Motohiko Nagayama

Conditional Kif3a ablation causes abnormal hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage formation during mouse skeletogenesis.

The motor protein Kif3a and primary cilia regulate important developmental processes, but their roles in skeletogenesis remain ill-defined. Here we created mice deficient in Kif3a in cartilage and focused on the cranial base and synchondroses. Kif3a deficiency caused cranial base growth retardation and dysmorphogenesis, which were evident in neonatal animals by anatomical and micro-computed tomography (μCT) inspection. Kif3a deficiency also changed synchondrosis growth plate organization and function, and the severity of these changes increased over time. By postnatal day (P)7, mutant growth plates lacked typical zones of chondrocyte proliferation and hypertrophy, and were instead composed of chondrocytes with an unusual phenotype characterized by strong collagen II (Col2a1) gene expression but barely detectable expression of Indian hedgehog (Ihh), collagen X (Col10a1), Vegf (Vegfa), MMP-13 (Mmp13) and osterix (Sp7). Concurrently, unexpected developmental events occurred in perichondrial tissues, including excessive intramembranous ossification all along the perichondrial border and the formation of ectopic cartilage masses. Looking for possible culprits for these latter processes, we analyzed hedgehog signalling topography and intensity by monitoring the expression of the hedgehog effectors Patched 1 and Gli1, and of the hedgehog-binding cell-surface component syndecan 3. Compared with controls, hedgehog signaling was quite feeble within mutant growth plates as early as P0, but was actually higher and was widespread all along mutant perichondrial tissues. Lastly, we studied postnatal mice deficient in Ihh in cartilage; their cranial base defects only minimally resembled those in Kif3a-deficient mice. In summary, Kif3a and primary cilia make unique contributions to cranial base development and synchondrosis growth plate function. Their deficiency causes abnormal topography of hedgehog signaling, growth plate dysfunction, and un-physiologic responses and processes in perichondrial tissues, including ectopic cartilage formation and excessive intramembranous ossification.

Garlic allyl derivatives interact with membrane lipids to modify the membrane fluidity

As a novel approach to the mode of medicinal action of garlic, its constituents were comparatively studied with respect to their interactions with membrane lipids to modify the membrane fluidity. Allyl derivatives rigidified tumor cell and platelet model membranes consisting of unsaturated phospholipids and cholesterol at 20-500 muM with the potency being diallyl trisulfide (DATS) > diallyl disulfide (DADS) by preferentially acting on the hydrocarbon cores of lipid bilayers. They were also effective in rigidifying candida cell model membranes prepared with ergosterol and phospholipids at 100-500 microM with the potency being DADS > DATS > diallyl sulfide (DAS), but not bacteria cell model membranes without ergosterol. Alliin, a precursor of these DASs, was not active on any membranes at 500 microM. Both relative intensity and selectivity in membrane effects correlated with those in antiproliferative, antiplatelet and antimicrobial effects. In cell culture experiments, membrane-active DASs inhibited the growth of tumor cells cultured for 24 and 48 h at 20-500 muM to show the potency being DATS > DADS, together with rigidifying cell membranes by acting on their deeper regions more intensively. However, membrane-inactive allyl derivatives were not growth-inhibitory on tumor cells. The membrane lipid interactions of DASs appear to be one of possible mechanisms underlying different effects of garlic.

Different antibacterial actions of isoflavones isolated from Erythrina poeppigiana against methicillin-resistant Staphylococcus aureus

Aims:  To screen six isoflavones isolated from Erythrina poeppigiana (Leguminosae) for their antibacterial activity against methicillin‐resistant Staphylococcus aureus (MRSA).

Evaluation of co-aggregation among Streptococcus mitis, Fusobacterium nucleatum and Porphyromonas gingivalis

Aims: To develop a semi‐quantitative method for evaluating co‐aggregation reactions among three bacterial species, and to examine the influence of Fusobacterium nucleatum on the adherence of Porphyromonas gingivalis.

Regulation of CCN2 gene expression and possible roles in developing tooth germs

CCN proteins are extracellular and cell-associated molecules involved in several developmental processes, but their expression patterns and regulation in tooth development remain unclear. Here we first determined the expression patterns of CCN genes in mouse tooth germs. We found that at early stages CCN2 was detected in dental lamina, dental mesenchyme, and primary enamel knot, while other CCN family members were expressed broadly. By the bell stage, all members were expressed in differentiating odontoblasts and ameloblasts, but CCN1 and CCN2 transcripts were conspicuous in differentiating osteoblasts in dental follicle. Next, we asked what signalling molecules regulate CCN2 expression and what roles CCN2 may have. We found that upon surgical removal of dental epithelium CCN2 was not longer expressed in dental mesenchyme in cultured bud stage germs. Implantation of beads pre-coated with BMPs and FGFs onto E12-13 mandibular explants induced CCN2 expression in dental mesenchyme. There was a dose-dependent effect of BMP-4 on CCN2 induction; a concentration of 100 ng/μl was able to induce strong CCN2 expression while a minimum concentration of 25 ng/μl was needed to elicit appreciable expression. Importantly, Noggin treatment inhibited endogenous and BMP-induced CCN2 expression, verifying that CCN2 expression in developing tooth germs requires BMP signalling. Lastly, we found that rCCN2 stimulated proliferation in dental mesenchyme in a dose-dependent manner. Together, the data indicate that expression of CCN genes is spatio-temporally regulated in developing tooth germs. CCN2 expression appears to depend on epithelial and mesenchymal-derived signalling factors, and CCN2 can elicit strong proliferation in dental mesenchyme.

Establishment of a novel dwarf rat strain: cartilage calcification insufficient (CCI) rats

Rats with dwarfism accompanied by skeletal abnormalities, such as shortness of the limbs, tail, and body (dwarf rats), emerged in a Jcl-derived Sprague-Dawley rat colony maintained at the Institute for Animal Experimentation, St. Marianna University Graduate School of Medicine. Since the dwarfism was assumed to be due to a genetic mutation based on its frequency, we bred the dwarf rats and investigated their characteristics in order to identify the causative factors of their phenotypes and whether they could be used as a human disease model. One male and female that produced dwarf progeny were selected, and reproduction was initiated by mating the pair. The incidence of dwarfism was 25.8% among the resultant litter, and dwarfism occurred in both genders, suggesting that it was inherited in an autosomal recessive manner. At 12 weeks of age, the body weights of the male and female dwarf rats were 40% and 57% of those of the normal rats, respectively. In soft X-ray radiographic and histological examinations, shortening and hypoplasia of the long bones, such as the tibia and femur, were observed, which were suggestive of endochondral ossification abnormalities. An immunohistochemical examination detected an aggrecan synthesis disorder, which might have led to delayed calcification and increased growth plate thickening in the dwarf rats. We hypothesized that the principal characteristics of the dwarf rats were systemically induced by insufficient cartilage calcification in their long bones; thus, we named them cartilage calcification insufficient (CCI) rats.

Intraneural perineurioma arising in the lateral border of the tongue

To the Editor: The term perineurioma (PN) was first suggested by Lazarus and Trombetta in 1978 to describe a rare peripheral nerve sheath tumor consisting of the proliferation of neoplastic perineurial cells. It is divided into four types on the basis of histological findings: soft tissue, sclerosing, reticular and intraneural PN. Soft tissue PN generally occurs as a nodular mass subcutaneously, especially in the extremities of adults. Sclerosing PN prefers the fingers of adults and reticular PN is generally observed within the upper limbs of adult women. Intraneural PN causes enlargement of the affected nerve, especially the thick nerves of the extremities of infants and young adults. Although the etiology of this lesion is unknown, the possibility of a clonal neoplastic disorder has been supported by a previous report. Intraneural PN in oral regions is extremely rare; only six cases have been reported in the literature. This report describes an unusual case of intraneural PN in the lateral border of the tongue with histological and immunohistochemical findings. A 14-year-old boy presented at his primary dental clinic because of a painless nodule in the right lateral border of the tongue. The patient had first noticed the nodule on the surface of the tongue 2 years ago and reported that it was gradually increasing in size. He had no prior history of trauma; intraoral examination revealed an elastic, wellcircumscribed nodule on the right lateral surface of the tongue (Fig. 1a). The lesion was approximately 4 × 3 mm in size, and the overlying epithelium was whitish and smooth. An excisional biopsy of the nodule was performed under local anesthesia (Fig. 1b). Histopathologically, the preparation disclosed a sessilebased and relatively well-circumscribed lesion composed of spindle-shaped cells arranged concentrically around the axons of a small peripheral nerve, forming numerous pseudo-onion bulb structures (Fig. 1c). In addition, there were many small peripheral nerves continuously from the bottom of the excisional fragment of the lesion (Fig. 1d). The pseudo-onion bulb structures consisted of a fibrous connective tissue with increased irregular collagen bundles with many capillary blood vessels and few inflammatory cells (Fig. 2a,b). Immunohistochemically, the spindle-shaped cells were positive for epithelial membrane antigen (EMA) (Fig. 2c). Glucose transporter 1 was positive within the tumor cell population, and Vimentin positivity was observed throughout. In contrast, S-100 protein immunostaining was positive in the Schwann cells and centrically located axons but negative in the pseudo-onion bulb structures (Fig. 2d). The tissue showed no positive staining for the other antigens tested (cytokeratin and CD34). These findings completely confirm a diagnosis of intraneural PN. The postoperative course was uneventful and 6 months after the operation there was no sign of recurrence. PN is a rare peripheral nerve sheath tumor usually affecting the extremities of infants to adults. Only six cases in the oral region have been reported and five of these cases occurred in the tongue. Four of the previous six cases were located in the lateral border of the tongue. Intraneural PN commonly involves a thick nerve. Among the described cases of oral intraneural PN, the mandibular cases involved the inferior alveolar nerve, while the other cases affecting the tongue and buccal mucosa arose from an unnamed nerve. Generally, intraneural PN causes several symptoms, such as sensory or motor deficits, muscle weakness and muscle atrophy. The present patient did not show any symptoms; this is observed in most cases arising in oral soft tissue suggesting that these cases including the present case had no relationship to major nerves. Most peripheral nerve sheath tumors in the oral region are Schwannoma and neurofibroma, and PN is extremely rare. Histopathologically, soft tissue PN shows bundles, whorls or storiform arrangement composed of spindle-shaped and/or wavy cells with long, tapered cytoplasmic processes and elongated nuclei, within a dense collagenous stroma. Sclerosing PN reveals a proliferation of cords, storiform or small island-like arrangements composed of the cuboidal or epithelioid tumor cells within an abundantly dense collagenous stroma. Reticular PN shows a proliferation of the reticularlike tumor cells within a myxoid or fibrous stroma. Intraneural PN reveals numerous pseudo-onion bulb structures composed of whorled proliferation of spindle-shaped cells around central axons, as observed in the present case. Immunohistochemically, the spindle-shaped cells are positive for EMA but negative for S-100 protein, indicating that PN is derived from perineural cells. Although Schwann cells are positive for S-100 protein, the ratio of S-100-positive cells is lower than that for EMA. These immunohistochemical findings are useful for distinguishing between PN and other peripheral nerve sheath tumors. In the differential diagnosis of intraneural PN, the hereditary hypertrophic neuropathies of Charcot-Marie-Tooth disease and Déjerine-Sottas should be considered. In these diseases, true onion-bulb structures Pathology International 2013; 63: 619–621 doi:10.1111/pin.12121 bs_bs_banner

Guided Bone Regeneration, Platelet Rich Plasma and Low-Intensity Ultrasound Irradiation for Dental Implants

Because a concept of an osteointegrated dental implant system was established, prognosis of a dental implant at treatment improved and the treatment of restoration for missing teeth was changed. However, a dental implant treatment into atrophic jaw bone requires bone augmentation, obviously. At this time, we studied for the purpose of establishing the evidence of each method for clinical application of these bone augmentation method, such as guided bone regeneration (GBR), and autogenous bone block graft (BBG). In addition, we pursued the basic study of the evidence about the bone formation with platelet rich plasma (PRP) which recognized the availability in clinic. Furthermore, we present the results of basic studies which we tested for the purpose of applying a low-intensity pulse ultrasound (LIPU) irradiation applied to a fracture treatment in orthopedics area to intra-oral area, specially the condition after implant placement. In the results of comparison with GBR site and BBG, the differences of labeling bands were observed with a fluorescence microscopy. There was much labeling bands on GBR sections in comparison with BBG. This meaning that the bone remodeling around implants at GBR site was more active than BBG site. And the new bone formation by PRP was identified on soft X-ray graphically at first week after PRP applied mandible bone defect (experimental side). At same region of first week specimen, we confirmed positive reactions of platelet derived growth factor

Pthlh, a promising cancer modifier gene in rat tongue carcinogenesis.

Susceptibly to the induction of rat tongue cancer (TC) by oral 4-nitroquinoline 1-oxide (4NQO) exposure is a polygenic trait. Among several quantitative trait loci identified by crosses between TC-susceptible Dark Agouti (DA) rats and TC-resistant Wistar-Furth (WF) rats, we focused on tongue cancer susceptibility locus (Tcas3) of chromosome 4. We examined tongue carcinogenesis in the reciprocal congenic strains DA.WF-Tcas3 and WF.DA-Tcas3 and in their parental strains. The Tcas3DA allele, and not the Tcas3WF allele, significantly favored tumor latency, incidence and TC number/size. In genomic DNA of TCs induced in (DA × WF) F1 rats, the resistant Tcas3WF allele was frequently and selectively lost, particularly in larger tumors. Thus, we searched the possible candidate genes in the Tcas3 region using microarray analysis of TCs in F1 rats and revealed significant upregulation of 2 cancer-related genes, parathyroid hormone-like hormone (Pthlh) and Kras2. The relevance of the WF allele of Pthlh as a cancer modifier was indicated by 3 single nucleotide polymorphisms specific to this strain. In contrast, no consistent strain-specific variations were found in Kras2. Moreover, the plasma Ca2+ level was consistently higher in DA rats when compared to the level in WF rats bearing TCs; moreover, the Pthlh-mRNA expression level was >30-fold higher in TCs when compared to this level in the normal tongue mucosa. Immunostaining experiments showed strong PTHrP protein expression in TCs of DA rats, and the signal was intensified in larger TCs. Kras2 was also upregulated in TCs, but to a lesser degree than PTHrP. Thus, Pthlh is a promising candidate modifier gene in the development and progression of rat TCs.