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Apigenin is a flavonoid with antioxidant and anticancer effects. It has been reported that apigenin inhibits proliferation, migration, and invasion and induces apoptosis in cultured lung cancer cells. However, there is little information on the involvement of microRNAs (miRNAs) in its effects. miRNA microarray analysis and polymerase-chain-reaction analysis of miRNAs revealed that treatment of human lung cancer A549 cells with apigenin up-regulated the level of miR-34a-5p. Furthermore, mRNA microarray analysis and the results of three microRNA target prediction tools showed that Snail Family Transcriptional Repressor 1 (SNAI1), which inhibits the induction of apoptosis, had its mRNA expression down-regulated in A549 cells treated with apigenin. Our findings suggest that apigenin might induce apoptosis by down-regulation of SNAI1 through up-regulation of miR-34a-5p in A549 cells.

Background Prasugrel inhibits platelet aggregation more potently than clopidogrel. In the global phase III trial, prasugrel [loading dose/maintenance dose (LD/MD), 60/10 mg] reduced the incidence of ischemic events but involved a higher risk of hemorrhage than clopidogrel in patients with acute coronary syndromes who were scheduled to undergo percutaneous coronary intervention (PCI). In the Japanese phase III trial for similar patients wherein the prasugrel dose regimen was adjusted (LD/MD, 20/3.75 mg), the efficacy of prasugrel and clopidogrel were comparable to that in the global trial; however, the safety could not be determined due to limited power. Given the strict enrollment criteria, the results of the Japanese phase III trial may not be applicable to routine clinical practice. We compared the safety and effectiveness of prasugrel and clopidogrel in the real-world setting in Japanese patients. Methods With an analysis of a large claims database prepared during the post-marketing stages in Japan, we identified patients undergoing PCI and compared the incidence of bleeding and ischemic coronary events between patients who received prasugrel and those receiving clopidogrel. Results Between January 1, 2014 and December 31, 2018, we identified 1977 patients who were scheduled to undergo urgent PCI (urgent PCI cohort) and 1922 who were scheduled to undergo elective PCI (elective PCI cohort). After propensity-score matching, there were no significant differences in the baseline clinical characteristics of the prasugrel and clopidogrel groups in the urgent (n = 1080) and elective PCI (n = 1626) cohorts. In Cox proportional hazard analyses, there were no significant differences in the incidence of bleeding or ischemic coronary events during the median 8-month follow-up in both cohorts. Conclusions The safety and effectiveness of prasugrel was comparable to that of clopidogrel in real-world Japanese patients scheduled to undergo PCI.

Gastric dysmotility has been reported in patients with long-standing diabetes mellitus (DM). Some patients with DM are diagnosed as diabetes gastroparesis and have several upper gastrointestinal (GI) symptoms such as appetite loss and abdominal pain. This study aimed to identify the relationship between gastric motility and upper GI symptoms in patients with long-standing DM.This study was conducted among 23 patients with DM and 15 healthy controls. All the patients with DM were receiving insulin treatment and had at least one history of incidence of diabetic nephropathy, retinopathy or neuropathy. Gastric motility was evaluated using electrogastrography (EGG) and gastric emptying using theCompared to healthy controls, patients with long-standing DM showed a significantly lower percentage of normogastria at the postprandial state with a lower power ratio in EGG. Gastric emptying was significantly delayed in patients with DM in the overall analysis. Sixteen patients with DM (69.6%) demonstrated abnormalities in either gastric myoelectrical activity or gastric emptying. Among patients with abnormal EGG or delayed gastric emptying, 12 had some GI symptoms, compared with 3 patients with normal gastric motility. No significant correlation was observed between the gastric emptying parameters and HbA1c values.Patients with long-standing DM showed gastric dysmotility, including impaired gastric myoelectrical activity and delayed gastric emptying. Gastric dysmotility appears to be closely correlated with upper GI symptoms in patients with long-standing DM.

Heart failure is a major public health problem. Although there have been significant advances in the management of heart failure, the mortality and morbidity in heart failure patients remain high. Heart failure patients are susceptible to influenza-related complications including acute heart failure exacerbations and secondary infections such as pneumonia, both of which lead to significant morbidity and mortality. An earlier meta-analysis of observational cohort studies reported that influenza vaccination was associated with reduced risk of mortality in heart failure patients. Although there are no published randomized controlled trials (RCTs) on the effect of influenza vaccination on clinical outcomes in heart failure patients, there are several on-going RCTs examining the effect in these patients. We aim to conduct a meta-analysis of RCTs to assess the efficacy and safety of influenza vaccination in heart failure patients.This meta-analysis will include RCTs examining the effect of influenza vaccination in heart failure patients. Information of studies will be collected from electronic databases. The primary outcome of interest will be cardiovascular death. The secondary outcomes of interest will be all-cause death, nonfatal myocardial infarction, nonfatal stroke, hospitalization for heart failure, and hospitalization for any cause.This meta-analysis will evaluate the efficacy and safety of influenza vaccination in heart failure patients, providing evidence to the use of influenza vaccine in these patients.INPLASY202210115.

Background: Prasugrel inhibits platelet aggregation more potently and exerts therapeutic action faster than clopidogrel. In the global phase III trial conducted in Western and South American countries that excluded Asian countries, prasugrel reduced ischemic events but increased hemorrhagic risk compared with clopidogrel in patients with acute coronary syndrome scheduled for percutaneous coronary intervention. In the Japanese phase III trial for similar patients, the efficacy of prasugrel compared with clopidogrel was comparable to the global trial, but the safety could not be confirmed because of an insufficient number of patients. Furthermore, given the strict enrollment criteria, the results of these trials may not be applicable to routine clinical practice. Accordingly, we compared the hemorrhagic risk of prasugrel and clopidogrel in real-world settings by analyzing adverse drug event reports in post-marketing stages provided by the Japanese regulatory authorities and the U.S. Food and Drug Administration (FDA). Methods: We analyzed a total of 3,970 reports for prasugrel (n = 518) or clopidogrel (n = 3,452) between 2014 and 2017 in the Japanese Adverse Drug Event Report (JADER) and a total of 91,914 reports for either prasugrel (n = 5,992) or clopidogrel (n = 85,922) between 2009 and 2019 in the FDA Adverse Event Reporting System (FAERS). Results: In JADER and FAERS, prasugrel was more frequently and significantly associated with hemorrhagic event reports than clopidogrel. After adjustment for known confounders including age, sex, and concomitant medications (aspirin, anticoagulants, and proton pump inhibitors), the hemorrhagic risk of prasugrel compared with clopidogrel remained significant (adjusted reporting odds ratios [95% CI] for total, intracranial, and gastrointestinal hemorrhagic events = 2.42 [1.97-2.96], 2.45 [1.85-3.24], and 2.27 [1.73-2.97] in JADER, and 2.21 [2.09-2.34], 1.21 [1.09-1.33], and 1.41 [1.29-1.54] in FAERS). Conclusions: The hemorrhagic risk was found to be greater with prasugrel than clopidogrel in real-world patients, including Japanese patients.

4-Hydroxyderricin (4-HD) is a major polyphenol of Angelica keiskei (Japanese name Ashitaba), exhibiting anti-allergic, anti-diabetic, anti-oxidant, and antitumor effects. The present study was designed to evaluate the effects of 4-HD on bone formation and maintenance by using cultured osteoclasts and osteoblasts. 4-HD did not affect cell proliferation of stromal ST2 cells and preosteoblast MC3T3-E1 cells at concentrations of 1–10 μM. This compound inhibited the formation of multinucleated osteoclasts from mouse splenic cells, and we identified a molecular pathway of osteoclast differentiation mediated by 4-HD, which led to inhibition of the expression of receptor activator of nuclear factor-κB ligand and macrophage-colony stimulating factor in ST2 cells. By contrast, 4-HD enhanced indices of osteoblast differentiation, such as alkaline phosphatase activity and calcium deposition by osteoblastic MC3T3-E1 cells, at concentrations of 1–10 μM. Furthermore, we found that 4-HD at 1 μM attenuated H2O2 levels in MC3T3-E1 cells. Our findings indicate that 4-HD may have critical effects on bone formation and maintenance.

Kidney disease (KD) is a serious risk factor for cardiovascular event, and it is important to protect the heart and kidneys during treatment of the high blood pressure to prevent cardiovascular event. Japanese guideline (JSH2014) suggests using combination therapy to reduce the risk of comorbidities rather than high-dose monotherapy for the patients with cardiovascular disease and KD. Therefore, the present study assessed antihypertensive prescription patterns in Japanese patients with ischemic heart disease related diseases (IHDRD) and KD, and evaluated whether the prescription patterns match with the guideline-suggested therapies by analyzing the national insurance claims database (NDB). We extracted antihypertensive prescription patterns among Japanese IHDRD patients from the data of October 2011 of NDB, and examined the effect of KD on the prescription patterns. The number of prescribed antihypertensive was associated with KD among patients regardless of IHDRD. Patients with IHDRD and KD were more frequently prescribed combination therapy (calcium channel blockers/angiotensin II receptor blockers) than the calcium channel blocker monotherapy, based on the JSH2014. On the other hand, we did not observe the standard use of diuretics for patients with heart failure, which is suggested by the JSH2014. These findings suggested that patients with IHDRD and KD were frequently prescribed combination therapy to achieve its cardioprotective and renoprotective effects, according to the JSH2014, but the prescription profile to the patients with heart failure didn't match that of guideline-suggested therapies. This study provided a clinically important information and demonstrated the utility of NDB for compliance assessment for therapeutics guideline.

The survival rate of cardiac arrest patients is less than 10%; therefore, development of a therapeutic strategy that improves their prognosis is necessary. Herein, we searched data collected from medical facilities throughout Japan for drugs that improve the survival rate of cardiac arrest patients. Candidate drugs, which could improve the prognosis of cardiac arrest patients, were extracted using “TargetMine,” a drug discovery tool. We investigated whether the candidate drugs were among the drugs administered within 1 month after cardiac arrest in data of cardiac arrest cases obtained from the Japan Medical Data Center. Logistic regression analysis was performed, with the explanatory variables being the presence or absence of the administration of those candidate drugs that were administered to ≥10 patients and the objective variable being the “survival discharge.” Adjusted odds ratios for survival discharge were calculated using propensity scores for drugs that significantly improved the proportion of survival discharge; the influence of covariates, such as patient background, medical history, and treatment factors, was excluded by the inverse probability-of-treatment weighted method. Using the search strategy, we extracted 165 drugs with vasodilator activity as candidate drugs. Drugs not approved in Japan, oral medicines, and external medicines were excluded. Then, we investigated whether the candidate drugs were administered to the 2,227 cardiac arrest patients included in this study. The results of the logistic regression analysis showed that three (isosorbide dinitrate, nitroglycerin, and nicardipine) of seven drugs that were administered to ≥10 patients showed significant association with improvement in the proportion of survival discharge. Further analyses using propensity scores revealed that the adjusted odds ratios for survival discharge for patients administered isosorbide dinitrate, nitroglycerin, and nicardipine were 3.35, 5.44, and 4.58, respectively. Thus, it can be suggested that isosorbide dinitrate, nitroglycerin, and nicardipine could be novel therapeutic agents for improving the prognosis of cardiac arrest patients.

Background Nearly half of patients with heart failure (HF) have preserved ejection fraction (EF) and the mortality and morbidity of patients with HF with preserved EF (HFpEF) are high. However, there is no established therapy to improve survival in these patients. HFpEF patients are often elderly and their primary chronic symptom is severe exercise intolerance. Thus, improvement of exercise capacity presents another important clinical outcome in HFpEF patients. Iron deficiency is common in HF patients, and the presence of iron deficiency, regardless of concomitant anemia, is associated with worse symptoms, impaired exercise capacity, and higher mortality and hospitalization in these patients. Several meta-analyses of randomized controlled trials reported that iron administration improved HF symptoms, exercise capacity, and clinical outcomes in iron-deficiency patients with HF with reduced EF. However, there is insufficient evidence as to the effect of iron administration in iron-deficiency HFpEF patients. Methods and results This meta-analysis will include randomized controlled trials on the effects of iron administration on HF symptoms, exercise capacity, and health-related quality of life in iron-deficiency HFpEF patients. Information of studies will be collected from PubMed, Web of Science, Cochrane Library, and ClinicalTrials.gov. The primary outcome will be exercise capacity (6-minute walking distance). The secondary outcomes will be HF symptoms, health-related quality of life, and mortality and hospitalization rates. Conclusion This meta-analysis will evaluate the effect of iron therapy in iron-deficiency HFpEF patients, providing evidence as to the iron administration in these patients. Systematic review registration PROSPERO CRD42020205297.

Summary What is known and objective We evaluated the effectiveness of warning letters published by the pharmaceutical regulatory agency in Japan on communication of drug safety and risk by quantitative analysis of the national health insurance claims database (NHICD). We then explored what factors may have affected risk communication. Methods We measured the implementation rate of the hepatitis virus-monitoring test among methotrexate (MTX)-treated patients; a warning letter had been issued regarding the use of MTX, as it apparently activates the hepatitis virus. Data from the NHICD, which include 99·3% of Japanese residents, were used. A total of 4 933 481 patients with rheumatoid arthritis (RA) (January–June, 2010) were the focus of this study. Results The implementation rate of the hepatitis virus-monitoring test increased from 1·4% before to 1·8% after the warning letter announcement. Logistic regression analysis suggested that the installation of a drug information management room is one of the important factors affecting risk communication. Further analysis revealed that the hepatitis virus monitoring rates in hospitals without drug information management rooms increased from 2·3% to 4·1% due to the issue of the warning letter. What is new and conclusion The warning letter from the regulatory agency plays an important role in risk communication in hospitals without drug information management rooms.

Polyphenol have been reported to have physiological effects with respect to alleviating diseases such as osteoporosis and osteopetrosis. We recently reported that the olive polyphenol hydroxytyrosol accelerates bone formation both in vivo and in vitro. The present study was designed to evaluate the in vivo and in vitro effects of apigenin (4',5,7-trihydroxyflavone), one of the major polyphenols in olives and parsley, on bone formation by using cultured osteoblasts and osteoclasts and ovariectomized (OVX) mice, respectively. Apigenin markedly inhibited cell proliferation and indices of osteoblast differentiation, such as collagen production, alkaline phosphatase activity, and calcium deposition in osteoblastic MC3T3-E1 cells at concentrations of 1-10 μM. At 10 μM, apigenin completely inhibited the formation of multinucleated osteoclasts from mouse splenic cells. Moreover, injection of apigenin at 10 mg kg(-1) body weight significantly suppressed trabecular bone loss in the femurs of OVX mice. Our findings indicate that apigenin may have critical effects on bone maintenance in vivo.

The effect of subchronic exposure of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), an active metabolite of trichloroethylene (TCE), was investigated in mice, as a part of mechanistic assessment of renal toxicity of TCE. To examine the subchronic effects of DCVC on kidney function, Balb/c male mice were administered DCVC orally and intraperitoneally once a week for 13 weeks at 1, 10 and 30 mg/kg (Main Study) and for 8 weeks at 30 mg/kg (PCR Study). At the terminal sacrifice, mice orally and intraperitoneally administered with 10 and 30 mg/kg showed significantly lower kidney weight and significantly higher blood urea nitrogen levels than the control group. Pathological examination revealed that a dose of 30 mg/kg delivered by both routes resulted in renal tubular degeneration characterized by tubular necrosis and interstitial fibrosis, and in degradation of the cortex. Degenerative changes were accompanied by the increased expression of tumor necrosis factor-α, interleukin-6 and cyclooxygenase-2 mRNAs in the kidney of mice treated with 30 mg/kg for 8 weeks. These pathohistological observations mostly corresponded to those in short-term toxicity studies on DCVC. DCVC might be a direct cause of renal toxicity, which is suggested from the aggravation in these symptoms with the dose increase.

Polyphenols reportedly exert physiological effects against diseases such as cancer, arteriosclerosis, hyperlipidemia and osteoporosis. The present study was designed to evaluate the effects of oleuropein, hydroxytyrosol and tyrosol, the major polyphenols in olives, on bone formation using cultured osteoblasts and osteoclasts, and on bone loss in ovariectomized mice. No polyphenols markedly affected the proliferation of osteoblastic MC3T3-E1 cells at concentrations up to 10μM. Oleuropein and hydroxytyrosol at 10 to 100μM had no effect on the production of type I collagen and the activity of alkaline phosphatase in MC3T3-E1 cells, but stimulated the deposition of calcium in a dose-dependent manner. In contrast, oleuropein at 10 to 100μM and hydroxytyrosol at 50 to 100μM inhibited the formation of multinucleated osteoclasts in a dose-dependent manner. Furthermore, both compounds suppressed the bone loss of trabecular bone in femurs of ovariectomized mice (6-week-old BALB/c female mice), while hydroxytyrosol attenuated H(2)O(2) levels in MC3T3-E1 cells. Our findings indicate that the olive polyphenols oleuropein and hydroxytyrosol may have critical effects on the formation and maintenance of bone, and can be used as effective remedies in the treatment of osteoporosis symptoms.

To determine the timing for follow-up study of power Doppler ultrasonography (PDUS) by evaluating the response of finger joint synovitis in patients with rheumatoid arthritis (RA) to treatment including infliximab, an antitumor necrosis factor alpha agent. Bilateral second/third metacarpo-phalangeal (MCP) joints and second proximal inter-phalangeal (PIP) joints (total of six joints) in 21 patients (18 women and three men; median age 53 years) with chronic active RA were assessed by PDUS before and after 2 weeks, 6 weeks, 14 weeks, 30 weeks, 38 weeks, 46 weeks, and 54 weeks of infliximab infusion. Pulse Doppler settings were standardized for each patient and optimized for the detection of synovial blood flow by adjustment of color gain, pulse repetition, and flow optimization. Power Doppler signal was graded for each joint [joint grade for power Doppler (JGPD) signals], and the sum of the grades of six joints was defined as the PDUS index [joint index for power Doppler signals (JIPD)] at each visit. PDUS and clinical parameters [28-joint disease activity score (DAS28), health assessment questionnaire, and C-reactive protein (CRP) level] were independently assessed and compared with baseline values. The American College of Rheumatology (ACR) core set responders and non-responders at week 54 were compared for clinical parameters and PDUS index at each visit. Fourteen patients completed the planned treatment for 1 year, while six patients dropped out for various reasons and one died suddenly. PDUS was performed a total of 146 times on 467 joints. DAS28 was assessed 127 times. Both DAS28 and JIPD had decreased at the follow-up. Comparative analysis between DAS28 and PDUS was available 125 times. The transverse correlation between the PDUS index and DAS28 was not significant throughout the follow-up period. When responders and non-responders were discriminated at week 54, a logistic regression model for the binary endpoint of responder vs non-responder, with PDUS index as explanatory variable at time point 0, and follow-up revealed statistical significance from week 38 and on. PDUS reflected infliximab’s effect on pannus vascular signals; this effect was observed as early as 2 weeks after treatment had begun. Also, the responders to treatment at 54 weeks tended to have fewer JIPD than non-responders in the follow-up period. PDUS may be performed at week 38 or later to foresee the response to the treatment at week 54.

Background and purpose: The connexin (Cx) 32 gene, a member of the gap junction gene family, acts as a tumour suppressor gene in human renal cell carcinoma (RCC) and is down-regulated by the hypermethylation of CpG islands in a promoter region of the Cx gene. The current study investigated whether the restoration of Cx32 silenced by hypermethylation in RCC by a DNA demethylating agent could be an effective treatment against RCC. Experimental approach: Using nude mice bearing Caki-1 cells (a human metastatic RCC cell line), the effects of 5-aza-2′-deoxycytidine (5-aza-CdR), a DNA demethylase inhibitor, on Cx32 mRNA expression and tumour growth were examined by RT-PCR, and by measuring tumour weight and volume. Cx32 expression in Caki-1 tumours was inhibited by Cx32 short interfering (si) RNA, and the effect of siRNA on 5-aza-CdR-dependent suppression of tumour growth in nude mice was evaluated. Key results: 5-aza-CdR treatment inhibited the growth of Caki-1 cells in nude mice by 70% and increased 7-fold the level of Cx32 mRNA. The intratumour injection of Cx32 siRNA almost totally inhibited the expression of Cx32 mRNA and significantly reduced the suppression of tumour growth in 5-aza-CdR-treated nude mice. Conclusions and implications: 5-aza-CdR suppressed the growth of Caki-1 tumours in a xenograft model, by restoring Cx32 expression. This finding suggests that treatment with 5-aza-CdR could be a new effective therapy against human metastatic RCC and that Cx32 could be a potential target for the treatment of RCC. British Journal of Pharmacology (2008) 153, 1373–1381; doi:10.1038/bjp.2008.17; published online 11 February 2008

We have previously reported that connexin (Cx) 32 gene, a member of gap junctions, was specifically downregulated in human renal cell carcinoma (RCC) and it acts as a tumor suppressor against RCC. Because there is no standard therapy for advanced RCC, we investigated the anti-metastatic effect of Cx32 to seek a possibility of new RCC therapy. In this study, we used human metastatic RCC cell (Caki-1), and established Cx32-expressed cell clone (Caki-1T) or only mock-transfected cell clone (Caki-1W). For experimental production of metastases, the cells were injected into the lateral tail vein of SCID mice. Seventy days after inoculation, metastatic colonies were observed in Caki-1W inoculated group, though none of them were in Caki-1T inoculated group. The plasma VEGF concentration was significantly lower in Caki-1T group compared to Caki-1W group. To investigate where Cx32 effects on, we also tried in vitro analysis and found that the malignant phenotypes involving metastasis steps were significantly decreased in Caki-1T under hypoxia, a mimic condition of internal tumor environment. After hypoxia treatment, the protein level of HIF-2alpha, which plays main role for hypoxia adaptation, was observed to increase in Caki-1W, whereas no expression was observed in Caki-1T. We investigated the activation of Src, which is required for stabilization of HIF-2alpha, is suppressed in Caki-1T compared to Caki-1W. These results suggest that Cx32 inhibits hypoxia adaptation governed by HIF-2alpha, and this may help to reduce the metastasis of RCC cells.

Bowman–Birk protease inhibitor (BBI) from soybean acts as a potential chemopreventive agent in several types of tumors. However, the mechanism is still unclear. The present study was undertaken to estimate a mechanism of BBI-dependent negative growth control of human osteosarcoma cell (U2OS cell). BBI had negative growth control of the cells via induction of connexin (Cx) 43, a tumor suppressor gene in U2OS cells. This negative growth control by BBI was abrogated under down-regulation of Cx43 induced by a Cx43 antisense nucleotide treatment. It was also found that the BBI-dependent induction of Cx43 was due to elevation of Cx43 mRNA and stabilization of Cx43 protein. Especially, BBI-dependent inhibition of chymotrypsin-like activity in proteasome contributed to stabilization of Cx43 protein. These results suggest that a major negative growth effect of BBI is based on the restoration of Cx43 expression in U2OS cells.

We isolated a novel peptide, calcitonin receptor-stimulating peptide-1 (CRSP-1), from porcine brain and found that the administration of this peptide into rats induced a transient decrease in plasma calcium concentration. Therefore, we investigated the effects of CRSP-1 on osteoclastogenesis. Osteoclast-like cells were formed from spleen cells or bone marrow cells by a combination of the receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CRSP-1 dose-dependently inhibited the formation of multinucleated osteoclast-like cells, and a calcitonin receptor inhibitor antagonized in part the inhibition of osteoclast formation by CRSP-1. Furthermore, CRSP-1 destroyed the actin ring that is a typical index of osteoclast resorption activity; it contributed to this action via the signaling pathway of protein kinase A. Our findings indicate that CRSP-1 inhibits osteoclastogenesis by inhibiting the formation and activity of multinucleated osteoclasts. The inhibitory effects of CRSP-1 on osteoclast metabolism were similar in degree to those of porcine calcitonin. CRSP-1 might provide a clue to the development of tools useful in the prevention and treatment of osteoporosis.

Connexin (Cx) genes exert negative growth effects on tumor cells with certain cell specificity, and tumor-suppressive effects of the Cx genes contribute to enhancement of chemotherapeutical agents-induced cytotoxicity in some cancer cells. Since we and others have been reported that Cx32 acts as a tumor suppressor gene in lung adenocarcinomas, this study was undertaken to estimate if the combination of Cx32-dependent tumor-suppressive effect and vinorelbine (VBN), a chemotherapeutic agent which has been utilized for clinical lung adenocarcinoma treatment, could be effective in enhancing the sensitivity of the lung cancer to VBN treatment. We established the A549 cells (a human lung adenocarcinoma cell line) which had stable expression of Cx32 and estimated effect of Cx32 on VBN-induced cytotoxicity in the established cells. Cx32 expression in A549 cells significantly potentiated VBN-induced cytotoxicity on the cells due to enhancement of apoptosis induction. The enhancing cytotoxicity in A549 cells by Cx32 mainly depended on a decrease in expression of multi-drug resistance-1 (MDR-1) gene responsible for reduction of VBN accumulation into the cells. We also observed that silencing of Cx32 by siRNA treatment elevated the expression level of MDR-1 mRNA in A549 cells and that inhibition of MDR-1 gene product-dependent function enhanced VBN-induced cytotoxicity in the cells. These results suggest that Cx32 contributes to the enhancement of VBN-induced cytotoxicity in A549 cells via the reduction of MDR-1 expression.

We have reported that connexin (Cx) 32 gene, a member of gap junction protein family, acts as a tumor suppressor gene in human renal cell carcinoma (RCC). Of solid tumors, RCC is one of the most chemoresistant cancers, and there is no effective cancer chemotherapy against RCC at present. In this study, we examined if the combination of Cx32-dependent tumor-suppressive effect and vinblastine (VBL), a chemotherapeutic agent which has been utilized for clinical RCC treatment, could be effective in enhancing the sensitivity of RCC to VBL treatment. Cx32 expression in a human metastatic RCC cell (Caki-1 cell) significantly enhanced in vitro and in vivo VBL-induced cytotoxicity on the cell. Cx32 expression in the RCC cells potentiated VBL-induced apoptosis compared to the Cx32-negative RCC cells in vitro as well as in vivo. The enhancing apoptosis in the RCC cells by Cx32 mainly depended on the decrease of P-glycoprotein (P-gp), a multidrug resistance gene-1 (MDR-1) product responsible for reduction of VBL accumulation into the cells. We also observed that silencing of Cx32 by short interfering RNA (siRNA) treatment elevated the level of P-gp in Caki-1 cells and that inhibition of P-gp function enhanced VBL-induced apoptosis in the RCC cells. These results suggest that Cx32 is effective to enhance VBL-induced cytotoxicity in Caki-1 cells via the reduction of P-gp. Overall, it seems that the combination of Cx32-dependent tumor-suppressive effect and VBL is promising as a new cancer therapy against RCC.

Zfp318, a mouse gene with a Cys2/His2 zinc finger motif, is mainly expressed in germ cells in the testis. It encodes two alternative transcripts, which regulate androgen receptor-mediated transcriptional activation or repression by overexpression of them. However, the role of Zfp318 is still obscure in vivo, especially in spermatogenesis. To elucidate the role of Zfp318 during gamete production, we established a knockout mouse line. Zfp318-null male mice exhibited infertility, whereas Zfp318-null female mice displayed normal fertility. ZFP318 was expressed during multiple stages of spermatogenesis, from spermatocytes to round spermatids. The nuclei of secondary spermatocytes showed high levels of expression. Histological analysis and quantitative analysis of DNA content showed decreased numbers of both spermatids in the seminiferous tubules and mature spermatozoa in the epididymides of Zfp318-null mice. These results suggest that Zfp318 is expressed as a functional protein in testicular germ cells and plays an important role in meiosis during spermatogenesis.

We previously reported that testicular zinc finger protein (TZF) is a corepressor for androgen receptor (AR). The present study demonstrated that a central portion (amino acids 512–663) of TZF, TZF(512–663), is responsible for both binding to AR and repressing the transactivation. TZF recruited endogenous histone deacetylase 2 (HDAC2) and formed a complex with agonist-bound AR. Imaging analyses showed that TZF and TZF(512–663) were recruited by AR and simultaneously impaired distinct AR foci formation. Quantification of the foci number using a three-dimensional imaging method revealed that the number of intranuclear AR foci was related to its transactivation activity. Moreover, increased levels of TZF dissociated a coactivator, TIF2, from the AR foci and vice versa. These results indicate that the ligand-dependent transactivation function of AR is quantitatively related to its intranuclear foci formation, and suggest that corepressors, such as TZF, act on these intranuclear events competitively with coactivators.

Steroid hormones control the transcriptional activity of target genes mediated by intracellular nuclear receptors, and these transcriptional activities are modulated by the combination with coactivators and corepressors. We found in this study that testicular zinc finger protein (TZF) that was a nuclear protein with a zinc finger motif of the Cys2-His2 type was a novel corepressor of androgen receptor (AR). Fusion protein with green fluorescence protein GFP formed the specific foci in nuclei and TZF-dependent foci were located close to the splicing factor compartment. In addition, TZF was recruited into AR subnuclear foci after the treatment of dihydrotestosterone. Furthermore, we revealed that TZF bound to the activation function-1 (AF-1) domain (N-terminal transactivating domain) of AR protein. Transient over-expression of TZF in COS-7 cells or LNCaP human prostatic cancer cell resulted in decreased AR activity in a ligand-dependent fashion. Moreover, a transcriptional corepressor N-CoR additively decreased the transcriptional activity of AR with TZF. These findings suggest that TZF might be a novel corepressor of AR.

Connexin (Cx) genes exert negative growth effects on tumor cells with certain cell specificity. We have recently reported that Cx32 acts as a tumor suppressor gene in renal cancer cells due to the inhibition of Src-dependent signaling. In line with the previous study, here we examined if a Src family inhibitor (PP1) could potentiate tumor-suppressive effect of Cx32 in Caki-2 cell from human renal cell carcinoma. In order to clarify the potentialization of PP1, using Cx32-transfected Caki-2 cells and mock-transfected Caki-2 cells, we estimated difference in cytotoxic effect of PP1 on the two cell clones in vitro as well as in vivo. PP1 showed more cytotoxic effect on Caki-2 cells having Cx32 positive expression than that of Cx32 negative expression at lower doses. This potentialization was also observed in xenograft model of nude mice. The potentialization of the effect mainly depended on the induction of apoptosis but not the control of cell growth. In conjugation with this event, the reduction of anti-apoptotic molecules (Bcl-2 and Bcl-xL) was caused by the combination of Cx32 expression and PP1 treatment in Caki-2 cells. These results suggest that PP1 potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells through the reduction of anti-apoptotic molecules.

Although the constitute activation of the Src family of kinases (Src) has been established as a poor prognostic factor in several types of cancer, the role of Src in renal cell carcinoma (RCC) has not been defined. This study aimed to determine whether Src could contribute to the appearance of malignant phenotypes in RCC. The role of Src in the appearance of malignant phenotypes in RCC was examined in two human renal cancer cell lines, Caki-1 from human metastatic RCC and ACHN from human primary RCC. Src activity in Caki-1 cells was higher than that in ACHN cells, and this difference corresponded to the difference of PP1 (a Src family inhibitor)-induced cytotoxicity on the two cells. The difference in cytotoxicity between the cells did not depend on cell cycle regulation but on the induction of apoptosis, and the difference in apoptosis particularly related to the reduction of the Bcl-xL level. Furthermore, in Caki-1 cells with higher Src activity, Src stimulated the production of vascular endothelial growth factor (VEGF), partially via the activation of Stat3, and the inhibition of Src activity caused a reduction of the VEGF level in serum, angiogenesis, and tumor development in a xenograft model. These results suggested that Src contributed to the appearance of malignant phenotypes in renal cancer cells, particularly due to the resistance against apoptosis by Bcl-xL and angiogenesis stimulated by Src-Stat3-VEGF signaling. © 2005 Wiley-Liss, Inc.

We have reported that connexin (Cx) 32 acts as a tumor suppressor gene in renal cancer cells partly due to Her-2 inactivation. Here, we determined if a Her-2/Her-1 inhibitor (PKI-166) can enhance the tumor-suppressive effect of Cx32 in Caki-2 cells from human renal cell carcinoma. The expression of Cx32 in Caki-2 cells was required for PKI-166-induced cytotoxic effect at lower doses. The cyctotoxicity was dependent on the occurrence of apoptosis and partly mediated by Cx32-driven gap junction intercellular communications. These results suggest that PKI-166 further supports the tumor-suppressive effect of the Cx32 gene in renal cancer cells through the induction of apoptosis. Keywords:: connexin 32, PKI-166, renal cancer cell

Tributyltin is ubiquitous in the environment and an endocrine disruptor for many wildlife species. However, minimal information is available regarding the effect of this chemical on bone formation. When tributyltin chloride (TBT) (1mg/kg body weight) was administered subcutaneously to pregnant mice at 10, 12, and 14 days post coitus (dpc), fetuses at 17.5 days post coitus revealed the inhibition of calcification of supraoccipital bone. In contrast, 1mg/kg body weight monobutyltin trichloride (MBT) did not affect the fetal skeleton. Therefore, we examined the effects of TBT and its metabolites (dibutyltin dichloride, DBT, and MBT) on bone metabolism using rat calvarial osteoblast-like cells (ROB cells). The viability of ROB cells was not affected by the exposure of the cells to 10(-10) to 10(-7)M TBT. However, TBT reduced the activity of alkaline phosphatase (ALPase) and the rate of deposition of calcium of ROB cells. In addition, the expression levels of mRNA for ALPase and osteocalcin, which are markers of osteoblastic differentiation, were depressed by the treatment with TBT. TBT inhibited ALPase activity and the deposition of calcium to a greater extent than did DBT. MBT had no effect on the osteoblast differentiation of ROB cells. Tributyltin is known to inhibit the activity of aromatase. However, the aromatase inhibitor aminoglutethimide did not reproduce the inhibitory effects of TBT on osteoblast differentiation. Our findings indicate that TBT might have critical effects on the formation of bone both in vivo and in vitro although its action mechanism is not clarified.

It is possible that the flavonoids that are found in many foods might have a protective effect against osteoclastic activity. However, little information is available about the effects of flavonoids on osteoblastogenesis. Therefore, we investigated the effects of quercetin, a flavonoid, on the metabolism of rat calvarial osteoblast-like cells (ROB cells) in culture. The proliferation of cells was markedly inhibited upon exposure of cells to quercetin at 5 x 10(-6) to 1 x 10(-5) M. Quercetin at 1 x 10(-5) M did not induce apoptosis in ROB cells but arrested cells at the G1 phase of the cell cycle. In addition, quercetin stimulated the expression of mRNA for p21(waf1/cip1), which inhibits the activity of cyclin-dependent kinases, and inhibited the phosphorylation of histone H1. Furthermore, after cells had ceased to proliferate, quercetin reduced the activity of alkaline phosphatase, the level of expression of mRNA for osteocalcin, the rate of deposition of Ca(2+), and the formation of mineralized nodules, all of which are markers of osteoblast differentiation. These findings indicate that quercetin inhibits the proliferation, differentiation, and mineralization of osteoblastic cells.

A method using the support vector machine (SVM) to detect local damages in a building structure with the limited number of sensors is proposed. The SVM is a powerful pattern recognition tool applicable to complicated classification problems. The method is verified to have capability to identify not only the location of damage but also the magnitude of damage with satisfactory accuracy. In our proposed method, feature vectors derived from the modal frequency patterns are used. The feature vectors contain the information on the location and magnitude of damages. As the method does not require modal shapes, typically only two vibration sensors are enough for detecting input and output signals to obtain the modal frequencies. The support vector machines trained for single damage is also effective for detecting damage in multiple stories.

The ability of mammalian central nervous system (CNS) neurons to survive and/or regenerate following injury is influenced by surrounding glial cells. To identify the factors that control glial cell function following CNS injury, we have focused on the endothelin B receptor (ETBR), which we show is expressed by the majority of astrocytes that are immunoreactive for glial acid fibrillary protein (GFAP) in both the normal and crushed rabbit optic nerve. Optic nerve crush induces a marked increase in ETBR and GFAP immunoreactivity (IR) without inducing a significant increase in the number of GFAP-IR astrocytes, suggesting that the crush-induced astrogliosis is due primarily to astrocyte hypertrophy. To define the role that endothelins play in driving this astrogliosis, artificial cerebrospinal fluid (CSF), ET-1 (an ETAR and ETBR agonist), or Bosentan (a mixed ETAR and ETBR antagonist) were infused via osmotic minipumps into noninjured and crushed optic nerves for 14 days. Infusion of ET-1 induced a hypertrophy of ETBR/GFAP-IR astrocytes in the normal optic nerve, with no additional hypertrophy in the crushed nerve, whereas infusion of Bosentan induced a significant decrease in the hypertrophy of ETBR/GFAP-IR astrocytes in the crushed but not in the normal optic nerve. These data suggest that pharmacological blockade of astrocyte ETBR receptors following CNS injury modulates glial scar formation and may provide a more permissive substrate for neuronal survival and regeneration. GLIA 41:180–190, 2003. © 2003 Wiley-Liss, Inc.

Background/aims: Liver regeneration after partial hepatectomy is thought to be regulated by various molecules including the components of the plasminogen activator (PA)-plasmin system. We have examined the role of fibrinolytic factors, i.e., tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), and their substrate, plasminogen, in the proliferation of hepatocytes in primary culture. Methods: Hepatocyte and nonparenchymal liver cells were isolated from Wistar strain rat by a method perfusing the liver with collagenase. DNA synthesis was assessed by measuring the incorporation of [3H]-thymidine into cellular DNA fraction. tPA, uPA and type-1 plasminogen activator inhibitor (PAI-1) gene expressions were measured by Northern blotting. PA activity was measured by fibrin/agarose plate method. Results: Cellular density-dependent DNA synthesis was observed in the primary cultured hepatocytes; DNA synthesis was lower at high cell density (1.0×105 cells/cm2) than that at low cell density (0.2×105 cells/cm2). DNA synthesis in the hepatocytes cultured at a low cell density was increased by co-culture with nonparenchymal liver cells. Under these growth-stimulated culture conditions, tPA and uPA mRNAs were induced and up-regulated. On the contrary, the PAI-1 mRNA level was decreased under these conditions, and total PA activity was augmented accordingly. The synthetic plasmin inhibitor tranexamic acid, a competitive inhibitor for the plasmin molecule, and PASI-535, a plasmin active center-directed inhibitor, both suppressed hepatocyte proliferation in a dose-dependent fashion. Anti-plasmin antibody also suppressed hepatocyte proliferation. Conclusions: The up-regulation of PA activity for ensuring plasmin activity should be an important mechanism in the proliferation of hepatocytes.

We reported previously that C-type natriuretic peptide (CNP) promotes the differentiation and mineralization of osteoblast-like cells. However, little information is available about the mechanism of action of CNP in differentiating osteoblastic cells. In this study, using the technique known as differential display-polymerase chain reaction, we identified a novel cDNA fragment that corresponded to a transcript whose level was increased by CNP in mouse clonal preosteoblastic MC3T3-E1 cells. Northern blotting analysis revealed transcripts of 1.3 kb and 2.3 kb in MC3T3-E 1 cells. Both these transcripts were also expressed at high levels in the heart and stomach. We isolated a full-length cDNA (2,135 bp) from a cDNA library derived from MC3T3-E1 cells using the original cDNA fragment. Analysis of the sequence and of products of transcription and translation in vitro indicated that the transcript of the gene did not include any extensive open reading frames. Enhanced expression, after transfection, of transcript in MC3T3-E1 cells stimulated the deposition of calcium of these cells and the formation of mineralized nodules, but did not affect the activity of alkaline phosphatase. Our results suggest that CNP promotes the expression of a novel transcript, which might stimulate the mineralization of MC3T3-E1 cells.

IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naive mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.

Analysis of the mammalian natriuretic peptide system has established the presence of three types of receptors with distinct structural and functional features and tissue distributions. To clarify the physiological role of each subtype, we studied the natriuretic peptide system in animals with specialized anatomical and physiological features. In this review, following a brief description of the comparative and evolutionary aspects of the ligands, we will analyze the structure and distribution of natriuretic peptide receptors in lower vertebrates, as well as those of rats with essential and salt-sensitive hypertension, and discuss the evolutionary aspects of the natriuretic peptide systems in mammals and fishes. Emphasis is placed on our series of studies with eel receptors that revealed (i) interesting variations in the pattern of intra- and inter-molecular disulfide bonding; (ii) dense chondrocyte localization of NPR-C, which opened a new field of study for natriuretic peptides and bone metabolism; and (iii) the presence of a new receptor subtype, NPR-D, which is abundant in the brain and a member of the receptor subfamily with a short cytoplasmic C-terminal tail.Key words: chloride cell, evolution, natriuretic peptide, osmoregulation, receptor.