Involvement of TRPV1 and AQP2 in hypertonic stress by xylitol in odontoblast cells.

AIM: To examine the responses of mouse odontoblast-lineage cell line (OLC) cultures to xylitol-induced hypertonic stress. METHODOLOGY: OLCs were treated with xylitol, sucrose, sorbitol, mannitol, arabinose and lyxose. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay. The expression of transient receptor potential vanilloids (TRPV) 1, 3 and 4 was detected using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The expression of aquaporin (AQP) 2 was detected using immunofluorescence and Western blotting analysis. The expression of interleukin-6 (IL-6) under xylitol-induced hypertonic stress was assessed using an enzyme-linked immunosorbent assay (ELISA). Small interfering ribonucleic acid (siRNA) for AQP-2 was used to inhibition assay. RESULTS: Xylitol-induced hypertonic stress did not decrease OLC viability, unlike the other sugars tested. OLCs expressed TRPV1, 3 and 4 as well as AQP2. Xylitol inhibited lipopolysaccharide (LPS)-induced IL-6 expression after 3 h of hypertonic stress. TRPV1 mRNA expression was upregulated by xylitol. Costimulation with HgCl2 (AQP inhibitor) and Ruthenium red (TRPV1 inhibitor) decreased cell viability with xylitol stimulation. OLCs treated with siRNA against TRPV1 exhibited decreased cell viability with xylitol stimulation. CONCLUSION: OLCs have high-cell viability under xylitol-induced hypertonic stress, which may be associated with TRPV1 and AQP2 expressions.

Transferability of microsatellites for studies on the social behavior of the tufted capuchin monkey (genus Sapajus).

Because of relevant results that indicated that molecular techniques can provide increased knowledge of animal social systems, they usually complement observational field studies. Despite the great utility of microsatellites, they are not available for all species. Gathering genetic information using microsatellites that were originally designed for other species is a time-saving procedure. The aim of this study was to test the transferability of microsatellites and their usefulness in studies of social behavior of black capuchin monkeys (Sapajus nigritus). We noninvasively sampled adult and subadult black capuchins of three wild groups in southeastern Brazil. Seventeen microsatellites, which were previously designed for and successfully amplified in multiple Neotropical primate species, were tested. Nine of the 17 microsatellite loci tested produced an average of 6.22 alleles (range 2-12) per locus. The allelic richness and the expected heterozygosity for all loci was 5.93 and 0.70, respectively. The combined non-exclusion probability for one candidate parent across all loci was 0.01. The nine microsatellite loci optimized in this study have a great potential for application in studies of social structure and dispersal patterns in S. nigritus populations and in other Neotropical primate species.

The expression of PTEN in the development of mouse cochlear lateral wall.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that regulates various cell processes including proliferation, growth, synaptogenesis, neural and glioma stem/progenitor cell renewal. In addition, PTEN can regulate sensory cell proliferation and differentiation of hair bundles in the mammalian cochlea. In this study we use immunofluorescence, Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) to reveal the expression of PTEN in the developing cochlear lateral wall, which is crucial for regulating K(+) homeostasis. Relatively high levels of PTEN are initially expressed in the marginal cells (MCs) of the lateral wall at embryonic day (E) 17.5 when they start to differentiate. Similarly high levels are subsequently expressed in differentiating root cells (RCs) at postnatal day (P) 3 and then in spiral ligament fibrocytes (SLFs) at P 10. In the mature cochlea, PTEN expression is low or undetectable in MCs and SLFs but it remains high in RCs and their processes. The expression pattern for PTEN in the developing lateral wall suggests that it plays a critical role in the differentiation of the cellular pathways that regulate K(+) homeostasis in the cochlea.

Immunoreactivity of glucose transporter 5 is located in epithelial cells of the choroid plexus and ependymal cells.

High fructose intake is associated with increased plasma triglyceride concentration, hepatic steatosis, impaired glucose tolerance, insulin resistance, and high blood pressure. In addition, increased fructose intake has recently been supposed to be a risk factor for dementia. However, direct effects of fructose on the brain function remain to be clarified. The localization of glucose transporter 5 (Glut5), a representative transporter of fructose, was immunohistochemically examined in the brains of humans, rats, and mice to clarify whether fructose was transported from the blood into the brain. Glut5 immunoreactivity was demonstrated to be located in the epithelial cells of the choroid plexus and the ependymal cells in the brains of humans and rats using commercial antibodies for Glut5. In addition, mRNA expression of mouse Glut5 was confirmed in the brains of mice. Immunohistochemical examination using a custom-made antibody against two regions of amino acid sequences of mouse Glut5 revealed that Glut5 immunoreactivity was also seen in the epithelial cells of the choroid plexus and the ependymal cells in the brains of mice. These findings show that Glut5 immunoreactivity is located in the epithelial cells of the choroid plexus and the ependymal cells, suggesting the possibility of the direct transportation of intravascular fructose into the brain parenchyma.

Thymus histology and concomitant autoimmune diseases in Japanese patients with muscle-specific receptor tyrosine kinase-antibody-positive myasthenia gravis.

BACKGROUND AND PURPOSE: The differences in the characteristics of thymus histology, coexisting autoimmune diseases and related autoantibodies between anti-muscle-specific receptor tyrosine kinase (MuSK)-antibody (Ab)-positive myasthenia gravis (MG) patients, and anti-acetylcholine receptor (AChR)-Ab-positive MG patients are not clearly defined. METHODS: The types of thymus histology, coexisting autoimmune diseases and associated Abs in 83 MuSK-Ab-positive patients nationwide were investigated and were compared with those in AChR-Ab-positive patients followed at our institute (n = 83). As for the autoantibodies associated with thymoma, titin Abs were measured. RESULTS: Thymoma was not present in any of the MuSK-Ab-positive patients but presented in 21 patients (25.3%) amongst the AChR-Ab-positive patients. Titin Abs were absent in MuSK-Ab-positive patients but positive in 25 (30.1%) of the AChR-Ab-positive patients. Concomitant autoimmune diseases were present in eight MuSK-Ab-positive patients (9.6%) amongst whom Hashimoto's thyroiditis and rheumatoid arthritis predominated, whereas 22 AChR-Ab-positive patients (26.5%) had one or more concomitant autoimmune diseases of which Graves' disease predominated. CONCLUSIONS: Differences in frequency of thymoma and thymic hyperplasia, coexisting autoimmune diseases and autoantibody positivity between MuSK-Ab-positive and AChR-Ab-positive MG were indicated, suggesting that, in contrast with AChR-Ab-positive MG, thymus does not seem to be involved in the pathogenic mechanisms of MuSK-Ab-positive MG.

Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing.

The IRE1α-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Therefore, the availability of small-molecule inhibitors targeting this pathway would offer a new chemotherapeutic strategy for MM. Here, we screened small-molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) activation. Furthermore, although toyocamycin was unable to inhibit IRE1α phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Toyocamycin inhibited not only ER stress-induced but also constitutive activation of XBP1 expression in MM lines as well as primary samples from patients. It showed synergistic effects with bortezomib, and induced apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM. Taken together, our results suggest toyocamycin as a lead compound for developing anti-MM therapy and XBP1 as an appropriate molecular target for anti-MM therapy.

Phosphatase and tensin homolog deleted on chromosome 10 regulates sensory cell proliferation and differentiation of hair bundles in the mammalian cochlea.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that regulates cell proliferation, differentiation and growth. It regulates neural and glioma stem/progenitor cell renewal and PTEN deletion can drive expansion of epithelial progenitors in the lung, enhancing their capacity for regeneration. Because it is expressed at relatively high levels in developing mammalian auditory hair cells we have analyzed the phenotype of the auditory epithelium in PTEN knock-out mice. PTEN(+/-) heterozygous littermates have only one functional copy of the gene and show clear evidence for haploinsufficiency in the organ of Corti. Auditory sensory epithelial progenitors withdraw from the cell cycle later than in wild-type animals and this is associated with increases in the numbers of both inner and outer hair cells. The cytoskeletal differentiation of hair cells was also affected. While many hair bundles on the hair cells appeared to develop normally, others were structurally disorganized and a number were missing, apparently lost after they had been formed. The results show that PTEN plays a novel role in regulating cell proliferation and differentiation of hair bundles in auditory sensory epithelial cells and suggest that PTEN signaling pathways may provide therapeutic targets for auditory sensory regeneration.

Significance of thrombomodulin release from gingival epithelial cells in periodontitis patients.

BACKGROUND AND OBJECTIVE: Thrombomodulin, a cell transmembrane glycoprotein, binds to thrombin and converts it from a procoagulant protease to an anticoagulant enzyme that activates protein C. Thrombomodulin is very important in regulating the function of thrombin. Elevated soluble thrombomodulin is present in the gingival crevicular fluid of subjects with periodontitis. The objective of the present study was to investigate the mechanisms about the elevated soluble thrombomodulin in gingival crevicular fluid. MATERIAL AND METHODS: Gingival sections from six patients with chronic periodontitis and from three periodontally healthy subjects were immunostained for thrombomodulin detection. Thrombomodulin levels were investigated in the gingival crevicular fluid of 11 subjects with chronic periodontitis. The effects of neutrophil enzymes on thrombomodulin release and on thrombomodulin in the gingival crevicular fluid were examined by an enzyme-linked immunosorbent assay or by Western blotting. RESULTS: The expression of gingival epithelial thrombomodulin was lost or decrease near infiltrating neutrophils. Thrombomodulin was rapidly released from gingival epithelial cells by neutrophil enzymes, and gingival crevicular fluid with periodontitis included the proteolytic cleavage thrombomodulin using immunoblotting analysis. The thrombomodulin release was not caused by rapid cell damage, on lactate dehydrogenase assay. There were significant differences in thrombomodulin content between gingival crevicular fluid samples from healthy and diseased sites, regardless of the degree of probing depth. CONCLUSION: Neutrophil enzymes induced rapid thrombomodulin release from the membrane surface of gingival epithelial cells. This might explain the thrombomodulin increase in gingival crevicular fluid with local diseased gingiva. Elevation of thrombomodulin in gingival crevicular fluid may be a potential marker of epithelial cell membrane injury.

The role of water channel aquaporin 3 in the mechanism of TNF-alpha-mediated proinflammatory events: Implication in periodontal inflammation.

Aquaporin 3 (AQP3) is the predominant water channel protein in human keratinocytes and acts as an inflammatory mediator in some lesions. A chronic, inflammatory process of periodontitis is related with a dramatic change of surrounding fluid homeostasis to plasma extravasation. The exact pattern of aquaporin (AQP) water channel expression and its mechanism in periodontal disease is still unknown. We describe herein an up-regulated AQP3 expression in the epithelial lesion with chronic periodontitis and its functional role. The levels of AQP3 expression in inflamed gingival epithelial tissues were significantly higher than those of healthy subjects. Consistent with these results, AQP3 expression (i.e., levels of mRNA and protein) in cultured rat primary gingival epithelial cells and the human gingival epithelial cell line Ca9-22 were strongly increased in response to TNF-alpha treatment through the 55 kDa TNF-alpha receptor (TNFR I). In this context, small interfering RNA- (siRNA)-mediated "aqp-3 gene silencing," which could reduce AQP3 expression by more than 65%, significantly attenuated selected proinflammatory events of ICAM-1 expression induced by TNF-alpha in Ca9-22. A sixfold increase in leukocyte adherence to TNF-alpha-stimulated epithelial cells was demonstrated by an adherence assay (P < 0.001) and pretreatment with AQP3 siRNA and anti-ICAM-1 antibody reduced leukocyte retention by 85% (P < 0.001). Our study indicates for the first time a novel important mode in the regulation of the inflammatory response through TNF-alpha/TNFR I ligation at the site of epithelial lesions by specialized membrane channel AQP3 and ICAM-1 protein, which is closely implicated in the development of periodontitis mechanisms.

[Mitral valve plasty for a 91-year-old woman with mitral valve prolapse].

We reported a successful mitral valve plasty for a 91-year-old woman with mitral valve prolapse. She has lived healthfully and independently without a big problem. She was admitted to another hospital for acute heart failure. Echo cardiography revealed prolapse of posterior mitral valve leaflet and severe mitral regurgitation. Drug therapy was not enough to control her complaint In spite of her age, the patient was able to support herself, and she and her family desired to have a surgical treatment. Therefore she referred to our hospital and underwent mitral valve plasty. Post operative course was almost uneventful. She discharged the hospital 3 months after the operation. If a selective criteria for individual patients is applied, the nonagenarian can safety undergo cardiac surgery.

Roxithromycin inhibits tumor necrosis factor-alpha-induced matrix metalloproteinase-1 expression through regulating mitogen-activated protein kinase phosphorylation and Ets-1 expression.

BACKGROUND AND OBJECTIVE: In periodontitis, matrix metalloproteinases (MMPs) are upregulated in response to locally released inflammatory cytokines, resulting in pathologic processes. Roxithromycin is a 14-membered ring macrolide antibiotic with broad-spectrum antibacterial effects against oral pathogens and immunomodulatory effects. Recently, we reported that roxithromycin inhibits tumor necrosis factor (TNF)-alpha-induced vascular endothelial growth factor expression in human periodontal ligament (HPDL) cell cultures. In the present study, we examined the effect of roxithromycin on TNF-alpha-induced MMP-1 production by HPDL cells. MATERIAL AND METHODS: Cultured cells were incubated with 1% fetal bovine serum for 24 h, followed by treatment with 10 ng/ml TNF-alpha, 10 microM roxithromycin, and mitogen-activated protein kinase inhibitor at various concentrations. Culture supernatants and sediments were collected at different time-points and used for enzyme-linked immunosorbent assays, and northern and western blot analyses. RESULTS: In HPDL cell cultures, roxithromycin strongly inhibited TNF-alpha-induced MMP-1 mRNA expression and production. The inhibition of MMP-1 gene expression by roxithromycin was dependent on de novo protein synthesis and was regulated at the transcriptional level. Roxithromycin significantly inhibited TNF-alpha-induced c-Jun N-terminal kinase activation (JNP) and marginally inhibited extracellular signal-regulated kinase (ERK) 1/2 activation, but not p38 mitogen-activated protein kinase activation. Furthermore, roxithromycin reduced the induction of Ets-1, one of the critical factors in MMP-1 transcription. CONCLUSION: Roxithromycin inhibits TNF-alpha-mediated MMP-1 induction through the downregulation of ERK1/2 and JNK activation and the subsequent reduction of Ets-1, suggesting that roxithromycin may have therapeutic use in periodontitis and other chronic inflammatory conditions involving MMP-1 induction.

Synthesis of precursor of anti-inflammatory agents by using highly reactive zinc.

Highly reactive zinc metal was prepared by electrolysis of a N,N-dimethylformamide (DMF) solution containing naphthalene and a supporting electrolyte in a one-compartment cell fitted with a platinum cathode and a zinc anode. This highly reactive electrogenerated zinc (EGZn/Naph) was used for transformation of ethyl 2-bromoacrylate into the corresponding organozinc compound, which can not be achieved by the use of usual zinc metals. Reaction of the organozinc compounds thus prepared with various aryl halides in the presence of 5 mol% of palladium catalyst gave the corresponding cross-coupling products in high yields. These cross-coupling reactions were successfully applied to a synthesis of the precursor of anti-inflammatory agents such as ibuprofen, naproxen, cicloprofen and suprofen.

Synthesis of precursor of anti-inflammatory agents by using highly reactive zinc

Highly reactive zinc metal was prepared by electrolysis of a N,N-dimethylformamide (DMF) solution containing naphthalene and a supporting electrolyte in a one-compartment cell fitted with a platinum cathode and a zinc anode. This highly reactive electrogenerated zinc (EGZn/Naph) was used for transformation of ethyl 2-bromoacrylate into the corresponding organozinc compound, which can not be achieved by the use of usual zinc metals. Reaction of the organozinc compounds thus prepared with various aryl halides in the presence of 5 mol% of palladium catalyst gave the corresponding cross-coupling products in high yields. These cross-coupling reactions were successfully applied to a synthesis of the precursor of anti-inflammatory agents such as ibuprofen, naproxen, cicloprofen and suprofen.

pp60c-src activation in lung adenocarcinoma.

Nine src family members are known including c-Src, c-Yes, c-Lck, c-Fyn, c-Hck, c-Lyn, c-Blk, c-Fgr and c-Yrk. They encode proteins with molecular weights of 55-62 kilodaltons (kDa), which are either cytoplasmic or membrane-associated protein tyrosine kinases. A close correlation exists between an elevated pp60c-src tyrosine kinase activity and cell transformation. However, the level of activation of pp60c-src in non-small cell lung cancers (NSCLC) remains obscure. The aim of this study was to examine the level of activity of pp60c-src in NSCLC. pp60c-src expression and in vitro protein tyrosine kinase activity in lung cancer tissue samples were measured by western blotting and in vitro kinase assays and compared with those in the surrounding non-tumour lung tissue from the same patient. pp60c-src phosphorylation was assessed by two-dimensional tryptic phosphopeptide mapping. The kinase activity of pp60c-src was significantly activated in NSCLC, especially in adenocarcinomas. In addition, the pp60c-src kinase activity increased with the size of the adenocarcinoma. Two-dimensional tryptic phosphopeptide mapping showed dephosphorylation of pp60c-src at Tyr 530 in adenocarcinomas. The proto-oncogene product, pp60c-src, was activated in NSCLC, especially in adenocarcinomas, in part through the dephosphorylation of Tyr 530. Our results suggest that activation of pp60c-src might play an important role in the progression of lung adenocarcinomas.

Confirmation by DNA analysis that Contarinia maculipennis (Diptera: Cecidomyiidae) is a polyphagous pest of orchids and other unrelated cultivated plants.

The cytochrome oxidase subunit I gene in mitochondrial DNA of 53 larvae of Contarinia maculipennis Felt from flower buds of various host plants, collected from Hawaii, Japan and Thailand was analysed. Monophyly of the clade including C. maculipennis from Hawaii, Thailand and Japan was supported. There was no sequential variation within the specimens from Hawaii and Japan, which differed from one another by 6 bp (1.37%). Three haplotypes were recognized in specimens from Thailand but differences from Hawaiian and Japanese specimens were small. Overall, there were no differences in the 146 deduced amino acid residues. It is therefore concluded that C. maculipennis is a polyphagous species that can develop on plant hosts representing at least seven botanical families. This pest of Dendrobium flower buds in glasshouses is considered to have entered Hawaii, Florida and Japan from Southeast Asia, and was recently intercepted in the Netherlands. Infestations have established and spread in orchid glasshouses, causing concern about the possibility of more extensive damage to orchids and to crops, such as bitter gourd, grown in close proximity to orchid glasshouses in Japan. The potential usefulness of DNA analysis in determining host plant ranges of morphologically identical cecidomyiid species that are currently identified solely on differences of host plant is emphasized.

Nonspecific interstitial pneumonia as pulmonary involvement of primary Sjögren's syndrome.

The pathologic patterns of lung involvement in nine patients with Sjögren's syndrome (SjS) are evaluated. The patients consisted of three males and six females, with a median age of 59 years. The SjS was diagnosed according to the criteria of the First International Seminar on SjS. In all patients, high-resolution computed radiographic scanning (HRCT) of the lungs was performed, and apparent honeycomb or microhoneycomb formation was observed in six patients. Pathologically, six patients were diagnosed with usual interstitial pneumonia (UIP), and three were diagnosed with nonspecific interstitial pneumonia/fibrosis (NSIP) (group II). There were no apparent honeycomb formations on HRCT in patients diagnosed with NSIP. In conclusion, NSIP is also a possible histologic classification of interstitial pneumonia associated with SjS.

Jab1 expression is associated with inverse expression of p27(kip1) and poor prognosis in epithelial ovarian tumors.

PURPOSE: Jab1 (Jun activation domain-binding protein 1) has been described as a coactivtor of AP1 transcription factor, and is a subunit of a large protein complex (called the COP9 signalosome). Recent study (K. Tomoda et al., Nature (Lond.), 398: 160-165, 1999) found that Jab1 protein can cause breakdown of p27(kip1) protein in mammalian cells. To investigate whether Jab1 expression is correlated with p27(kip1) protein levels as well as how it might be clinically relevant, we evaluated the expression of Jab1 in a group of epithelial ovarian tumors. EXPERIMENTAL DESIGN: Immunohistochemical analysis was performed in 80 cases of ovarian tumors (33 benign ovarian tumors and 47 ovarian carcinomas). Twenty-six of the 80 cases were evaluated by Western blot analysis. RESULTS: Jab1 overexpression was detected in 68.1% (32 of 47) of malignant tumors and 33.3% (11 of 33) of benign tumors. The positive ratio of Jab1 was increased from benign to malignant ovarian tumors (P = 0.002). A negative correlation between Jab1 and p27(kip1) expression was found in both benign (P = 0.003) and malignant (P = 0.002) ovarian tumors. No significant correlation was observed between Jab1 overexpression and clinicopathological parameters. Kaplan-Meier survival analysis showed that Jab1 overexpression was significantly associated with poor prognosis of patients (P = 0.049). CONCLUSIONS: Jab1 expression is inversely correlated with p27(kip1) expression levels, and Jab1, as a negative regulator of p27(kip1), may be associated with the progression and prognosis of epithelial ovarian tumors.

Implication of malignancy and prognosis of p27(kip1), Cyclin E, and Cdk2 expression in epithelial ovarian tumors.

OBJECTIVE: The aim of this study was to further evaluate whether the expression of p27(kip1), cyclin E, and cdk2 is related to the malignancy of ovarian tumors and whether their expressions, alone or in combination, are associated with prognosis in epithelial ovarian carcinoma. METHODS: Immunohistochemical analysis using anti-p27(kip1), anti-cyclinE, and anti-cdk2 antibodies was carried out for 103 cases consisting of benign, borderline, and malignant ovarian tumors, and Western blot analysis and cdk2 activity assay were performed in 26 fresh ovarian tumor samples. RESULTS: p27(kip1) expression was reduced in ovarian carcinomas in contrast to benign and borderline tumors. The expression of cyclin E and cdk2 gradually increased from benign to borderline to malignant tumors. Kaplan-Meier survival analysis showed that patients with p27(kip1) expression had a high overall survival rate. Patients with cyclin E overexpression had a low overall survival rate. When the combination of these proteins was analyzed, patients with the p27(kip1) (-)/cyclin E (++)/cdk2 (++) phenotype were significantly associated with the poorest overall survival. In multivariate Cox regression analysis, the combined phenotype of p27(kip1) (-)/cyclin E (++)/cdk2 (++) was independently related to poor prognosis. CONCLUSIONS: Our results suggest that loss of p27(kip1) expression and overexpression of cyclin E or cdk2 were significantly associated with malignancy in ovarian tumors. p27(kip1) and cyclin E proteins may be valuable prognostic factors for epithelial ovarian carcinoma patients. Furthermore, the combined evaluation of p27(kip1)/cyclin E/cdk2 may provide the most important prognostic implication.

Clinical features of polymyositis/dermatomyositis associated with silicosis and a review of the literature.

There are few reports which describe the association of polymyositis/dermatomyositis (PM/DM) and silicosis. The purpose of this study is to describe the clinical features of PM/DM associated with silicosis. We first describe clinical features of two cases and provide a review of the literature. Finally, seven patients with PM/DM associated with silicosis are retrospectively evaluated. There were one female and six males. Histological specimens were obtained by open lung biopsy in five cases and not examined in two. The mechanism of the association between silicosis and PM/DM is discussed.