Repression of hTERT transcription by the introduction of chromosome 3 into human oral squamous cell carcinoma.

Telomerase is a ribonucleoprotein enzyme that maintains telomere length. Telomerase activity is primarily attributed to the expression of telomerase reverse transcriptase (TERT). It has been reported that introduction of an intact human chromosome 3 into the human oral squamous cell carcinoma cell line HSC3 suppresses the tumorigenicity of these cells. However, the mechanisms that regulate tumorigenicity have not been elucidated. To determine whether this reduction in tumorigenicity was accompanied by a reduction in telomerase activity, we investigated the transcriptional activation of TERT in HSC3 microcell hybrid clones with an introduced human chromosome 3 (HSC3#3). HSC#3 cells showed inhibition of hTERT transcription compared to that of the parental HSC3 cells. Furthermore, cell fusion experiments showed that hybrids of HSC3 cells and cells of the RCC23 renal carcinoma cell line, which also exhibits suppression of TERT transcription by the introduction of human chromosome 3, also displayed suppressed TERT transcription. These results suggested that human chromosome 3 may carry functionally distinct, additional TERT repressor genes.

Cancer-associated fibroblasts and CD163-positive macrophages in oral squamous cell carcinoma: their clinicopathological and prognostic significance.

BACKGROUND: Stromal cells are believed to affect cancer invasion and metastasis. The purpose of this study was to evaluate the distribution of cancer-associated fibroblasts (CAFs) and the incidence of tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC), focusing on clinicopathological factors and patient prognosis, as well as cancer invasion. METHODS: The study included 108 patients with OSCC. Anti-α-smooth muscle actin, CD68, and CD163 antibodies were used to identify CAFs and TAMs. CAFs were divided into 4 grades on the basis of staining intensity: negative (0), scanty (1), focal (2), and abundant (3). The most intensive areas of macrophage concentration in each tumor invasive stroma were also evaluated. RESULTS: The cancer specimens were divided into Grade 0/1, Grade 2, and Grade 3 on the basis of CAF grade. In addition, they were divided into low- and high-grade groups on the basis of the number of CD68-positive and CD163-positive macrophages. The latter were significantly increased in the Grade 2 CAF group compared to the Grade 0/1 group (P = 0.009). Kaplan-Meier and multivariate survival analyses revealed that Grade 2 CAFs (P = 0.003) and high CD163-positive macrophage levels (P = 0.007) significantly correlated with a poor outcome in patients with OSCC, and that a high CD163-positive macrophage level was a significant and an independent prognostic factor (P = 0.045). CONCLUSIONS: Cancer-associated fibroblasts and CD163-positive macrophages may be potential prognostic predictors of OSCC.

[Successful outcome from treatment modality with low-dose S-1 for three oldest old patients with advanced oral cancer].

We herein present three oldest old patients with advanced oral cancer for whom low-dose S-1 treatment was effective and improved QOL. The first patient is a 90-year-old female with cancer of the maxilla(T4N0Mx). Because of her generally poor condition, her family did not desire radical therapy. S-1(40mg/m2)was administered for 2 weeks followed by a 1-week interval. Because of a partial response without serious side effects, we increased the dose of S-1 to 50mg/m2. The tumor currently shows a tendency toward reduction. The second patient is a 96-year-old female with cancer of the mandible(T4N0Mx). Because of her old age, her family desired palliative therapy. S-1(40mg/m2)was administered for 2 weeks followed by a 1-week interval. A complete response was obtained at the end of the second course. There has been no recurrence. The third patient is a 94-year-old female with cancer of the maxilla(T3N0M0). Her family selected palliative therapy. S-1(50mg/m2)was administered for 2 weeks followed by a 1-week interval. The tumor size decreased after administration of S-1, without serious side effects. However, the tumor increased after the end of the fifth course. Considering the patient's condition, S-1 administration was discontinued at the end of the eighth course. S-1 is considered to be an effective and safe treatment for the maintenance or improvement of the QOL of old and oldest old patients with advanced oral cancer.

Minichromosome maintenance-7 and geminin are reliable prognostic markers in patients with oral squamous cell carcinoma: immunohistochemical study.

BACKGROUND: Minichromosome maintenance (MCM) proteins act as the origin licensing machinery that regulates initiation of DNA replication. Geminin is a licensing repressor and prevents reinitiation of DNA replication by blocking reloading of MCM proteins at replication origins. Recent studies have proposed that MCM7 and geminin may be sensitive proliferative and prognostic markers of various malignant tumors. This study aimed to analyze the expression of MCM7 and geminin to clarify pathobiological significance in human oral squamous cell carcinomas (OSCCs). METHODS: We performed immunohistochemical analysis on 10 specimens of normal oral epithelia, 50 lesions with dysplasia and 113 OSCCs. Labeling indices (LIs) for MCM7, geminin and Ki-67 were evaluated, comparing with clinicopathological profiles. RESULTS: The mean LIs for MCM7 were 29.2% for normal epithelia, 32.2% for dysplasias, and 51.1% for OSCCs; the value was significantly higher in the last than in the former two (P < 0.01). The mean LIs for geminin were 6.8% for normal epithelia, 9.2% for dysplasias, and 21.3% for OSCCs; the value was significantly higher in the OSCCs (P < 0.01). The MCM7 LIs were correlated with the histological grade of OSCCs, in which the highest LIs were noted in the poorly differentiated type (P < 0.01). The survival rate was significantly lower in patients with a higher MCM7 LI (>49.5%) than in those with a lower LI (P < 0.05) at stage III-IV. However, the survival rate in the patients with a higher geminin LI (>19.5%) was significantly higher than in those with a lower LI (P < 0.05) at stage IV.

Bisphosphonate-induced reactive oxygen species inhibit proliferation and migration of oral fibroblasts: A pathogenesis of bisphosphonate-related osteonecrosis of the jaw.

BACKGROUND: The onset mechanism for bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been reported, with a focus on bone remodeling, biofilm formation, and epithelial cell proliferation and migration. However, the involvement of stromal cells, especially fibroblasts, in the oral cavity is unclear. Therefore, this study was focused on how bisphosphonates (BPs) affect orthotopic periodontal ligament fibroblasts from the viewpoint of oxidative stress compared with ectopically obtained fibroblasts. METHODS: Normal human periodontal ligament fibroblasts (HPdLFs) and normal human dermal fibroblasts (NHDFs) were used to gain insight into the functional differences in sensitivity and reactions to BPs. Cell growth assay, measurement of reactive oxygen species (ROS) and nitric oxide (NO) production, and wound-healing assay in vitro were performed. Maxillary first molars were extracted in C57BL/6 mice and either BP, N-acetyl-cysteine (NAC), and BP or saline were administered. RESULTS: BP-induced IC50 values were significantly lower in HPdLFs (30.6 µM) than in NHDFs (109.7 µM). BP resulted in an increase in ROS, but not NO generation in HPdLFs. BPs also inhibited proliferation and migration of HPdLFs but not NHDFs, while the addition of a ROS inhibitor, NAC, reversed those inhibitions. A BRONJ mouse model in which BP was administered and then the tooth was extracted, impaired wound healing of the socket was observed. When NAC was administered before tooth extraction, wound healing was significantly improved. CONCLUSION: These results suggest that BP causes fibroblasts obtained from the oral cavity but not from skin to generate ROS and that the subsequent ROS-mediated inhibition of fibroblast growth and migration definitely delays wound healing, thereby contributing to BRONJ pathogenesis.

Involvement of N-acetyltransferase human in the cytotoxic activity of 5-fluorouracil.

N-acetyltransferase human (NATH) participates in a posttranslational modification of the proteins and has been reported to play a role in apoptosis. In this study, the involvement of NATH in the cytotoxic action of 5-fluorouracil (5-FU) in human squamous cell carcinoma HEp-2 cells was examined. We found that 5-FU decreased NATH expression in a dose-dependent and time-dependent manner. No change was observed after treatment with bleomycin, nedaplatin, mitomycin C, or methotrexate. Interestingly, knockdown of NATH by small interfering RNA resulted in the downregulation of thymidylate synthase mRNA expression and induced apoptosis. Conversely, NATH overexpression facilitated cell proliferation independent of the presence of 5-FU. The effect of NATH knockdown on the expression of proteins in HEp-2 cells was examined using two-dimensional gel electrophoresis and mass spectrometry. Profilin 1, CutA, ras-related nuclear protein, annexin A5, enolase 1, and elongation factor 1 alpha 1 were found to be upregulated and 14-3-3eta, tublin, nuclear auto antigenic sperm protein, heat shock protein 70, and heat shock protein 90 were downregulated by knockdown of NATH. The results of this study suggest that NATH plays an important role in the cytotoxic activity of 5-FU.

Runt-related transcription factor 3 expression in human oral squamous cell carcinomas; implication for tumor progression and prognosis.

Runt-related transcription factor 3 (RUNX3) is a tumor suppressor factor of gastric cancer and appears to be an important component of the transforming growth factor-beta (TGF-beta)-induced tumor suppression pathway. This study aimed to analyze the expression of the RUNX3 protein in human oral normal epithelia, dysplasia and squamous cell carcinomas (SCCs), comparing it with clinicopathological profiles. Western blot analysis revealed the RUNX3 protein as a single band at 44kDa in oral non-neoplastic mucosa and SCC. The expression of RUNX3 protein was also examined in 10 normal epithelia, 51 dysplasias and 108 oral SCCs. The labeling indices (LIs) of RUNX3, Ki-67, P21, P27 and the apoptotic index (AI) were evaluated using immunohistochemistry and the TUNEL method. The LI of RUNX3 was 7.7+/-1.6 in the normal epithelia, 20.8+/-2.7 in the dysplasias and 9.0+/-1.3 in the SCCs. The LI of RUNX3 was significantly highest in the dysplasias, followed by the SCCs (p<0.05) and normal epithelia (p<0.05). The RUNX3 LI correlated with the histological differentiation of SCCs, being the highest in the well differentiated SCCs (p<0.01). In addition, RUNX3 expression was significantly related to the lower Ki-67 LI, but not to LI of P21 and P27, and AI in the SCCs. The survival rate was significantly lower in the patients with lower RUNX3 expression (<5%) than in those with higher expression (5%) (p<0.05). These results indicate that the expression of RUNX3 is correlated with histological differentiation, and inversely with cellular proliferation of the oral SCCs, and might be a new prognostic marker in the patients with oral SCC.

Palonosetron Prevents Highly Emetogenic Chemotherapy-induced Nausea and Vomiting in Oral Cancer Patients.

BACKGROUND/AIM: To evaluate the efficacy of palonosetron in preventing acute and delayed nausea and vomiting in patients receiving highly emetogenic chemotherapy (HEC) in oral cancer patients. PATIENTS AND METHODS: Oral cancer patients receiving HEC were enrolled; among the 40 patients, 87 courses of chemotherapy were administered. On day 1, 0.75 mg palonosetron was intravenously administrated just before chemotherapy. RESULTS: The primary endpoint was the proportion of patients with a complete response (CR) and the secondary endpoint was the proportion of patients with complete control (CC) during the acute and delayed phase. During the acute phase, 86 of 87 courses (98.9%) had CR and 84 of 87 courses (96.6%) had CC. During the delayed phase, 84 of 87 courses (96.6%) had CR and 70 of 87 courses (80.5%) had CC. CONCLUSION: Palonosetron is effective at preventing HEC-induced chemotherapy-induced nausea and vomiting (CINV) in oral cancer chemotherapeutic regimens in the acute and delayed phases.

Associations between Systemic Markers of Bone Turnover or Bone Mineral Density and Anti-Resorptive Agent-Related Osteonecrosis of the Jaw in Patients Treated with Anti-Resorptive Agents.

BACKGROUND: Some previous studies have examined anti-resorptive agent-related osteonecrosis of the jaw (ARONJ) prediction using systemic markers of bone turnover as risk factors. Radiographic imaging is also effective at detecting ARONJ. In this study, computed tomography (CT)-derived bone mineral density (BMD) values and the levels of systemic markers of bone turnover were evaluated, and then each parameter was compared between patients that developed ARONJ and those who did not after treatment with systemic anti-resorptive agents. The aim of this study was to determine whether systemic markers of bone turnover and/or BMD values can be used to predict the risk of ARONJ. METHODS: The subjects' serum levels of cross-linked N-terminal telopeptide of type I collagen (NTX) and bone alkaline phosphatase (BAP) (systemic markers of bone turnover) were measured. BMD was calibrated to CT values using a medical imaging phantom. Then, the subjects' BMD were assessed using quantitative computed tomography. Fifty-six patients who had received systemic anti-resorptive agents were included in this study. Thirty-two of the patients developed ARONJ after receiving the drugs whereas the remaining 24 did not. RESULTS: No correlation was observed between the serum levels of the systemic markers of bone turnover and the incidence of ARONJ. On the other hand, the ARONJ patients exhibited higher mandibular BMD values than the control group. BMD was not associated with healing or the clinical stage of ARONJ. CONCLUSION: These results suggest that increased mandibular BMD values are associated with ARONJ. Furthermore, mandibular BMD might serve as a novel marker for predicting the risk of ARONJ in patients that are taking anti-resorptive agents and are about to undergo tooth extraction. Accordingly, mandibular BMD could be a useful tool for aiding risk assessments and guiding treatment decisions.

In Vitro Inhibition of Cytopathic Effect of Influenza Virus and Human Immunodeficiency Virus by Bamboo Leaf Extract Solution and Sodium Copper Chlorophyllin.

BACKGROUND: Although the link between oral and oropharyngeal health status and susceptibility to infection has long been recognized, there is a limit to the selection of antiseptics for oral care. METHODS: Madin-Darby canine kidney (MDCK) cells were exposed to influenza virus and cultured in the presence or absence of test reagents: bamboo leaf extract solution and sodium copper chrolophyllin. MDCK cells were pre-incubated with the reagents to assess the inhibitory activity at adsorption (viral attachment). Similarly, anti-HIV activity and the inhibitory mechanism at adsorption were assessed by MT-2 cell culture system. Mixture of HIV and bamboo leaf extract solution was fixed and examined by transmission electron microscopy. RESULTS: The 50% inhibitory concentration (IC50) of bamboo leaf extract solution against influenza virus and the 50% cytotoxic concentration (CC50) in MDCK cells of the solution lay between 0.0313-0.0625% and 0.5-1.0%. The solution inhibited the influenza virus adsorption at the concentration of 0.5% (P < 0.05). The values of IC50 and CC50 of sodium copper chlorophyllin lay between 50-100 µM and 200-400 µM, respectively. This inhibited the virus adsorption at 200 µM (P < 0.05). The bamboo leaf extract solution showed values of IC50 against HIV and CC50 in MT-2 cells at around 0.0313% and between 0.25-0.5%, respectively. This solution inhibited HIV adsorption at 1.25% (P < 0.05). The IC50 and CC50 of sodium copper chlorophyllin lay between 50-100 µM and 200-400 µM, respectively. Sodium copper chlorophyllin inhibited HIV adsorption at 2.5 mM (P < 0.05). HIV particles survived after the exposure to 0.5% bamboo leaf extract solution. CONCLUSION: Sodium copper chlorophyllin exerted antiviral activities against influenza virus and HIV as the major ingredient of bamboo leaf extract solution by blocking adsorption. This mechanism of action is different completely from the one of povidone-iodine.

A novel PAX3 mutation in a Japanese boy with Waardenburg syndrome type 1.

Waardenburg syndrome type 1 (WS1) is a rare autosomal dominant disorder characterized by hair hypopigmentation, abnormal iris pigmentation, and congenital hearing loss. WS1 is caused by mutations in paired box gene 3 (PAX3). We identified a novel PAX3 mutation (c.1107 C>G, p.Ser369Arg) in a Japanese WS1 patient showing abnormal right iris pigmentation, right-sided congenital hearing loss, synophrys, incomplete left cleft lip, and cryptorchidism.

Wnt/Beta-Catenin Signal Inhibitor HC-1 Sensitizes Oral Squamous Cell Carcinoma Cells to 5-Fluorouracil through Reduction of CD44-Positive Population.

BACKGROUND: Oral squamous cell carcinoma is a prevalent and frequently lethal malignancy worldwide. Existence of treatment-resistant cancer stem cells is considered to be associated with tumor formation, recurrence and metastasis. Wnt/beta-catenin signal is one of the crucial signaling pathways for cancer stem cells. Wnt/beta-catenin signal inhibitor may reduce the population of cancer stem cells and improve therapeutic effects on the cancers. METHODS: The effects of three derivatives of Wnt/beta-catenin signal inhibitors, HC-1, IC-2 and PN3-13, which we recently developed, on oral squamous cell carcinoma cell line HSC2, were examined by luciferase reporter assay, WST assay, cell sorting assay and apoptosis assay. RESULTS: The reporter assay showed that these small molecule compounds reduced Wnt/beta-catenin transcriptional activity in HSC2 cells. Of these compounds, IC-2 and PN3-13 inhibited cell viability in a dose-dependent manner, whereas HC-1 did not at even higher concentrations. Notably, however, the cell-sorting assay revealed that HC-1 significantly reduces the CD44-positive population of oral squamous cell carcinoma cells, compared to other compounds without affecting cell viability. In addition, HC-1 increases the cytotoxicity of HSC2 cells to 5-fluorouracil. The combination treatment of HC-1 with 5-fluorouracil significantly increased the apoptotic cells whereas treatment by either compound did not. CONCLUSION: These data suggest that HC-1 is an effective compound to target cancer stem cells, and the combination treatment of HC-1 and 5-fluorouracil can stimulate the tumor suppressive effect on oral squamous cell carcinoma cells.

Cyclo-oxygenase-1 and -2 expression in human oral mucosa, dysplasias and squamous cell carcinomas and their pathological significance.

Cyclo-oxygenase (COX) is a key enzyme in the conversion of arachidonic acid to prostanoids. The expression of its isoforms, COX-1 and -2 is found in many human malignancies. This study analyzed the correlation between COX expression and the pathobiological nature of human oral mucosa, dysplasias and squamous cell carcinomas (SCCs). We examined 9 specimens of normal oral epithelia, 65 lesions with dysplasias and 50 SCCs. Labeling indices (LIs) for COX-1, COX-2, Ki-67 and P53, microvessel density (MVD) and apoptotic index (AI) were evaluated using immunohistochemistry and TUNEL methods. Western blot analysis of COX-1 and -2 was performed on four human oral SCC cell lines, all of which showed expression. The LIs for COX-1 and -2 were higher for the dysplasias than the SCCs. LIs of COX-2 but not COX-1 correlated with the histological grade of dysplasia, being highest for the severe dysplasias (p < 0.05). In contrast, the COX-2 LIs as well as COX-1 were significantly (p < 0.05) inversely correlated with the histological differentiation of the SCCs. COX-2 expression was significantly correlated with LIs of COX-1 for dysplasia (p < 0.05), but not for the SCCs. In addition no significant relationship was noted between COX-2 expression and the Lis of Ki-67, P53, AI as well as MVD for the dysplasias and SCCs. The expression of COX-1 and -2 is correlated with early stage tumorigenesis and cellular differentiation of SCCs in the oral dysplasia-carcinoma sequence.

The Effect of Bamboo Leaf Extract Solution and Sodium Copper Chlorophyllin Solution on Growth and Volatile Sulfur Compounds Production of Oral Malodor Associated Some Anaerobic Periodontal Bacteria.

BACKGROUND: Bamboo leaf extract solution (BLES) and sodium copper chlorophyllin solution (SCCS) are known for their anti-oxidant activities. Oral malodor is often related with periodontal pathogens. The present study was undertaken to investigate the anti-bacterial effect of both BLES and SCCS on anaerobic periodontal bacteria producing oral malodorous volatile sulfur compounds (VSC). METHODS: Porphyromonas gingivalis W83 (PG), Prevotella intermidai TDC19B (PI), Fusobacterium nucleatum ATCC25586 (FN) and Prevotella nigrescence ATCC33563 (PN) were investigated as oral isolated bacteria. VSC production ability of the oral strains was investigated by gas chromatography. With serial dilution of BLES or SCCS, the strains PG, PI, FN or PN were cultured anaerobically with AnaeroPack at 37 ℃ for 3 days. For the determination of anti-bacterial action of BLES or SCCS, the inoculum was cultured with original concentrations of BLES 0.16% (w/v) or SCCS 0.25% (w/v). RESULTS: Gas chromatography exhibited that all strains, PG, PI, FN and PN were responsible for producing a high range of H2S and a moderate range of CH3SH. Anti-bacterial effect of BLES or SCCS on the strains was observed. Inhibition of BLES or SCCS on the strains was revealed as concentration dependent. BLES or SCCS inhibited bacterial proliferation at higher concentrations (PG; 0.04% BLES or 0.03% SCCS, PI; 0.002% BLES or 0.03% SCCS, FN; 0.005% BLES or 0.01% SCCS, PN; 0.01% BLES or 0.015% SCCS). No viable bacterial colony observed at original concentration of BLES 0.16% or SCCS 0.25%. Strain growth was eliminated from inhibition at lower concentrations (PG; 0.02% BLES or 0.015% SCCS, PI; 0.001% BLES or 0.015% SCCS, FN; 0.002% BLES or 0.007% SCCS, PN; 0.005% BLES or 0.007% SCCS). CONCLUSION: High concentrations of both BLES (0.16%) and SCCS (0.25%) show superior inhibiting capability on all four oral malodor associated periodontal anaerobes during testing, suggesting that these compounds might have a beneficial effect on oral health care.

Low prevalence of Merkel cell polyomavirus with low viral loads in oral and maxillofacial tumours or tumour-like lesions from immunocompetent patients: Absence of Merkel cell polyomavirus-associated neoplasms.

It was recently demonstrated that ~80% of Merkel cell carcinomas (MCCs) harbour a novel polyomavirus, Merkel cell polyomavirus (MCPyV). MCPyV has been detected in various human tissue samples. However, previous studies on the prevalence of MCPyV in oral tumours or tumour-like lesions are incomplete. To address this issue, we measured MCPyV DNA quantity using quantitative polymerase chain reaction (qPCR) in 327 oral tumours or tumour-like lesions and 54 jaw tumours or cyst lesions from 381 immunocompetent patients, as well as in 4 oral lesions from 4 immunosuppressed patients. qPCR revealed a low MCPyV prevalence (25/381, 6.6%) with low viral loads (0.00024-0.026 copies/cell) in oral and maxillofacial tumours and tumour-like lesions from immunocompetent patients. The prevalence was 7/176 (4.0%) in invasive squamous cell carcinomas (SCCs) [2/60 (3.33%) SCCs of the tongue, 4/52 (7.7%) SCCs of the gingiva and 1/19 (5.3%) SCCs of the floor of the mouth], 1/10 (10%) in dysplasias, 1/5 (20%) in adenocarcinomas, 2/13 (15.4%) in adenoid cystic carcinomas, 1/10 (10%) in non-Hodgkin's lymphomas, 3/10 (30%) in lipomas, 3/5 (60%) in neurofibromas, 1/3 (33.3%) in Schwannomas, 2/12 (16.7%) in Warthin's tumours, 2/11 (18.2%) in pyogenic granulomas, 1/14 (7.1%) in radicular cysts and 1/12 (8.3%) in ameloblastomas. The prevalence in lesions from immunosuppressed patients (1/4, 25%) was higher compared with that in lesions from immunocompetent patients (25/381, 6.6%), but the difference was not statistically significant. To the best of our knowledge, this study was the first to report prevalence data of MCPyV in tumours and cysts of the jaws (2/54, 3.7%). These data indicated absence of MCPyV-related tumours or tumour-like lesions in the oral cavity and jaws and suggested that the detected MCPyV DNA was derived from non-neoplastic background tissues with widespread low-level MCPyV infection.

Involvement of glucocorticoid in induction of lingual T1R3 in rodents.

We previously reported that in rats, chronic exposure to stress inhibits the induction of the common receptor (T1R3) for sweet and umami tastes. Here, we investigated whether endogenous glucocorticoids (GCs) might be responsible for this inhibition. In addition, we used mouse taste-bud cells (TB cells) expressing T1R3 to examine the effect of exogenous GC on T1R3 induction. Both adrenal glands were removed from rats [adrenalectomized (ADX) rats] and T1R3 mRNA expression in fungiform papillae was examined by real-time RT-PCR. T1R3 mRNA expression was significantly reduced in the ADX rats (versus sham-ADX rats). The reduced mRNA expression was restored to the level seen in the sham-ADX rats by administration of dexamethasone (DEX) at the smallest dose tested (0.1ng/kg, i.p.). However, with larger doses of DEX (10 and 1000ng/kg, i.p.) there was no such restoration (i.e., the expression level did not differ from that seen in ADX rats). Expression of the mRNA for the GC receptor-α was detected in mouse TB cells by RT-PCR. Significantly reduced T1R3 mRNA expression, as measured by real-time RT-PCR, was observed in TB cells at 24h after application of DEX (0.1, 1.0, or 10µM). These results suggest that in rodents: (a) a low concentration of endogenous GC is necessary and sufficient for induction of T1R3 expression, and that higher concentrations may actually inhibit such induction, and (b) this inhibitory effect may be due, at least in part, to a direct action of GC on taste cells.

Construction of a Luciferase Reporter System to Monitor Osteogenic Differentiation of Mesenchymal Stem Cells by Using a Mammalian Artificial Chromosome Vector.

BACKGROUND: Mesenchymal stem cells (MSCs) hold promise for application in adult stem cell-mediated regenerative medicine in bone remodeling and fracture repair. MSCs in vitro can be directed to osteogenic lineage by dexamethasone (DEX); however, the use of DEX is not practical in clinical settings because of adverse side effects such as glucocorticoid-induced osteoporosis. For identifying substances that facilitate osteogenesis, a monitoring system, which detects the osteogenic differentiation stage of MSCs accurately and easily, is required. METHODS: By focusing on the human osteocalcin (OC) gene whose expression profile is described along with osteogenic differentiation, we constructed the luciferase (Luc) reporter gene driven by the enhancer/promoter sequence of the human OC gene (OC-Luc) utilizing a mammalian artificial chromosome. Mammalian artificial chromosome is a suitable platform for loading reporter constructs, because of its stable episomal maintenance in host cells, transferability into any cell and assurance of long-term physiological transgene expression. We loaded the OC-Luc on a mammalian artificial chromosome vector engineered from mouse chromosome (designated as mouse artificial chromosome, MAC) in Chinese hamster ovary cells (OC-Luc/MAC) and transferred this into human MSC cells via chromosome transfer. RESULTS: OC-Luc/MAC in human MSC cells are responsive to positive and negative stimulation by 1 alpha,25-dihydroxyvitamin D3 and DEX in differentiation stage of MSCs to osteoblasts, reflecting the manner of physiological expression. CONCLUSION: The OC-Luc/MAC reporter system may contribute not only to monitoring the osteogenic differentiation stage from MSC but also to identify novel osteogenic drugs.

Frequent Isolations of Influenza A Viruses (H1N1)pdm09 with Identical Hemagglutinin Sequences for More Than Three Months in Japan.

BACKGROUND: Although it has been suggested that antigenic drift does not occur in a single epidemic season in temperate countries, there is not enough evidence on the circulation period of influenza virus with identical nucleotide sequences. Therefore, strains of influenza virus were isolated sequentially during five consecutive epidemic seasons in Japan and their nucleotide sequences were determined. METHODS: Nasal swabs or aspirated nasal discharges were collected from influenza A virus antigen-positive individuals living in Tottori Prefecture, Japan for five consecutive winters starting in 2009-2010, and subjected to viral isolation, determination of hemagglutinin nucleotide sequence and phylogenic analyses. The nucleotide sequences were compared with each other and also with those of foreign strains in the International Nucleotide Sequence Database. RESULTS: Totally 288 A(H1N1)pdm09 strains were tested and those composed 38 clusters with identical ones displaying 100% nucleotide homology. One strain showed sequential infections more than three months without any detectable mutation, and a maximum interval of two detection timings of strains was 94 days. This implies that influenza viruses mutate rarely in an epidemic season in Japan if they can be hypothesized, mutation frequency of influenza viruses being mostly the same among strains. Among these identical strains, two strains were not only identical to other Japanese isolates, but also to those isolated in Mongolia and Thailand in the same epidemic season. CONCLUSION: These results suggest that genetic drift has occurred infrequently in Japan as shown in some other countries. The drifted strains may have generated somewhere else and entered into Japan. These results support the proposed 'sink-source' model of viral ecology in which new lineages are seeded from a persistent influenza reservoir in tropical countries to 'sink' populations in temperate regions including Japan.

Reduction of osteoclasts in a critical embryonic period is essential for inhibition of mouse tooth eruption.

Alveolar bone resorption by osteoclasts is essential for tooth eruption. Osteoclast-deficient Csfm(op) homozygous (op/op) mice, which lack functional macrophage colony-stimulating factor (M-CSF), suffer from osteopetrosis and completely lack tooth eruption. Although osteoclasts appear, and osteopetrosis is cured with age in op/op mice, tooth eruption is never seen. This fact suggests that there is a critical period when osteoclasts are required for tooth eruption. In this study, to detect the critical period, we administered an antagonistic antibody directed against c-Fms, a receptor for M-CSF, to inbred C57BL/6 mice for various periods. Administration of this antibody decreased tartrate-resistant acid phosphatase-positive (TRAP) osteoclasts, and incisor eruption was completely inhibited by continual administration of this antibody from embryonic day 15.5 (E15.5) until postnatal day 12.5 (D12.5). A 1-day delay of this administration abolished the inhibition of incisor eruption. The number of TRAP-positive osteoclasts was significantly reduced between E16.5 and E18.5 in the mice treated with antibody from E15.5 compared with those treated from E16.5. These results indicate that this period, during which the number of osteoclasts decreases significantly, is critical for inhibiting incisor eruption in C57BL/6 mice.

Usefulness as guided bone regeneration membrane of the alginate membrane.

Alginate membrane is a new bioabsorbable, guided bone regeneration (GBR) membrane, which is placed directly on the surface of the bone defect. It is designed to drop a calcium chloride aqueous solution into the bone defect, which is filled with sodium alginate aqueous solution. Alginate membrane is an excellent agent for this procedure due to its close assimilation to the surface of the bone. In this study, we evaluated the short-term biocompatibility of alginate membrane in the bone defects of rat tibiae. GBR membrane availability was also examined. Consequently, we found that the healing process in bone defects covered with an alginate membrane was delayed in comparison with that of controls, however, the defect was restored to nearly original condition. In contrast, in the controls, bone defect repairs exhibited partitioning as a result of connective tissue involvement. Furthermore, we observed a relation between the sodium alginate concentration and the rate of absorption of the sodium alginate membrane. Absorption of a 1.5% sodium alginate membrane was slow. As a result, the compound was not absorbed completely and bone repairs resembled an hourglass. Moreover, the inflammatory response was absent surrounding the alginate membrane. The present findings suggested that the alginate membrane functions effectively as a GBR membrane. In addition, the alginate membrane derived from 3% calcium chloride and 1% sodium alginate was most suitable as a GBR membrane.