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1.  Tumor-Derived Interleukin-1 Promotes Lymphangiogenesis and Lymph Node Metastasis through M2-Type Macrophages 
PLoS ONE  2014;9(6):e99568.
Tumors formed by a highly metastatic human lung cancer cell line are characterized by activated signaling via vascular endothelial growth factor (VEGF)-C through its receptor (VEGFR-3) and aggressive lymph node metastasis. In this study, we examined how these highly metastatic cancers acquired aggressive lymph node metastasis. Compared with their lower metastatic counterparts, the highly metastatic tumors formed by this cell line expressed higher amounts of interleukin (IL)-1α, with similarly augmented expression of IL-1α and IL-1β by tumor stromal cells and of VEGF-A and VEGF-C by tumor-associated macrophages. These tumor-associated macrophages were mainly of the M2 type. Administration of a macrophage-targeting drug suppressed the production of these potent angiogenic and lymphangiogenic factors, resulting in decreased tumor growth, angiogenesis, lymphangiogenesis, and lymph node metastasis. In Matrigel plug assays, the highly metastatic cells formed tumors that were extensively infiltrated by M2-type macrophages and exhibited enhanced angiogenesis and lymphangiogenesis. All of these responses were suppressed by the IL-1 receptor (IL-1R) antagonist anakinra. Thus, the IL-1α-driven inflammatory activation of angiogenesis and lymphangiogenesis seems to provide a highly metastatic tumor microenvironment favorable for lymph node metastasis through cross-talk with macrophages. Accordingly, the IL-1R/M2-type macrophage axis may be a good therapeutic target for patients with this form of lung cancer.
PMCID: PMC4055709  PMID: 24924428
2.  PCNA Mono-ubiquitination and Activation of Translesion DNA Polymerases by DNA Polymerase α 
Journal of biochemistry  2009;146(1):13-21.
Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol α, PCNA was spontaneously mono-ubiquitinated. Pol α L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol α errors, pol ζ participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol δ mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol η) suppressed this defect. These data suggest that nucleotide misincorporation by pol α induces exposure of single-stranded DNA, PCNA mono-ubiquitination, and activates TLS pols.
PMCID: PMC3892766  PMID: 19279190
DNA polymerase; translesion DNA synthesis; mutagensis; PCNA; ubiquitination
3.  Quantitative Proteomic Profiling Identifies DPYSL3 as Pancreatic Ductal Adenocarcinoma-Associated Molecule That Regulates Cell Adhesion and Migration by Stabilization of Focal Adhesion Complex 
PLoS ONE  2013;8(12):e79654.
Elucidation of how pancreatic cancer cells give rise to distant metastasis is urgently needed in order to provide not only a better understanding of the underlying molecular mechanisms, but also to identify novel targets for greatly improved molecular diagnosis and therapeutic intervention. We employed combined proteomic technologies including mass spectrometry and isobaric tags for relative and absolute quantification peptide tagging to analyze protein profiles of surgically resected human pancreatic ductal adenocarcinoma tissues. We identified a protein, dihydropyrimidinase-like 3, as highly expressed in human pancreatic ductal adenocarcinoma tissues as well as pancreatic cancer cell lines. Characterization of the roles of dihydropyrimidinase-like 3 in relation to cancer cell adhesion and migration in vitro, and metastasis in vivo was performed using a series of functional analyses, including those employing multiple reaction monitoring proteomic analysis. Furthermore, dihydropyrimidinase-like 3 was found to interact with Ezrin, which has important roles in cell adhesion, motility, and invasion, while that interaction promoted stabilization of an adhesion complex consisting of Ezrin, c-Src, focal adhesion kinase, and Talin1. We also found that exogenous expression of dihydropyrimidinase-like 3 induced activating phosphorylation of Ezrin and c-Src, leading to up-regulation of the signaling pathway. Taken together, the present results indicate successful application of combined proteomic approaches to identify a novel key player, dihydropyrimidinase-like 3, in pancreatic ductal adenocarcinoma tumorigenesis, which may serve as an important biomarker and/or drug target to improve therapeutic strategies.
PMCID: PMC3855176  PMID: 24339867
4.  SGOL1 variant B induces abnormal mitosis and resistance to taxane in non-small cell lung cancers 
Scientific Reports  2013;3:3012.
Mitosis is the most conspicuous cell cycle phase and Shugoshin-like 1 (SGOL1) is a key protein in protecting sister chromatids from precocious separation during mitosis. We studied the role of SGOL1 and its splice variants in non-small cell lung cancer (NSCLC) using 82 frozen NSCLC tissue samples. SGOL1-B expression was prevalent in smokers, in cases with a wild-type (WT) EGFR status, and in cases with the focal copy number amplification of genes that are known to be important for defining the biological behaviors of NSCLC. The overexpression of SGOL1-B1 in an NSCLC cell line induced aberrant chromosome missegregation, precociously separated chromatids, and delayed mitotic progression. A higher level of SGOL1-B mRNA was related to taxane resistance, while the forced downregulation of SGOL1-B increased the sensitivity to taxane. These results suggest that the expression of SGOL1-B causes abnormal mitosis and taxane resistance in NSCLC cells.
PMCID: PMC3804856  PMID: 24146025
5.  Inhibitory Effects of Salinomycin on Cell Survival, Colony Growth, Migration, and Invasion of Human Non-Small Cell Lung Cancer A549 and LNM35: Involvement of NAG-1 
PLoS ONE  2013;8(6):e66931.
A major challenge for oncologists and pharmacologists is to develop more potent and less toxic drugs that will decrease the tumor growth and improve the survival of lung cancer patients. Salinomycin is a polyether antibiotic used to kill gram-positive bacteria including mycobacteria, protozoans such as plasmodium falciparum, and the parasites responsible for the poultry disease coccidiosis. This old agent is now a serious anti-cancer drug candidate that selectively inhibits the growth of cancer stem cells. We investigated the impact of salinomycin on survival, colony growth, migration and invasion of the differentiated human non-small cell lung cancer lines LNM35 and A549. Salinomycin caused concentration- and time-dependent reduction in viability of LNM35 and A549 cells through a caspase 3/7-associated cell death pathway. Similarly, salinomycin (2.5–5 µM for 7 days) significantly decreased the growth of LNM35 and A549 colonies in soft agar. Metastasis is the main cause of death related to lung cancer. In this context, salinomycin induced a time- and concentration-dependent inhibition of cell migration and invasion. We also demonstrated for the first time that salinomycin induced a marked increase in the expression of the pro-apoptotic protein NAG-1 leading to the inhibition of lung cancer cell invasion but not cell survival. These findings identify salinomycin as a promising novel therapeutic agent for lung cancer.
PMCID: PMC3689654  PMID: 23805285
6.  Met Is the Most Frequently Amplified Gene in Endometriosis-Associated Ovarian Clear Cell Adenocarcinoma and Correlates with Worsened Prognosis 
PLoS ONE  2013;8(3):e57724.
Clear cell adenocarcinoma of the ovary (OCC) is a chemo-resistant tumor with a relatively poor prognosis and is frequently associated with endometriosis. Although it is assumed that oxidative stress plays some role in the malignant transformation of this tumor, the characteristic molecular events leading to carcinogenesis remain unknown. In this study, an array-based comparative genomic hybridization (CGH) analysis revealed Met gene amplification in 4/13 OCC primary tumors and 2/8 OCC cell lines. Amplification of the AKT2 gene, which is a downstream component of the Met/PI3K signaling pathway, was also observed in 5/21 samples by array-based CGH analysis. In one patient, both the Met and AKT2 genes were amplified. These findings were confirmed using fluorescence in situ hybridization, real-time quantitative PCR, immunoblotting, and immunohistochemistry. In total, 73 OCC cases were evaluated using real-time quantitative PCR; 37.0% demonstrated Met gene amplification (>4 copies), and 8.2% had AKT2 amplification. Furthermore, stage 1 and 2 patients with Met gene amplification had significantly worse survival than patients without Met gene amplification (p<0.05). Met knockdown by shRNA resulted in reduced viability of OCC cells with Met amplification due to increased apoptosis and cellular senescence, suggesting that the Met signaling pathway plays an important role in OCC carcinogenesis. Thus, we believe that targeted inhibition of the Met pathway may be a promising treatment for OCC.
PMCID: PMC3587638  PMID: 23469222
7.  Frondoside A Suppressive Effects on Lung Cancer Survival, Tumor Growth, Angiogenesis, Invasion, and Metastasis 
PLoS ONE  2013;8(1):e53087.
A major challenge for oncologists and pharmacologists is to develop less toxic drugs that will improve the survival of lung cancer patients. Frondoside A is a triterpenoid glycoside isolated from the sea cucumber, Cucumaria frondosa and was shown to be a highly safe compound. We investigated the impact of Frondoside A on survival, migration and invasion in vitro, and on tumor growth, metastasis and angiogenesis in vivo alone and in combination with cisplatin. Frondoside A caused concentration-dependent reduction in viability of LNM35, A549, NCI-H460-Luc2, MDA-MB-435, MCF-7, and HepG2 over 24 hours through a caspase 3/7-dependent cell death pathway. The IC50 concentrations (producing half-maximal inhibition) at 24 h were between 1.7 and 2.5 µM of Frondoside A. In addition, Frondoside A induced a time- and concentration-dependent inhibition of cell migration, invasion and angiogenesis in vitro. Frondoside A (0.01 and 1 mg/kg/day i.p. for 25 days) significantly decreased the growth, the angiogenesis and lymph node metastasis of LNM35 tumor xenografts in athymic mice, without obvious toxic side-effects. Frondoside A (0.1–0.5 µM) also significantly prevented basal and bFGF induced angiogenesis in the CAM angiogenesis assay. Moreover, Frondoside A enhanced the inhibition of lung tumor growth induced by the chemotherapeutic agent cisplatin. These findings identify Frondoside A as a promising novel therapeutic agent for lung cancer.
PMCID: PMC3540099  PMID: 23308143
8.  Fenton Reaction Induced Cancer in Wild Type Rats Recapitulates Genomic Alterations Observed in Human Cancer 
PLoS ONE  2012;7(8):e43403.
Iron overload has been associated with carcinogenesis in humans. Intraperitoneal administration of ferric nitrilotriacetate initiates a Fenton reaction in renal proximal tubules of rodents that ultimately leads to a high incidence of renal cell carcinoma (RCC) after repeated treatments. We performed high-resolution microarray comparative genomic hybridization to identify characteristics in the genomic profiles of this oxidative stress-induced rat RCCs. The results revealed extensive large-scale genomic alterations with a preference for deletions. Deletions and amplifications were numerous and sometimes fragmented, demonstrating that a Fenton reaction is a cause of such genomic alterations in vivo. Frequency plotting indicated that two of the most commonly altered loci corresponded to a Cdkn2a/2b deletion and a Met amplification. Tumor sizes were proportionally associated with Met expression and/or amplification, and clustering analysis confirmed our results. Furthermore, we developed a procedure to compare whole genomic patterns of the copy number alterations among different species based on chromosomal syntenic relationship. Patterns of the rat RCCs showed the strongest similarity to the human RCCs among five types of human cancers, followed by human malignant mesothelioma, an iron overload-associated cancer. Therefore, an iron-dependent Fenton chemical reaction causes large-scale genomic alterations during carcinogenesis, which may result in distinct genomic profiles. Based on the characteristics of extensive genome alterations in human cancer, our results suggest that this chemical reaction may play a major role during human carcinogenesis.
PMCID: PMC3430702  PMID: 22952676
9.  The Epstein-Barr Virus Latent Membrane Protein 1 and Transforming Growth Factor–β1 Synergistically Induce Epithelial–Mesenchymal Transition in Lung Epithelial Cells 
The histopathology of idiopathic pulmonary fibrosis (IPF) includes the presence of myofibroblasts within so-called fibroblastic foci, and studies suggest that lung myofibroblasts may be derived from epithelial cells through epithelial–mesenchymal transition (EMT). Transforming growth factor (TGF)–β1 is expressed and/or activated in fibrogenesis, and induces EMT in lung epithelial cells in a dose-dependent manner. A higher occurrence of Epstein-Barr virus (EBV) has been reported in the lung tissue of patients with IPF. EBV expresses latent membrane protein (LMP) 1 during the latent phase of infection, and may play a role in the pathogenesis of pulmonary fibrosis inasmuch as LMP-1 may act as a constitutively active TNF-α receptor. Our data show a remarkable increase in mesenchymal cell markers, along with a concurrent reduction in the expression of epithelial cell markers in lung epithelial cells cotreated with LMP-1, and very low doses of TGF-β1. This effect was mirrored in lung epithelial cells infected with EBV expressing LMP1 and cotreated with TGF-β1. LMP1 pro-EMT signaling was identified, and occurs primarily through the nuclear factor–κB pathway and secondarily through the extracellular signal–regulated kinase (ERK) pathway. Activation of the ERK pathway was shown to be critical for aspects of TGF-β1–induced EMT. LMP1 accentuates the TGF-β1 activation of ERK. Together, these data demonstrate that the presence of EBV-LMP1 in lung epithelial cells synergizes with TGF-β1 to induce EMT. Our in vitro data may help to explain the observation that patients with IPF demonstrating positive staining for LMP1 in lung epithelial cells have a more rapid demise than patients in whom LMP1 is not detected.
PMCID: PMC3135845  PMID: 20693406
latent membrane protein 1; transforming growth factor–β1; epithelial–mesenchymal transition; idiopathic pulmonary fibrosis; Epstein-Barr virus
10.  Seven-Signal Proteomic Signature for Detection of Operable Pancreatic Ductal Adenocarcinoma and Their Discrimination from Autoimmune Pancreatitis 
There is urgent need for biomarkers that provide early detection of pancreatic ductal adenocarcinoma (PDAC) as well as discrimination of autoimmune pancreatitis, as current clinical approaches are not suitably accurate for precise diagnosis. We used mass spectrometry to analyze protein profiles of more than 300 plasma specimens obtained from PDAC, noncancerous pancreatic diseases including autoimmune pancreatitis patients and healthy subjects. We obtained 1063 proteomic signals from 160 plasma samples in the training cohort. A proteomic signature consisting of 7 mass spectrometry signals was used for construction of a proteomic model for detection of PDAC patients. Using the test cohort, we confirmed that this proteomic model had discrimination power equal to that observed with the training cohort. The overall sensitivity and specificity for detection of cancer patients were 82.6% and 90.9%, respectively. Notably, 62.5% of the stage I and II cases were detected by our proteomic model. We also found that 100% of autoimmune pancreatitis patients were correctly assigned as noncancerous individuals. In the present paper, we developed a proteomic model that was shown able to detect early-stage PDAC patients. In addition, our model appeared capable of discriminating patients with autoimmune pancreatitis from those with PDAC.
PMCID: PMC3361197  PMID: 22675630
11.  Comprehensive predictions of target proteins based on protein-chemical interaction using virtual screening and experimental verifications 
BMC Chemical Biology  2012;12:2.
Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis.
We applied our protocol of predicting target proteins combining in silico screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by in vitro binding of incednine to seven proteins, whose expression can be confirmed in our cell system.
As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins.
This study revealed that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.
PMCID: PMC3471015  PMID: 22480302
12.  A Novel Network Profiling Analysis Reveals System Changes in Epithelial-Mesenchymal Transition 
PLoS ONE  2011;6(6):e20804.
Patient-specific analysis of molecular networks is a promising strategy for making individual risk predictions and treatment decisions in cancer therapy. Although systems biology allows the gene network of a cell to be reconstructed from clinical gene expression data, traditional methods, such as Bayesian networks, only provide an averaged network for all samples. Therefore, these methods cannot reveal patient-specific differences in molecular networks during cancer progression. In this study, we developed a novel statistical method called NetworkProfiler, which infers patient-specific gene regulatory networks for a specific clinical characteristic, such as cancer progression, from gene expression data of cancer patients. We applied NetworkProfiler to microarray gene expression data from 762 cancer cell lines and extracted the system changes that were related to the epithelial-mesenchymal transition (EMT). Out of 1732 possible regulators of E-cadherin, a cell adhesion molecule that modulates the EMT, NetworkProfiler, identified 25 candidate regulators, of which about half have been experimentally verified in the literature. In addition, we used NetworkProfiler to predict EMT-dependent master regulators that enhanced cell adhesion, migration, invasion, and metastasis. In order to further evaluate the performance of NetworkProfiler, we selected Krueppel-like factor 5 (KLF5) from a list of the remaining candidate regulators of E-cadherin and conducted in vitro validation experiments. As a result, we found that knockdown of KLF5 by siRNA significantly decreased E-cadherin expression and induced morphological changes characteristic of EMT. In addition, in vitro experiments of a novel candidate EMT-related microRNA, miR-100, confirmed the involvement of miR-100 in several EMT-related aspects, which was consistent with the predictions obtained by NetworkProfiler.
PMCID: PMC3110206  PMID: 21687740
13.  Pandemic (H1N1) 2009–associated Pneumonia in Children, Japan 
Emerging Infectious Diseases  2011;17(2):279-282.
To describe clinical aspects of pandemic (H1N1) 2009 virus–associated pneumonia in children, we studied 80 such children, including 17 (21%) with complications, who were admitted to 5 hospitals in Japan during August–November 2009 after a mean of 2.9 symptomatic days. All enrolled patients recovered (median hospitalization 6 days). Timely access to hospitals may have contributed to favorable outcomes.
PMCID: PMC3204745  PMID: 21291606
Pandemic (H1N1) 2009 virus; viruses; influenza; pediatric inpatients; pneumonia; children; complicating illnesses; Japan; dispatch
14.  Clinically Relevant Characterization of Lung Adenocarcinoma Subtypes Based on Cellular Pathways: An International Validation Study 
PLoS ONE  2010;5(7):e11712.
Lung adenocarcinoma (AD) represents a predominant type of lung cancer demonstrating significant morphologic and molecular heterogeneity. We sought to understand this heterogeneity by utilizing gene expression analyses of 432 AD samples and examining associations between 27 known cancer-related pathways and the AD subtype, clinical characteristics and patient survival. Unsupervised clustering of AD and gene expression enrichment analysis reveals that cell proliferation is the most important pathway separating tumors into subgroups. Further, AD with increased cell proliferation demonstrate significantly poorer outcome and an increased solid AD subtype component. Additionally, we find that tumors with any solid component have decreased survival as compared to tumors without a solid component. These results lead to the potential to use a relatively simple pathological examination of a tumor in order to determine its aggressiveness and the patient's prognosis. Additional results suggest the ability to use a similar approach to determine a patient's sensitivity to targeted treatment. We then demonstrated the consistency of these findings using two independent AD cohorts from Asia (N = 87) and Europe (N = 89) using the identical analytic procedures.
PMCID: PMC2908611  PMID: 20661423
15.  Bronchial Casts and Pandemic (H1N1) 2009 Virus Infection 
Emerging Infectious Diseases  2010;16(2):344-346.
PMCID: PMC2958032  PMID: 20113579
Bronchial casts; plastic bronchitis; atelectasis; influenza; H1N1; pandemic; virus; pediatric patients; expedite; letter
16.  Traditional Chinese Medicine for Chronic Fatigue Syndrome 
More and more patients have been diagnosed as having chronic fatigue syndrome (CFS) in recent years. Western drug use for this syndrome is often associated with many side-effects and little clinical benefit. As an alternative medicine, traditional Chinese medicine (TCM) has provided some evidences based upon ancient texts and recent studies, not only to offer clinical benefit but also offer insights into their mechanisms of action. It has perceived advantages such as being natural, effective and safe to ameliorate symptoms of CFS such as fatigue, disordered sleep, cognitive handicaps and other complex complaints, although there are some limitations regarding the diagnostic standards and methodology in related clinical or experimental studies. Modern mechanisms of TCM on CFS mainly focus on adjusting immune dysfunction, regulating abnormal activity in the hypothalamic-pituitary-adrenal (HPA) axis and serving as an antioxidant. It is vitally important for the further development to establish standards for ‘zheng’ of CFS, i.e. the different types of CFS pathogenesis in TCM, to perform randomized and controlled trials of TCM on CFS and to make full use of the latest biological, biochemical, molecular and immunological approaches in the experimental design.
PMCID: PMC2816380  PMID: 18955323
chronic fatigue syndrome; herbal therapy; traditional Chinese medicine
17.  Mao-to Prolongs the Survival of and Reduces TNF-α Expression in Mice with Viral Myocarditis 
Goal of this study was to evaluate effects of Mao-to on development of myocarditis induced by encephalomyocarditis (EMC) virus in mice. Mice were randomly divided into five groups. Group N included uninfected controls (n = 18), while group A, B and C underwent intraperitoneal injection of EMC virus. Group A was administered oral saline from day 0 to day 4. Group B was administered oral Mao-to (500 mg−1 kg−1 day−1) from day 0 to day 4. Group C was administered Mao-to from day 2 to day 6. Group D was administered Mao-to from day 5 to day 10. Treated mice were followed for survival rates during 2 weeks after infection. Body weight (BW) and organ weights including heart (HW), lungs, thymus and spleen were examined on days 4, 6 and 14. Survival rate of group C (36.4%) was significantly improved compared with group A, B or D (0% of each, P < 0.05). HW and HW/BW ratio in group C was significantly (P < 0.05) lower than those in group A, B or D. Viral titers of hearts were significantly different among groups A, B and C. Cardiac expression in tumor necrosis factor-α (TNF-α) was significantly reduced in group C in comparison with group A, B or D on day 6 by immunohistochemical study. Administration of Mao-to starting on day 2 improves mortality resulting from viral myocarditis in mice with reduced expression of cardiac TNF-α. These findings suggest that timing of Mao-to is crucial for preventing cardiac damage in mice with viral myocarditis.
PMCID: PMC2887330  PMID: 20671770
Mao-to; viral myocarditis; tumor necrosis factor-α
18.  Acupuncture Regulates Leukocyte Subpopulations in Human Peripheral Blood 
Acupuncture has recently been attracting more and more people throughout the world as an alternative treatment, however little is known about its physiological activities (i.e. immune system). We examined acupuncture both quantitatively and qualitatively by measuring CD-positive cell counts and cytokine expression levels in the blood, to determine the activity of T cells, B cells, macrophages and natural killer (NK) cells. Fifteen milliliters of peripheral blood obtained from 17 healthy volunteers aged 21–51 years, were analyzed using flow cytometry before and after acupuncture treatment. There was a statistically significant increase in the number of CD2+, CD4+, CD8+, CD11b+, CD16+, CD19+, CD56+ cells as well as IL-4, IL-1β and IFN-γ levels in the cells after acupuncture stimulation of meridian points. These observations indicate that acupuncture may regulate the immune system and promote the activities of humoral and cellular immunity as well as NK cell activity. In this article, we discussed how acupuncture regulated leukocyte numbers and functions since they are considered to be potential indicators for evaluating complementary and alternative medicine.
PMCID: PMC2176150  PMID: 18227912
acupuncture; CD positive cells; cytokine expression; Eastern medicine; leukocyte subset
19.  A Kampo Medicine, Boi-ogi-to, Inhibits Obesity in Ovariectomized Rats 
In women facing menopause, end of menstrual activity is accompanied by lower levels of estrogen and gradual weight gain. Postmenopausal weight gain sounds an alarm for women's health and may lead to hyperlipidemia, a lipid increase and glucose intolerance. These phenomena are connected to lifestyle-related diseases such as hypertension, type II diabetes mellitus, arteriosclerosis and metabolic syndrome, making it essential to prevent weight gain in women. A Kampo medicine, Boi-ogi-to, is traditionally used to treat obese conditions, but the mechanism has not yet been investigated. In this experiment, we tested the antiobesity properties of Boi-ogi-to in ovariectomized rats by measuring changes of serum cytokine levels and adipocytokines in fat cells. After treatment with this extract for 6 weeks (20-week-old rats), we found that there was a significant weight decrease in rats treated with Boi-ogi-to as compared with that in the control group. Serum tumor necrosis factor (TNF)-α levels increased significantly in a dose-dependent manner. Gene expression of adipose tissue in uterus also dose dependently showed a significant increase of TNF-α levels, suggesting that secretion of TNF-α by fat cells might play a role in the ability of Boi-ogi-to to inhibit weight gain. While peroxisome proliferators-activated receptor-γ and adiponectin levels did not show a significant difference as compared with those in the control, levels of mRNA expression showed a tendency to increase dose dependently. Resistin did not show any significant change. These results suggest that Boi-ogi-to might be useful for the prevention of obesity that occurs in women with reduction of estrogen.
PMCID: PMC2816381  PMID: 18955282
Adipocytokines; Boi-ogi-to; obesity; ovariectomized rats
20.  hDREF Regulates Cell Proliferation and Expression of Ribosomal Protein Genes▿  
Molecular and Cellular Biology  2007;27(6):2003-2013.
Although ribosomal proteins (RPs) are essential cellular constituents in all living organisms, mechanisms underlying regulation of their gene expression in mammals remain unclear. We have established that 22 out of 79 human RP genes contain sequences similar to the human DREF (DNA replication-related element-binding factor; hDREF) binding sequence (hDRE) within 200-bp regions upstream of their transcriptional start sites. Electrophoretic gel mobility shift assays and chromatin immunoprecipitation analysis indicated that hDREF binds to hDRE-like sequences in the RP genes both in vitro and in vivo. In addition, transient luciferase assays revealed that hDRE-like sequences act as positive elements for RP gene transcription and cotransfection of an hDREF-expressing plasmid was found to stimulate RP gene promoter activity. Like that of hDREF, expression of RP genes is increased during the late G1 to S phases, and depletion of hDREF using short hairpin RNA-mediated knockdown decreased RP gene expression and cell proliferation in normal human fibroblasts. Knockdown of the RPS6 gene also resulted in impairment of cell proliferation. These data suggest that hDREF is an important transcription factor for cell proliferation which plays roles in cell cycle-dependent regulation of a number of RP genes.
PMCID: PMC1820502  PMID: 17220279
21.  Expression profiling of genes regulated by TGF-beta: Differential regulation in normal and tumour cells 
BMC Genomics  2007;8:98.
TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations. Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells.
Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated by TGF-beta in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells. In addition, 267 genes are commonly regulated in both the cell-lines. Semi-quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data. In order to identify the signalling pathways that influence TGF-beta mediated gene regulation, we used specific inhibitors of p38 MAP kinase, ERK kinase, JNK kinase and integrin signalling pathways. The data suggest that regulation of majority of the selected genes is dependent on at least one of these pathways and this dependence is cell-type specific. Interestingly, an integrin pathway inhibitor, RGD peptide, significantly affected TGF-beta regulation of Thrombospondin 1 in A549 cells.
These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells and significant role of non-canonical TGF-beta pathways in the regulation of many genes by TGF-beta.
PMCID: PMC1858692  PMID: 17425807
22.  Beneficial Effect of Brewers' Yeast Extract on Daily Activity in a Murine Model of Chronic Fatigue Syndrome 
The aim of this study was to assess the effect of Brewers' yeast extract (BYE) on daily activity in a mouse model of chronic fatigue syndrome (CFS). CFS was induced by repeated injection of Brucella abortus (BA) antigen every 2 weeks. BYE was orally administered to mice in a dose of 2 g per kg per day for 2 weeks before injecting BA and for 4 weeks thereafter. We evaluated daily running activity in mice receiving BYE as compared with that in untreated mice. Weekly variation of body weight (BW) and survival in both groups was monitored during the observation period. Spleen weight (SW), SW/BW ratio, percent splenic follicular area and expression levels of interferon-γ (IFN-γ) and interleukin-10 (IL-10) mRNA in spleen were determined in both groups at the time of sacrifice. The daily activity during 2 weeks after the second BA injection was significantly higher in the treated group than in the control. There was no difference in BW between both groups through the experimental course. Two mice in the control died 2 and 7 days after the second injection, whereas no mice in the treated group died. Significantly decreased SW and SW/BW ratio were observed in the treated mice together with elevation of splenic follicular area. There were suppressed IFN-γ and IL-10 mRNA levels in spleens from the treated mice. Our results suggest that BYE might have a protective effect on the marked reduction in activity following repeated BA injection via normalization of host immune responses.
PMCID: PMC1375235  PMID: 16550231
Brewers' yeast extract; chronic fatigue syndrome; daily activity; spleen
23.  Effect of Hochu-ekki-to (TJ-41), a Japanese Herbal Medicine, on Daily Activity in a Murine Model of Chronic Fatigue Syndrome 
We aimed to evaluate the effect of a Japanese herbal medicine, Hochu-ekki-to (TJ-41), on daily activity in a murine model of chronic fatigue syndrome (CFS). CFS was induced by repeated injection of Brucella abortus (BA) antigen every 2 weeks. TJ-41 was orally administered to mice in a dose of 500 mg/kg/day for 1 week before injecting BA and for 4 weeks thereafter. We evaluated daily running activity in mice receiving TJ-41 as compared with that in untreated mice. Survival of both mouse groups was also monitored during the observation period. Body weight (BW), spleen weight (SW), SW/ BW ratio and expression levels of interleukin-10 (IL-10) mRNA in spleen were determined in both groups at the time of sacrifice. The daily activity was significantly higher in the treated group than in the control. Two mice in the untreated group died 2 days after the second injection of BA, whereas no mice in the group treated with TJ-41 died. The SW and SW/BW ratio were significantly lower in the treated mice than in the control. Suppressed IL-10 mRNA levels were observed in the spleens of the mice treated with TJ-41. Our data suggest that Hochu-ekki-to might possess an inhibitory effect on the marked decrease in running activity following BA injection.
PMCID: PMC516453  PMID: 15480446
Hochu-ekki-to; herbal medicine; daily activity; chronic fatigue syndrome
24.  Molecular Analysis of Human Herpesvirus 8 by Using Single Nucleotide Polymorphisms in Open Reading Frame 26 
Journal of Clinical Microbiology  2003;41(6):2492-2497.
Human herpesvirus 8 (HHV-8) can be classified into distinct subtypes on the basis of sequence polymorphisms in several open reading frames (ORFs). We analyzed the subtypes of HHV-8 in 59 human immunodeficiency virus-infected Japanese patients by using polymorphisms in ORF26 and found that over two-thirds of the HHV-8 isolates fell into major subtype A. We also found that single nucleotide polymorphisms (SNPs) at nucleotide positions 1032 (C-to-A substitution) and 1055 (G-to-T substitution) in HHV-8 ORF26 were correlated with increased susceptibility to Kaposi's sarcoma, compared to the results obtained with HHV-8 with wild-type nucleotides at these positions (P = 0.0106). This observation suggests that molecular heterogeneity of the HHV-8 genome affects the biological properties of HHV-8, resulting in different clinical phenotypes of HHV-8 infection. Since sensitive PCR of ORF26 allowed us to analyze the SNPs by using peripheral blood from HHV-8-infected patients, the ORF26 SNPs will be a potent tool for investigating the pathogenesis of HHV-8 infection.
PMCID: PMC156527  PMID: 12791871
25.  Relationship between Mutations in Dihydropteroate Synthase of Pneumocystis carinii f. sp. hominis Isolates in Japan and Resistance to Sulfonamide Therapy 
Journal of Clinical Microbiology  2000;38(9):3161-3164.
We examined mutations in the dihydropteroate synthase (DHPS) genes of Pneumocystis carinii f. sp. hominis (P. carinii) strains isolated from 24 patients with P. carinii pneumonia (PCP) in Japan. DHPS mutations were identified at amino acid positions 55 and/or 57 in isolates from 6 (25.0%) of 24 patients. The underlying diseases for these six patients were human immunodeficiency virus type 1 infection (n = 4) or malignant lymphoma (n = 2). This frequency was almost the same as those reported in Denmark and the United States. None of the six patients whose isolates had DHPS mutations were recently exposed to sulfa drugs before they developed the current episode of PCP, suggesting that DHPS mutations not only are selected by the pressure of sulfa agents but may be incidentally acquired. Co-trimoxazole treatment failed more frequently in patients whose isolates had DHPS mutations than in those whose isolates had wild-type DHPS (n = 4 [100%] versus n = 2 [11.1%]; P = 0.002). Our results thus suggest that DHPS mutations may contribute to failures of co-trimoxazole treatment for PCP.
PMCID: PMC87344  PMID: 10970350

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