An invasive micropapillary variant (IMPV) has recently been described in several organs but has not been reported in the gallbladder. It has been mentioned to have aggressive behavior with a high propensity for lymphovascular invasion, lymph node metastasis and poor clinical outcome. We analyzed the clinicopathologic findings of IMPV and compared them with those of a conventional adenocarcinoma in the gallbladder to clarify the highly aggressive potential of IMPV of gallbladder carcinoma. Ninety consecutive cases of surgically resected gallbladder carcinomas were studied for age, gender, type, depth of invasion and lymph node and distant metastases. Histologically, IMPV of gallbladder carcinoma was characterized by a small cluster of tumor cells lying within clear stromal spaces. This pattern mimicked extensive lymphatic invasion, but the cluster of tumor cells showed a distinctive retraction artifact from the surrounding stroma. In total, 20 (22.2%) cases had foci of IMPV, which ranged from 5% to 10% of the primary tumor tissue. Of those 20 cases, 17 (85.0%) carcinomas with IMPV also included lymph node metastasis, which was more frequent than in conventional carcinoma (32.8%, P < 0.001). Carcinomas with IMPV had a more advanced tumor status and showed severe lymphatic invasion (P = 0.001, P < 0.001, respectively). A multivariate regression analysis demonstrated that the presence of IMPV is an independent predictor of regional nodal metastasis (Odds ratio: 9.995, 95% confidence interval: 1.996-50.052, P = 0.005). IMPV is a useful predictor of regional lymph node metastasis in gallbladder adenocarcinoma.

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Ceramide is generated by the hydrolysis of membrane sphingomyelin by sphingomyelinase and is implicated in multiple signaling pathways, including those regulating differentiation, inflammation and immune responses. Excess formation of prostaglandin E₂ (PGE₂) is thought to increase susceptibility to infection, rheumatoid arthritis and inflammation, including periodontal diseases. We investigated the inhibitory effect of C₂-ceramide, a short-chain ceramide analog, on the PGE₂-stimulated accumulation of cAMP in human gingival fibroblasts. In human gingival fibroblasts pre-treated with C₂-ceramide for 18 h, the PGE₂-stimulated accumulation of cAMP was reduced, but an inactive C₂-ceramide analog had no such effect. The accumulation of cAMP induced by EP2 and EP4 receptor agonists (ONO-AE1-259 and ONO-AE1-329, respectively) was inhibited in cells treated with C₂-ceramide. However, treatment with C₂-ceramide had no effect on the expression of mRNAs encoding the EP2 and EP4 receptors. Accumulation of cAMP could be induced by cAMP-elevating agents (forskolin, isobutylmethylxanthine and mastparan) but was not reduced by treatment with C₂-ceramide. These observations suggest that C₂-ceramide attenuates PGE₂ receptor function and consequently inhibits the accumulation of cAMP in human gingival fibroblasts.

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Esophageal squamous cell carcinoma (ESCC) has been generally considered as one of the most aggressive cancers with poor prognosis. Vimentin is the major protein constituent of intermediate filaments in normal and neoplastic mesenchymal cells, and has been regarded as a marker of epithelial-mesenchymal transition (EMT). However, little is known about ESCC with vimentin expression as a marker of EMT. In this study, we analyzed vimentin expression in 129 cases of ESCC in order to elucidate whether vimentin expression is correlated with clinicopathological features and aggressive behavior of ESCC. Vimentin expression was identified in 96 of the 129 cases (74.4%). The cases with vimentin-positive carcinoma cells showed a significantly higher incidence of lymph node metastasis (P = 0.001). Carcinomas with vimentin expression were more advanced in terms of tumor status and lymphatic invasion (P = 0.001, P = 0.009, respectively), and associated with stronger stromal α-smooth muscle actin (α-SMA) expression (P < 0.001). Vimentin expression was also associated with distant metastasis, including distant node metastasis (P = 0.014). Vimentin expression in both primary and metastatic carcinomas was found in 68.6% (48/70) of the cases, while no vimentin expression in both primary and metastatic carcinomas comprised 92.3% of the cases (12/13) (P < 0.001). In conclusion, we demonstrated that vimentin expression in ESCC is an independent predictor of lymph node metastasis (multivariate analysis, P = 0.014, odds ratio: 3.314, 95% confidence interval: 1.276-8.605). In addition, vimentin expression was frequently retained in metastatic carcinoma of the lymph node.

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We recently demonstrated that short time exposure to hypoxia (15 min) in H9c2 cardiomyocytes protected cells against cell death, and longer exposure to hypoxia induced cell death. To understand the molecular mechanism concerning cell death and survival, it is intriguing to identify survival factors against cell death. Using proteomics analysis, levels of proteins derived from H9c2 cells exposed to hypoxia and normoxia were compared and candidates for survival factor were identified. One of the candidates was a prohibitin. Overexpression of prohibitin inhibited H9c2 cell death induced by hypoxia for longer hours. We further clarified the mechanism of cell death. Overexpression of prohibitin inhibited decrease of mitochondrial membrane potential levels, decrease of Bcl-2 level in mitochondria and cytochrome c release to cytosol from mitochondria induced by hypoxia. The mechanism for survival was that overexpression of prohibitin inhibited cytochrome c release by decrease of mitochondrial membrane potential levels and decrease of Bcl-2 level. Taken together, identified prohibitin may function as a survival factor against hypoxiainduced cell death.

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BRAK/CXCL14 is a chemokine that is expressed in many normal cells and tissues but is absent from or expressed at very low levels in transformed cells and cancerous tissues including head and neck squamous cell carcinoma (HNSCC). We reported previously that the forced expression of BRAK/CXCL14 in HNSCC cells decreased the rate of tumor formation and size of tumor xenografts in athymic nude mice and SCID mice, suggesting that expression level of the gene is important for tumor suppression. In order to study the regulatory mechanisms governing the expression of this gene, we determined the transcriptional start site and promoter motifs of the gene. The major transcriptional start site determined by 5'rapid amplification of cDNA end method was located 283 bp downstream of the first proposed site of the gene. Determination of luciferase activities of reporter gene constructs with various deletions or mutations showed that an atypical TATA-like sequence, TATTAA was essential for the transcription of the gene and that the AP-1 binding sequence and tandem GC box were necessary for stimulating the expression of the gene in human squamous epithelial cells. The human DNA region was highly homologous (95% base identity) to the mouse gene. In addition, okadaic acid, an inhibitor of serine/threonine phosphatases 1, 2A and 2B, stimulated TATTAA sequence and AP-1 binding-sequence dependent promoter activity as well as increased the level of BRAK/CXCL14 mRNA, indicating that these sequences are essential for the regulation of BRAK/CXCL14 gene expression in the cells.

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All multi-cellular animals, including hydra, insects and vertebrates, develop gap junctions, which communicate directly with neighboring cells. Gap junctions consist of protein families called connexins in vertebrates and innexins in invertebrates. Connexins and innexins have no homology in their amino acid sequence, but both are thought to have some similar characteristics, such as a tetra-membrane-spanning structure, formation of a channel by hexamer, and transmission of small molecules (e.g. ions) to neighboring cells. Pannexins were recently identified as a homolog of innexins in vertebrate genomes. Although pannexins are thought to share the function of intercellular communication with connexins and innexins, there is little information about the relationship among these three protein families of gap junctions. We phylgenetically and bioinformatically examined these protein families and other tetra-membrane-spanning proteins using a database and three analytical softwares. The clades formed by pannexin families do not belong to the species classification but do to paralogs of each member of pannexins. Amino acid sequences of pannexins are closely related to those of innexins but less to those of connexins. These data suggest that innexins and pannexins have a common origin, but the relationship between innexins/pannexins and connexins is as slight as that of other tetra-membrane-spanning members.

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In this study, we examined the effect of the difference in time of going to sleep on the numerical values of leukocyte subsets and various hormones. Subjects consisted of 26 healthy adults (15 men, 11 women) with a mean age of 37.6 years. Among the 26 individuals, 12 persons (Group E) were of the habit of going to sleep before midnight consistently, while 14 persons (Group L) were of the habit of staying up late, consistently going to sleep after 2 am. For Group E, it was found that the ratio of lymphocytes was remarkably high in comparison with Group L (Group E 41.6 ± 2.54%, Group L 31.7 ± 2.03%, P < 0.01). On the other hand, for Group L it was found that the ratio of granulocytes was remarkably high in comparison with Group E (Group E 53.0 ± 2.51%, Group L 62.3 ± 2.22%, P < 0.01). However, no difference was observed in lymphocyte and granulocyte ratios due to the duration of the sleep. As the excessive quantity of granulocytes was not corrected through longer sleep, these findings suggest that the time when first going to sleep is more important than the total hours of sleep achieved.

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Titanium dioxide (TiO₂) is known to confer photocatalytic bactericidal effects under ultraviolet (UV) irradiation. Few reports are available, however, on the clinical applications of TiO₂ particle mixtures. Our objective in the present research was to evaluate the in vitro bactericidal effects of a TiO₂ particle mixture in a nutrition-rich biological environment. A bacterial suspension of Staphylococcus aureus and epidermidis 3 × 10³ CFU/mL was added to a TiO₂ particle mixture (0.038 mg/mL) containing mainly sodium percarbonate and citric acid. To simulate a biological environment, 40 μL of 10% bovine serum albumin was added and the culture temperature was maintained at 37°C. The resulting product was irradiated by UV light and the bacterial survival rate was calculated for each time of UV irradiation. In the control sample treated with distilled water + UV, the bacteria survived at a high rate even after 180 min. In the TiO₂ mixture + UV sample, meanwhile, the bacterial survival rate dropped to 43.8% and 6.0% of the baseline values in S. aureus and S. epidermidis, respectively, after 60 min of UV irradiation. The photocatalytic antibacterial action of the TiO₂ particle mixture was high even in a protein-rich biological environment.

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Renin is the most important enzyme in the renin-angiotensin system. Our previous study led to the identification of soyasaponin I, the first renin inhibitor isolated from soybean. In the present study, the effects of saponins and sapogenols on human renin activities were investigated. Soyasaponins I and II, glycyrrhizin, monoglucuronyl glycyrrhetic acid (MGGA), chikusetsusaponin IV, and Kochia scoparia fruit saponins (momordins) were found to inhibit renin activity. On the other hand, sapogenols (soyasapogenol B and glycyrrhetic acid), saikosaponins b2 and c, and ginsenoside Rb₁ had no effect on renin activity. These results clearly indicate that the 3-O-β-dglucopyranosiduronic moiety in saponins (glucuronide saponin) is essential for renin inhibition.

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We used capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) to characterize the metabolic profiles of the seed, pulp, stem and leaf of Illicium anisatum. CE-TOFMS detected more than 1000 polar metabolites within 40 min, of which 58 were annotated and quantified. Seed had higher levels of glycolytic metabolites than pulp, stem and leaf, while leaf had higher levels of TCA cycle and nucleoside metabolites. Among amino acid metabolites, levels of Gln, Glu, and Asp were higher in almost every organ. Levels of shikimic acid, the source compound for Tamiflu^®, were high in all organs, ranging from 6.84% to 28.82%. These results indicate that CETOFMS-based metabolomics offers an efficient, convenient method for comprehensive metabolite profiling, and may be a powerful tool for the screening of drug discovery.

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We investigated the feasibility of measuring the partial pressure of oxygen (pO₂) in cultured mouse oocytes by electron paramagnetic resonance (EPR) oximetry. Approximately 15 pL (0.015 μL) of Ox063, an oxygen-sensing paramagnetic material, at a concentration of 500 mM was injected into the ooplasm of mouse oocytes. When one, five and 20 oocytes were used, the sample of 20 oocytes was sufficient to yield an effective EPR spectrum for determining the pO₂. The mean pO₂ levels in oocytes cultured under tensions of 5 and 20% O₂ were 60.2 and 153.1 mmHg, respectively. The present study indicates that it is possible to utilize EPR oximetry with Ox063 for the measurement of pO₂ in cultured oocytes.

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