings suggest that the metabolism of the cell wall matrix polysaccharides of the drought-tolerant wheat cultivar is far different from that of the drought-sensitive wheat cultivar during seedling development.     

Expression of Set-alpha during morphogenesis of mouse lower first molar

The detailed in situ expression pattern of the Set- gene has been studied. Previously we showed that Set- is a differentially expressed gene in the embryonic mouse mandible at day 10.5 (E10.5) gestational age. Cells expressing Set- were widely distributed in both the epithelial and underlying ectomesenchymal cells at E10.5. At E12, they were slightly aggregated in an area where tooth germ of the lower first molar is estimated to be formed. At E13.5, Set- was strongly expressed in the tooth germ. At the cap stage, Set- was expressed in the enamel organ and dental papilla. At the bell stage, Set- was distinctly expressed in the inner enamel epithelial and dental papilla cells facing the inner enamel epithelial layer, which were intended to differentiate into ameloblasts and odontoblasts, respectively. Interestingly, Set- was also expressed in several embryonic craniofacial tissues derived from the ectoderm. This study is the first report that Set- is distinctly expressed in the developing tooth germ, and suggests that Set- plays an important role in both the initiation and the growth of the tooth germ, as well as in the differentiation of ameloblasts and odontoblasts.