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Background and objective According to basic studies, hot spring use has positive effects on the mind and body. However, the association between habitual hot spring use and prevention of long-term care is unknown. Using long-term care insurance data for the residents of Atami City, Japan, who can choose to install hot spring water supply in their homes, this study aimed to determine the association between the installation of a hot spring water supply in the home and prevention of long-term care. Methods 1. Study design: case-control study 2. Subjects: 2719 residents (754 men, 1965 women) of Atami City, Shizuoka Prefecture, Japan, who received long-term care insurance and were certified as “Needing Support” or “Needing Long-Term Care” as of March 2017. 3. Survey methods: Information on long-term care insurance certification was linked to subjects' care level dating back to their initial certification. Also, the installation (or lack thereof) of hot spring water supply in each subject's home was linked to information on Atami household water use as of March 2017. 4. Analysis methods: The age distribution of the subjects was determined. Initial care status and care status as of March 2017 were then compared for the 2194 subjects who received long-term care certification at least twice. These subjects were classified into two groups: those whose care level had not changed or had improved (no change/improvement group) and those whose care level had worsened (worsening group). Subjects were then compared by sex and initial care level in terms of hot spring installation and percentages of no change/improvement or worsening of care level; odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using the chi-square test. Lastly, the same analysis was performed for all subjects grouped together, and ORs and 95% CIs were calculated using the Mantel-Haenszel test. Results Hot springs were installed in the homes of 2359 subjects overall (86.8%). The no change/improvement group and the worsening group comprised 1192 and 1002 subjects, respectively. Overall, improvement or no change in care level was observed in 1050 subjects (55.2%) in the hot spring group and 142 subjects in the no hot spring group (48.5%). Sex-adjusted OR (95% CI) was 1.311 (1.025–1.677, p = 0.036), which represented a significant association. Having a hot spring water supply in the home may be associated with preventing worsening of care level. Conclusion Having a hot spring water supply in the home may be associated with preventing worsening of care level.

Objectives Sex hormone receptors are reported to be present in human dental pulp (HDP) cells. The purpose of this study was to examine the biological significance of oestrogen and androgen receptors (ER and AR, respectively) in HDP cells. Design We isolated HDP cells expressing ER- and AR-mRNAs and investigated the expression status of the receptors and the response to sex hormones in the cells. Results HDP cells expressing ER- and/or AR-mRNAs had the ability to form alizarin red S-positive nodules in which calcium and phosphorus were deposited in vitro and to differentiate into odontoblasts-like cells and dentine-like tissue in vivo. Individual clones isolated from HDP cells exhibited a different expression pattern of mRNA for ER and AR. Some clones expressed ERα- and/or ERβ-mRNAs and the others coexpressed ER- and AR-mRNAs. Using the Ingenuity software, we found that 17β-estradiol (E2) and dihydrotestosterone (DHT) could act directly on HDP cells through ER-or androgen signalling-mediated mechanisms. E2 or DHT stimulated the mRNA expression for genes related to odontogenesis of dentine-containing teeth and odontoblast differentiation, suggesting that ER and AR in HDP cells may be involved in dentinogenesis. Conclusions Our findings provide new insights into the biological significance of sex hormone receptors in HDP cells.

Purpose: The potential of sucrose fatty acid esters (SEs) to enhance intestinal absorption was investigated in order to identify their utility for the intestinal absorption of water-soluble high molecular weight compounds. Methods: Fluorescein isothiocyanate-labeled dextran (FD) with a molecular weight (MW) of 4,000 (FD-4) was used as a model compound, and several SEs were tested as absorption enhancers. After FD-4 was administered intra-duodenally at 10 % (w/v) with the non-loop method in situ in rats in the absence or presence of SEs, the plasma concentration-time profiles of FD-4 were examined. As to sucrose laurate (L-1695), the relationship between concentration and enhancement effect was investigated. In addition, the enhancement effect after dosing into the different small intestinal regions, the effect on FDs with different MWs and the influence of N-acetyl-cysteine (NAC) co-existence were examined. Results: Low watersoluble SEs exhibited slight and/or slow absorption enhancement effects, while L-1695, being highly water-soluble, had good potential to enhance the absorption rate and extent. The enhancement effect became greater as the concentration of L1695 increased. L-1695 displayed high enhancement potential in wide intestinal areas. The enhancement effect of L-1695 (10 %, w/v) depended on MWs of FDs; the mean values of the area under the plasma concentration curve from 0-120 min (AUC0-120 mins) increased by 14 and 8 times for FD-4 and FD-10 (MW 10,000), while it was hardly changed as for FD-70 (MW 70,000). The enhancement effect of L-1695 (10 %, w/v) was similar to that of sodium caprate (10 %, w/v), and was influenced to some extent by the co-existence of NAC (5 %, w/v). Conclusion: The absorption enhancement potential of SEs depended on their water-solubility. L-1695, being highly water-soluble, showed a good enhancement effect, and its absorption profiles were elucidated. This study proposes the possibility of SEs, in particular, L-1695, as intestinal absorption enhancers. As far as the present non-loop method is concerned, the intestinal damage was not observed macroscopically with the addition of L1695 at 2.5-20 % (w/v).

The lubricant property of sucrose esters of fatty acids (SEs) having different particle size and Hydrophile-Lipophile Balance (HLB) was evaluated with a model formulation in tableting. The binding tendency of the model formulation composed of isomalt powder was depressed by adding SEs in the formulation. Tablets containing SE showed faster disintegration time compared to those containing magnesium stearate or calcium stearate, while the tablets containing SEs indicated a high tensile strength. Milled SE (S-370F) exhibited better tablet properties than original SE (S-370). The SEs having different HLB showed different effects on the resultant tablet properties such as the magnitude of tensile strength, disintegration time, and spreading properties. The relatively hydrophilic SE was found to much improve the resultant disintegrating properties of tablet compared to magnesium stearate, calcium stearate and hydrophobic SEs. Consequently, SEs are able to use as lubricants on tablet formulation by appropriately controlling its particle size, content, and HLB.

Fast-disintegrating tablets of furosemide (FS) were prepared by the novel direct compression method. FS, microcrystalline cellulose (MC), croscarmellose sodium (CC), xylitol (XL) and sucrose stearic acid esters (SSEs) with an hydrophilic-lipophilic balance (HLB) of 16, 15 and 11, named S1670, S1570 and S1170, were used. An FS/SSE/MC mixed powder was obtained by solvent evaporation of a suspension of MC in ethanol solution containing FS and SSE, and the resultant mixed powder was mixed with CC and XL, and directly compressed. The tablets with hardness of more than 40 N and disintegration time of less than 20 s were obtained at the addition of SSE at 0--0.5% (w/w). A tablet with S1670 at 0.1% (w/w), named TA2, dissolved faster than a commercial FS tablet, Lasix. TA2 tended to show higher plasma concentration than Lasix after intragastric administration to rats. It was demonstrated that the present direct compression using homogeneous FS/S1670/MC powder mixture could give an excellent fast-disintegrating tablet of FS.

Previous studies have shown that submandibular glands suffering interruption of the blood circulation from the main supplying artery have more surviving parenchymal cells in their peripheral portion than in their central portion. Although the reason for this difference between the peripheral and central portions has not yet been clarified, the existence of collateral circulation has been suspected. The present study was designed to examine whether or not the vessels binding the gland proper to the capsule provide such collateral circulation. Silicone rubber or methacrylate was injected into the main artery supplying the mouse submandibular gland, in which the gland proper is wrapped by a capsule similar to that of the human submandibular gland, and then the gland was observed by both stereoscopic and scanning electron microscopy. Three-dimensional observations showed no communicating vessels between the gland proper and the capsule. Therefore, it is suggested that the parenchymal cells surviving in the ischemic peripheral portion of the submandibular gland are nourished by permeation of tissue fluid contained in the capsule.

OBJECTIVE AND BACKGROUND Our previous studies suggest that little adverse effect on the growth of the periodontal ligament would be expected, if tetracycline, minocycline, ofloxacin, roxithromycin, clarithromycin, and azithromycin were topically administered to the periodontal pocket at their MIC90 doses required to inhibit the growth of 90% of periodontopathic bacteria, including Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans. In the present study, we investigated the cytocidal effects of eight antibacterial agents on the human gingival epithelial cell line NDUSD-1. We also used NDUSD-1 cells to examine the effects of these agents on the mRNA and protein expressions of genes associated with the proliferation, differentiation, or cellular adhesion important to the epithelial regeneration of the periodontal attachment. METHODS The cytocidal effect of the test agents was measured as a decrease in cell survival. To obtain a quantitative measure of the cytocidal effect, the LD50, i.e. the concentration which results in a 50% decrease in cell survival relative to the controls, was extrapolated from the concentration-response curves. The effects of the agents on the mRNA and protein expressions in NDUSD-1 cells were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses, respectively. RESULTS The cytocidal effect increased in a concentration-dependent manner as the concentration of each of the eight test agents increased. The order of the agents according to their cytocidal effects (LD50) was minocycline > tetracycline > enoxacin > clarithromycin > roxythromycin approximately ofloxacin > azithromycin > erythromycin. The cytocidal effects of minocycline, tetracycline, enoxacin, clarithromycin, roxythromycin, ofloxacin, and azithromycin ranged from 1.2 to 23.2 times greater than that of erythromycin. The maximum non-cytocidal concentrations (MNCCs) of these agents for NDUSD-1 cells were: 0.3 microm for minocycline, 1 microm for tetracycline, 3 microm for ofloxacin and erythromycin, 10 microm for enoxacin, clarithromycin, and azithromycin, and 100 microm for roxythromycin. The MNCCs of ofloxacin, azithromycin, clarithromycin, and roxythromycin were greater than their MIC90 concentrations for periodontopathic bacteria described above. The effects on the mRNA and protein expressions of epithelial-cell- or cell-adhesion-related genes were examined in NDUSD-1 cells exposed to clarithromycin, roxythromycin, ofloxacin, and azithromycin at their MNCCs. None of the agents affected the mRNA expressions of five genes: keratinocyte growth factor receptor, keratin 18, integrin beta1, integrin beta4, and laminin 5gamma2. Clarithromycin and ofloxacin slightly decreased the protein expression of integrin beta4. Roxythromycin markedly decreased the protein expressions of integrin beta4 and laminin 5gamma2. Azithromycin had little inhibitory effects on the protein expressions of any of the five genes. CONCLUSIONS These results suggest that little, if any, adverse effects on growth, differentiation, and adhesion of basal epithelial cells would be expected with topical administration of clarithromycin, ofloxacin or azithromycin to the periodontal pocket at a dose equivalent to the MIC90. It is important to note, however, that the extrapolation of these findings to in vivo conditions has yet to be undertaken.

The ability of the human papillomavirus type 16 (HPV-16) E6 or E7 gene to induce immortalization of normal human embryonic fibroblast WHE-7 cells was examined. WHE-7 cells at 9 population doublings (PD) were infected with retrovirus vectors encoding either HPV-16 E6 or E7 alone or both E6 and E7 (E6/E7). One of 4 isolated clones carrying E6 alone became immortal and is currently at >445 PD. Four of 4 isolated clones carrying E7 alone escaped from crisis and are currently at >330 PD. Three of 5 isolated clones carrying E6/E7 were also immortalized and are currently at >268 PD. The immortal clone carrying E6 only and 2 of the 3 immortal clones carrying E6/E7 expressed a high level of E6 protein, and all the immortal clones carrying E7 alone and the other immortal clone carrying E6/E7 expressed a high level of E7 protein when compared to their mortal or precrisis clones. The immortal clones expressing a high level of E6 or E7 protein were positive for telomerase activity or an alternative mechanism of telomere maintenance, respectively, known as ALT (alternative lengthening of telomeres). All the mortal or precrisis clones were negative for both phenotypes. All the immortal clones exhibited abrogation of G1 arrest after DNA damage by X-ray irradiation. The expression of INK4a protein (p16(INK4a)) was undetectable in the E6-infected mortal and immortal clones, whereas Rb protein (pRb) was hyperphosphorylated only in the immortal clone. The p16(INK4a) protein was overexpressed in all the E7-infected immortal clones and their clones in the pre-crisis period as well as all the E6/E7-infected mortal and immortal clones, but the pRb expression was downregulated in all of these clones. These results demonstrate for the first time to our knowledge that HPV-16 E6 or E7 alone can induce immortalization of normal human embryonic fibroblasts. Inactivation of p16(INK4a)/pRb pathways in combination with activation of a telomere maintenance mechanism is suggested to be necessary for immortalization of normal human embryonic fibroblasts by these viral oncogenes. The susceptibility of human cells to immortalization may be related to the state of differentiation of the cells.

To examine the telomere maintenance mechanism in the process of human fibroblast immortalization, we studied telomere length, telomerase activity, chromosome instability, and minisatellite alterations in human fibroblasts following the introduction of the human papillomavirus type 16 E6 gene or E7 gene, or both. One cell line immortalized by E6 alone maintained short telomeres, and its telomerase activity was positive. Fairly long and heterogeneous telomeres were maintained in all four E7-immortalized cell lines lacking telomerase activity. Of the three clones immortalized by both E6 and E7, two cell lines with telomerase activity maintained short telomeres, and the other cell line, which lacked telomerase activity, maintained long and heterogeneous telomeres. Although all immortal cell lines expressed mRNAs for human telomerase RNA component and telomerase-associated protein, expression of mRNA for human telomerase reverse transcriptase was detected only in the telomerase-positive cell lines. All immortal cell lines showed both chromosomal abnormalities, including structural and numerical changes, and minisatellite alterations detected by DNA fingerprinting. These findings indicate the existence of different telomere maintenance mechanisms in telomerase-positive and -negative fibroblast cell lines immortalized by E6, E7, or E6/ E7, and the possible involvement of chromosome instability and minisatellite alterations in the activation of the telomere maintenance mechanisms.

Loss of heterozygosity in two in vitro-immortalized human oral keratinocyte cell lines was analysed by polymerase chain reaction using 42 polymorphic microsatellite markers on chromosomes 4, 6, 7 and 15. These chromosomes are regarded as candidates for harbouring genes involved in the immortalization of human cells or tumour-suppressor genes in several tumours, including oral cancers, and karyotypic analysis has revealed that both cell lines have non-random alterations in these chromosomes. No allele losses were detected at any informative loci on chromosomes 4 and 6 in the cell lines, including genomic regions adjacent to putative human tumour-suppressor genes and putative senescence genes. When analysed for loss of heterozygosity on chromosomes 7 and 15, allele losses common to both cell lines were detected in the regions at 7q11.2, 7q21.1–21.3 and 7q31.1. High frequencies of loss of heterozygosity on chromosome 7q in at least two distinct regions, particularly centred around 7q31, are observed in a variety of tumours, including oral squamous-cell carcinoma, suggesting that multiple genes involved in immortalization of these cell lines might be present on chromosome 7q.

本研究は, 顎下腺の主導管結紮後と解除後における腺房細胞と筋上皮細胞の組織学的変化と再生過程を明らかにする目的で行われた. ラット顎下腺の主導管を1週間結紮し, その組織学的変化, 次いで結紮解除後の経日的変化を調べた. 免疫染色やTUNEL染色を施した試料について光学顕微鏡と透過電子顕微鏡による観察を行った.正常なラット顎下腺では, 筋上皮細胞に被われた導管様構造物は認められなかったが, 主導管結紮後, このような組織像が多数観察された. 光顕下では, アポトーシスを思わせるTUNEL染色陽性腺房細胞が若干観察されたが, 電顕観察では確認できなかった. このことから, 細胞死が生じたとしても少数であると思われた. 結紮解除後4週で顎下腺全体にほぼ正常な腺房が観察された. これらの所見から, 腺房の回復は主として導管様構造物が腺房へ回復することによるものと思われた.