Kyoichi Kishi, Arinobu Yamauchi, Fujiko Shizuka, Yasuhiro Kido, Takeshi Nikawa and Kazuhito Rokutan : Serotonin in the Central Nervous System and Periphery, --- Effect of macronutrient intakes on brain serotonin metabolite as measured by in vivo microdialysis in the rat ---, Elsevier, Tokyo, Apr. 1995.
学術論文(審査論文):
1.
Tatsuya Nishikawa, Yuki Kuwano, Mayu Nakata, Kazuhito Rokutan and Kensei Nishida : Multiple G-quadruplexes in the LMNA promoter regulate LMNA variant 6 transcription and promote colon cancer cell growth., Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Vol.1864, No.10, 2021.
(要約)
Lamin A/C proteins, major components of the nuclear lamina, are encoded by the LMNA gene. These proteins have multiple cellular functions, including DNA transcription and replication, chromatin organization, regulation of the cell cycle, and apoptosis. Mutations in LMNA are associated with a variety of diseases called laminopathies. LMNA has implications in cancer; however, its mechanisms of dysregulation in cancer cells are not yet fully understood. In this study, among the LMNA transcript variants, we focused on a transcriptional variant 6 (termed LMNA-V6), which contains unique 3 exons upstream of exon 1 of LMNA. The promoter region of LMNA-V6 formed multiple G-quadruplexes and increased its transcriptional activity. Moreover, LMNA-V6 negatively regulated other LMNA mRNA variants, lamin A and lamin C, via direct interaction with their promoter. Knockdown of LMNA-V6 decreased the proliferation of colon cancer cells, whereas overexpression of the unique 3 exons of LMNA-V6 increased cell growth. Furthermore, microarray gene expression profiling showed that alteration of LMNA-V6 levels influenced the expression of p53 in colon cancer cells. Taken together, the results suggest that LMNA-V6 may be a novel functional RNA whose expression is regulated through multiple G-quadruplexes in colon cancer cells.
Kensei Nishida, Yuki Kuwano and Kazuhito Rokutan : The MicroRNA-23b/27b/24 Cluster Facilitates Colon Cancer Cell Migration by Targeting FOXP2, Cancers, Vol.12, No.1, 174, 2020.
(要約)
Acquisition of cell migration capacity is an early and essential process in cancer development. The aim of this study was to identify microRNA gene expression networks that induced high migration capacity. Using colon cancer HCT116 cells subcloned by transwell-based migrated cell selection, microRNA array analysis was performed to examine the microRNA expression profile. Promoter activity and microRNA targets were assessed with luciferase reporters. Cell migration capacity was assessed by either the transwell or scratch assay. In isolated subpopulations with high migration capacity, the expression levels of the cluster increased in accordance with the increased expression of the short transcript, a host gene of the cluster. E2F1-binding sequences were involved in the basic transcription activity of the short expression, and E2F1-small-interfering (si)RNA treatment reduced the expression of both the and clusters. Overexpression experiments showed that and promoted cell migration, but the opposite effect was observed with . Forkhead box P2 (FOXP2) mRNA and protein levels were reduced by both/either and . Furthermore, FOXP2 siRNA treatment significantly promoted cell migration. Our findings demonstrated a novel role of the cluster in cell migration through targeting FOXP2, with potential implications for the development of microRNA-based therapy targeted at inhibiting cancer migration.
Kensei Nishida, Sawada Daisuke, Yuki Kuwano, Hiroki Tanaka and Kazuhito Rokutan : Health Benefits of Lactobacillus gasseri CP2305 Tablets in Young Adults Exposed to Chronic Stress: A Randomized, Double-Blind, Placebo-Controlled Study, Nutrients, Vol.11, No.8, 1859, 2019.
(要約)
Short-term administration of CP2305 improves stress-associated symptoms and clinical symptoms in healthy young adults and in patients with irritable bowel syndrome, respectively. We evaluated the efficacy and health benefits of the long-term use of a tablet containing heat-inactivated, washed CP2305 (CP2305) in healthy young adults. Sixty Japanese medical students (41 men and 19 women) preparing for the national examination for medical practitioners ingested CP2305-containing or placebo tablets once daily for 24 weeks. Intake of the CP2305 tablet significantly reduced anxiety and sleep disturbance relative to placebo, as quantitated by the Spielberger State-Trait Anxiety Inventory and the Pittsburgh Sleep Quality Index. Single-channel sleep electroencephalograms show that CP2305 significantly shortened sleep latency and wake time after sleep onset and increased the delta power ratio in the first sleep cycle. CP2305 also significantly lowered salivary chromogranin A levels compared with placebo. Furthermore, 16S rRNA gene sequencing of participant feces demonstrated that CP2305 administration attenuated the stress-induced decline of spp. and the stress-induced elevation of spp. We conclude that the long-term use of CP2305-containing tablets may improve the mental state, sleep quality, and gut microbiota of healthy adults under stressful conditions.
Tatsuya Nishikawa, Yuki Kuwano, Yumiko Takahara, Kensei Nishida and Kazuhito Rokutan : HnRNPA1 interacts with G-quadruplex in the TRA2B promoter and stimulates its transcription in human colon cancer cells., Scientific Reports, Vol.9, No.1, 2019.
(要約)
The human TRA2B gene consists of 10 exons and 9 introns and produces 5 splice isoforms (TRA2β1 to TRA2β5). TRA2B exon 2 encodes multiple premature termination codons. TRA2β1 lacks exon 2 and is translated into a functional transformer 2β (Tra2β) protein, whereas TRA2β4 contains 10 exons and works as a functional RNA. Overexpressed Tra2β and ectopic expression of TRA2β4 may be oncogenic. We found that heterogeneous nuclear ribonucleoprotein (hnRNP)A1 and hnRNPU interacted with TRA2β4 exon 2. Minigene assays revealed that hnRNPA1 facilitated inclusion of exon 2, whereas hnRNPU promoted its skipping. However, knockdown of hnRNPA1 or hnRNPU reduced both TRA2β1 and TRA2β4 levels, and overexpression of these hnRNPs increased levels of both isoforms, suggesting that hnRNPA1 and hnRNPU mainly regulate the transcription of TRA2B. In fact, hnRNPA1 and hnRNPU positively regulated the promoter activity of TRA2B. Circular dichroism analyses, electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated the presence of G-quadruplex (G4) formation in the promoter of TRA2B. Formation of G4 suppressed TRA2B transcription, whereas hnRNPA1, but not hnRNPU, interacted with the G4 to facilitate transcription. Our results suggest that hnRNPA1 may modulate TRA2B transcription through its regulation of G4 formation in its promoter in colon cancer cells.
Saki Saijo, Yuki Kuwano, Shoichiro Tange, Kazuhito Rokutan and Kensei Nishida : A novel long non-coding RNA from the HOXA6-HOXA5 locus facilitates colon cancer cell growth., BMC Cancer, Vol.19, No.1, 2019.
(要約)
Our results suggested that HOXA5 short RNA, a novel lncRNA, may play a crucial role in colon tumor growth through activation of EGF signaling.
Diversification of transcriptomic and epigenomic states may occur during the expansion of colorectal cancers. Certain cancer cells lose their epithelial characters and gain mesenchymal properties, known as epithelial-mesenchymal transition (EMT), and they aggressively migrate into the non-tumorigenic extracellular matrix. In this study, we isolated a subpopulation with accelerated baseline motility (MG cells) and an immotile one (non-MG cells) from a colon cancer cell line (HCT116). Gene expression signatures of the MG cells indicated that this subpopulation was likely an EMT hybrid. The MG cells substantially lost their migratory properties after treatment with a methyltransferase inhibitor, 5-azacytidine, suggesting a role of DNA methylation in this process. Global transcriptome assays of both types of cells with or without 5-azacytidine treatment identified 640 genes, whose expression might be methylation-dependently down-regulated in the MG cells. Global methylation analysis revealed that 35 out of the 640 genes were hyper-methylated in the MG cells. Among them, we focused on the anti-oncogene ZNF350, which encodes a zinc-finger and BRCA1-interacting protein. Notably, ZNF350 knockdown accelerated migration of the non-MG cells, while overexpression of ZNF350 in the MG cells significantly impaired their migration. Finally, pyrosequence analysis together with dual luciferase assays of serially truncated fragments of the ZNF350 promoter (-268 to +49 bp) indicated that three hyper-methylated sites were possibly responsible for the basal promoter activity of ZNF350. Taken together, our results suggest that hyper-methylation of the ZNF350 proximal promoter may be one of the crucial determinants for acquiring increased migratory capabilities in colon cancer cells.
Satake Yuzuru, Yuki Kuwano, Tatsuya Nishikawa, Fujita Kinuyo, Saijo Saki, Miki Itai, Tanaka Hiroki, Kensei Nishida and Kazuhito Rokutan : Nucleolin facilitates nuclear retention of an ultraconserved region containing TRA24 and accelerates colon cancer cell growth., Oncotarget, Vol.9, No.42, 26817-26833, 2018.
(要約)
Transcribed-ultraconserved regions (T-UCRs), which contain conserved sequences with 100% identity across human, rat and mouse species, are a novel category of functional RNAs. The human gene () encodes a UCR that spans exon 2 (276 bp) and its neighboring introns. Among five spliced RNA variants () transcribed from the gene, only contains the conserved exon 2. is overexpressed in colon cancer cells and accelerates cell growth by blocking the transcription of . However, the mechanisms underlying the overexpression of in colon cancer cells are unknown. Using biotinylated RNA pull-down assays followed by liquid chromatography-mass spectrometric analysis, we identified nucleolin as a -binding protein. Knockdown of nucleolin reduced the nuclear retention of and accelerated its degradation in the cytoplasm, whereas nucleolin overexpression increased levels and its mitogenic activity. Nucleolin directly bound to exon 2 via the glycine/arginine-rich (GAR) domain. Overexpression of GAR-deficient nucleolin failed to increase expression and growth of colon cancer cells. RNA fluorescence hybridization showed that co-localized with nucleolin in nuclei but not with the mutant lacking GAR. Our results suggest that specific interactions between nucleolin and UCR-containing may be associated with abnormal growth of colon cancer cells.
Miki Itai, Yuki Kuwano, Tatsuya Nishikawa, Kazuhito Rokutan and Kensei Nishida : Geranylgeranylacetone prevents stress-induced decline of leptin secretion in mice., The Journal of Medical Investigation : JMI, Vol.65, No.1.2, 103-109, 2018.
(要約)
Geranylgeranylacetone (GGA) is a chaperon inducer that protects various types of cell and tissue against stress. We examined whether GGA modulated energy intake and expenditure under stressful conditions. After mice were untreated or treated orally with GGA (0.16 g per kg body weight per day) for 10 days, they were subjected to 2-h restraint stress once or once a day for 5 consecutive days. GGA administration did not affect corticosterone response to the stress. Restraint stress rapidly decreased plasma leptin levels in control mice. GGA significantly increased circulating leptin levels without changing food intake and prevented the stress-induced decline of circulating leptin. However GGA-treated mice significantly reduced food intake during the repeated stress, compared with control mice. GGA prevented the stress-induced decline of leptin mRNA and its protein levels in epidydimal adipose tissues. We also found that GGA decreased ghrelin mRNA expression in gastric mucosa before the stress, whereas GGA-treated mice recovered the ghrelin mRNA expression to the baseline level after the repeated stress. Leptin and ghrelin are now recognized as regulators of anxiety and depressive mood. Our results suggest that GGA may regulate food intake and relief stress-induced mood disturbance through regulating leptin and ghrelin secretions. J. Med. Invest. 65:103-109, February, 2018.
Healthy students (21 males and 11 females) took paraprobiotic CP2305 daily for 5 weeks during a cadaver dissection course. The General Health Questionnaire and the Pittsburgh Sleep Quality Index were employed to assess stress-related somatic symptoms and sleep quality respectively. The aggravation of stress-associated somatic symptoms was observed in female students (P = 0·029). Sleep quality was improved in the paraprobiotic CP2305 group (P = 0·038), particularly in men (P = 0·004). Among men, paraprobiotic CP2305 shortened sleep latency (P = 0·035) and increased sleep duration (P = 0·048). Diarrhoea-like symptoms were also effectively controlled with CP2305 (P = 0·005) in men. Thus, we observed sex-related differences in the effects of paraprobiotic CP2305. In addition, CP2305 affected the growth of faecal Bacteroides vulgatus and Dorea longicatena, which are involved in intestinal inflammation.
(キーワード)
Adult / Feces / Female / Humans / Lactobacillus gasseri / Male / Probiotics / Sex Factors / Sleep / Stress, Psychological / Students, Medical
Shizue Masuki, Kensei Nishida, Shigenari Hashimoto, Mayuko Morikawa, Satoshi Takasugi, Masashi Nagata, Shun'ichiro Taniguchi, Kazuhito Rokutan and Hiroshi Nose : Effects of milk product intake on thigh muscle strength and NFKB gene methylation during home-based interval walking training in older women: A randomized, controlled pilot study., PLoS ONE, Vol.12, No.5, e0176757, 2017.
(要約)
Muscle atrophy with aging is closely associated with chronic systemic inflammation and lifestyle-related diseases. In the present study, we assessed whether post-exercise milk product intake during 5-month interval walking training (IWT) enhanced the increase in thigh muscle strength and ameliorated susceptibility to inflammation in older women. Subjects [n = 37, 66±5 (standard deviation) yrs] who had been performing IWT for >6 months participated in this study. They were randomly divided into the following 3 groups: IWT alone (CNT, n = 12), IWT + low-dose post-exercise milk product intake (LD, n = 12; 4 g protein and 3 g carbohydrate) or IWT + a 3-times higher dose of milk product intake than the LD group (HD, n = 13). They were instructed to repeat ≥5 sets of fast and slow walking for 3 min each at ≥70% and 40% peak aerobic capacity for walking, respectively, per day for ≥4 days/week. After IWT, thigh muscle strength increased in the HD group (8±2%) more than in the CNT group (-2±3%, P = 0.022), despite similar IWT achievements between the groups (P>0.15). Pyrosequencing analysis using whole blood showed that methylation of NFKB1 and NFKB2, master genes of inflammation, was enhanced in the HD group (29±7% and 44±11%, respectively) more than in the CNT group (-20±6% and -10±6%, respectively; P<0.001). Moreover, the genome-wide DNA methylation analysis showed that several inflammation-related genes were hyper-methylated in the HD group compared with that in the CNT group, suggesting greater pro-inflammatory cytokine gene suppression in the HD group. HD milk product intake after exercise produced a greater percent increase in thigh muscle strength and NFKB1 and NFKB2 gene methylation during IWT in physically active older women. UMIN-CTR No. UMIN000024544 and No. UMIN000024912.
(キーワード)
Age Factors / Aged / Animals / Blood Cells / CpG Islands / DNA Methylation / Dairy Products / Exercise / Female / Gene Expression Profiling / Genome-Wide Association Study / Humans / Middle Aged / Milk / Muscle Strength / NF-kappa B / Patient Compliance / Physical Fitness / Pilot Projects / Promoter Regions, Genetic / Signal Transduction / Thigh / Time Factors / Walking
Shinji Kitamura, Toshihito Tanahashi, Eriko Aoyagi, Tadahiko Nakagawa, Koichi Okamoto, Tetsuo Kimura, Hiroshi Miyamoto, Yasuhiro Mitsui, Kazuhito Rokutan, Naoki Muguruma and Tetsuji Takayama : Response Predictors of S-1, Cisplatin, and Docetaxel Combination Chemotherapy for Metastatic Gastric Cancer: Microarray Analysis of Whole Human Genes., Oncology, Vol.93, No.2, 127-135, 2017.
(要約)
The aim of this study was to identify biomarkers for predicting the efficacy of docetaxel, cisplatin, and S-1 (DCS) therapy for advanced gastric cancer using microarrays of biopsy specimens before chemotherapy. Nineteen samples were taken from 19 patients with unresectable metastatic gastric cancer who received DCS as a first-line therapy. Laser capture microdissection was performed, and total cellular RNA was extracted from each microdissected sample. Whole-gene expression was analyzed by microarray, and the difference in mRNA expression observed with the microarrays was confirmed by quantitative real-time PCR. Immunohistochemical staining was performed using clinical tissue sections obtained by endoscopic biopsy. Eleven patients were identified as early responders and 8 patients as nonresponders to DCS therapy. Twenty-nine genes showed significant differences in relative expression ratios between tumor and normal tissues. A classifier set of 29 genes had high accuracy (94.7%) for distinguishing gene expression between 11 early responders and 8 nonresponders. Decreasing the size of the classifier set to 4 genes (PDGFB, PCGF3, CISH, and ANXA5) increased the accuracy to 100%. Expression levels by real-time PCR for validation were well correlated with those 4 genes in microarrays. The genes identified may serve as efficient biomarkers for personalized cancer-targeted therapy.
Mai Takada, Kensei Nishida, Y. Gondo, H. Kikuchi-Hayakawa, H. Ishikawa, K. Suda, M. Kawai, R. Hoshi, Yuki Kuwano, K. Miyazaki and Kazuhito Rokutan : Beneficial effects of Lactobacillus casei strain Shirota on academic stress-induced sleep disturbance in healthy adults: a double-blind, randomised, placebo-controlled trial., Beneficial Microbes, Vol.8, No.2, 153-162, 2017.
(要約)
The present study examined whether Lactobacillus casei strain Shirota (LcS) improves sleep quality under psychological stress. A double-blind, placebo-controlled trial was conducted in healthy 4(th) year medical students exposed to academic examination stress. The trial was repeated over two consecutive years in different groups of students, and the data were pooled. For 8 weeks prior to and 3 weeks after a national standardised examination, a total of 48 and 46 subjects received a daily dose of 100 ml of LcS-fermented milk or non-fermented placebo milk, respectively. Study measures included subjective anxiety, overnight single-channel electroencephalography (EEG) recordings, and the Oguri-Shirakawa-Azumi (OSA) sleep inventory scores of subjective sleep quality. Total OSA scores were significantly lower than baseline on the day before the exam and recovered after the exam, indicating a stress-induced decline in sleep quality. There was a significant positive effect of LcS treatment on OSA factors for sleepiness on rising and sleep length. Sleep latency measured by EEG lengthened as the exam approached in the placebo group but was significantly suppressed in the LcS group. The percentage of stage 3 non-REM (N3) sleep decreased in the placebo group as the exam approached, whereas it was maintained in the LcS group throughout the trial. Delta power during the first sleep cycle, measured as an index of sleep intensity, increased as the exam approached in the LcS group and was significantly higher than in the placebo group. These findings suggest that daily consumption of LcS may help to maintain sleep quality during a period of increasing stress. The observed retention of N3 sleep and increased delta power in the LcS group may have contributed to higher perceived sleep satisfaction.
Daisuke Sawada, Tomoko Kawai, Kensei Nishida, Yuki Kuwano, Shigeru Fujiwara and Kazuhito Rokutan : Daily intake of Lactobacillus gasseri CP2305 improves mental, physical, and sleep quality among Japanese medical students enrolled in a cadaver dissection course., Journal of Functional Foods, Vol.31, 188-197, 2017.
Tomoko Kawai, Yuki Kuwano, Kiyoshi Masuda, Kinuyo Fujita, Hiroki Tanaka, Tatsuya Nishikawa, Kazuhito Rokutan and Kensei Nishida : Adverse parenting is associated with blunted salivary cortisol awakening response and altered expression of glucocorticoid receptor and 2-adrenergic receptor mRNAs in leukocytes in Japanese medical students., Stress, Vol.20, No.2, 159-166, 2017.
(要約)
Adverse parenting is associated with an increased risk for the development of mood and behavioral disorders. In this study, we assessed the perceived parental bonding of 232 medical students using the parental bonding instrument (PBI) and extracted 22 students who reported their parents' rearing attitudes as affectionless control (LOW; low care, high overprotection). Using the 28-item general health questionnaire, the Zung self-rating depression scale (Zung-SDS), the hospital anxiety and depression scale (HADS), and the Spielberger state-trait-anxiety-inventory (STAI), physical and mental state of the LOW students were compared with those of 30 students who reported their parental bonding as optimal (OPT; high care and low overprotection). These questionnaire measurements demonstrated significantly higher anxiety and depressive mood in the LOW students versus the OPT students. Compared with the OPT students, the LOW students also exhibited a significantly reduced salivary cortisol awakening response (CAR) without changes across the rest of the diurnal salivary cortisol profile. Among glucocorticoid-related genes examined (GR, ADRB2, IκBα, IL10, IL1R2, IL1RN, MR, MC2R, TGFB1, TGFB2 and FASLG), real-time reverse transcription-PCR showed that the LOW students significantly increased expression of a dominant negative glucocorticoid receptor β (GRβ) mRNA and decreased β2-adrenergic receptor (ADRB2) mRNA levels in circulating leukocytes. These results suggest that negative perception of parents' child-rearing attitudes may be associated with anxiety and depressive mood and altered glucocorticoid signaling even in healthy young adults.
Yasuteru Fujino, Shunsaku Takeishi, Kensei Nishida, Koichi Okamoto, Naoki Muguruma, Tetsuo Kimura, Shinji Kitamura, Hiroshi Miyamoto, Akiko Fujimoto, Jun Higashijima, Mitsuo Shimada, Kazuhito Rokutan and Tetsuji Takayama : Downregulation of miR-100/miR-125b is associated with lymph node metastasis in early colorectal cancer with submucosal invasion., Cancer Science, Vol.108, No.3, 390-397, 2017.
(要約)
A majority of early colorectal cancers (CRCs) with submucosal invasion undergo surgical operation, despite a very low incidence of lymph node metastasis. Our study aimed to identify microRNAs (miRNAs) specifically responsible for lymph node metastasis in submucosal CRCs. MicroRNA microarray analysis revealed that miR-100 and miR-125b expression levels were significantly lower in CRC tissues with lymph node metastases than in those without metastases. These results were validated by quantitative real-time PCR in a larger set of clinical samples. The transfection of a miR-100 or miR-125b inhibitor into colon cancer HCT116 cells significantly increased cell invasion, migration, and MMP activity. Conversely, overexpression of miR-100 or miR-125b mimics significantly attenuated all these activities but did not affect cell growth. To identify target mRNAs, we undertook a gene expression array analysis of miR-100-silenced HCT116 cells as well as negative control cells. The Ingenuity Pathway Analysis, TargetScan software analyses, and subsequent verification of mRNA expression by real-time PCR identified mammalian target of rapamycin (mTOR) and insulin-like growth factor 1 receptor (IGF1R) as direct, and Fas and X-linked inhibitor-of-apoptosis protein (XIAP) as indirect candidate targets for miR-100 involved in lymph node metastasis. Knockdown of each gene by siRNA significantly reduced the invasiveness of HCT116 cells. These data clearly show that downregulation of miR-100 and miR-125b is closely associated with lymph node metastasis in submucosal CRC through enhancement of invasion, motility, and MMP activity. In particular, miR-100 may promote metastasis by upregulating mTOR, IGF1R, Fas, and XIAP as targets. Thus, miR-100 and miR-125b may be novel biomarkers for lymph node metastasis of early CRCs with submucosal invasion.
K. Nobutani, Daisuke Sawada, Shiferu Fujiwara, Yuki Kuwano, Kensei Nishida, J. Nakayama, H. Kutsumi, T. Azuma and Kazuhito Rokutan : The effects of administration of the Lactobacillus gasseri strain CP2305 on quality of life, clinical symptoms and changes in gene expression in patients with irritable bowel syndrome., Journal of Applied Microbiology, Vol.122, No.1, 212-224, 2017.
(要約)
To clarify the effects of Lactobacillus gasseri CP2305 (CP2305) on quality of life and clinical symptoms and its functional mechanisms in patients with irritable bowel syndrome (IBS). After the patients were administered CP2305 daily for 4 weeks, the IBS-severity index score was significantly improved compared with that of the placebo group, and this improvement was accompanied by a reduction in health-related worry and changes in intestinal microbiota. The gene expression profiling of the peripheral blood leucocytes showed that CP2305 treatment significantly up-regulated genes related to eukaryotic initiation factor 2 (EIF2) signalling. Eighty-two genes were down-regulated in IBS patients compared with healthy controls. The expression of 23 of these genes exhibited a CP2305-dependent increase associated with an improvement in IBS severity. The majority of the restored genes were related to EIF2 signalling. CP2305 administration is a potential candidate therapeutic option for patients with IBS. Although probiotics have been proposed to benefit IBS patients, objective clinical evidence and elucidation of the functional mechanism remain insufficient. Our study demonstrated that CP2305 administration beneficially influences IBS patients in both subjective and objective evaluations, and gene expression profiling provided insights into the functional mechanism.
(キーワード)
Adult / Female / Humans / Irritable Bowel Syndrome / Lactobacillus gasseri / Male / Middle Aged / Probiotics / Quality of Life / Treatment Outcome
Yuki Kuwano, Kensei Nishida, Yoko Akaike, Ken Kurokawa, Tatsuya Nishikawa, Kiyoshi Masuda and Kazuhito Rokutan : Homeodomain-Interacting Protein Kinase-2: A Critical Regulator of the DNA Damage Response and the Epigenome., International Journal of Molecular Sciences, Vol.17, No.10, 2016.
(要約)
Homeodomain-interacting protein kinase 2 (HIPK2) is a serine/threonine kinase that phosphorylates and activates the apoptotic program through interaction with diverse downstream targets including tumor suppressor p53. HIPK2 is activated by genotoxic stimuli and modulates cell fate following DNA damage. The DNA damage response (DDR) is triggered by DNA lesions or chromatin alterations. The DDR regulates DNA repair, cell cycle checkpoint activation, and apoptosis to restore genome integrity and cellular homeostasis. Maintenance of the DDR is essential to prevent development of diseases caused by genomic instability, including cancer, defects of development, and neurodegenerative disorders. Recent studies reveal a novel HIPK2-mediated pathway for DDR through interaction with chromatin remodeling factor homeodomain protein 1. In this review, we will highlight the molecular mechanisms of HIPK2 and show its functions as a crucial DDR regulator.
Akito Kato-Kataoka, Kensei Nishida, Mai Takada, Mitsuhisa Kawai, Hiroko Kikuchi-Hayakawa, Kazunori Suda, Hiroshi Ishikawa, Yusuke Gondo, Kensuke Shimizu, Takahiro Matsuki, Akira Kushiro, Ryoutaro Hoshi, Osamu Watanabe, Tomoki Igarashi, Kouji Miyazaki, Yuki Kuwano and Kazuhito Rokutan : Fermented Milk Containing Lactobacillus casei Strain Shirota Preserves the Diversity of the Gut Microbiota and Relieves Abdominal Dysfunction in Healthy Medical Students Exposed to Academic Stress., Applied and Environmental Microbiology, Vol.82, No.12, 3649-3658, 2016.
(要約)
A novel clinical trial was conducted with healthy medical students under examination stress conditions. It was demonstrated that the daily consumption of lactic acid bacteria provided health benefits to prevent the onset of stress-associated abdominal symptoms and a good change of gut microbiota in healthy medical students.
K Kajita, Yuki Kuwano, Y Satake, S Kano, K Kurokawa, Y Akaike, Kiyoshi Masuda, Kensei Nishida and Kazuhito Rokutan : Ultraconserved region-containing Transformer 24 controls senescence of colon cancer cells., Oncogenesis, Vol.5, 2016.
M Takada, Kensei Nishida, A Kataoka-Kato, Y Gondo, H Ishikawa, K Suda, M Kawai, R Hoshi, O Watanabe, T Igarashi, Yuki Kuwano, K Miyazaki and Kazuhito Rokutan : Probiotic Lactobacillus casei strain Shirota relieves stress-associated symptoms by modulating the gut-brain interaction in human and animal models., Neurogastroenterology and Motility, 2016.
T. Kawamura, K. Uno, K. Tanaka, Y. Ueda, N. Sakiyama, Kensei Nishida, Kazuhito Rokutan and K. Yasuda : Morphological Characteristics and Location of Missed, Advanced Colorectal Neoplasms after Colonoscopy., The Journal of Medical Investigation : JMI, Vol.63, No.3-4, 163-170, 2016.
(要約)
This retrospective study aimed to clarify the clinical characteristics of advanced colorectal neoplasms after colonoscopy, likely to have been missed on the previous colonoscopy. We reviewed a total of 5,768 consecutive colonoscopies performed from April 2010 to September 2013 in 4,841 patients, and analyzed advanced colorectal neoplasms after colonoscopy, particularly focusing on their morphological characteristics and locations, as compared with primary lesions, defined as lesions detected in their first colonoscopy or in a subsequent colonoscopy >5 years after the previous one. Of the 5,768 examinations, 922 advanced neoplasms (including 217 cancers with ≥T2) were detected, and 167 lesions (18.1%) were diagnosed within 5 years after a previous colonoscopy (post-colonoscopy advanced neoplasms). The incidence of right-sided lesions in the post-colonoscopy advanced neoplasms (48.5%, 81/167) was significantly higher than in the primary lesions (34.0%, 257/755; p <0.001). We excluded 217 cancers with ≥T2 from the morphological analysis to characterize early-stage post-colonoscopy advanced neoplasms. The incidence of non-polypoid lesions in the post-colonoscopy advanced neoplasms (25.6%, 41/160) was significantly higher than that in the primary lesions (12.3%, 67/545; p <0.001). These findings suggest that extra attention should be paid to non-polypoid, right-sided advanced colorectal neoplasms during screening and surveillance colonoscopy. J. Med. Invest. 63: 163-170, August, 2016.
(キーワード)
Aged / Aged, 80 and over / Colonoscopy / Colorectal Neoplasms / Female / Humans / Male / Middle Aged / Retrospective Studies
Saki Saijo, Yuki Kuwano, Kiyoshi Masuda, Tatsuya Nishikawa, Kazuhito Rokutan and Kensei Nishida : Serine/arginine-rich splicing factor 7 regulates p21-dependent growth arrest in colon cancer cells., The Journal of Medical Investigation : JMI, Vol.63, No.3-4, 219-226, 2016.
(要約)
Serine/arginine-rich splicing factors (SRSFs) play wide-ranging roles in gene expression through post-transcriptional regulation as well as pre-mRNA splicing. SRSF7 was highly expressed in colon cancer tissues, and its knockdown inhibited cell growth in colon cancer cells (HCT116) in association with altered expression of 4,499 genes. The Ingenuity Pathway Analysis revealed that cell cycle-related canonical pathways were ranked as the highly enriched category in the affected genes. Western blotting confirmed that p21, a master regulator in cell cycle, was increased without any induction of p53 in SRSF7 knockdown cells. Furthermore, cyclin-dependent kinase 2 and retinoblastoma protein were remained in the hypophosphorylated state. In addition, the SRSF7 knockdown-induced cell growth inhibition was observed in p53-null HCT116 cells, suggesting that p53-independent pathways were involved in the SRSF7 knockdown-induced cell growth inhibition. The reduction of SRSF7 stabilized cyclin-dependent kinase inhibitor 1A (CDKN1A) mRNA without any activation of the CDKN1A promoter. Interestingly, SRSF7 knockdown also blocked p21 degradation. These results suggest that the reduction of SRSF7 post-transcriptionally regulates p21 induction at the multistep processes. Thus, the present findings disclose a novel, important role of SRSF7 in cell proliferation through regulating p21 levels. J. Med. Invest. 63: 219-226, August, 2016.
A Kato-Kataoka, Kensei Nishida, M Takada, K Suda, M Kawai, K Shimizu, A Kushiro, R Hoshi, O Watanabe, T Igarashi, K Miyazaki, Yuki Kuwano and Kazuhito Rokutan : Fermented milk containing Lactobacillus casei strain Shirota prevents the onset of physical symptoms in medical students under academic examination stress., Beneficial Microbes, Vol.7, No.2, 153-156, 2016.
(要約)
This pilot study investigated the effects of the probiotic Lactobacillus casei strain Shirota (LcS) on psychological, physiological, and physical stress responses in medical students undertaking an authorised nationwide examination for promotion. In a double-blind, placebo-controlled trial, 24 and 23 healthy medical students consumed a fermented milk containing LcS and a placebo milk, respectively, once a day for 8 weeks until the day before the examination. Psychophysical state, salivary cortisol, faecal serotonin, and plasma L-tryptophan were analysed on 5 different sampling days (8 weeks before, 2 weeks before, 1 day before, immediately after, and 2 weeks after the examination). Physical symptoms were also recorded in a diary by subjects during the intervention period for 8 weeks. In association with a significant elevation of anxiety at 1 day before the examination, salivary cortisol and plasma L-tryptophan levels were significantly increased in only the placebo group (P<0.05). Two weeks after the examination, the LcS group had significantly higher faecal serotonin levels (P<0.05) than the placebo group. Moreover, the rate of subjects experiencing common abdominal and cold symptoms and total number of days experiencing these physical symptoms per subject were significantly lower in the LcS group than in the placebo group during the pre-examination period at 5-6 weeks (each P<0.05) and 7-8 weeks (each P<0.01) during the intervention period. Our results suggest that the daily consumption of fermented milk containing LcS may exert beneficial effects preventing the onset of physical symptoms in healthy subjects exposed to stressful situations.
Yuki Kuwano, Kensei Nishida, Keisuke Kajita, Yuzuru Satake, Yoko Akaike, Kiyuyo Fujita, Shizuka Kano, Kiyoshi Masuda and Kazuhito Rokutan : Transformer 2 and miR-204 regulate apoptosis through competitive binding to 3 UTR of BCL2 mRNA, Cell Death and Differentiation, Vol.22, No.5, 815-825, 2015.
(要約)
RNA-binding proteins and microRNAs are potent post-transcriptional regulators of gene expression. Human transformer 2β (Tra2β) is a serine/arginine-rich-like protein splicing factor and is now implicated to have wide-ranging roles in gene expression as an RNA-binding protein. RNA immunoprecipitation (RIP) with an anti-Tra2β antibody and microarray analysis identified a subset of Tra2β-associated mRNAs in HCT116 human colon cancer cells, many of which encoded cell death-related proteins including Bcl-2 (B-cell CLL/lymphoma 2). Tra2β knockdown in HCT116 cells decreased Bcl-2 expression and induced apoptosis. Tra2β knockdown accelerated the decay of BCL2α mRNA that encodes Bcl-2 and full-length 3' UTR, while it did not affect the stability of BCL2β mRNA having a short, alternatively spliced 3' UTR different from BCL2α 3' UTR. RIP assays with anti-Tra2β and anti-Argonaute 2 antibodies, respectively, showed that Tra2β bound to BCL2α 3' UTR, and that Tra2β knockdown facilitated association of miR-204 with BCL2α 3' UTR. The consensus sequence (GAA) for Tra2β-binding lies within the miR-204-binding site of BCL2 3' UTR. Mutation of the consensus sequence canceled the binding of Tra2β to BCL2 3' UTR without disrupting miR-204-binding to BCL2 3' UTR. Transfection of an anti-miR-204 or introduction of three-point mutations into the miR-204-binding site increased BCL2 mRNA and Bcl-2 protein levels. Inversely, transfection of precursor miR-204 reduced their levels. Experiments with Tra2β-silenced or overexpressed cells revealed that Tra2β antagonized the effects of miR-204 and upregulated Bcl-2 expression. Furthermore, TRA2β mRNA expression was significantly upregulated in 22 colon cancer tissues compared with paired normal tissues and positively correlated with BCL2 mRNA expression. Tra2β knockdown in human lung adenocarcinoma cells (A549) increased their sensitivity to anticancer drugs. Taken together, our findings suggest that Tra2β regulates apoptosis by modulating Bcl-2 expression through its competition with miR-204. This novel function may have a crucial role in tumor growth.
Y Akaike, Yuki Kuwano, Kensei Nishida, K Kurokawa, K Kajita, S Kano, Kiyoshi Masuda and Kazuhito Rokutan : Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1., Oncogene, Vol.34, No.26, 3463-3473, 2014.
(要約)
Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1β, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent phosphorylation of HP1γ may participate in the regulation of dynamic interaction between HP1γ and histone H3K9me3 to promote DNA damage repair. This HIPK2/HP1γ pathway may uncover a new functional aspect of HIPK2 as a tumor suppressor.
Yoko Akaike, Kiyoshi Masuda, Yuki Kuwano, Kensei Nishida, Keisuke Kajita, Ken Kurokawa, Yuzuru Satake, Katsutoshi Shoda, Issei Imoto and Kazuhito Rokutan : HuR Regulates Alternative Splicing of the TRA2 Gene in Human Colon Cancer Cells under Oxidative Stress., Molecular and Cellular Biology, Vol.34, No.15, 2857-2873, 2014.
(要約)
Hu antigen R (HuR) regulates stress responses through stabilizing and/or facilitating the translation of target mRNAs. The human TRA2 gene encodes splicing factor transformer 2 (Tra2) and generates 5 mRNA isoforms (TRA21 to -5) through alternative splicing. Exposure of HCT116 colon cancer cells to sodium arsenite stimulated checkpoint kinase 2 (Chk2)- and mitogen-activated protein kinase p38 (p38(MAPK))-mediated phosphorylation of HuR at positions S88 and T118. This induced an association between HuR and the 39-nucleotide (nt) proximal region of TRA2 exon 2, generating a TRA24 mRNA that includes exon 2, which has multiple premature stop codons. HuR knockdown or Chk2/p38(MAPK) double knockdown inhibited the arsenite-stimulated production of TRA24 and increased Tra2 protein, facilitating Tra2-dependent inclusion of exons in target pre-mRNAs. The effects of HuR knockdown or Chk2/p38(MAPK) double knockdown were also confirmed using a TRA2 minigene spanning exons 1 to 4, and the effects disappeared when the 39-nt region was deleted from the minigene. In endogenous HuR knockdown cells, the overexpression of a HuR mutant that could not be phosphorylated (with changes of serine to alanine at position 88 [S88A], S100A, and T118A) blocked the associated TRA24 interaction and TRA24 generation, while the overexpression of a phosphomimetic HuR (with mutations S88D, S100D, and T118D) restored the TRA24-related activities. Our findings revealed the potential role of nuclear HuR in the regulation of alternative splicing programs under oxidative stress.
Ken Kurokawa, Yoko Akaike, Kiyoshi Masuda, Yuki Kuwano, Kensei Nishida, Naoko Yamagishi, Keisuke Kajita, Toshihito Tanahashi and Kazuhito Rokutan : Downregulation of serine/arginine-rich splicing factor 3 induces G1 cell cycle arrest and apoptosis in colon cancer cells, Oncogene, Vol.33, No.11, 1407-1417, 2014.
(要約)
Serine/arginine-rich splicing factor 3 (SRSF3) likely has wide-ranging roles in gene expression and facilitation of tumor cell growth. SRSF3 knockdown induced G1 arrest and apoptosis in colon cancer cells (HCT116) in association with altered expression of 833 genes. Pathway analysis revealed 'G1/S Checkpoint Regulation' as the most highly enriched category in the affected genes. SRSF3 knockdown did not induce p53 or stimulate phosphorylation of p53 or histone H2A.X in wild-type HCT116 cells. Furthermore, the knockdown induced G1 arrest in p53-null HCT116 cells, suggesting that p53-dependent DNA damage responses did not mediate the G1 arrest. Real-time reverse transcription-polymerase chain reaction and western blotting confirmed that SRSF3 knockdown reduced mRNA and protein levels of cyclins (D1, D3 and E1), E2F1 and E2F7. The decreased expression of cyclin D and E2F1 likely impaired the G1-to-S-phase progression. Consequently, retinoblastoma protein remained hypophosphorylated in SRSF3 knockdown cells. The knockdown also induced apoptosis in association with reduction of BCL2 protein levels. We also found that SRSF3 knockdown facilitated skipping of 81 5'-nucleotides (27 amino acids) from exon 8 of homeodomain-interacting protein kinase-2 (HIPK2) and produced a HIPK2 Δe8 isoform. Full-length HIPK2 (HIPK2 FL) is constantly degraded through association with an E3 ubiquitin ligase (Siah-1), whereas HIPK2 Δe8, lacking the 27 amino acids, lost Siah-1-binding ability and became resistant to proteasome digestion. Interestingly, selective knockdown of HIPK2 FL induced apoptosis in various colon cancer cells expressing wild-type or mutated p53. Thus, these findings disclose an important role of SRSF3 in the regulation of the G1-to-S-phase progression and alternative splicing of HIPK2 in tumor growth.
Shizuka Kano, Kensei Nishida, Hiroyuki Kurebe, Chihiro Nishiyama, Kentaro Kita, Yoko Akaike, Keisuke Kajita, Ken Kurokawa, Kiyoshi Masuda, Yuki Kuwano, Toshihito Tanahashi and Kazuhito Rokutan : Oxidative stress-inducible truncated serine/arginine-rich splicing factor 3 regulates interleukin-8 production in human colon cancer cells, American Journal of Physiology, Cell Physiology, Vol.306, No.3, C250-C262, 2014.
(要約)
Serine/arginine-rich splicing factor 3 (SRSF3) is a member of the SR protein family and plays wide-ranging roles in gene expression. The human SRSF3 gene generates two alternative splice transcripts, a major mRNA isoform (SRSF3-FL) encoding functional full-length protein and a premature termination codon (PTC)-containing isoform (SRSF3-PTC). The latter is degraded through nonsense-mediated mRNA decay (NMD). Treatment of a human colon cancer cell line (HCT116) with 100 μM sodium arsenite increased SRSF3-PTC mRNA levels without changing SRSF3-FL mRNA levels. A chemiluminescence-based NMD reporter assay system demonstrated that arsenite treatment inhibited NMD activity and increased SRSF3-PTC mRNA levels in the cytoplasm, facilitating translation of a truncated SRSF3 protein (SRSF3-TR) from SRSF3-PTC mRNA. SRSF3-TR lacked two-thirds of the Arg/Ser-rich (RS) domain whose phosphorylation state is known to be crucial for subcellular distribution. SRSF3-FL was localized in the nucleus, while overexpressed SRSF3-TR was diffusely distributed in the cytoplasm and the nucleus. A part of SRSF3-TR was also associated with stress granules in the cytoplasm. Interestingly, treatment of HCT116 cells with a small interference RNA specifically targeting SRSF3-PTC mRNA significantly attenuated arsenite-stimulated induction of c-JUN protein, its binding activity to the AP-1 binding site (-126 to 120 bp) in the interleukin (IL)-8 gene promoter, and AP-1 promoter activity, resulting in significant reduction of arsenite-stimulated IL-8 production. Our results suggest that SRSF3-TR may function as a positive regulator of oxidative stress-initiated inflammatory responses in colon cancer cells.
Keisuke Kajita, Yuki Kuwano, Naruka Kitamura, Yuzuru Satake, Kensei Nishida, Ken Kurokawa, Yoko Akaike, Manami Honda, Kiyoshi Masuda and Kazuhito Rokutan : Ets1 and heat shock factor 1 regulate transcription of the Transformer 2β gene in human colon cancer cells, Journal of Gastroenterology, Vol.48, No.11, 1222-1233, 2013.
(要約)
Transformer (Tra) 2β is a member of the serine/arginine-rich (SR)-like protein family that regulates alternative splicing of numerous genes in a concentration-dependent manner. Several types of cancer cells up-regulate Tra2β expression, while the regulatory mechanism of Tra2β expression remains to be elucidated. In this study, we examined the transcriptional regulation and possible functions of Tra2β in human colon cancer cells. We cloned 959 bp-upstream of the human TRA2β 5'-flank into luciferase constructs. Chromatin immunoprecipitation (ChIP) was employed to identify crucial cis element(s) and trans activator(s) of the TRA2β promoter. Tra2β expression in the human colon and colon cancer tissues was examined by immunohistochemistry. In response to sodium arsenite, colon cancer cells (HCT116) increased levels of TRA2β1 mRNA encoding a functional, full-length Tra2β with a peak around 6 h without changing its mRNA stability. Transient expression assays using a reporter gene driven by serially truncated TRA2β promoters and Chip assay demonstrated that an Ets1-binding site present at -64 to -55 bp was crucial for basal transcription, while three heat shock elements (HSEs) located at -145 to -99 bp mediated the oxidant-induced transactivation of TRA2β. Tra2β knockdown caused apoptosis of HCT116 cells. Tra2β were preferentially expressed in proliferative compartment of normal human colonic glands and adenocarcinomas, where Ets1 and heat shock factor 1 were also highly expressed. Our results suggest that oxidative stress-responsive Tra2β may play an important role in colon cancer growth.
Manami Honda, Yuki Kuwano, Sakurako Katsuura-Kamano, Kinuyo Fujita, Yoko Akaike, Shizuka Kano, Kensei Nishida, Kiyoshi Masuda and Kazuhito Rokutan : Chronic academic stress increases a group of microRNAs in peripheral blood, PLoS ONE, Vol.8, No.10, e75960, 2013.
(要約)
MicroRNAs (miRNAs) play key roles in regulation of cellular processes in response to changes in environment. In this study, we examined alterations in miRNA profiles in peripheral blood from 25 male medical students two months and two days before the National Examination for Medical Practitioners. Blood obtained one month after the examination were used as baseline controls. Levels of seven miRNAs (miR-16, -20b, -26b, -29a, -126, -144 and -144*) were significantly elevated during the pre-examination period in association with significant down-regulation of their target mRNAs (WNT4, CCM2, MAK, and FGFR1 mRNAs) two days before the examination. State anxiety assessed two months before the examination was positively and negatively correlated with miR-16 and its target WNT4 mRNA levels, respectively. Fold changes in miR-16 levels from two days before to one month after the examination were inversely correlated with those in WNT4 mRNA levels over the same time points. We also confirmed the interaction between miR-16 and WNT4 3'UTR in HEK293T cells overexpressing FLAG-tagged WNT4 3'UTR and miR-16. Thus, a distinct group of miRNAs in periheral blood may participate in the integrated response to chronic academic stress in healthy young men.
(キーワード)
Adult / Carrier Proteins / Humans / Male / MicroRNAs / Protein-Serine-Threonine Kinases / Receptor, Fibroblast Growth Factor, Type 1 / Stress, Psychological / Teaching / Wnt4 Protein
Kiyoshi Masuda, Yuki Kuwano, Kensei Nishida, Kazuhito Rokutan and Issei Imoto : NF90 in Posttranscriptional Gene Regulation and MicroRNA Biogenesis., International Journal of Molecular Sciences, Vol.14, No.8, 17111-17121, 2013.
(要約)
Gene expression patterns are effectively regulated by turnover and translation regulatory (TTR) RNA-binding proteins (RBPs). The TTR-RBPs control gene expression at posttranscriptional levels, such as pre-mRNA splicing, mRNA cytoplasmic export, turnover, storage, and translation. Double-stranded RNA binding proteins (DSRBPs) are known to regulate many processes of cellular metabolism, including transcriptional control, translational control, mRNA processing and localization. Nuclear factor 90 (NF90), one of the DSRBPs, is abundantly expressed in vertebrate tissue and participates in many aspects of RNA metabolism. NF90 was originally purified as a component of a DNA binding complex which binds to the antigen recognition response element 2 in the interleukin 2 promoter. Recent studies have provided us with interesting insights into its possible physiological roles in RNA metabolism, including transcription, degradation, and translation. In addition, it was shown that NF90 regulates microRNA expression. In this review, we try to focus on the function of NF90 in posttranscriptional gene regulation and microRNA biogenesis.
Masahiro Toda, Tomoko Kawai, Keiko Takeo, Kazuhito Rokutan and Kanehisa Morimoto : Associations between Chronotype and Salivary Endocrinological Stress Markers., Endocrine Research, Vol.38, No.1, 1-7, 2013.
(要約)
Objectives. To investigate the effect of chronotype on salivary cortisol or salivary -amylase (sAA). Methods. From 108 male university students, saliva samples were collected in the afternoon (between 15:00 and 17:00). The salivary cortisol and sAA levels were determined with commercial kits. Chronotype was quantitatively evaluated using the Horne and Östberg Morningness-Eveningness Questionnaire. Subjects were categorized into morning types and evening types. Results. The sAA levels were lower in the morning types than in the evening types. We found no significant difference in salivary cortisol levels between the two groups. Conclusions: These findings suggest that the sAA levels may be associated with chronotype.
Naoko Yamagishi, Shigetada Kondo, Kiyoshi Masuda, Kensei Nishida, Yuki Kuwano, Duyen T Dang, Long H Dang, Takeshi Nikawa and Kazuhito Rokutan : Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells, BMC Cancer, Vol.13, 229, 2013.
(要約)
Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Our findings suggest that chronic inhibition of tumor cell-derived VEGF accelerates tumor cell malignant phenotypes.
Masato Komatsu, Tetsuro Yoshimaru, Taisuke Matsuo, Kazuma Kiyotani, Yasuo Miyoshi, Toshihito Tanahashi, Kazuhito Rokutan, Rui Yamaguchi, Ayumu Saito, Seiya Imoto, Satoru Miyano, Yusuke Nakamura, Mitsunori Sasa, Mitsuo Shimada and Toyomasa Katagiri : Molecular features of triple negative breast cancer cells by genome-wide gene expression profiling analysis., International Journal of Oncology, Vol.42, No.2, 478-506, 2013.
(要約)
Triple negative breast cancer (TNBC) has a poor outcome due to the lack of beneficial therapeutic targets. To clarify the molecular mechanisms involved in the carcinogenesis of TNBC and to identify target molecules for novel anticancer drugs, we analyzed the gene expression profiles of 30 TNBCs as well as 13 normal epithelial ductal cells that were purified by laser-microbeam microdissection. We identified 301 and 321 transcripts that were significantly upregulated and downregulated in TNBC, respectively. In particular, gene expression profile analyses of normal human vital organs allowed us to identify 104 cancer-specific genes, including those involved in breast carcinogenesis such as NEK2, PBK and MELK. Moreover, gene annotation enrichment analysis revealed prominent gene subsets involved in the cell cycle, especially mitosis. Therefore, we focused on cell cycle regulators, asp (abnormal spindle) homolog, microcephaly-associated (Drosophila) (ASPM) and centromere protein K (CENPK) as novel therapeutic targets for TNBC. Small-interfering RNA-mediated knockdown of their expression significantly attenuated TNBC cell viability due to G1 and G2/M cell cycle arrest. Our data will provide a better understanding of the carcinogenesis of TNBC and could contribute to the development of molecular targets as a treatment for TNBC patients.
Kiyoshi Masuda, Yuki Kuwano, Kensei Nishida and Kazuhito Rokutan : Application of DNA microarray technology to gerontological studies, Methods in Molecular Biology, Vol.1048, 285-308, 2013.
(要約)
Gene expression patterns change dramatically in aging and age-related events. The DNA microarray is now recognized as a useful device in molecular biology and widely used to identify the molecular mechanisms of aging and the biological effects of drugs for therapeutic purpose in age-related diseases. Recently, numerous technological advantages have led to the evolution of DNA microarrays and microarray-based techniques, revealing the genomic modification and all transcriptional activity. Here, we show the step-by-step methods currently used in our lab to handling the oligonucleotide microarray and miRNA microarray. Moreover, we introduce the protocols of ribonucleoprotein [RNP] immunoprecipitation followed by microarray analysis (RIP-chip) which reveal the target mRNA of age-related RNA-binding proteins.
Shizuka Kano, Kensei Nishida, Chihiro Nishiyama, Yoko Akaike, Keisuke Kajita, Ken Kurokawa, Kiyoshi Masuda, Yuki Kuwano, Toshihito Tanahashi and Kazuhito Rokutan : Truncated serine/arginine-rich splicing factor 3 accelerates cell growth through up-regulating c-Jun expression, The Journal of Medical Investigation : JMI, Vol.60, No.3-4, 228-235, 2013.
(要約)
Serine/arginine-rich splicing factor 3 (SRSF3), a member of the SRSF family, plays a wide-ranging role in gene expression. The human SRSF3 gene generates a major mRNA isoform encoding a functional, full-length protein and a PTC-containing isoform (SRSF3-PTC). The latter is expected to be degraded through the nonsense-mediated mRNA decay system. However, it was reported that SRSF3-PTC mRNA was produced under stressful conditions and translated into a truncated SRSF3 protein (SRSF3-TR). To disclose unknown functions of SRSF3-TR, we established Flp-In-293 cells stably expressing SRSF3-TR. The SRSF3-TR-expressing cells increased mRNA and protein levels of positive regulators for G1 to S phase transition (cyclin D1, cyclin D3, CDC25A, and E2F1) and accelerated their growth. c-Jun is required for progression through the G1 phase, the mechanism by which involves transcriptional control of the cyclin D1 gene. We also found that the JUN promoter activity was significantly increased in the Flp-In-293 cells stably expressing SRSF3-TR, compared with mock-transfected control cells. The SRSF3-TR-expressing cells increased c-Jun and Sp-1 levels, which are important for the positive autoregulation and basal transcription of JUN, respectively. Our results suggest that stress-inducible SRSF3-TR may participate in the acceleration of cell growth through facilitating c-Jun-mediated G1 progression under stressful conditions.
(キーワード)
Alternative Splicing / Base Sequence / Cell Line / Cell Proliferation / DNA / G1 Phase Cell Cycle Checkpoints / Genes, bcl-1 / Genes, jun / Humans / Molecular Sequence Data / Oxidative Stress / Peptide Fragments / Promoter Regions, Genetic / RNA-Binding Proteins / Up-Regulation
Yoshiko Kamezaki, Sakurako Katsuura, Yuki Kuwano, Toshihito Tanahashi and Kazuhito Rokutan : Circulating cytokine signatures in healthy medical students exposed to academic examination stress., Psychophysiology, Vol.49, No.7, 991-997, 2012.
(要約)
Stress-induced production of proinflammatory cytokines in the brain and periphery is associated with mental distress. In this study, we measured changes in levels of salivary cortisol and 50 circulating immune mediators in 28 4th-grade medical students (19 males and 9 females) 7 weeks before, 1 day before, immediately after, and 1 week after an authorized nationwide examination for promotion. Repeated measures ANOVA with multiple testing correction and post hoc tests revealed that the examination significantly increased levels of proinflammatory cytokines (granulocyte colony-stimulating factor, interferon-, interleukin (IL)-1, and tumor necrosis factor-), Th2 cytokines (IL-4, IL-5, and IL-13), and -nerve growth factor in association with significant decreases in salivary cortisol levels and anxiety after the examination. These mediators may have a negative impact on the mental state of healthy young adults exposed to naturalistic stressors.
Sakurako Katsuura, Yuki Kuwano, Naoko Yamagishi, Ken Kurokawa, Keisuke Kajita, Yoko Akaike, Kensei Nishida, Kiyoshi Masuda, Toshihito Tanahashi and Kazuhito Rokutan : MicroRNAs miR-144/144* and miR-16 in peripheral blood are potential biomarkers for naturalistic stress in healthy Japanese medical students., Neuroscience Letters, Vol.516, No.1, 79-84, 2012.
(要約)
Non-coding microRNAs (miRNAs) are suggested to serve fundamental roles in cellular stress responses and in coping with sudden environmental changes in experimental animals. We examined whether naturalistic stressor-responsive miRNAs were detectable in whole blood. Blood and saliva were collected between 16:00 and 17:00 from 10 healthy medical students (5 males and 5 females; aged 22.4±0.8 years, mean±SD) 7 weeks before, one day before, immediately after, and one week after a nationally administered examination for academic promotion. Samples obtained one week after the examination were used as baseline controls. State anxiety and salivary cortisol levels reached maximum levels the day before the examination. Eleven candidate miRNAs (miR-144, -144*, -16, -15a, -19a, -19b, -26b, -30b, -106b, -126, and -142-3p) were extracted using a human miRNA microarray, and quantitative real-time reverse transcription PCR confirmed significant elevation of miR-144/144* and miR-16 levels immediately after finishing the examination. miR-16 levels in individual students were positively correlated with those of serum tumor necrosis factor (TNF)- measured immediately after the examination. Percentage changes in miR-144* and miR-16 levels from immediately after to one week after the examination were significantly correlated with percentage changes in circulating interferon- and/or TNF- levels over the same time points. Our results suggest that miR-144/144* and miR-16 may constitute a part of an integrated response to naturalistic stressors in healthy young adults.
(キーワード)
Biological Markers / Female / Humans / Interferon-gamma / 日本 (Japan) / Male / MicroRNAs / Prevalence / Stress, Psychological / Students, Medical / Tumor Necrosis Factor-alpha / Young Adult
Ken Kurokawa, Toshihito Tanahashi, Tsutomu Iima, Yuta Yamamoto, Yoko Akaike, Kensei Nishida, Kiyoshi Masuda, Yuki Kuwano, Yoshiki Murakami, Masakazu Fukushima and Kazuhito Rokutan : Role of miR-19b and its target mRNAs in 5-fluorouracil resistance in colon cancer cells., Journal of Gastroenterology, Vol.47, No.8, 883-895, 2012.
(要約)
BACKGROUND: Drug resistance in colorectal cancers is assumed to be mediated by changes in the expression of microRNAs, but the specific identities and roles of microRNAs are largely unclear. We examined the effect of 5-fluorouracil (5-FU) resistance on microRNA expression. METHODS: Two types of 5-FU-resistant colon cancer cells were derived from the DLD-1 and KM12C cell lines. The expressions of microRNAs were profiled with a microarray containing 723 microRNAs and validated by quantitative real-time polymerase chain reaction (qRT-PCR). To survey the downstream mediators of microRNA, we used a microRNA:mRNA immunoprecipitation (RIP)-Chip and pathway analysis tool to identify potential direct targets of microRNA. RESULTS: In response to 5-FU, miR-19b and miR-21 were over-expressed in 5-FU-resistant cells. Of note, miR-19b was up-regulated 3.47-fold in the DLD-1 resistant cells, which exhibited no alteration in cell cycle profiles despite exposure to 5-FU. After transfection of miR-19b, specific mRNAs were recruited to microRNA:mRNA complexes isolated with Ago2 antibody and subjected to whole-genome transcriptional analysis. In this analysis, 66 target mRNAs were enriched by at least 5.0-fold in the microRNA:mRNA complexes from DLD-1 resistant cells. Ingenuity pathway analysis of mRNA targets significantly (P < 0.05) indicated the category "Cell Cycle" as a probable area of the molecular and cellular function related with 5-FU resistance. Among candidate mRNA targets, SFPQ and MYBL2 have been linked to cell cycle functions. CONCLUSIONS: We revealed up-regulation of miR-19b in response to 5-FU and potential targets of miR-19b mediating the cell cycle under treatment with 5-FU. Our study provides an important insight into the mechanism of 5-FU resistance in colorectal cancers.
Kiyoshi Masuda, Yuki Kuwano, Kensei Nishida and Kazuhito Rokutan : General RBP expression in human tissues as a function of age., Ageing Research Reviews, Vol.11, No.4, 423-431, 2012.
(要約)
Gene expression patterns vary dramatically in a tissue-specific and age-dependent manner. RNA-binding proteins that regulate mRNA turnover and/or translation (TTR-RBPs) critically affect the subsets of expressed proteins. Although many proteins implicated in age-related processes are encoded by mRNAs that are targets of TTR-RBPs, very little is known regarding the tissue- and age-dependent expression of TTR-RBPs in humans. Recent analysis of TTR-RBPs expression using human tissue microarray has provided us interesting insight into their possibly physiologic roles as a function of age. This analysis has also revealed striking discrepancies between the levels of TTR-RBPs in senescent human diploid fibroblasts (HDFs), widely used as an in vitro model of aging, and the levels of TTR-RBPs in tissues from individuals of advancing age. In this article, we will review our knowledge of human TTR-RBP expression in different tissues as a function of age.
Yuki Kuwano, Yoko Kamio, Tomoko Kawai, Sakurako Katsuura, Naoko Inada, Akiko Takaki and Kazuhito Rokutan : Autism-associated gene expression in peripheral leucocytes commonly observed between subjects with autism and healthy women having autistic children., PLoS ONE, Vol.6, No.9, e24723, 2011.
(要約)
Autism spectrum disorder (ASD) is a severe neuropsychiatric disorder which has complex pathobiology with profound influences of genetic factors in its development. Although the numerous autism susceptible genes were identified, the etiology of autism is not fully explained. Using DNA microarray, we examined gene expression profiling in peripheral blood from 21 individuals in each of the four groups; young adults with ASD, age- and gender-matched healthy subjects (ASD control), healthy mothers having children with ASD (asdMO), and asdMO control. There was no blood relationship between ASD and asdMO. Comparing the ASD group with control, 19 genes were found to be significantly changed. These genes were mainly involved in cell morphology, cellular assembly and organization, and nerve system development and function. In addition, the asdMO group possessed a unique gene expression signature shown as significant alterations of protein synthesis despite of their nonautistic diagnostic status. Moreover, an ASD-associated gene expression signature was commonly observed in both individuals with ASD and asdMO. This unique gene expression profiling detected in peripheral leukocytes from affected subjects with ASD and unaffected mothers having ASD children suggest that a genetic predisposition to ASD may be detectable even in peripheral cells. Altered expression of several autism candidate genes such as FMR-1 and MECP2, could be detected in leukocytes. Taken together, these findings suggest that the ASD-associated genes identified in leukocytes are informative to explore the genetic, epigenetic, and environmental background of ASD and might become potential tools to assess the crucial factors related to the clinical onset of the disorder.
Yoko Akaike, Ken Kurokawa, Keisuke Kajita, Yuki Kuwano, Kiyoshi Masuda, Kensei Nishida, Seung Kang Wan, Toshihito Tanahashi and Kazuhito Rokutan : Skipping of an alternative intron in the srsf1 3' untranslated region increases transcript stability., The Journal of Medical Investigation : JMI, Vol.58, No.3-4, 180-187, 2011.
(要約)
The srsf1 gene encodes serine/arginine-rich splicing factor 1 (SRSF1) that participates in both constitutive and alternative splicing reactions. This gene possesses two ultraconserved elements in the 3' untranslated region (UTR). Skipping of an alternative intron between the two elements has no effect on the protein-coding sequence, but it generates a premature stop codon (PTC)-containing mRNA isoform, whose degradation is considered to depend on nonsense-mediated mRNA decay (NMD). However, several cell lines (HCT116, RKO, HeLa, and WI38 cells) constitutively expressed significant amounts of the srsf1 PTC variant. HCT116 cells expressed the PTC variant nearly equivalent to the major isoform that includes the alternative intron in the 3' UTR. Inhibition of NMD by silencing a key effecter UPF1 or by treatment with cycloheximide failed to increase amounts of the PTC variant in HCT116 cells, and the PTC variant was rather more stable than the major isoform in the presence of actinomycin D. Our results suggest that the original stop codon may escape from the NMD surveillance even in skipping of the alternative intron. The srsf1 gene may produce an alternative splice variant having truncated 3' UTR to relief the microRNA- and/or RNA-binding protein-mediated control of translation or degradation. J. Med. Invest. 58: 180-187, August, 2011.
Sakurako Katsuura, Yoshiko Kamezaki, Naoko Yamagishi, Yuki Kuwano, Kensei Nishida, Kiyoshi Masuda, Toshihito Tanahashi, Tomoko Kawai, Kokichi Arisawa and Kazuhito Rokutan : Circulating vascular endothelial growth factor is independently and negatively associated with trait anxiety and depressive mood in healthy Japanese university students., International Journal of Psychophysiology, Vol.81, No.1, 38-43, 2011.
(要約)
Anxiety and depressive mood are sometimes accompanied by modulation of neuroendocrine and immune functions. The aim of this study was to identify circulating immune mediators reflecting anxiety and depressive mood in healthy young adults. Anxiety and depressive mood in 209 healthy medical students (125 males and 84 females, aged 20.7±2.7years (mean±SD)) were assessed by the Spielberger state-trait anxiety inventory (STAI) and the Zung self-rating depression scale (Zung-SDS), respectively. Cortisol and chromogranin A (CgA) levels in saliva were measured using enzyme immunoassay kits, and 50 different mediators in sera were measured by a multiplex-suspension array system. The level of statistical significance was set at =0.05. Forty-four mediators were measurable in sera, and each mediator showed substantial individual variations. After determining Pearson correlation coefficients, we selected candidate cytokines whose levels were associated with STAI-state (2 cytokines), STAI-trait (8 cytokines), or SDS scores (8 cytokines). The candidate cytokines plus interleukin (IL)-1, IL-6, tumor necrosis factor-, and macrophage migration inhibitory factor were then subjected to multiple regression analysis adjusted for gender, BMI, and salivary concentrations of cortisol and CgA. Vascular endothelial growth factor (VEGF) was independently and negatively associated with both trait anxiety (p<0.05) and depressive mood (p<0.01). IL-1 showed independently positive association with depressive mood (p<0.05). Interactions between these two cytokines and gender or BMI were not observed. Besides IL-1, circulating VEGF may be a potential biomarker for negative mood states in healthy young adults.
(キーワード)
Adolescent / Anxiety / Body Mass Index / 細胞質分裂 (cytokinesis) / Depressive Disorder / Endothelial Growth Factors / 女性 (female) / Humans / Hydrocortisone / 日本 (Japan) / 男性 (male) / Psychiatric Status Rating Scales / Questionnaires / Regression Analysis / Saliva / Sex Factors / Students / Universities / Vascular Endothelial Growth Factor A / Young Adult
Ken Kurokawa, Toshihito Tanahashi, Akiho Murata, Yoko Akaike, Sakurako Katsuura, Kensei Nishida, Kiyoshi Masuda, Yuki Kuwano, Tomoko Kawai and Kazuhito Rokutan : Effects of chronic academic stress on mental state and expression of glucocorticoid receptor α and β isoforms in healthy Japanese medical students., Stress, Vol.14, No.4, 431-438, 2011.
(要約)
Chronic academic stress responses were assessed by measuring mental state, salivary cortisol levels, and the glucocorticoid receptor (GR) gene expression in healthy Japanese medical students challenging the national medical license examination. Mental states of 17 male and 9 female medical undergraduates, aged 25.0 ± 1.2 years (mean ± SD), were assessed by the State and Trait Anxiety Inventory (STAI) and the Self-Rating Depression Scale (SDS) 2 months before, 2 days before, and 1 month after the examination. At the same time points, saliva and blood were collected. STAI-state scores peaked 2 days before the examination. Scores on STAI-trait and SDS, and salivary cortisol levels were consistently higher during the pre-examination period. One month after the examination, all these measures had significantly decreased to baseline levels. Real-time reverse transcription PCR showed that this chronic anxious state did not change the expression of the functional GRα mRNA isoform in peripheral leukocytes, while it resulted in reduced expression of the GRβ isoform 2 days before the examination. Our results replicate and extend a significant impact of chronic academic stressors on the mental state of healthy Japanese medical students and suggest a possible association of GRβ gene in response to psychological stress.
(キーワード)
Adult / Asian Continental Ancestry Group / Female / Humans / Hydrocortisone / Male / Protein Isoforms / RNA, Messenger / Receptors, Glucocorticoid / 唾液腺 (salivary glands) / Stress, Psychological / Students, Medical
The human genome sequence has been decoded, and the more complicated regulation of gene function is revealed in the post-genome era. In the various mechanisms of epigenome, RNA dramatically controls gene expression through the various post-transcriptional processing including transcription, splicing, cap addition, polyadenylation, nuclear export, translation. Especially, the alternative splicing is involved in all of those post-transcriptional regulations, as well as splicing of pre-mRNA. However, there were few reports, how the alternative splicing contributes to the regulations of cellular functions because of its difficulty of the analysis. This review discusses the molecular mechanism of alternative splicing and its regulator ; Serine/arginine-rich splicing factor (SRSF). We also discuss how the SRSF genes sustain their own proper expressions and functions.
Stress plays an important role in both mental and physical problems. Stressful life events initiate a coordinated physiological process that is produced by interactions between the hypothalamuspituitary- adrenal axis, sympathetic nervous system, and immune system. The response to psychological stress varies considerably and depends on a wide range of environmental and genetic factors. Establishment of a new biomental tool for objectively assessing stress response is required. We focus on high-throughput analysis of gene expression using microarray system to study the complex stress responses. Alternative splicing(AS)regulates the gene expression program in response to surrounding environment. However, acute psychological stress-initiated AS events have not been documented yet. Academic examinations are one of the brief naturalistic stressors and have been shown to change gene expression in peripheral leukocytes, which is postulated to be involved in the psychological response. Using the GeneChip human exon1．0ST array, AS events of 27 genes with splicing indices >1．0could be detected immediately after the examination among healthy university students. Real-time reverse transcription PCR validated the stress-initiated skipping of exon 63 of SMG -1 that encodes a phosphatidylinositol 3-kinase-related protein kinase crucial for activations of p 53-dependent pathways and mRNA decay system. These results indicate that AS mediated regulation of gene expression in response to brief naturalistic stressors in peripheral leukocytes, and suggest the SMG -1 splice variant as a potential biomarker for acute psychological stress.
Ken Kurokawa, Yuki Kuwano, Kumiko Tominaga, Tomoko Kawai, Sakurako Katsuura, Naoko Yamagishi, Yuzuru Satake, Keisuke Kajita, Toshihito Tanahashi and Kazuhito Rokutan : Brief naturalistic stress induces an alternative splice variant of SMG-1 lacking exon 63 in peripheral leukocytes., Neuroscience Letters, Vol.484, No.2, 128-132, 2010.
(要約)
Alternative splicing (AS) not only regulates the gene expression program in response to surrounding environment, but also produces protein isoforms with unique properties under stressful conditions. However, acute psychological stress-initiated AS events have not been documented in human studies. After assessments of changes in salivary cortisol levels and anxiety among 28 fourth-grade medical students 7 weeks prior to, 1 day before, immediately after, and 1 week after an examination for promotion, we selected 5 male students, who showed a typical stress response, and screened AS events in their circulating leukocytes using the GeneChip human exon 1.0 ST array. AS events of 27 genes with splicing indices >1.0 could be detected between immediately after and either 7 weeks before, 1 day before, or 1 week after the examination. The examination stress preferentially caused skipping rather than inclusion: 21 out of the 27 pre-mRNAs underwent skipping of exons, and skipping in 3'UTR was observed in 8 genes. Among the candidate genes, real-time reverse transcription PCR validated the stress-initiated skipping of exon 63 of SMG-1 that encodes a phosphatidylinositol 3-kinase-related protein kinase crucial for activations of p53-dependent pathways and nonsense-mediated mRNA decay. Our results indicate a significant impact of brief naturalistic stressors on AS-mediated regulation of gene expression in peripheral leukocytes, and suggest the SMG-1 splice variant as a potential biomarker for acute psychological stress.
(キーワード)
Alternative Splicing / Analysis of Variance / Exons / Female / Gene Expression Profiling / Gene Expression Regulation / Humans / Hydrocortisone / Leukocytes / Male / Oligonucleotide Array Sequence Analysis / Phosphatidylinositol 3-Kinases / Psychiatric Status Rating Scales / Salvia / Stress, Psychological / Young Adult
Sakurako Katsuura, Yoshiko Kamezaki, Kumiko Tominaga, Kiyoshi Masuda, Kensei Nishida, Yuta Yamamoto, Keiko Takeo, Naoko Yamagishi, Toshihito Tanahashi, Tomoko Kawai and Kazuhito Rokutan : High-throughput screening of brief naturalistic stress-responsive cytokines in university students taking examinations., International Journal of Psychophysiology, Vol.77, No.2, 135-140, 2010.
(要約)
This study was designed to prospectively examine the impact of a brief naturalistic stressor (academic examination) on salivary/serum cortisol, measures of anxiety and depressive mood, and 50 circulating immune mediators assessed 7 days before, the first day of, and 2 days after the first term examination period (5 days) among 20 male and 6 female medical students (19.7+/-3.1 years, mean+/-SD). Of 42 serum factors detected, repeated measures ANOVA and Bonferroni post hoc testing indicated that concentrations of macrophage migration inhibitory factor (MIF), monocyte chemoattractant protein (MCP)-3, and beta-nerve growth factor (beta-NGF) were significantly decreased 2 days after finishing examinations, compared with the levels on the first day of examinations (p<0.05) in association with a concomitant post-examination decreases (p<0.05) in anxiety and salivary cortisol levels. In contrast, interleukin (IL)-16 was reciprocally increased between the two time points (p<0.05). However, after correction for multiple comparisons, only changes in MIF were significant (p<0.05/42=0.00119), and MIF levels peaked on the first day of examinations was significantly higher than those measured both 7 days before and 2 days after the examination. The present high-throughput analysis with multiplex cytokine panels reconfirms the impact of brief naturalistic stressors on immune outcomes, and suggests a potential role of MIF in the acute stress response.
Mai Kamizato, Kensei Nishida, Kiyoshi Masuda, Keiko Takeo, Yuta Yamamoto, Tomoko Kawai, Shigetada Teshima-Kondo, Toshihito Tanahashi and Kazuhito Rokutan : Interleukin 10 inhibits interferon gamma- and tumor necrosis factor alpha-stimulated activation of NADPH oxidase 1 in human colonic epithelial cells and the mouse colon., Journal of Gastroenterology, Vol.44, No.12, 1172-1184, 2009.
(要約)
BACKGROUND: NADPH oxidase 1 (Nox1) is preferentially expressed in the colon, but its functional role is not fully understood. This study was designed to elucidate a potential role of Nox1 in inflammation of the colon. METHODS: Superoxide production by T84 cells was measured by the cytochrome c method. Protein and mRNA levels of Nox1 and Nox organizer 1 (NOXO1) in the cells were measured by real-time reverse transcriptase PCR and Western blotting, respectively. Expression of Nox1, Nox2, dual oxidase 2 (Duox2), NOXO1, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha mRNAs was measured in proximal, middle, and distal portions of colonic mucosas from male wild-type C57BL/6J and interleukin (IL)-10 knockout mice at 6, 10, and 16 weeks of age. Grading of inflammation was done by scoring histological changes. RESULTS: IL-10 significantly inhibited IFN-gamma- or TNF-alpha-induced up-regulation of superoxide-producing activity in T84 cells by suppressing expression of Nox1 mRNA and protein. IL-10 also inhibited TNF-alpha-stimulated induction of NOXO1 and p38 MAPK phosphorylation. Levels of Nox1, but not Nox2 or Duox2 mRNA, was age-dependently increased following a gradient with low levels in the proximal colon and high levels in the distal colon of the wild-type mice. The absence of IL-10 significantly facilitated Nox1 expression in association with increased IFN-gamma mRNA expression before the development of spontaneous colitis and age-dependently accelerated their mRNA expression. CONCLUSIONS: IL-10 may be a possible down-regulator of the Nox1-based oxidase in the colon, suggesting a potential role of reactive oxygen species (ROS) derived from Nox1-based oxidase in inflammation of the colon.
Satoru Dakeshita, Tomoko Kawai, Hirokazu Uemura, Mineyoshi Hiyoshi, Etsuko Oguma, Hyogo Horiguchi, Fujio Kayama, Keiko Aoshima, Satoshi Shirahama, Kazuhito Rokutan and Kokichi Arisawa : Gene expression signatures in peripheral blood cells from Japanese women exposed to environmental cadmium, Toxicology, Vol.257, No.1,2, 25-32, 2009.
(要約)
The objective of this study was to examine the effects of environmental cadmium (Cd) exposure on the gene expression profile of peripheral blood cells, using an original oligoDNA microarray. The study population consisted of 20 female residents in a Cd-polluted area (Cd-exposed group) and 20 female residents in a non-Cd-polluted area individually matched for age (control group). The mRNA levels in Cd-exposed subjects were compared with those in respective controls, using a microarray containing oligoDNA probes for 1867 genes. Median Cd concentrations in blood (3.55 microg/l) and urine (8.25 microg/g creatinine) from the Cd-exposed group were 2.4- and 1.9-times higher than those of the control group, respectively. Microarray analysis revealed that the Cd-exposed group significantly up-regulated 137 genes and down-regulated 80 genes, compared with the control group. The Ingenuity Pathway Analysis Application (IPA) revealed that differentially expressed genes were likely to modify oxidative stress and mitochondria-dependent apoptosis pathways. Among differentially expressed genes, the expression of five genes was positively correlated with Cd concentrations in blood or urine. Quantitative real-time PCR (RT-PCR) analysis validated the significant up-regulation of CASP9, TNFRSF1B, GPX3, HYOU1, SLC3A2, SLC19A1, SLC35A4 and ITGAL, and down-regulation of BCL2A1 and COX7B. After adjustment for differences in the background characteristics of the two groups, we finally identified seven Cd-responsive genes (CASP9, TNFRSF1B, GPX3, SLC3A2, ITGAL, BCL2A1, and COX7B), all of which constituted a network that controls oxidative stress response by IPA. These seven genes may be marker genes useful for the health risk assessment of chronic low level exposure to Cd.
Sachiko Chikahisa, Kumiko Tominaga, Tomoko Kawai, Kazuyoshi Kitaoka, Katsutaka Oishi, Norio Ishida, Kazuhito Rokutan and Hiroyoshi Sei : Bezafibrate, a PPARs agonist, decreases body temperature and enhances EEG delta oscillation during sleep in mice., Endocrinology, Vol.149, No.10, 5262-5271, 2008.
(要約)
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor family. PPARs play a critical role in lipid and glucose metabolism. We examined whether chronic treatment with bezafibrate, a PPAR agonist, would alter sleep and body temperature (BT). Mice fed with a control diet were monitored for BT, electroencephalogram (EEG), and electromyogram for 48 h under light-dark conditions. After obtaining the baseline recording, the mice were provided with bezafibrate-supplemented food for 2 wk, after which the same recordings were performed. Two-week feeding of bezafibrate decreased BT, especially during the latter half of the dark period. BT rhythm and sleep/wake rhythm were phase advanced about 2-3 h by bezafibrate treatment. Bezafibrate treatment also increased the EEG delta-power in nonrapid eye movement sleep compared with the control diet attenuating its daily amplitude. Furthermore, bezafibrate-treated mice showed no rebound of EEG delta-power in nonrapid eye movement sleep after 6 h sleep deprivation, whereas values in control mice largely increased relative to baseline. DNA microarray, and real-time RT-PCR analysis showed that bezafibrate treatment increased levels of Neuropeptide Y mRNA in the hypothalamus at both Zeitgeber time (ZT) 10 and ZT22, and decreased proopiomelanocortin-alpha mRNA in the hypothalamus at ZT10. These findings demonstrate that PPARs participate in the control of both BT and sleep regulation, which accompanied changes in gene expression in the hypothalamus. Activation of PPARs may enhance deep sleep and improve resistance to sleep loss.
Yuki Kuwano, Kumiko Tominaga, Tsukasa Kawahara, Hidekazu Sasaki, Keiko Takeo, Kensei Nishida, Kiyoshi Masuda, Tomoko Kawai, Shigetada Teshima-Kondo and Kazuhito Rokutan : Tumor necrosis factor alpha activates transcription of the NADPH oxidase organizer 1 (NOXO1) gene and upregulates superoxide production in colon epithelial cells., Free Radical Biology and Medicine, Vol.45, No.12, 1642-1652, 2008.
(要約)
NADPH oxidase 1 (Nox1) is a multicomponent enzyme consisting of p22(phox), Nox organizer 1 (NOXO1), Nox1 activator 1, and Rac1. Interleukin-1beta, flagellin, interferon-gamma, and tumor necrosis factor alpha (TNF-alpha) similarly induced Nox1 in a colon cancer cell line (T84), whereas only TNF-alpha fully induced NOXO1 and upregulated superoxide-producing activity by ninefold. This upregulation was canceled by knockdown of NOXO1 with small interfering RNAs. TNF-alpha rapidly phosphorylated p38 mitogen-activated protein kinase and c-Jun N-terminal kinase 1/2, followed by phosphorylation of c-Jun and c-Fos and appearance of an AP-1 binding activity within 30 min. We cloned the 5' flank of the human NOXO1 gene (-3888 to +263 bp), and found that the region between -585 and -452 bp, which contains consensus elements of YY-1, AP-1, and Ets, and the GC-rich region encoding three putative binding sites for SP-1, was crucial for TNF-alpha-dependent promoter activity. Serial mutation analysis of the elements identified an AP-1 binding site (from -561 to -551 bp, agtAAGtcatg) as a crucial element for TNF-alpha-stimulated transcription of the human NOXO1 gene, which was also confirmed by the AP-1 decoy experiments. Thus, TNF-alpha acts as a potent activator of Nox1-based oxidase in colon epithelial cells, suggesting a potential role of this oxidase in inflammation of the colon.
Takuya Saiki, Tomoko Kawai, Kyoko Morita, Masayuki Ohta, Toshiro Saito, Kazuhito Rokutan and Nobutaro Ban : Identification of marker genes for differential diagnosis of chronic fatigue syndrome., Molecular Medicine, Vol.14, No.9-10, 599-607, 2008.
(要約)
Chronic fatigue syndrome (CFS) is a clinically defined condition characterized by long-lasting disabling fatigue. Because of the unknown mechanism underlying this syndrome, there still is no specific biomarker for objective assessment of the pathological fatigue. We have compared gene expression profiles in peripheral blood between 11 drug-free patients with CFS and age- and sex-matched healthy subjects using a custom microarray carrying complementary DNA probes for 1,467 stress-responsive genes. We identified 12 genes whose mRNA levels were changed significantly in CFS patients. Of these 12 genes, quantitative real-time PCR validated the changes in 9 genes encoding granzyme in activated T or natural killer cells (GZMA), energy regulators (ATP5J2, COX5B, and DBI), proteasome subunits (PSMA3 and PSMA4), putative protein kinase c inhibitor (HINT ), GTPase (ARHC), and signal transducers and activators of transcription 5A (STAT5A). Next, we performed the same microarray analysis on 3 additional CFS patients and 20 other patients with the chief complaint of long-lasting fatigue related to other disorders (non-CFS patients) and found that the relative mRNA expression of 9 genes classified 79% (11/14) of CFS and 85% (17/20) of the non-CFS patients. Finally, real-time PCR measurements of the levels of the 9 involved mRNAs were done in another group of 18 CFS and 12 non-CFS patients. The expression pattern correctly classified 94% (17/18) of CFS and 92% (11/12) of non-CFS patients. Our results suggest that the defined gene cluster (9 genes) may be useful for detecting pathological responses in CFS patients and for differential diagnosis of this syndrome.
Hirofumi Kanemura, Kenji Kusumoto, Hidenori Miyake, Seiki Tashiro, Kazuhito Rokutan and Mitsuo Shimada : Geranylgeranylacetone prevents acute liver damage after massive hepatectomy in rats through suppression of a CXC chemokine GRO1 and induction of heat shock proteins., Journal of Gastrointestinal Surgery, Vol.13, No.1, 66-73, 2008.
(要約)
BACKGROUND AND METHODS: Acute liver failure after massive hepatectomy remains a challenging problem. In this study, using a microarray designed to monitor the side effects of drugs, we examined changes in gene expression in the remnant liver during the 24 h after hepatectomy and the effects of a nontoxic heat shock protein (HSP) 70 inducer, geranylgeranylacetone (GGA), after 90% hepatectomy in rats. RESULTS: A single oral administration of 100 mg/kg GGA significantly suppressed the release of aminotransferases and improved survival compared with vehicle administration. The hepatectomy upregulated 74 genes and downregulated 95. Interestingly, ten cytokine genes were upregulated, while no cytokine-related gene was downregulated. Among the ten cytokine genes, a potent chemoattractant for neutrophils, GRO1, was most rapidly and markedly upregulated after 90% hepatectomy. GGA effectively suppressed the up-regulation of GRO1 messenger ribonucleic acid, and this was validated by Northern hybridization. Microarray and immunoblot analyses showed that, in addition to HSP70 and HSP27, GGA preferentially induced an endoplasmic reticulum chaperone, BIP. CONCLUSION: Considering hemodynamic and metabolic overloading as a primary cause of acute lever failure, the ER stress response enhanced by GGA may also play an important role in the prevention of overload-induced liver damage.
Eun Ju Jung, Tomoko Kawai, Hwan Ki Park, Yoshiaki Kubo, Kazuhito Rokutan and Seiji Arase : Identification of ultraviolet B-sensitive genes in human peripheral blood cells., The Journal of Medical Investigation : JMI, Vol.55, No.3-4, 204-210, 2008.
(要約)
Ultraviolet B (UVB) is a serious irritant for the skin and increases a risk for skin cancer. To identify UVB-sensitive genes in peripheral blood, 11 healthy male volunteers were exposed to 0.3 J/cm(2) of narrow-band (NB)-UVB, about half of minimal erythema dose (MED) in Japanese, and gene expression in blood was analyzed at 4 h, 24 h, 4 d and 7 d after the irradiation using microarray carrying oligonucleotide probes for 2,000 stress-responsive genes. RNA prepared before the irradiation was used as a reference control. Microarray analysis identified 21 genes as UVB-responsive genes with a peak at 24 h in 6 subjects, and real-time PCR validated the significant down-regulation of 9 (ABCB10, ATF1, ABCD3, TANK, FAS, SLC30A9, CHUK, CASP1, and ABCE1) out of the 21 genes in 11 subjects. Considering sensitive and characteristic features of 9 marker genes, they may be useful indicators for monitoring systemic response to UVB irradiation.
Kazuhito Rokutan, Tsukasa Kawahara, Yuki Kuwano, Kumiko Tominaga, Keisei Nishida and Shigetada Teshima-Kondo : Nox enzymes and oxidative stress in the immunopathology of the gastrointestinal tract., Seminars in Immunopathology, Vol.30, No.3, 315-327, 2008.
(要約)
Chronic inflammation caused by Helicobacter pylori infection or inflammatory bowel disease (IBD) is closely linked to cancer development. Innate immune abnormalities and enhanced production of reactive oxygen species through a phagocyte NADPH oxidase (Nox2) are key issues in understanding the pathogenesis of inflammation-dependent carcinogenesis. Besides Nox2, functionally distinct homologues (Nox1, Nox3, Nox4, Nox5, Duox1, and Duox2) have been identified. Nox1 and Duox2 are highly expressed in the gastrointestinal tract. Although the functional roles of Nox/Duox in the gastrointestinal tract are still unclear, we will review their potential roles in the gastrointestinal immunopathology, particularly in H. pylori-induced inflammation, IBD, and malignancy.
Kiyoshi Masuda, Shigetada Teshima-Kondo, Mina Mukaijo, Naoko Yamagishi, Yoshiko Nishikawa, Kensei Nishida, Tomoko Kawai and Kazuhito Rokutan : A novel tumor-promoting function residing in the 5' non-coding region of vascular endothelial growth factor mRNA., PLoS Medicine, Vol.5, No.5, e94, 2008.
(要約)
BACKGROUND: Vascular endothelial growth factor-A (VEGF) is one of the key regulators of tumor development, hence it is considered to be an important therapeutic target for cancer treatment. However, clinical trials have suggested that anti-VEGF monotherapy was less effective than standard chemotherapy. On the basis of the evidence, we hypothesized that vegf mRNA may have unrecognized function(s) in cancer cells. METHODS AND FINDINGS: Knockdown of VEGF with vegf-targeting small-interfering (si) RNAs increased susceptibility of human colon cancer cell line (HCT116) to apoptosis caused with 5-fluorouracil, etoposide, or doxorubicin. Recombinant human VEGF165 did not completely inhibit this apoptosis. Conversely, overexpression of VEGF165 increased resistance to anti-cancer drug-induced apoptosis, while an anti-VEGF165-neutralizing antibody did not completely block the resistance. We prepared plasmids encoding full-length vegf mRNA with mutation of signal sequence, vegf mRNAs lacking untranslated regions (UTRs), or mutated 5'UTRs. Using these plasmids, we revealed that the 5'UTR of vegf mRNA possessed anti-apoptotic activity. The 5'UTR-mediated activity was not affected by a protein synthesis inhibitor, cycloheximide. We established HCT116 clones stably expressing either the vegf 5'UTR or the mutated 5'UTR. The clones expressing the 5'UTR, but not the mutated one, showed increased anchorage-independent growth in vitro and formed progressive tumors when implanted in athymic nude mice. Microarray and quantitative real-time PCR analyses indicated that the vegf 5'UTR-expressing tumors had up-regulated anti-apoptotic genes, multidrug-resistant genes, and growth-promoting genes, while pro-apoptotic genes were down-regulated. Notably, expression of signal transducers and activators of transcription 1 (STAT1) was markedly repressed in the 5'UTR-expressing tumors, resulting in down-regulation of a STAT1-responsive cluster of genes (43 genes). As a result, the tumors did not respond to interferon (IFN)alpha therapy at all. We showed that stable silencing of endogenous vegf mRNA in HCT116 cells enhanced both STAT1 expression and IFNalpha responses. CONCLUSIONS: These findings suggest that cancer cells have a survival system that is regulated by vegf mRNA and imply that both vegf mRNA and its protein may synergistically promote the malignancy of tumor cells. Therefore, combination of anti-vegf transcript strategies, such as siRNA-based gene silencing, with anti-VEGF antibody treatment may improve anti-cancer therapies that target VEGF.
Sawako Kinouchi, Junichi Iga, Shu-ichi Ueno, Ken Yamauchi, Shusuke Numata, Hongwei Song, Satsuki Sumitani, Sumiko Shibuya-Tayoshi, Mari Haku, Toshiyuki Yasui, Minoru Irahara, Kyoko Morita, Kazuhito Rokutan and Tetsuro Ohmori : FKBP5, SERT and COMT mRNA expressions in the peripheral leukocytes during menstruation cycle in healthy reproductive females., Neuroscience Letters, Vol.434, No.1, 124-128, 2008.
(要約)
There have been several evidences that the mRNA expressions in the peripheral leukocytes may indicate not only physical but also psychological states. The purpose of this study is whether the mRNA expressional changes in the leukocytes are related to the mental states across the menstrual cycle in reproductive healthy female subjects. Thirty-eight female subjects (22.4+/-1.4 year-old) were participated in this study at three menstruation cycle periods (menstrual, follicular and luteal phase). The FKBP5 (FK506-binding protein gene), SERT (serotonin transporter gene) and COMT (catechol-o-methyltransferase gene) mRNA expressions in the leukocytes were determined with hormonal data. The psychological changes were assessed with self-rating hospital anxiety and depression scale (HADS). Only one thirds of subjects (n=12) had regular menstrual cycles during the experiment. So we analyzed the data from these 12 subjects. The anxiety score of each subject was changed across the menstrual cycle (Friedman test: P<0.05). The FKBP5 mRNA expression was significantly lower in the follicular phase than in the other phases but no changes were seen in either SERT or COMT mRNA expressions among the phases. In conclusion, there are differences of HADS anxiety score and FKBP5 mRNA expression in the leukocytes across the menstrual cycle but there is no correlation between anxiety scores and FKBP5 mRNA.
Tomoko Kawai, Kyoko Morita, Kiyoshi Masuda, Kensei Nishida, Michiyo Shikishima, Masayuki Ohta, Toshiro Saito and Kazuhito Rokutan : Gene expression signature in peripheral blood cells from medical students exposed to chronic psychological stress., Biological Psychology, Vol.76, No.3, 147-155, 2007.
(要約)
To assess response to chronic psychological stress, gene expression profiles in peripheral blood from 18 medical students confronting license examination were analyzed using an original microarray. Total RNA was collected from each subject 9 months before the examination and mixed to be used as a universal control. At that time, most students had normal scores on the state-trait anxiety inventory (STAI). However, STAI scores were significantly elevated at 2 months and at 2 days before the examination. Pattern of the gene expression profile was more uniform 2 days before than 2 months before the examination. We identified 24 genes that significantly and uniformly changed from the universal control 2 days before the examination. Of the 24 genes, real-time PCR validated changes in mRNA levels of 10 (PLCB2, CSF3R, ARHGEF1, DPYD, CTNNB1, PPP3CA, POLM, IRF3, TP53, and CCNI). The identified genes may be useful to assess chronic psychological stress response.
Tomoko Kawai, Kyoko Morita, Kiyoshi Masuda, Kensei Nishida, Atsuo Sekiyama, Shigetada Kondo, Masayuki Ohta, Toshiro Saito and Kazuhito Rokutan : Physical exercise-associated gene expression signatures in peripheral blood., Clinical Journal of Sport Medicine, Vol.17, No.5, 375-383, 2007.
(要約)
To assess response to physical stress, gene expression profiles in peripheral blood cells were analyzed using an original microarray carrying 1467 stress-responsive complementary DNA probes. Gene expression was analyzed at 4, 24, and 48 hours after exercising on a cycle ergometer at 60% VO2 max for 1 hour (aerobic exercise) or until exhausted (exhaustive exercise). Institute of Health Biosciences, University of Tokushima Graduate School. Twelve healthy male students of the postgraduate or undergraduate school. The volunteers performed the aerobic or exhaustive exercise on a cycle ergometer. Detection of aerobic exercise-responsive or exhaustive exercise-responsive genes in peripheral blood cells. Aerobic and exhaustive exercise transiently changed the expression of 21 and 16 genes, respectively, with the peak at 4 hours. Only 2 genes significantly responded to both types of exercise. Exhaustive but not aerobic exercise produced a secondary response with significantly altered expression of 14 genes at 24 hours. Five of those genes encode receptors for neurotransmitters (HTR1A, CHRNB2, GABRB3, GABRG3, and LOC51289). The behavior of the individual genes shown here may be informative to objectively assess acute physical stress and exhaustion-associated responses.
Fusako Teramoto, Kazuhito Rokutan, Yasuko Sugano, Kazuyuki Oku, Eriko Kishino, Koki Fujita, Kozo Hara, Kyouichi Kishi, Masao Fukunaga and Tetsuro Morita : Long-term administration of 4G-beta-D-galactosylsucrose (lactosucrose) enhances intestinal calcium absorption in young women: a randomized, placebo-controlled 96-wk study., Journal of Nutritional Science and Vitaminology, Vol.52, No.5, 337-346, 2006.
(要約)
This study determined the effect of long-term administration of 4(G)-beta-D-galactosylsucrose (lactosucrose; LS) on intestinal calcium absorption. In a randomized, single-blind, parallel-group study, LS (n=9, 6.0 g twice daily) or a placebo (maltose; n=8, 6.0 g twice daily) was administered to healthy young women for 92 wk: the study also included a 4-wk post-administration period. All participants completed the study. Dietary nutrient intake; fecal weight, pH, and moisture content; fecal concentrations of short-chain fatty acids (SCFA), putrefactive products, ammonia, and minerals (calcium, magnesium, phosphorus, and iron); and serum calcium and osteocalcin concentrations were measured every 24 wk. Urinary pyridinoline (PYR) and deoxypyridinoline (DPD), and urinary calcium excretion were measured every 12 wk. Significant effects of oligosaccharide treatment, time, and the interaction between oligosaccharide treatment and time were observed for fecal pH, SCFA, ammonia, and putrefactive product values (p<0.05). Fecal pH, ammonia, and putrefactive product values decreased in the LS group, and the fecal SCFA concentration significantly increased during the administration period; these changes were not observed 4 wk post-administration. To examine the mineral balance of calcium, magnesium, and phosphorus in detail, all the participants completed a 6-d mineral balance study, sometime between week 56 and 60 of the longer study. During the mineral balance study, the daily calcium intake was set at 400 mg; all feces and urine were collected each day for 6 d after an 8-d acclimation period. In the balance study, fecal calcium excretion was significantly lower in the LS group than in the placebo group (p<0.05), and apparent calcium absorption and retention, apparent magnesium and phosphorus absorption, and magnesium retention were significantly higher in the LS group than in the placebo group (p<0.05). Our results suggest that the administration of LS produces a long-term enhancement of intestinal calcium absorption in healthy young women with lower than recommended calcium intakes.
Kazuhito Rokutan, Tsukasa Kawahara, Yuki Kuwano, Kumiko Tominaga, Atsuo Seiyama and Shigetada Teshima-Kondo : NADPH Oxidase in the gastrointestinal tract: A potential role of Nox1 in innate immune response and carcinogenesis., Antioxidants & Redox Signaling, Vol.8, No.9,10, 1573-1582, 2006.
(要約)
The gastrointestinal epithelium functions as physical and innate immune barriers against commensal or pathogenic microbes. NADPH oxidase 1 (Nox1) and dual oxidase 2 (Duox2), highly expressed in the colon, are suggested to play a potential role in host defense. Guinea-pig gastric pit cells and human colonic epithelial cells (T84 cells) express Nox1. With regard to activation of Nox1, the gastric epithelial cells are primed with Helicobacter pylori lipopolysaccharide, whereas T84 cells preferentially use the Toll-like receptor (TLR) 5, rather than TLR4, against Salmonella enteritidis infection. Thus, gastric and colonic epithelial cells may use different TLR members to discern pathogenicities among bacteria, depending on their environments and to activate Nox1 appropriately for host defense. Nox1-derived reactive oxygen species (ROS) have been implicated in the pathogenesis of inflammation-associated tumor development. The human stomach does not express Nox1. Helicobacter pylori infection alone does not induce it, whereas Nox1 is specifically expressed in gastric adenocarcinomas. In the human colon, Nox1 is differentiation-dependently expressed, and its expression is upregulated in adenomas and well-differentiated adenocarcinomas. Although Nox1 expression may not be directly linked to mitogenic activity, Nox1-derived ROS may exert a cancer-promoting effect by increasing resistance to programmed cell death of tumor cells.
Rie Shimooka, Yasuhiro Kido, Naoko Chiba, Junko Tanaka, Kazuhito Rokutan, Harumi Furochi, Katsuya Hirasaka, Takeshi Nikawa and Kyoichi Kishi : Soy protein diet prevents hypermethioninemia caused by portacaval shunt in rats., The Journal of Medical Investigation : JMI, Vol.53, No.3-4, 255-263, 2006.
(要約)
In hepatic disorders, abnormal plasma amino acid profiles are observed. In this study, we examined whether soy protein isolate (SPI) improved plasma methionine concentration in the model animals. Portacaval shunt (PCS) increased alanine aminotransferase (ALT) activity and methionine concentration in blood of rats fed a 40% casein diet supplemented with 0.6% methionine (casein-M diet). A 40% SPI diet supplemented with 1.28% methionine (SPI-M diet), which contained the same amount of methionine as that in 40% casein-M diet, normalized plasma ALT activity and methionine level in PCS rats. These effects of a SPI diet may be due to its amino acid composition, since an amino acid mixture diet mimicking a 40% SPI-M diet was also effective to hypermethioninemia of PCS rats. To find key enzymes for the beneficial effect of soy protein, we examined effects of a 40% SPI-M or casein-M diet on the activities of three methionine-metabolizing enzymes in liver of PCS rats. A SPI-M diet stimulated only the activity of cystathionine gamma-lyase, compared with a casein-M diet. A SPI diet has a preventive effect on hypermethioninemia, at least in part, by stimulating cystathionine gamma-lyase activity in liver and may be used for nutritional management of liver disorders with hypermethioninemia.
Junichi Iga, Shu-ichi Ueno, Ken Yamauchi, Shusuke Numata, Ikuyo Motoki, Sumiko Tayoshi, Sawako Kinouchi, Koshi Ohta, Hongwei Song, Kyoko Morita, Kazuhito Rokutan, Hirotaka Tanaba, Akira Sano and Tetsuro Ohmori : Gene expression and association analysis of LIM (PDLIM5) in major depression., Neuroscience Letters, Vol.400, No.3, 203-207, 2006.
(要約)
LIM (PDLIM5) is a small protein that interacts with protein kinase C-epsilon and the N-type calcium channel alpha-1B subunit and modulates neuronal calcium signaling. Recently, the LIM mRNA expression in postmortem brains and immortalized lymphoblastoid cells from mood disorder patients was reported to be changed and seems to be involved in its pathophysiology. We hypothesized that the expression of the LIM mRNA in the native peripheral leukocytes may be a good candidate for the biological marker for mood disorders. Twenty patients with major depression and age- and sex-matched control subjects were included in this expression study. The LIM mRNA levels in the peripheral leukocytes from drug-naive depressive patients were significantly lower than those from control subjects and increased significantly after 4-week paroxetine treatments, to almost the same level as controls'. Hamilton depressive scores (HAM-D) were improved about 50% after 4-week treatment but neither paroxetine concentrations nor the changes of HAM-D scores showed significant correlation with the change of the mRNA levels. Then, we genotyped three single nucleotide polymorphic markers of LIM gene, which were reported to be associated with bipolar disorder in patients with major depression and control subjects (n=130, each), but there were no associations between these SNPs and major depression. Our investigation indicates that the lower expression levels of LIM mRNA in the peripheral leukocytes are associated with the depressive state and that its recovery after treatment may be an adaptive change induced by the antidepressant.
Tadashi Wada, Hiroyoshi Sei, Kenji Kusumoto, Kazuyoshi Kitaoka, Sachiko Chikahisa, Kazuhito Rokutan and Yusuke Morita : Geranylgeranylacetone, an inducer of HSP 70, attenuates REM sleep rebound after sleep deprivation., Brain Research Bulletin, Vol.69, No.4, 388-392, 2006.
(要約)
The effect of pretreatment of geranylgeranylacetone (GGA), an inducer of heat shock protein (HSP) 70, on responses in sleep and core body temperature (Tcore) against sleep deprivation (SD) was examined in rats. After 3 days of GGA or vehicle injection, a 6-h period of SD was performed. During the recovery period, both rapid-eye movement (REM) and non-REM (NREM) sleep were increased in both GGA- and vehicle-injected rats. However, in GGA-injected rats, REM-sleep rebound was significantly suppressed, while NREM-sleep rebound remained unaffected. In addition, the increase of Tcore caused by SD was also attenuated in GGA-injected rats. In the hippocampus, both SD and the GGA pretreatment induced an increase in the expression of HSP70 mRNA, indicating that the SD functions as a stress for hippocampal neurons and that the GGA induces HSP70 expression. The findings suggest that pretreatment with GGA suppresses REM sleep rebound and the response of Tcore against SD.
Yuki Kuwano, Tsukasa Kawahara, Hironori Yamamoto, Shigetada Kondo, Kumiko Tominaga, Kiyoshi Masuda, Kyoichi Kishi, Kyoko Morita and Kazuhito Rokutan : Interferon-γ activates transcription of NADPH oxidase 1 gene and upregulates production of superoxide anion by human large intestinal epithelial cells, American Journal of Physiology, Cell Physiology, Vol.290, No.2, C433-C443, 2006.
(要約)
NADPH oxidase 1 (Nox1), a homolog of gp91(phox), is dominantly expressed in large intestinal epithelium, and reactive oxygen species derived from Nox1 are suggested to serve a role in host defense. We report that interferon (IFN)-gamma, a crucial transactivator of the gp91(phox) gene, also stimulates expression of Nox1 mRNA and protein in large intestinal epithelium (T84 cells), leading to fourfold upregulation of superoxide anion (O(2)(-)) generation. Introduction of small interfering Nox1 RNA completely blocked this priming. We cloned the region from -4,831 to +195 bp of the human Nox1 gene. To reveal IFN-gamma-responsive cis elements, we performed transient expression assays using a reporter gene driven by serially truncated Nox1 promoters in T84 cells. IFN-gamma-responsive elements were located between -4.3 and -2.6 kb, and one gamma-activated sequence (GAS) element present at -3,818 to -3,810 bp exhibited this IFN-gamma-dependent promoter activity. IFN-gamma caused tyrosine phosphorylation of signal transducer and activator of transcription 1 (STAT1) and produced a protein-GAS complex that was recognized by anti-STAT1 antibody. The introduction of three-point mutation of GAS, which did not interact with STAT1, completely canceled the IFN-gamma-dependent promoter activity of the region from -4,831 to +195 bp. A Janus protein tyrosine kinase 2 inhibitor (AG490) blocked the IFN-gamma-stimulated tyrosine phosphorylation of STAT1, promoter activity of the -4,831 to +195 bp region, Nox1 mRNA expression, and O(2)(-) production, also suggesting a crucial role of STAT1 and GAS in the IFN-gamma-stimulated transcription of the Nox1 gene. Our results support a potential contribution of Nox1 to mucosal host defense and inflammation in the colon.
Atsuo Sekiyama, Haruyasu Ueda, Shin-ichiro Kashiwamura, Kensei Nishida, Seiko Yamaguchi, Hideyuki Sasaki, Yuki Kuwano, Kaori Kawai, Shigetada Kondo, Kazuhito Rokutan and Haruki Okamura : A role of the adrenal gland in stress-induced up-regulation of cytokines in plasma, Journal of Neuroimmunology, Vol.171, No.1-2, 38-44, 2006.
(要約)
To reveal a pathway by which psychological/physical stresses influence host defense capability, responses to immobilization stress in mice were investigated, focusing on a multifunctional cytokine, interleukin-18 (IL-18). Immobilization stress induced interleukin-18 accumulation in plasma and in the adrenal gland. Inhibition on ACTH resulted in suppressed levels of IL-18 both in plasma and the adrenal gland. In hemi-adrenalectomized mice, plasma IL-18 levels after stress were lower than in sham-operated mice. This, together with the observation in stressed hemi-adrenalectomized mice that IL-6 levels in plasma were suppressed but up-regulated by recombinant IL-18, showed that the adrenal gland plays a crucial role in stress-related elevation of IL-6 in plasma via IL-18. Adrenal gland is highlighted as an organ connecting the psychological, endocrine, and immune systems. Controlling the secretion of IL-18 from the adrenal gland may serve as a possible preventative means against a stress-related disruption of host defenses.
Kazunori Sekine, Kazuhito Rokutan and Noriaki Takeda : Microarray Analysis of Stress-Related Gene Expression in Patients with Ménière's Disease, ORL; Journal for Oto-Rhino-Laryngology and its Related Specialties, Vol.67, No.5, 294-299, 2005.
(要約)
We developed an original microarray carrying 1,467 cDNAs of stress-related genes for the assessment of stress responses. In this study, we used microarray analysis to assess the stress-related gene expression profiles in peripheral leukocytes in 2 patients with definite Ménière's disease. In the attack and active phases, mRNA expression levels of 57 genes and 163 genes were either up-regulated more than twofold or down-regulated by less than half in patient 1 and patient 2, respectively. Patient 1 had sporadic episodes of vertigo attack, while patient 2 had an intractable course with frequent vertigo attacks, suggesting that the magnitude of changes in gene expression is correlated with the severity of the disorder in Ménière's disease. The expression of a total of 26 genes commonly changed in both patients in the attack and active phases and returned to the baseline levels in the remission phase, suggesting the involvement of the distinct group of stress-related genes in the development of vertigo attacks in Ménière's disease. We then examined the effects of caloric stimulation on the stress-related gene expression profiles in peripheral leukocytes in 5 healthy volunteers. Although unilateral caloric stimulation with cold water caused acute vertigo with nystagmus, the expression profiles of stress-related genes did not significantly change after this experiment. This finding indicated that the up- or down-regulated genes in the attack and active phases in patients with Ménière's disease are not secondary to vertigo or vertigo-associated anxiety. All these findings suggested that the distinct group of stress-related genes contributed to the development of vertigo attacks of Ménière's disease and that stress-related gene expression profiles in peripheral leukocytes can be a predictive and therapeutic tool for episodic vertigo attacks in patients with Ménière's disease.
Junichi Iga, Shu-ichi Ueno, K Yamauchi, I Motoki, S Tayoshi, K Ohta, H Song, Kyoko Morita, Kazuhito Rokutan and Tetsuro Ohmori : Serotonin transporter mRNA expression in peripheral leukocytes of patients with major depression before and after treatment with paroxetine., Neuroscience Letters, Vol.389, No.1, 12-16, 2005.
(要約)
Serotonin transporter (5HTT) is thought to be involved in the pathophysiology of major depression and the target of antidepressants. We hypothesized that 5HTT mRNA levels in peripheral leukocytes may be associated with depressive states and the therapeutic response to antidepressant treatments. Fifteen patients with major depression and age-, sex-matched control subjects were studied. 5HTT mRNA levels were determined with quantitative real-time PCR method. 5HTT mRNA levels in leukocytes were significantly higher in depressive patients at baseline (before medication) than in control subjects. 5HTT mRNA levels were decreased significantly after 8 weeks of paroxetine medication compared with those at baseline. Our investigation suggested that the increased expression of 5HTT mRNA in peripheral leukocytes may be related with the pathophysiology of depression and its reduction after treatment may reflect the adaptive change induced by the antidepressant.
Yutaka Nakaya, Y Hata, K Ishida, Akira Takahashi, Kyoko Morita and Kazuhito Rokutan : Approach to novel functional foods for stress control 2. Microarray assessment of exercise in healthy volunteers., The Journal of Medical Investigation : JMI, Vol.52, No.Suppl, 242-243, 2005.
(要約)
DNA microarray was used to measure stress response by exercise in peripheral blood leukocytes. Aerobic exercise did not alter mRNA pattern or urinary secretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Strenuous exercise increased urinary secretion of 8-OHdG and altered mRNA pattern in microarray. These results suggest that moderate exercise, i. e. aerobic exercise, did not show any change in 8-OHdG, an oxidative stress marker, or mRNA expression in the leukocytes, which might reflect whole body neurohormanal changes. In addition, strenuous exercise produced quite different expression pattern from those of psychological stress.
In this study, we have developed a microarray including 1467 cDNAs that were selected to specifically measure stress response in peripheral blood leukocytes. Venous blood was collected from 10 graduate students 2 h before and 2 or 24 h after an open presentation for their Ph.D. The mRNA levels in leukocytes were compared with those prepared 4 weeks before the presentation. Hierarchical cluster showed that distinct groups of genes uniformly changed their expression values in response to the stress. Bayesian t test identified significantly up-regulated 49 genes and down-regulated 21 genes. Most of them are categorized into cytokines, cytokine receptors, growth- or apoptosis-related molecules, and heat shock proteins, suggesting that stressful life events trigger acute responses in leukocytes. Our results suggest that gene expression profile in peripheral blood leukocytes may be a potentially useful method for the assessment of complex stress responses.
Psychological/physical stresses are known to cause relapses of autoimmune and inflammatory diseases. To reveal a mechanism by which noninflammatory stresses affect host defenses, responses to immobilization stress in mice were investigated, focusing on the role of a multifunctional cytokine, interleukin-18 (IL-18). In the adrenal cortex, the stress induced IL-18 precursor proteins (pro-IL-18) via adrenocorticotropic hormone (ACTH) and a superoxide-mediated caspase-1 activation pathway, resulting in conversion of pro-IL-18 to the mature form, which was released into plasma. Inhibitors of caspase-1, reactive oxygen species, and P38 mitogen-activated protein kinase (MAPK) suppressed stress-induced accumulation of plasma IL-18. These inhibitors also blocked stress-induced IL-6 expression. This, together with the observation that IL-6 was not induced in IL-18-deficient mice, showed that IL-6 induction by stress is dependent on IL-18. In stressed organisms, IL-18 may influence pathological and physiological processes. Controlling the caspase-1 activating pathway to suppress IL-18 levels may provide preventative means against stress-related disruption of host defenses.
Reactive oxygen species (ROS) may play a critical role in the regulation of vascular tone and development of vascular diseases, such as stroke. NAD(P)H oxidase is a major source of ROS in vascular cells, including endothelial cells. It has been considered that Nox2 and Nox4 are exclusively expressed among Nox homologues in the endothelial cells of noncerebral blood vessels. However, the precise molecular identity of the NAD(P)H oxidase in the endothelial cells of the cerebral arteries is not fully understood. We examined the expression of Nox homologues and their activation mechanism in the endothelial cells of the cerebral arteries. We isolated and cultured basilar artery endothelial cells (BAECs) of Sprague-Dawley rats. Expression of NAD(P)H oxidase was examined by reverse-transcription-polymerase chain reaction (RT-PCR) and immunohistological staining. RT-PCR disclosed abundant expression of Nox4 with marginal Nox2 in BAEC. In addition, Nox1 was expressed highly both at mRNA and protein levels in BAECs. Immunohistological staining also showed the prominent expression of Nox1 in the endothelial cells of the basilar artery. With respect to the cytosolic components of NAD(P)H oxidases, BAECs expressed p67phox and, to a lesser extent, p47phox, Noxo1, and Noxa1. Both NADH and NADPH induced superoxide production of the BAEC membranes. The phagocyte-type cytosolic components, p47phox and p67phox, significantly enhanced the NADH-induced superoxide production of the BAEC membranes, whereas the components failed to increase the NADPH-induced superoxide production. Nox1 is highly expressed in the endothelial cells of the cerebral arteries along with Nox2 and Nox4, and the endothelial NAD(P)H oxidase of the cerebral arteries may have a unique activation mechanism by the phagocyte-type cytosolic components.
Mitsutoshi Fukuyama, Kazuhito Rokutan, Toshiaki Sano, Hidenori Miyake, Mitsuo Shimada and Seiki Tashiro : Overexpression of a novel superoxide-producing enzyme,NADPH oxidase 1,in adenoma and well differentiated adenocarcinoma of the human colon, Cancer Letters, Vol.221, No.1, 97-104, 2005.
(要約)
A superoxide-producing enzyme, NADPH oxidase 1 (Nox1), dominantly expressed in the colon, is implicated in the pathogenesis of colon cancer. Immunohistochemistry showed that Nox1 was constitutively expressed in surface mucous cells. Adenomas and well differentiated adenocarcinomas up-regulated Nox1 expression. Ki-67-negative, well differentiated tumor cells contained abundant Nox1, whereas Ki-67-positive, proliferating cells did not express it. This differentiation-dependent expression in normal as well as tumor tissues suggests distinct roles of Nox1 besides mitogenic function. Nuclear factor (NF)-kappaB was predominantly activated in adenoma and adenocarcinoma cells expressing abundant Nox1, suggesting that Nox1 may stimulate NF-kappaB-dependent antiapoptotic pathways in colon tumors.
Kenji Kusumoto, Tsukasa Kawahara, Yuki Kuwano, Shigetada Kondo, Kyoko Morita, Kyoichi Kishi and Kazuhito Rokutan : Ecabet sodium inhibits Helicobacter pylori lipopolysaccharide-induced activation of NADPH oxidase 1 or apoptosis of guinea pig gastric mucosal cells, American Journal of Physiology, Gastrointestinal and Liver Physiology, Vol.288, No.2, G300-G307, 2005.
(要約)
Helicobacter pylori LPS activates a homolog of gp91(phox), NADPH oxidase 1 (Nox1), in guinea pig gastric mucosal cells cultured in 10% FBS-containing medium. RT-PCR and Northern hybridization demonstrated that H. pylori LPS stimulated expression of Nox1 and a novel p47(phox) homolog (Noxo1) mRNAs with a peak at 4 h, followed by upregulation of superoxide anion (O2-) generation. Pretreatment with 10 mg/ml of a nonabsorbable antigastric ulcer drug, ecabet sodium (ecabet), completely blocked these two mRNA expressions and the upregulation of O2- production. Under low (0.1%)-FBS conditions, H. pylori LPS predominantly caused apoptosis of the cells. Ecabet completely blocked the LPS-triggered phosphorylation of transforming growth factor-beta-activated kinase 1 (TAK1) and TAK1-binding protein 1, activation of caspase 8, loss of mitochondrial membrane potential, release of cytochrome c, activation of caspase 3, and appearance of apoptotic cells. In contrast, ecabet had no effect on ethanol- or etoposide-initiated apoptosis. The ecabet-pretreated cells exhibited the responsiveness to H. pylori LPS, similarly as untreated control cells did, when ecabet was removed by washing before the addition of H. pylori LPS. Incubation of H. pylori LPS with ecabet eliminated the toxic effects of the LPS, and nondenatured polyacrylamide gel electrophoresis indicated the formation of higher molecular mass complexes between H. pylori LPS and ecabet, suggesting that ecabet may interact with H. pylori LPS and block the activation of Toll-like receptor 4 (TLR4). Our results suggest that ecabet may suppress TLR4-mediated inflammation or accelerated apoptosis caused H. pylori infection.
Tsukasa Kawahara, Motoyuki Kohjima, Yuki Kuwano, Hisano Mino, Shigetada Kondo, Ryu Takeya, Shohko Tsunawaki, Akihiro Wada, Hideki Sumimoto and Kazuhito Rokutan : Helicobacter pylori lipopolysaccharide activates Rac1 and transcription of NADPH oxidase Nox1 and its organizer NOXO1 in guines pig gastric mucosal cells, American Journal of Physiology, Cell Physiology, Vol.288, No.2, 450-457, 2005.
(要約)
Primary cultures of guinea pig gastric mucosal cells express NADPH oxidase 1 (Nox1), a homolog of gp91(phox), and produce superoxide anion (O2-) at a rate of approximately 100 nmol.mg protein(-1).h(-1) in response to Helicobacter pylori (H. pylori) lipopolysaccharide (LPS) from virulent type I strains. The upregulated O2- production also enhances H. pylori LPS-stimulated tumor necrosis factor-alpha or cyclooxygenase-2 mRNA expression, which suggests a potential role for Nox1 in the pathogenesis of H. pylori-associated diseases. The H. pylori LPS-stimulated O2- production in cultured gastric mucosal cells was inhibited by actinomycin D as well as cycloheximide, suggesting that the induction is regulated at the transcriptional level. The LPS treatment not only increased the Nox1 mRNA to a greater extent but also induced expression of the message-encoding, Nox-organizing protein 1 (NOXO1), a novel p47phox homolog required for Nox1 activity. In addition, H. pylori LPS activated Rac1; i.e., it converted Rac1 to the GTP-bound state. A phosphoinositide 3-kinase inhibitor, LY-294002, blocked H. pylori LPS-induced Rac1 activation and O2- generation without interfering with the expression of Nox1 and NOXO1 mRNA. O2- production inhibited by LY-294002 was completely restored by transfection of an adenoviral vector encoding a constitutively active Rac1 but not an inactive Rac1 or a constitutively active Cdc42. These findings indicate that Rac1 plays a crucial role in Nox1 activation. Thus the H. pylori LPS-stimulated O2- production in gastric mucosal cells appears to require two distinct events: 1) transcriptional upregulation of Nox1 and NOXO1 and 2) activation of Rac1.
Shigetada Kondo, Kazumi Kondo, Prado-Lourenco Leonel, Gonzalez-Herrera Gabriela Irma, Kazuhito Rokutan, bayard Francis, Arnal Jean-Francois and Prats Anne-Catherine : Hyperglycemia upregulates translation of the fibroblast growth factor 2 mRNA in mouse aorta via internal ribosome entry site, The FASEB journal, Vol.18, No.13, 1583-1585, 2004.
(要約)
Fibroblast growth factor 2 (FGF-2) is normally synthesized at low levels but is elevated in various pathophysiological conditions including diabetes-associated vascular diseases. FGF-2 expression is regulated translationally through an internal ribosome entry site (IRES) located in its mRNA, which allows a nonclassical cap-independent translation. We addressed the pathophysiological regulation of the IRES in vivo by using a streptozotocin-induced hyperglycemic model known to suppress markedly overall translation. Evaluation of FGF-2 IRES-dependent translation was performed with transgenic mice expressing dual luciferase bicistronic mRNA containing the FGF-2 IRES. FGF-2 IRES-dependent reporter activity increased 240% of control in the diabetic aorta although the reporter mRNA levels significantly decreased. Expression of endogenous FGF-2 protein in the aorta closely correlated with the IRES activity but not with FGF-2 mRNA levels. Moreover, the biosynthesis of endogenous FGF-2 protein was stimulated in an IRES-dependent manner by high glucose that significantly suppressed global protein synthesis in aortic smooth muscle cells from the transgenic mice. These results suggest that IRES-dependent translational regulation could play a pathological role in FGF-2 expression in vivo, especially in the cardiovascular consequences of diabetes.
Eiji Takeda, Junji Terao, Yutaka Nakaya, Ken-ichi Miyamoto, Yoshinobu Baba, Hiroshi Chuman, Ryuji Kaji, Tetsuro Ohmori and Kazuhito Rokutan : Stress control and human nutrition, The Journal of Medical Investigation : JMI, Vol.51, No.3-4, 139-145, 2004.
(要約)
Stress is a pervasive factor in everyday life that critically affects development and functioning. Severe and prolonged stress exposure impairs homeostatic mechanisms, particularly associated with the onset of depressive illness. Brain food is aimed at preventing as well as treating a growing number of stress-related mental disorders. Some topics on the association of stress and nutrition is reviewed. (1) An increased activity of serotonergic neurons in the brain is an established consequence of stress. An increase in brain tryptophan levels on the order of that produced by eating a carbohydrate-rich/protein-poor meal causes parallel increases in the amounts of serotonin released into synapses. (2) Eating is thought to be suppressed during stress, due to anorectic effects of corticotrophin releasing hormone, and increased during recovery from stress, due to appetite stimulating effects of residual cortisol. (3) A strong inverse association between coffee intake and risk of suicide. (4) Night eating syndrome has been found to occur during periods of stress and is associated with poor results at attempts to lose weight and disturbances in the hypothalamic-pituitary-adrenal axis. (5) Dietary antioxidants present in fruits and vegetables may improve cognitive function. Therefore, it is concluded that the establishment of functional foods that correctly regulate stress response must be firmly based upon scientific knowledge and legal regulation.
Chamulitrat Walee, Stermmel Wolfgang, Tsukasa Kawahara, Kazuhito Rokutan, Hirotada Fujii, Wingler Kirstin, Schmidt H.H.W. Harald and Schmidt Rainer : A Constitutive NADPH Oxidase-Like System Containing gp91 phox Homologs in Human Keratinocytes, The Journal of Investigative Dermatology, Vol.122, No.4, 1000-1009, 2004.
(要約)
In non-phagocytic cells, superoxide has been implicated in physiological and pathological cellular functions in the skin and mucosa, such as, host defense, mitogenic responses, and malignant conversion. Here, we identify a constitutively expressed heme-flavoprotein NADPH oxidase (Nox) system as a source of superoxide in human skin (HaCaT) and gingival mucosal (GM16) keratinocyte cell lines. Western blot analysis showed that both cell lines expressed the phagocyte oxidase (phox) cytosolic proteins Rac1, p40phox, and p67phox. With respect to the catalytic flavoheme protein subunit, HaCaT membranes, which expressed p22phox, showed an absorbance peak at 558 nm indicative of a b-type cytochrome. At mRNA levels, both GM16 and HaCaT cells expressed gp91phox homologs Nox1, Nox2, and Nox4, however, HaCaT cells expressed very low levels of Nox1 mRNA. At protein levels, Nox1 was readily detected in HaCaT but was nearly undetectable in GM16 cells. Consistently, Nox activity of HaCaT membranes was demonstrated by electron paramagnetic resonance spin-trapping and cytochrome c reduction, and the activity was sensitive to the flavoprotein inhibitor diphenylene iodonium. V(max) values were 20-fold lower than those reported for phagocytic oxidase. In conclusion, keratinocytes expressed a Nox distinct from the phox isoform of phagocytes providing molecular evidence for a source of superoxide that may regulate cell proliferation and host defense in skin and oral mucosa.
(キーワード)
human skin and oral epithelial cells / oxidoreductase / p67phox / spin trapping / superoxide radical
Tsukasa Kawahara, Yuki Kuwano, Shigetada Kondo, Ryu Takeya, Hideki Sumimoto, Kyoichi Kishi, Shohko Tsunawaki, Toshiya Hirayama and Kazuhito Rokutan : Role of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 1 in Oxidative Burst Response to Toll-Like Receptor 5 Signaling in Large Intestinal Epithelial Cells, The Journal of Immunology, Vol.172, No.5, 3051-3058, 2004.
(要約)
The NADPH oxidase 1 (Nox1) is a gp91(phox) homologue preferentially expressed in the colon. We have established primary cultures of guinea pig large intestinal epithelial cells giving 90% purity of surface mucous cells. These cells spontaneously released superoxide anion (O(2)(-)) of 160 nmol/mg protein/h and expressed the Nox1, p22(phox), p67(phox), and Rac1 mRNAs, but not the gp91(phox), Nox4, p47(phox), p40(phox), and Rac2 mRNAs. They also expressed novel homologues of p47(phox) and p67(phox) (p41(nox) and p51(nox), respectively). Human colon cancer cell lines (T84 and Caco2 cells) expressed the Nox1, p22(phox), p51(nox), and Rac1 mRNAs, but not the other NADPH component mRNAs, and secreted only small amounts of O(2)(-) (<2 nmol/mg protein/h). Cotransfection of p41(nox) and p51(nox) cDNAs in T84 cells enhanced PMA-stimulated O(2)(-) release 5-fold. Treatment of the transfected T84 cells with recombinant flagellin (rFliC) from Salmonella enteritidis further augmented the O(2)(-) release in association with the induction of Nox1 protein. The enhanced O(2)(-) production by cotransfection of p41(nox) and p51(nox) vectors further augmented the rFliC-stimulated IL-8 release from T84 cells. T84 cells expressed the Toll-like receptor 5, and rFliC rapidly phosphorylated TGF-beta-activated kinase 1 and TGF-beta-activated kinase 1-binding protein 1. A potent inhibitor for NF-kappaB (pyrrolidine dithiocarbamate) significantly blocked the rFliC-primed increase in O(2)(-) production and induction of Nox1 protein. These results suggest that p41(nox) and p51(nox) are involved in the Nox1 activation in surface mucous cells of the colon, and besides that, epithelial cells discern pathogenicities among bacteria to appropriately operate Nox1 for the host defense.
Satiko Lucia Yoshida, Satoshi Nishida, Takashi Shimoyama, Tsukasa Kawahara, Shigetada Kondo, Kazuhito Rokutan, Toshihiro Kobayashi and Shohko Tsunawaki : Superoxide Generation by Nox1 in Guinea Pig Gastric Mucosal Cells Involves a Component with p67phox-Ability, Biological & Pharmaceutical Bulletin, Vol.27, No.2, 147-155, 2004.
(要約)
Nox1, a homologue of gp91(phox) subunit of the phagocyte NADPH oxidase, is responsible for spontaneous superoxide (O(2)(-)) generation in guinea pig gastric mucosal cells (GMC), but involvement of regulatory components (p67(phox), p47(phox), and Rac) which are essential in phagocytes remains unknown. Here, we aimed to figure out how Nox1 of GMC achieves an active oxidase status. GMC in primary culture show low O(2)(-) generation but acquire a 9-fold higher activity when cultured with Helicobacter pylori lipopolysaccharide (LPS), in correlation with a far increased Nox1 expression. Investigation into the O(2)(-)-generating ability of LPS-induced Nox1 in cell-free reconstitution assays showed that: 1) Nox1 is unable to generate O(2)(-) per se; 2) the combination of Nox1 with GMC cytosol is insufficient for a significant O(2)(-) generation; 3) the combination with neutrophil cytosol enables Nox1 to act like gp91(phox), i.e., to produce O(2)(-) appreciably in response to myristate stimulation; 4) Nox1 prefers NADPH to NADH under the in vitro assay with neutrophil cytosol plus myristate (K(m)=10.4 microM); 5) substitution of neutrophil cytosol by a set of recombinant cytosolic components (rp67(phox), rp47(phox), Rac2) is, however, ineffective and still requires GMC cytosol. Thus, Nox1 probably requires an additional cytosolic factor(s). In contrast, GMC cytosol enables cytochrome b(558) to generate plenty of O(2)(-), on condition that rp47(phox) is added. This result suggests that GMC cytosol contains a component with p67(phox)-ability, and also Rac, but lacks p47(phox). These data indicate that GMC Nox1 requires at least a p67(phox) counterpart and Rac to acquire NADPH oxidase activity.
Kazuo Yoneda, Kazuhito Rokutan, Yoichi Nakamura, Hiroaki Yanagawa, Shigetada Kondo and Saburo Sone : Stimulation of human bronchial epithelial cells by IgE-dependent histamine-releasing factor, American Journal of Physiology. Lung Cellular and Molecular Physiology, Vol.286, No.1, 174-181, 2004.
(要約)
An IgE-dependent histamine-releasing factor (HRF p23; also known as translationally controlled tumor protein or p23) stimulates the release of histamine, IL-4, and IL-13 from a subpopulation of highly allergic donor basophils. It has also been shown to act as a chemoattractant for eosinophils. To elucidate novel functions of HRF p23 in airway inflammation, we examined the effects of human recombinant HRF p23 (hrHRF) on bronchial epithelium and found that hrHRF stimulated the secretions of IL-8 and granulocyte/macrophage colony-stimulating factor by both primary cultures of human bronchial epithelial cells and BEAS-2B cells. In response to hrHRF, these cells induced IL-8 mRNA expression within 4 h. H2O2, but not IL-1 beta or tumor necrosis factor-alpha, stimulated secretion of HRF p23 by BEAS-2B cells, suggesting that oxidative stress may trigger the release of HRF p23 from bronchial epithelial cells. Bronchoalveolar lavage (BAL) from healthy volunteers contained only trivial or undetectable amounts of HRF p23. Significantly higher amounts of HRF p23 were recovered from BAL fluid taken from asthmatic patients, and the amounts of HRF p23 were further elevated in patients with idiopathic eosinophilic pneumonia. Our results demonstrate for the first time that HRF p23 can stimulate nonimmune epithelium. HRF p23 derived from bronchial epithelial cells may regulate complex cytokine networks in eosinophil-dependent inflammation of the human airway.
Hidenori Miyake, Masahiko Fujii, Katsuya Sasaki, Tsutomu Ando, Shizuo Ikeyama, Takashi Iwata, Kazuhito Rokutan and Seiki Tashiro : Heat Shock Protein 70 Induction in Hepatocytes after Right Portal Vein Embolization, Hepato-Gastroenterology, Vol.50, No.54, 2084-2087, 2003.
(要約)
Preoperative right portal vein embolization enhances remnant liver function following massive hepatectomy. Several studies have reported an increase in the volume of the left hepatic lobe after right portal vein embolization, but little information exists regarding heat shock protein induction in hepatocytes after right portal vein embolization. The objective of this study is to determine whether heat shock protein is induced in hepatocytes after right portal vein embolization in patients who underwent extended right hepatic lobectomy. Four patients with gallbladder cancer and one patient with intrahepatic cholangiocellular carcinoma who underwent extended right hepatic lobectomy combined with caudate lobectomy and resection of the extrahepatic bile duct after right portal vein embolization were enrolled in this study. Operation was performed 21-36 days after right portal vein embolization. At operation, small liver specimens were taken immediately after laparotomy from both the right anterior segment (embolized lobe) and lower part of the left medial segment (non-embolized lobe) and heat shock protein 70 was induction in these specimens was measured by Western blotting. Heat shock protein 70 was induced in the left lobe relative to the right lobe in four patients, three of whom had an uneventful postoperative course. This paper is the first report to show the induction of heat shock protein 70 in the non-embolized hepatic lobe after right portal vein embolization in the clinical cases.
(キーワード)
Heat shock protein / Right portal vein embolization / Hepatectomy / Liver failure
(文献検索サイトへのリンク)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 14696469
Chamulitrat Walee, Schmidt Rainer, Tomakidi Pascal, Stremmel Wolfgang, Chunglok Warangkana, Tsukasa Kawahara and Kazuhito Rokutan : Association of gp91phox homolog Nox1 with anchorage-independent growth and MAP kinase-activation of transformed human keratinocytes, Oncogene, Vol.22, No.38, 6045-6053, 2003.
(要約)
Among five members of the NADPH oxidase (Nox) family, Nox1 confers mitogenic properties and is implicated to participate in the process of cell transformation. We have established two phenotypes of carcinogenesis model by ethanol treatment of human gingival keratinocytes immortalized with E6/E7 oncogenes of human papillomavirus type16: immortalized (EPI) nontransformed cells with epithelium-like morphology and more advanced transformed (FIB) cells with spindle fibroblastic-shape morphology. FIB membranes possessed a 63-kDa Nox1 protein at higher levels and exhibited 2.8-fold higher capability for superoxide and hydroxyl radical generation, compared with EPI membranes. Both EPI and FIB cells expressed more abundant Nox1 protein at a proliferating stage than that at a quiescent confluent phase. Immunofluorescence staining with an anti-Nox1 antibody showed that immunoreactive materials were distributed in the whole interior of both types of cells, while they were preferentially localized in the nuclei of FIB cells. Nuclei isolated from EPI and FIB cells contained a 63 kDa-Nox1 protein. Compared with EPI cells, FIB cells expressed elevated levels of Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase proteins. Furthermore, JNK2 was constitutively phosphorylated in FIB cells. Together, our data strongly implicate Nox1 in redox-mediated signaling related to cellular activation of human keratinocytes at a more advanced stage of transformation.
L.S. Yoshida, S. Nishida, T. Shimoyama, T. Kawahara, Kazuhito Rokutan and S. Tsunawaki : Expression of a p67phox homolog in Caco-2 cells giving O2-reconstituting ability to cytochrome b558 together with recombinant p47phox, Biochemical and Biophysical Research Communications, Vol.296, No.5, 1322-1328, 2002.
(要約)
Human normal and transformed (Caco-2) colon tissues as well as guinea pig gastric mucosal cells express Nox1, which is a homolog of the phagocyte NADPH oxidase subunit, gp91(phox) of membrane-bound cytochrome b(558). It was reported that Nox1-transfection to NIH 3T3 cells could provide O(2)(-)-generating ability, independently of regulatory cytosolic factors (Rac2, p67(phox), and p47(phox)) that are obligatory in the phagocyte oxidase system. Here, we detected and sequenced a p67(phox) homolog in Caco-2 almost identical to the neutrophil sequence, except for three nucleotide substitutions, two of which changed lysines 181 and 328 to arginines. Investigation of its ability to support O(2)(-)-generation in cell-free reconstitution experiments combining with neutrophil cytochrome b(558) showed O(2)(-)-generation, provided that recombinant p47(phox) was added. This result demonstrates that the intrinsic p67(phox) homolog of Caco-2 was able to function as a phagocyte p67(phox) for cytochrome b(558). The requirement of p47(phox) addition suggested that this component was absent in Caco-2 cells. Caco-2 membranes, used as a source of Nox1 in place of cytochrome b(558), did not show significant O(2)(-)-generation, which was mainly explained by their very little Nox1 expression.
Takeshi Nikawa, Madoka Ikemoto, Takashi Sakai, Mihoko Kano, Takako Kitano, Tsukasa Kawahara, Shigetada Teshima, Kazuhito Rokutan and Kyoichi Kishi : Effects of a Soy Protein Diet on Exercise-Induced Muscle Protein Catabolism in Rats, Nutrition, Vol.18, No.6, 490-495, 2002.
(要約)
We examined effects of dietary soy protein isolate on muscle calpain activity and myosin heavy chain (MHC) degradation in rats performing an acute running exercise. In rats fed a 20% casein diet, the treadmill running exercise, fixed at 80 kg/m, transiently increased calpain activity in gastrocnemius muscles in parallel with the release of creatine kinase into plasma. The fixed running also caused an accumulation of immunoreactive degradation fragments of MHC in the muscle. Feeding a 20% soy protein isolate diet as opposed to the control casein diet to rats significantly suppressed the running-induced activation of mu- and m-calpains, fragmentation of MHC, and release of creatine kinase into plasma (P < 0.05). Rats fed the soy protein isolate diet had significantly higher calpastatin activity in gastrocnemius muscle than did rats fed the casein diet (P < 0.05), indicating that this increase inhibits the exercise-induced autoactivation of calpain. Activities of proteasome, cathepsin B + L, and antioxidant enzymes and the levels of glutathione and thiobarbituric acid-reactive substances in the muscle did not differ between the diet groups at the end of the exercise. Our results suggest that diets containing soy protein prevent exercise-induced protein degradation in skeletal muscle, possibly through inhibiting the calpain-mediated proteolysis.
(キーワード)
soy protein isolate / treadmill running / calpain / myosin heavy chain / creatine kinase
Shinji Tsutsumi, Wataru Tomisato, Tatsunori Takano, Kazuhito Rokutan, Tomofusa Tsuchiya and Tohru Mizushima : Gastric irritant-induced apoptosis in guinea pig gastric mucosal cells in primary culture, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol.1589, No.2, 168-180, 2002.
(要約)
When the gastric mucosa is exposed to various irritants, apoptosis and subsequent gastric mucosal lesion can result in vivo. We here show that gastric irritants induced apoptosis in gastric mucosal cells in primary culture and examined its molecular mechanism. Ethanol, hydrogen peroxide, and hydrochloric acid all induced, in a dose-dependent manner, cell death, apoptotic DNA fragmentation, and chromatin condensation, suggesting that each of these gastric irritants induced apoptosis in vitro. Since each of these irritants decreased the mitochondrial membrane potential and stimulated the release of cytochrome c from mitochondria, gastric irritant-induced apoptosis seems to be mediated by mitochondrial dysfunction. Caspase-3, caspase-8, and caspase-9-like activities were all activated simultaneously by each of these irritants and the activation was concomitantly with cell death and apoptotic DNA fragmentation. Furthermore, pre-treatment of gastric mucosal cells with an inhibitor of caspase-8 suppressed the onset of cell death as well as the stimulation of caspase-3- and caspase-9-like activities caused by each of these gastric irritants. Based on these results, we consider that caspase-8, an initiator caspase, plays an important role in gastric irritant-induced apoptosis.
Kosuke Oikawa, Tetsuya Ohbayashi, Junsei Mimura, Yoshiaki Fujii-Kuriyama, Shigetada Teshima, Kazuhito Rokutan, Kiyoshi Mukai and Masahiko Kuroda : Dioxin Stimulates Synthesis and Secretion of IgE-Dependent Histamine-Releasing Factor, Biochemical and Biophysical Research Communications, Vol.290, No.3, 984-987, 2002.
(要約)
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin) is the most toxic man-made member of the class of environmental pollutants represented by polychlorinated dibenzo-p-dioxins and dibenzofurans. TCDD produces a wide variety of toxic effects. However, the downstream genes targeted by TCDD and their relation to the diversity of dioxin toxicity symptoms are poorly understood. To identify the target genes of TCDD, we used a cDNA representational difference analysis (RDA) to compare the mRNA patterns of mouse embryonic stem (ES) cells that had and had not been exposed to TCDD. Here we show that TCDD stimulated the expression of IgE-dependent histamine-releasing factor (HRF) mRNA via an aryl hydrocarbon receptor (AhR)-dependent pathway. TCDD also induced the synthesis and secretion of HRF. To our knowledge, this is the first example of HRF being a direct transcriptional target of a toxic agent. HRF has previously been shown to induce histamine release in a dose-dependent manner, at least in vitro. Thus, our data suggest that "endocrine-disrupting" agents may have the potential to influence allergic disorders in the human body.
Wataru Tomisato, Shinji Tsutsumi, Kazuhito Rokutan, Tomofusa Tsuchiya and Tohru Mizushima : NSAIDs induce both necrosis and apoptosis in guinea pig gastric mucosal cells in primary culture, American Journal of Physiology, Gastrointestinal and Liver Physiology, Vol.281, No.4, 1092-1100, 2001.
(要約)
A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin is gastropathy. In this study, we examined, using guinea pig gastric mucosal cells in primary culture, how NSAIDs damage gastric mucosal cells. The short-term treatment of cells with high concentrations of indomethacin decreased cell viability in the absence of apoptotic DNA fragmentation, chromatin condensation, or caspase activation. Cells lost membrane integrity with this short-term indomethacin treatment, suggesting that indomethacin induced necrosis under these conditions. In contrast, the long-term treatment of cells with low concentrations of indomethacin decreased cell viability and was accompanied by apoptotic DNA fragmentation, chromatin condensation, and caspase activation. Pretreatment of cells with inhibitors of caspases or protein synthesis suppressed cell death caused by long-term indomethacin treatment, suggesting that apoptosis was induced when the inhibitors were not present. These results imply that NSAIDs cause gastric mucosal damage through both necrosis and apoptosis of gastric mucosal cells.
Tsukasa Kawahara, Shigetada Teshima, Yuki Kuwano, Ayuko Oka, Kyoichi Kishi and Kazuhito Rokutan : Helicobacter pylori lipopolysaccharide induces apoptosis of cultured guinea pig gastric mucosal cells, American Journal of Physiology, Gastrointestinal and Liver Physiology, Vol.281, No.3, 726-734, 2001.
(要約)
Helicobacter pylori lipopolysaccharide (LPS) is generally accepted as a low-toxicity virulence. Primary cultures of guinea pig gastric mucosal cells expressed the Toll-like receptor 4 and were sensitive to H. pylori LPS as well as Escherichia coli LPS. H. pylori LPS stimulated phosphorylation of transforming growth factor-beta-activated kinase 1 (TAK1), TAK1-binding protein 1 (TAB1), and c-Jun NH(2)-terminal kinase (JNK) 2. H. pylori LPS at >2.1 endotoxin unit/ml (>1 ng/ml) activated caspase-8, stimulated cytochrome c release from mitochondria, and subsequently activated caspases-9 and -3, leading to apoptosis. Epidermal growth factor blocked all of these apoptotic processes and inhibited apoptosis, whereas it did not modify the phosphorylation of TAK1, TAB1, and JNK2. A comparatively specific inhibitor of caspase-8 or -9 blocked apoptosis, whereas cytochrome c release was prevented only with a caspase-8-like inhibitor. Our results suggest that caspase-8 and mitochondria may play crucial roles in H. pylori LPS-induced apoptosis and that this accelerated apoptosis may be involved in abnormal cell turnover of H. pylori-infected gastric mucosa.
Tsukasa Kawahara, Yuki Kuwano, Shigetada Kondo, Tomoko Kawai, Takeshi Nikawa, Kyoichi Kishi and Kazuhito Rokutan : Toll-like receptor 4 regulates gastric pit cell responses to Helicobacter pylori infection, The Journal of Medical Investigation : JMI, Vol.48, No.3,4, 190-197, 2001.
(要約)
Gastric pit cells express mitogen oxidase1 (Mox1) and essential components for the phagocyte NADPH oxidase (p67-, p47-, p40-, and p22-phoxes). Helicobacter pylori (Hp) lipopolysaccharide (LPS) is a potent up-regulator of the Mox 1 oxidase. In this study, we examined the expression levels of several key members of the Toll-like receptor (TLR) family in primary cultures of guinea pig gastric pit cells. These cells expressed the TLR4 mRNA. Immunoblot analysis and immunofluorescence histochemistry with an anti-TLR4 antibody showed that gastric pit cells possessed significant amounts of TLR4 protein preferentially on the plasma membrane. In contrast, the cells did not express the TLR2 and TLR9 transcripts and did not contain detectable amounts of TLR2 protein. Neither peptidoglycan from Staphylococcus aureus nor Hp DNA with the CpG motif up-regulated Mox1 oxidase activity. Hp LPS activated nuclear factor-kappa B in association with the expression of cyclooxygenase II and tumor necrosis factor alpha transcripts. These findings suggest that TLR4 may play a crucial role in the initiation of inflammatory responses of gastric pit cells against Hp infection.
Tsukasa Kawahara, Yuki Kuwano, Shigetada Kondo, Toshiro Sugiyama, Tomoko Kawai, Takeshi Nikawa, Kyoichi Kishi and Kazuhito Rokutan : Helicobacter pylori lipopolysaccharide from type 1, but not type 2 strains, stimulates apoptosis of cultured gastric mucosal cells, The Journal of Medical Investigation : JMI, Vol.48, No.3,4, 167-174, 2001.
(要約)
The cag pathogenicity island (cag PAI) genes are a major determinant of virulence of Helicobacter pylori (Hp). Lipopolysaccharide (LPS) purified from the cag PAI-positive (type I) strains induced apoptosis of primary cultures of guinea pig gastric mucosal cells. Lipid A catalyzed this apoptosis. These cells expressed the Toll-like receptor 4 (TLR4) mRNA and its protein, and type I Hp LPS phosphorylated transforming growth factor beta-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1) in association with up-regulation of the TLR4 expressions, suggesting that type I Hp LPS evoked distinct TLR4 signaling. In contrast, Hp LPS from type II strains with complete or partial deletion of the cag PAI genes did not phosphorylate TAK1 and TAB1 and failed to induce apoptosis. Accelerated apoptosis of gastric epithelial cells is one of the important events relevant to chronic, atrophic gastritis caused by Hp infection. The difference in proapoptotic action of LPS between the type I and II strains may support an important role of the cag PAI genes in the pathogenesis of gastric lesions caused by Hp infection.
Takeshi Nikawa, Madoka Ikemoto, Mihoko Kano, Kaori Tokuoka, Katsuya Hirasaka, Shoji Uehara, Kiyoshi Takatsu, Kazuhito Rokutan and Kyoichi Kishi : Impaired Vitamin A-Mediated Mucosal IgA Response in IL-5 Receptor-Knockout Mice, Biochemical and Biophysical Research Communications, Vol.285, No.2, 546-549, 2001.
(要約)
To clarify actions of vitamin A on mucosal immunity associated with interleukin-5 (IL-5), we examined effects of vitamin A on mucosal IgA level in IL-5 receptor alpha-chain-knockout (IL-5Ralpha(-/-)) mice. Daily supplementation of retinyl acetate (1 mg/mouse) increased Th2 cytokine levels and a number of their positive cells in the small intestinal mucosa of IL-5Ralpha(-/-) mice, as observed in wild-type or IL-5Ralpha(+/-) mice. Wild-type and heterozygous mice increased the IgA level and a number of IgA-containing cells in the mucosa in response to the vitamin A treatment, but not in IL-5Ralpha(-/-) mice. Retinyl acetate increased anti-cholera toxin (CT) IgA level in the mucosa of wild-type mice, improving their survival rate after an exposure to 0.4 mg of CT. However, retinyl acetate failed to induce resistance to CT toxicity in IL-5Ralpha(-/-) mice. Our results suggest that IL-5 may play an important role in an action of vitamin A on mucosal IgA system.
(キーワード)
ビタミンA (vitamin A) / IgA / IL-4 / IL-5 / IL-6 / small intestinal mucosa / IL-5 receptor α-chain-deficient mice / cholera toxin
Tsukasa Kawahara, Shigetada Teshima, Ayuko Oka, Toshiro Sugiyama, Kyoichi Kishi and Kazuhito Rokutan : Type 1 Helicobacter pylori Lipopolysaccharide Stimulates Toll-Like Receptor 4 and Activates Mitogen Oxidase 1 in Gastric PIt Cells, Infection and Immunity, Vol.69, No.7, 4382-4389, 2001.
(要約)
Guinea pig gastric pit cells express an isozyme of gp91-phox, mitogen oxidase 1 (Mox1), and essential components for the phagocyte NADPH oxidase (p67-, p47-, p40-, and p22-phox). Helicobacter pylori lipopolysaccharide (LPS) and Escherichia coli LPS have been shown to function as potent activators for the Mox1 oxidase. These cells spontaneously secreted about 10 nmol of superoxide anion (O(2)(-))/mg of protein/h under LPS-free conditions. They expressed the mRNA and protein of Toll-like receptor 4 (TLR4) but not those of TLR2. LPS from type I H. pylori at 2.1 endotoxin units/ml or higher stimulated TLR4-mediated phosphorylations of transforming growth factor beta-activated kinase 1 and its binding protein 1 induced TLR4 and p67-phox and up-regulated O(2)(-) production 10-fold. In contrast, none of these events occurred with H. pylori LPS from complete or partial deletion mutants of the cag pathogenicity island. Lipid A was confirmed to be a bioactive component for the priming effects, while removal of bisphosphates from lipid A completely eliminated the effects, suggesting the importance of the phosphorylation pattern besides the acylation pattern for the bioactivity. H. pylori LPS is generally accepted as having low toxicity; however, our results suggest that type I H. pylori lipid A may be a potent stimulator for innate immune responses of gastric mucosa by stimulating the TLR4 cascade and Mox1 oxidase in pit cells.
Shizuo Ikeyama, Kenji Kusumoto, Hidenori Miyake, Kazuhito Rokutan and Seiki Tashiro : A non-toxic heat shock protein 70 inducer, geranylgeranylacetone,suppresses apoptosis of cultured rat hepatocytes caused by hydrogen peroxide and ethanol, Journal of Hepatology, Vol.35, No.1, 53-61, 2001.
(要約)
A stress-inducible heat shock protein 70 (HSP70) is one of the best-known endogenous factors protecting cell injury under various pathological conditions. The aim of this study was to examine anti-apoptotic actions of a non-toxic HSP70 inducer, geranylgeranylacetone (GGA), on hepatocytes exposed to hydrogen peroxide (H2O2) or ethanol. Primary cultures of rat hepatocytes were treated with different concentrations of GGA and exposed to 0.5 mM H202 or 100 mM ethanol. The heat shock response was assessed by measuring the activation of heat shock factor 1 (HSF1), HSP70 mRNA expression, and accumulations of HSP70, HSP90, and HSP27. Apoptosis was evaluated by DNA fragmentation. Pretreatment with 1 microM GGA for 2 h enhanced nuclear translocation and phosphorylation of HSF1, HSF1-DNA binding, HSP70 mRNA expression, and its accumulation, when the cells were exposed to H202 or ethanol. In association with this accelerated response, GGA suppressed the insult-induced activation of c-Jun N-terminal kinases, caspase 9, and caspase 3-like proteases, leading to significant inhibition of apoptosis. GGA exerted anti-apoptotic actions, at least in part, by priming hepatocytes for enhanced HSP70 induction. Our results suggest that GGA may have a potential benefit for the treatment of alcoholic and ischemia-reperfusion liver injuries.
Madoka Ikemoto, Takeshi Nikawa, Shinichi Takeda, Chiho Watanabe, Takako Kitano, Baldwin M. Kenneth, Ryutaro Izumi, Ikuya Nonaka, Takae Towatari, Shigetada Teshima, Kazuhito Rokutan and Kyoichi Kishi : Space Shuttle flight (STS-90) enhances dagradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway, The FASEB journal, Vol.15, No.7, 1279-1281, 2001.
Takeshi Nikawa, Madoka Ikemoto, Kaori Tokuoka, Shigetada Teshima, Alpers H. David, Yoshihiro Masui, Kyoichi Kishi and Kazuhito Rokutan : Interleukin-1β enhances retinoic acid-mediated expression of bone-type alkaline phosphatase in rat IEC-6 cells, American Journal of Physiology, Gastrointestinal and Liver Physiology, Vol.280, No.3, 510-517, 2001.
(要約)
We previously showed that vitamin A upregulated the expression of bone-type alkaline phosphatase (ALP) in fetal rat small intestine and rat intestinal IEC-6 cells. In this study, we examined interactions between retinoic acid (RA) and several growth factors/cytokines on the isozyme expression in IEC-6 cells. Epidermal growth factor and interleukins (ILs)-2, -4, -5, and -6 completely blocked the RA-mediated increase in ALP activity. In contrast, IL-1beta markedly increased the activity, protein, and mRNA of the bone-type ALP only when RA was present. IL-1beta and/or RA did not change the type 1 IL-1 receptor transcript level, whereas IL-1beta enhanced the RA-induced expressions of retinoic acid receptor-beta (RAR-beta) and retinoid X receptor-beta (RXR-beta) mRNAs and RA-mediated RXR response element binding. The synergism of IL-1beta and RA on ALP activity was completely blocked by protein kinase C (PKC) inhibitors. Our results suggest that IL-1beta may modify the ALP isozyme expression in small intestinal epithelial cells by stimulating PKC-dependent, RAR-beta- and/or RXR-beta-mediated signaling pathways.
(キーワード)
Alkaline Phosphatase / Animals / Bone and Bones / Cell Line / DNA-Binding Proteins / Drug Synergism / Enzyme Inhibitors / Epidermal Growth Factor / Interleukin-1 / Interleukin-2 / Interleukin-4 / Interleukin-5 / Interleukin-6 / Intestine, Small / Isoenzymes / Protein Kinase C / RNA, Messenger / Rats / Receptors, Retinoic Acid / Transcriptional Activation / Tretinoin
Shigetada Teshima, Hiromu Kutsumi, Tsukasa Kawahara, Kyoichi Kishi and Kazuhito Rokutan : Regulation of growth and apoptosis of cultured guinea pig gastric mucosal cells by mitogenic oxidase 1, American Journal of Physiology, Gastrointestinal and Liver Physiology, Vol.279, No.6, G1169-G1176, 2000.
(要約)
We previously reported that primary cultures of guinea pig gastric pit cells expressed all of the phagocyte NADPH oxidase components (gp91-, p22-, p67-, p47-, and p40-phox) and could spontaneously release superoxide anion (O(2)(-)). We demonstrate here that pit cells express a nonphagocyte-specific gp91-phox homolog (Mox1) but not gp91-phox. Inclusion of catalase significantly inhibited [(3)H]thymidine uptake during the initial 2 days of culture. Pit cells, matured on day 2, slowly underwent spontaneous apoptosis. Scavenging O(2)(-) and related oxidants by superoxide dismutase plus catalase or N-acetyl cysteine (NAC) and inhibiting Mox1 oxidase by diphenylene iodonium activated caspase 3-like proteases and markedly enhanced chromatin condensation and DNA fragmentation. This accelerated apoptosis was completely blocked by a caspase inhibitor, z-Val-Ala-Asp-CH(2)F. Mox1-derived reactive oxygen intermediates constitutively activated nuclear factor-kappaB, and inhibition of this activity by nuclear factor-kappaB decoy oligodeoxynucleotide accelerated their spontaneous apoptosis. These results suggest that O(2)(-) produced by the pit cell Mox1 oxidase may play a crucial role in the regulation of their spontaneous apoptosis as well as cell proliferation.
Kazuhito Rokutan, Mami Miyoshi, Shigetada Teshima, Tomoko Kawai, Tsukasa Kuwahara and Kyoichi Kishi : Phenylarsine oxide inhibits heat shock protein 70 induction in cultured guinea pig gastric mucosal cells, American Journal of Physiology, Cell Physiology, Vol.279, No.5, C1506-C1515, 2000.
(要約)
Phenylarsine oxide (PAO) forms a stable ring complex with vicinal dithiols that can be reversed with 2,3-dimercaptopropanol (DMP) but not by dithiothreitol (DTT) or 2-mercaptoethanol (2-ME). PAO at 2 microM or higher inhibited heat shock protein 70 (HSP70) induction within minutes in cultured guinea pig gastric mucosal cells exposed to heat (43 degrees C) for 30 min. PAO did not affect the nuclear translocation and phosphorylation of heat shock factor 1 (HSF1) induced by heat stress, but it completely blocked the binding activity of HSF1 to the heat shock element (HSE), leading to the block of expression of HSP70 mRNA and accumulation of HSP70 in the cells. These inhibitions were completely reversed with 2 microM DMP but not with 0.1 mM DTT or 1 mM 2-ME, suggesting specific interactions between PAO and vicinal dithiol-containing molecules. Thioredoxin (Trx) reversed the inhibition of the binding activity of HSF1 in whole cell extracts prepared from PAO-treated, heat-stressed cells. Our results suggest that PAO may react with vicinal-containing molecules including Trx and specifically block the interaction between HSF1 and HSE.
Tomoko Kawai, Shigetada Teshima, Kenji Kusumoto, Tsukasa Kawahara, Kazumi Kondo, Kyoichi Kishi and Kazuhito Rokutan : A non-toxic heat shock protein 70 inducer, geranyl-geranyl-acetone,restores the heat shock response in gastric mucosa of protein-malnourished rats, The Journal of Laboratory and Clinical Medicine, Vol.136, No.2, 138-148, 2000.
(要約)
Acute gastric mucosal lesions caused by stress or noxious stimuli are important to consider in the management of critically or chronically ill patients. Protein malnutrition has been implicated as a risk factor for stress ulcer and subsequent complications in those patients. When male Wistar rats fed a 5% or 20% casein diet for 3 weeks were exposed to restraint and water-immersion stress, the low-protein diet significantly increased the ulcer index. The low-protein diet did not change the level of heat shock factor 1 (HSF1) in gastric mucosa but it did attenuate the HSF1 activation after exposure to the stress, resulting in the inhibition of HSP70 mRNA expression and HSP70 induction in gastric mucosa. HSP70 is crucial for the maintenance of cell integrity during pathophysiologic conditions; therefore the impaired HSP70 induction appeared to at least in part aggravate stress ulcer. We also tested whether a non-toxic HSP70 inducer, geranyl-geranyl-acetone (GGA), effectively improved the mucosal integrity by stimulating HSP70 induction under protein malnutrition. Intragastric administration of GGA (200 mg/kg twice a day) to the protein-malnourished rats for up to 1 week failed to stimulate the HSP70 induction. However, the administration of GGA (200 mg/kg twice a day) for 3 weeks restored HSP70 induction and induced higher resistance against stress ulcer as compared with results in vehicle-treated, normally nourished rats. Our results suggest that GGA may have a potential benefit for the prevention of stress ulcer in chronically or critically ill patients with protein malnutrition.
Kazuhito Rokutan, Shigetada Teshima, Tomoko Kawai, Tsukasa Kawahara, Kenji Kusumoto, Tohru Mizushima and Kyoichi Kishi : Geranylgeranylacetone stimulates mucin synthesis in cultured guinea pig gastric pit cells by inducing a neuronal nitric oxide synthase, Journal of Gastroenterology, Vol.35, No.9, 673-681, 2000.
(要約)
Nitric oxide (NO) has been considered to play an important role in the regulation of blood flow, mucosal integrity, and mucus production in the stomach. We investigated the stimulatory actions of epidermal growth factor (EGF) and a cytoprotective compound, geranylgeranylacetone (GGA), on mucin synthesis in guinea pig gastric pre-pit cells, maintained in a serum-free culture system. GGA increased [3H]glucosamine uptake and the accumulation of mucus granules positive for galactose oxidase-Schiff reaction in the cells. This stimulatory action of GGA was equivalent to that of EGF, but GGA did not stimulate the cell growth. Both EGF and GGA increased the release of NO degeneration products, NO2- and NO3-. The [3H]glucosamine uptake was completely inhibited by the non-selective NO synthase (NOS) inhibitors, N(G)-nitro-L-arginine and N(G)-monomethyl-L-arginine, and it was only partially inhibited by a more selective inhibitor for inducible NOS isoform (iNOS), aminoguanidine. Northern blotting with a cDNA probe for rat iNOS, and Western blotting with a polyclonal antibody against iNOS, demonstrated that GGA did not up-regulate the iNOS mRNA expression nor induce its protein. In contrast, GGA and EGF induced neuronal NOS, but not endothelial NOS, which was confirmed by immunoblot analyses with antibodies against these constitutive NOS isoforms. Thus, the present experiments suggests that GGA, as well as EGF, stimulates mucin synthesis at least in part through an NO-dependent pathway, leading to an increase in the integrity of the gastric mucosa.
Shigetada Teshima, Shohko Tsunawaki and Kazuhito Rokutan : Helicobacter pylori lipopolysaccharide enhances the expression of NADPH oxidase components in cultured guinea pig gastric mucosal cells, FEBS Letters, Vol.452, No.3, 243-246, 1999.
(要約)
Recently, we showed that cultured guinea pig gastric pit cells possess a phagocyte NADPH oxidase-like activity, which was up-regulated by Helicobacter pylori lipopolysaccharide. We demonstrate here that these cells express all of the phagocyte NADPH oxidase components (gp91-, p22-, p67-, p47-, and p40-phoxes). Treatment with lipopolysaccharide increased the expression of gp91-, p22-, and p67-phoxes, but not that of p47- and p40-phoxes. Intriguingly, the p67-phox expression consistently correlated with up-regulation of superoxide anion-producing ability. Thus, the gastric pit cell NADPH oxidase may play an important role in regulation of the inflammatory response associated with H. pylori infection.
Takeshi Nikawa, Kenji Odahara, Hiroyuki Koizumi, Yasuhiro Kido, Shigetada Teshima, Kazuhito Rokutan and Kyoichi Kishi : Vitamin A Prevents the Decline in Immunoglobulin A and Th2 Cytokine Levels in Small Intestinal Mucosa of Protein-Malnourished Mice1, Biochemical and Molecular Action of Nutrients, Vol.129, 934-941, 1999.
(キーワード)
vitamin A / protein-malnutrition / Immunoglobulin A / Th2 cytokine / mice
110.
Shigetada Teshima, Kazuhito Rokutan, Takeshi Nikawa and Kyoichi Kishi : Macrophage Colony-Stimulating Factor Stimulates Synthesis and Secretion of a Mouse Homolog of a Human IgE-Dependent Histamine-Releasing Factor by Macrophages In Vitro and In Vivo, The Journal of Immunology, Vol.161, No.11, 6356-6366, 1998.
111.
Shigetada Kondo, Kazuhito Rokutan, Takeshi Nikawa and Kyoichi Kishi : Macrophage colony-stimulating factor stimulates synthesis and secretion of a mouse homolog of a human IgE-dependent histamine-releasing factor by macrophages in vitro and in vivo., The Journal of Immunology, Vol.161, No.11, 6356-6366, 1998.
(要約)
Treatment of murine resident peritoneal macrophages with macrophage-CSF (M-CSF) up-regulated the synthesis of a discrete set of proteins, including a 26-kDa protein (p26). The sequence of 20 NH2-terminal amino acids of the purified p26 was identical with the mouse homolog of a human IgE-dependent histamine-releasing factor (HRF). Among macrophage activators tested (M-CSF, granulocyte-macrophage-CSF, IL-3, TNF-alpha, IFN-gamma, and LPS), only M-CSF could up-regulate the p26 HRF synthesis by cultured macrophages. M-CSF not only increased the levels of p26 HRF mRNA and protein, but also stimulated the secretion of an N-glycosylated p26 HRF with a m.w. of 30 kDa. Repeated injections of M-CSF into mouse peritoneal cavity for 4 days elicited macrophages expressing abundant p26 HRF. A single i.p. injection of M-CSF failed to increase the p26 HRF level in peritoneal macrophages of thioglycollate-, LPS-, or adjuvant-treated mice, while M-CSF challenge to OVA-immunized mice caused macrophage infiltration and overproduction of p26 HRF, similarly as did OVA challenge. The Ag-specific priming for enhanced synthesis and secretion of p26 HRF by M-CSF was also demonstrated in cultured macrophages prepared from OVA-immunized mice. An i.p. injection of M-CSF or recombinant p26 HRF triggered eosinophil recruitment, even in the absence of the Ag, in the sensitized mice, but not in normal mice. Furthermore, recombinant p26 HRF could induce eosinophilia without marked macrophage and lymphocyte infiltrations. Our results suggest that p26 HRF secreted by M-CSF-stimulated macrophages may be an important mediator for the late phase allergic inflammation.
Shigetada Teshima, Kazuhito Rokutan, Takeshi Nikawa and Kyoichi Kishi : Guinea pig gastric mucosal cells produce abundant superoxide anion through an NADPH oxidase-like system, Gastroenterology, Vol.115, No.5, 1186-1196, 1998.
(要約)
Superoxide anion (O2-) plays an important role in gastric pathophysiology. The aims of this study were to identify O2--producing activity in gastric mucosal cells and to elucidate its possible roles in inflammatory responses of the cells. The amount of O2- was measured by the reduction of cytochrome c, and O2--producing cells were visualized by nitroblue tetrazolium reaction. Cytosolic components of the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were detected by immunoblotting and immunocytochemical analyses with antibodies against p47-phox and p67-phox. Gastric pit cells, but not parietal cells, spontaneously released O2- at 50 nmol . mg protein-1 . h-1. NADPH or guanosine 5'-O-(3-thiotriphosphate) increased the release more than threefold, whereas diphenylene iodonium inhibited it. A reconstituted cell-free system showed that both membrane fraction and neutrophil-related cytosolic components were required for the activity. p47-phox and p67-phox were expressed in the cells. Live Helicobacter pylori organisms and their culture supernatants significantly increased the O2- release. Furthermore, H. pylori lipopolysaccharide could enhance the release more effectively than Escherichia coli lipopolysaccharide. The O2--dependent activation of nuclear factor κB occurred in these primed cells. Gastric pit cells may actively regulate inflammatory responses of gastric mucosa through a phagocyte NADPH oxidase-like activity.
Kazuhito Rokutan, Shigetada Teshima, Mami Miyoshi, Tomoko Kawai, Takeshi Nikawa and Kyoichi Kishi : Glutathione depletion inhibits oxidant-induced activation of nuclear factor-kappa B,AP-1,and c-Jun/ATF-2 in cultured guinea-pig gastric epithelial cells, Journal of Gastroenterology, Vol.33, No.5, 646-655, 1998.
(要約)
The aim of this study was to reveal the role of intracellular glutathione in the oxidative stress responses of gastric epithelial cells. Metabolic radiolabeling with L-[35S]methionine and analysis of synthesized proteins by gel electrophoresis and fluorography showed that upon exposure to hydrogen peroxide (H2O2) or diamide, primary cultures of guinea-pig gastric epithelial cells rapidly induced several undefined proteins, as well as heat shock proteins. When intracellular glutathione was depleted to less than 10% of the control value by treatment with buthionine-[S,R]-sulfoximine, these inductions were completely inhibited. Gel mobility shift assay demonstrated that H2O2 and diamide rapidly activated nuclear factor-kappa B (NF-kappaB), and diamide activated activator protein (AP)-1, and c-Jun/activating transcription factor (ATF)-2, suggesting that the response may be coupled to these reduction-oxidation (redox)-sensitive transcription factors, as well as heat shock transcription factor 1. The activations of NF-kappaB, AP-1, and c-Jun/ATF-2 by the oxidants did not occur in glutathione-depleted cells. Northern blot analysis showed that glutathione depletion markedly or completely suppressed the diamide-induced expression of c-fos and c-jun mRNAs. These results suggest that intracellular glutathione redox may participate in the initiation of oxidative stress responses; thereby, it plays an important role in gastric mucosal defense.
Shigetada Kondo, Kazuhito Rokutan, Takeshi Nikawa and Kyoichi Kishi : Cultured guinea pig gastric mucosal cells produce abundant superoxide anion through an NADPH oxidase-like system., Gastroenterology, Vol.115, No.5, 1186-1196, 1998.
(キーワード)
superoxide / gastoric cells / host defense
115.
Takeshi Nikawa, Kazuhito Rokutan, Kayo Nanba, Kaori Tokuoka, Shigetada Teshima, Michael J Engle, David H Alpers and Kyoichi Kishi : Vitamin A Up-Regulates Expression of Bone-Type Alkaline Phosphatase in a Rat Small Intestinal Crypt Cell Line and Fetal Rat Small Intestine, Biochemical and Molecular Roles of Nutrients, Vol.128, 1869-1877, 1998.
(キーワード)
vitamin A / liver/bone/kidney alkaline phosphatase / IEC-6 cells / retinoid receptors / fetal rat small intestine
116.
Kazuhito Rokutan, Shigetada Teshima, Mami Miyoshi, Takeshi Nikawa and Kyoichi Kishi : Oxidant-lnduced Activation of Nuclear Factor-Kappa B in Cultured Guinea Pig Gastric Epithelial Cells, Digestive Diseases and Sciences, Vol.42, No.9, 1880-1889, 1997.
Yasuhiro Kido, Takayoshi Tsukahara, Kazuhito Rokutan, Fujiko Shizuka and Kyoichi Kishi : Japanese allowance is sufficient for moderate physical exercise in young men., Journal of Nutritional Science and Vitaminology, Vol.43, No.1, 59-71, 1997.
(キーワード)
運動 / タンパク質 (protein) / 必要量
118.
Eiji Takeda, K. Takata, H. Yamanaka, Yutaka Taketani, K. Morita, Ken-ichi Miyamoto, Masayuki Shono, S. Teshima and Kazuhito Rokutan : Vitamin D3 elicits calcium response and activates blood monocyte-derived macrophages from patients with vitamin D dependent rickets type II., FEBS Letters, Vol.4, No.364, 157-160, 1996.
(キーワード)
Vitamin D3 / monocyte
119.
Tetsuya Hirakawa, Kazuhito Rokutan, Takeshi Nikawa and Kyoichi Kishi : Geranylgeranylacetone induces heat shock proteins. in cultured guinea pig gastric mucosal cells and rat gastric mucosa, Gastroenterology, Vol.111, No.2, 345-357, 1996.
120.
S. Ogihara, M. Yamada, T. Saito, Masayuki Shono and Kazuhito Rokutan : Insulin potentiates mitogenic effect of epidermal growth factor on cultured guinea pig gastric mucous cells., American Journal of Physiology, Gastrointestinal and Liver Physiology, Vol.271, No.1, 104-112, 1996.
(キーワード)
epidermal growth factor / guinea pig
121.
Kazuhito Rokutan, Tetsuya Hirakawa, Shigetada Teshima, Soichi Honda and Kyoichi Kishi : Glutathione Depletion Impairs Transcriptional Activation of Heat Shock Genes in Primary Cultures of Guinea Pig Gastric Mucosal Cells, The Journal of Clinical Investigation, Vol.97, No.10, 2242-2250, 1996.
122.
Eiji Takeda, Kazumi Takata, Hisami Yamanaka, Yutaka Taketani, Kyoko Morita, Ken-ichi Miyamoto, Masayuki Shono, Shigetada Teshima and Kazuhito Rokutan : Vitamin D3 elicits calcium response and activates blood monocyte-derived macrophages from patients with vitamin D dependent rickets type 2, FEBS Letters, Vol.396, 157-160, 1996.
Shigetada Teshima, Kazuhito Rokutan, Masayuki Takahashi, Takeshi Nikawa and Kyoichi Kishi : Induction of heat shock proteins and their possible roles in macrophages during activation by macrophage colony-stimulating factor, The Biochemical Journal, Vol.315, No.Pt 2, 497-504, 1996.
Kazuhiro Bandou, S. Kannuki, Kazuhito Rokutan, Masayuki Shono and K. Matsumoto : Effects of Trapidil and Suramin on Growth Factor-induced Calcium Response and Tyrosin Phosphorylation in Human Glioma Cells, Neurologia Medico-Chirurgica, Vol.35, No.9, 631-638, 1995.
(要約)
Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) induce the proliferation of glioma cells in vitro. Trapidil and suramin inhibit this growth factor-stimulated glioma cell growth, but the mechanisms are not fully understood. The effects of trapidil and suramin on PDGF- and EGF-induced early biochemical events in T98G cells were studied. PDGF induced a rapid increase of intracellular free calcium concentration ([Ca2+]i) in fura-2/acetoxymethyl ester-loaded single glioma (T98G) cells. This increase was completely inhibited by removal of extracellular Ca2+ with ethylene glycol bis(beta-aminoethyl ether)-N,N,N,N-tetraacetic acid but not by an L-type calcium channel blocker (nicardipine), suggesting that PDGF may cause calcium influx through voltage-independent calcium channels in T98G cells. Trapidil and suramin blocked the PDGF-induced calcium response and inhibited the PDGF-initiated tyrosine phosphorylation of the PDGF receptor as detected by Western blot analysis using an antibody specific for phosphotyrosine. Trapidil and suramin also inhibited EGF-initiated calcium response in T98G cells, but only partially inhibited EGF-initiated tyrosine phosphorylation at the same concentrations. Our results suggest that trapidil and suramin inhibit PDGF- and EGF-initiated early biochemical events, and thus suppress growth factor-induced cell proliferation.
Arinobu Yamauchi, Fujiko Shizuka, Takashi Yamamoto, Takeshi Nikawa, Yasuhiro Kido, Kazuhito Rokutan and Kyoichi Kishi : Amino acids and glucose differentially increased extracellular 5-hydroxyindoleacetic acid of the rat brain., Journal of Nutritional Science and Vitaminology, Vol.41, No.3, 325-340, 1995.
(キーワード)
amino acid / serotonin / 5-HIAA / brain / glucose
127.
Shigetada Teshima, Kazuhito Rokutan, Masayuki Takahashi, Takeshi Nikawa, Yasuhiro Kido and Kyoichi Kishi : Alteration of the Respiratory Burst and Phagocytosis of Macrophages under Protein Malnutrition, Journal of Nutritional Science and Vitaminology, Vol.41, No.1, 127-137, 1995.
Kensei Nishida, Yuki Kuwano, Tatsuya Nishikawa, Kiyoshi Masuda and Kazuhito Rokutan : RNA Binding Proteins and Genome Integrity., International Journal of Molecular Sciences, Vol.18, No.7, Jun. 2017.
(要約)
Genome integrity can be threatened by various endogenous or exogenous events. To counteract these stressors, the DNA damage response network contributes to the prevention and/or repair of genomic DNA damage and serves an essential function in cellular survival. DNA binding proteins are involved in this network. Recently, several RNA-binding proteins (RBPs) that are recruited to DNA damage sites have been shown to be direct players in the prevention or repair of DNA damage. In addition, non-coding RNAs, themselves, are involved in the RNA-mediated DNA repair system. Furthermore, RNA modification such as m6A methylation might also contribute to the ultraviolet-responsive DNA damage response. Accumulating evidence suggests that RNA metabolism is more deeply involved in diverse cellular functions than previously expected, and is also intricately associated with the maintenance of genome integrity. In this review, we highlight the roles of RBPs in the maintenance of genome integrity.
Kazuhito Rokutan, Kyoko Morita, Kiyoshi Masuda, Kumiko Tominaga, Michiyo Shikishima, Shigetada Kondo, Tetsuro Omori and Atsuo Sekiyama : Gene expression profiling in peripheral blood leukocytes as a new approach for assessment of human stress response, The Journal of Medical Investigation : JMI, Vol.52, No.3,4, 137-144, Aug. 2005.
(要約)
Stress is the coordinated physiological processes to maintain a dynamic equilibrium under stressful conditions. The equilibrium is threatened by certain physiological and psychological stressors. Stressors trigger physiological, behavioural, and metabolic responses that are aimed at reinstating homeostasis. The hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic nervous system play an essential role in the stress response. Excessive,prolonged, or inadequate response that is termed as "allostasis" or "allostatic load" leads to pathological outcomes. Dysregulation of the HPA axis activity is involved in the pathogenesis of stress-related disorders including major depression. The complex brain-immune-endocrine network regulates the HPA axis, and hereditary predisposition as well as environmental factors such as traumatic experiences in early life also modifies the capacity of an individual to cope. Therefore, it is difficult to correctly assess the complex stress response. We have developed a microarray carrying 1,467 cDNAs that were selected to specifically measure stress response in peripheral blood leukocytes. Using this tool, we have succeeded to objectively assess individual response to acute psychological stress and to detect unique expression profiles in patients with depression. Gene expression profile in peripheral blood leukocytes may be a potentially useful for the detection of disease-associated, abnormal stress responses.
Kazuhito Rokutan, Tetsuya Hirakawa, Shigetada Teshima, Yoko Nakano, Mami Miyoshi, Tomoko Kawai, Emi Konda, Hiroko Morinaga, Takeshi Nikawa and Kyoichi Kishi : Implications of heat shock/stress proteins for medicine and disease, The Journal of Medical Investigation : JMI, Vol.44, No.3-4, 137-147, Feb. 1998.
(要約)
Heat shock/stress proteins (HSPs) are crucial for maintenance of cellular homeostasis during normal cell growth and for survival during and after various cellular stresses. The HSP70 family functions as molecular chaperones and reduces stress-induced denaturation and aggregation of intracellular proteins. In addition to the chaperoning activities, HSP70 has been suggested to exert its protective action by protecting mitochondria and by interfering with the stress-induced apoptotic program. The biochemical and functional properties of HSPs observed in cultured cells may be relevant to organs and tissues in whole animals. The activation of the hypothalamic-pituitary-adrenal axis and the sympathetic nerve system elicits the stress response in selected peripheral tissues; the HSP70 expression in the vasculature and stomach increases resistance against hemodynamic stress and stress-induced mucosal damage, respectively. Gastric mucosa pretreated with mild irritants acquires a tolerance against subsequent mucosal-damaging insults. This phenomenon is known as "adaptive cytoprotection". Transient ischemia also induces ischemic tolerance in the brain and heart, which is called "ischemic preconditioning". The heat shock response is believed to contribute to the acquisition of the tolerance. The therapeutic applications of chaperone inducers that induce HSPs without any toxic effect are also introduced.
Yuki Kuwano, K kajita, S Kano, Y Satake, M Fujita, M Itai, Kensei Nishida and Kazuhito Rokutan : Ultraconserved region-containing transformer 24 inhibits senesces of colon cancer cells., Cell symposia-Human genomics, Singapore, Nov. 2015.
6.
Shizuka Kano, Kensei Nishida, Yuki Kuwano, Takuya Naruto and Kazuhito Rokutan : Analysis of functional transcribed-ultraconserved regions in SR protein family., Cold Spring Harbor Laboratory Meeting on Eukaryotic mRNA processing., Sep. 2015.
7.
Saki Saijo, Kensei Nishida, Shizuka Kano, Takuya Naruto, Yuki Kuwano and Kazuhito Rokutan : A role of serine/arginine-rich splicing factor 7 in cell cycle progression, Cold Spring Harbor Laboratory Meeting on Eukaryotic mRNA processing, Aug. 2015.
8.
Kensei Nishida, Saki Saijo, Shizuka Kano, Takuya Naruto, Yuki Kuwano and Kazuhito Rokutan : Analysis of 3 end processing factors expression during epithelial-mesenchymal transition, Cold Spring Harbor Laboratory Meeting on Eukaryotic mRNA processing, Aug. 2015.
9.
Yuki Kuwano, Y Satake, S Kano, K Fujita, M Itai, Kensei Nishida, Takuya Naruto, Kiyoshi Masuda and Kazuhito Rokutan : Transformer 2 and miR-204 regulate cell death through competitive binding to 3 UTR of BCL2 mRNA, Cell Symposia Regulatory RNAs, San Francisco, Oct. 2014.
10.
S Kano, Kensei Nishida, Y Satake, K Fujita, M Itai, Takuya Naruto, Kiyoshi Masuda, Yuki Kuwano and Kazuhito Rokutan : Analysis of function transcribed ultraconserved regions of SR protein family, Cell Symposia Regulatory RNAs, San Francisco, Oct. 2014.
11.
Kazuhito Rokutan : Lactobaccillus gasseri CP2305 and brain-gut interaction, Short Lecture in 11th International Symposium on Lactic Acid Bacteria, Egmond aan Zee, Netherland, Sep. 2014.
12.
Shizuka Kano, Kensei Nishida, Yuzuru Satake, Yoko Akaike, Kinuyo Fujita, Kiyoshi Masuda, Yuki Kuwano and Kazuhito Rokutan : Arsenite stress-inducible truncated serine/arginine-rich splicing factor 3 regulates interleukin-8 in human colon cancer cells, The 5th EMBO Meeting 2013, Amsterdam, Sep. 2013.
13.
Yoko Akaike, Kiyoshi Masuda, Yuzuru Satake, Kinuyo Fujita, Shizuka Kano, Kensei Nishida, Yuki Kuwano and Kazuhito Rokutan : Homeodomain interacting protein kinase 2 regulates interaction between heterochromatin protein 1 gamma and trimethylated histone H3 Lys9, The 5th EMBO Meeting 2013, Amsterdam, Sep. 2013.
14.
Kinue Fujita, Yuki Kuwano, Shizuka Kano, Yuzuru Satake, Kensei Nishida, Kiyoshi Masuda and Kazuhito Rokutan : Socioeconomic status-related gene expression profiles in peripheral leukocytes from medical staffs, The international conference on social stratification and health, Tokyo, Sep. 2013.
15.
Yuki Kuwano, Sakurako Katsuura-Kamano, Tomoko Kawai, YoKo Kamio and Kazuhito Rokutan : Autism-associated gene expression was commonly observed in peripheral blood leukocytes from subjects with autism and healthy mothers having autistic children., 11th World Congress of Biological Psychiatry, Kyoto, Jun. 2013.
16.
Kiyoshi Masuda, Y Akaike, K Fujita, M Honda, Y Satake, K Kajita, Kensei Nishida, Yuki Kuwano and Kazuhito Rokutan : Hu antigen R (HuR) Regulates an Alternative Splicing of Transformer 2-beta (Tra2beta) and Induces lncRNA (Tra2beta4) under Oxidative Stress, Cell Symposia Regulatory RNAs, Sitges, Spain, Dec. 2012.
17.
Yuki Kuwano, K. Kajita, Y. Satake, Y. Akaike, M. Honda, F. Fujita, Kensei Nishida, Kiyoshi Masuda and Kazuhito Rokutan : The RNA binding protein Transformer-2 beta modulates processing of microRNAs, Cell symposia-Functional RNAs, Dec. 2012.
18.
Y Akaike, Kiyoshi Masuda, K Fujita, M Honda, Y Satake, K Kajita, Kensei Nishida, Yuki Kuwano and Kazuhito Rokutan : Hu antigen R (HuR) Functions as An Alternative Splicing Regulator of Transformer 2-beta (TRA2beta) in response to Oxidative Stress, 4th EMBO meeting 2012, Niece, Sep. 2012.
19.
K Kajita, Yuki Kuwano, Y Satake, Y Akaike, M Honda, K Fujita, Kensei Nishida, Kiyoshi Masuda and Kazuhito Rokutan : Ultraconserved element-containing Transformer 24 mRNA regulates cellular senescence, The 4th EMBO Meeting 2012, Niece, Sep. 2012.
20.
Kazuhito Rokutan : Detection and clinical application of novel stress biomarkers in peripheral blood, Invited morning lecture, The 11th International Congress of Hyperthermic Oncology, Kyoto, Aug. 2012.
21.
Yuki Kuwano, 神尾 陽子, 河合 智子, Sakurako Katsuura, 稲田 尚子, 高木 晶子 and Kazuhito Rokutan : Autism-associated gene expression signatures in peripheral blood leucocytes, Joint Academic Conference on ASD 2011, Tokyo, Dec. 2011.
22.
Naoko Yamagishi, Shigetada Kondo, Kiyoshi Masuda, Yuki Kuwano, Toshihito Tanahashi and Kazuhito Rokutan : Identification of a novel tumor promoting non-coding RNA encoded in the Vegf gene, Cell Symposia Regulatory RNAs, Chicago, Oct. 2011.
23.
Yuki Kuwano, Keisuke Kajita, Yuzuru Satake, Ken Kurokawa, Naoko Yamagishi, Yoko Akaike, Manami Honda, Kensei Nishida, Kiyoshi Masuda, Toshihito Tanahashi and Kazuhito Rokutan : Transfomer-2beta regulates apoptosis through post-transcriptional regulation of bcl-2, Cell Symposia Regulatory RNAs, Chicago, Oct. 2011.
24.
Kiyoshi Masuda, Naoko Yamagishi, Ken Kurokawa, Yuzuru Satake, Keisuke Kajita, Yoko Akaike, Manami Honda, Kensei Nishida, Yuki Kuwano, Toshihito Tanahashi and Kazuhito Rokutan : Hu antigen R (huR) functions as an alternative pre-mRNA splicing enhancer of transformer 2-beta (Tra2beta) on exon definition under oxidative stress, Cell Symposia Regulatory RNAs, Chicago, Oct. 2011.
Manami Honda, Sakurako Katsuura, Yuki Kuwano, Naoko Yamagishi, Ken Kurokawa, Yuzuru Satake, Keisuke Kajita, Yoko Akaike, Kensei Nishida, Kiyoshi Masuda, Toshihito Tanahashi and Kazuhito Rokutan : High-throughput screening of immunomodulators identifies VEGF as a potential biomarker for trait anxiety and depressive mood in healthy Japanese university students, The International Conference on Social Stratification and Health 2011, Tokyo, Aug. 2011.
28.
Sakurako Katsuura, Kamezaki Yoishiko, Yuki Kuwano, Yamagishi Naoko, Kurokawa Ken, Satake Yuzuru, Toshihito Tanahashi and Kazuhito Rokutan : High-throughput screening of brief naturalistic stress-responsive cytokines in Japanese university students taking examinations, The 7th World Congress on Stress, Leiden, Netherland, Aug. 2010.
29.
Sakurako Katsuura, Yoshiko Kamezaki, Yuki Kuwano, Naoko Yamagishi, Ken Kurokawa, Keisuke Kajita, Toshihito Tanahashi and Kazuhito Rokutan : High-throughput screening of immunomodulators identifies VEGF as a potential biomarker for trait anxiety and depressive mood in healthy Japanese university students., The 7th World Congress on Stress, Leiden, Netherlands, Aug. 2010.
30.
Yuki Kuwano, Ken Kurokawa, Sakurako Katsuura, Naoko Yamagishi, Yuzuru Satake, Keisuke Kajita, Toshihito Tanahashi and Kazuhito Rokutan : Alternative splice variants of SMG-1 as a potential marker for brief naturalistic stressors in peripheral leukocytes., The 7th World Congress on Stress, Leiden, Netherlands, Aug. 2010.
31.
Sakurako Katuura, Yoshiko Kamezaki, Yuki Kuwano, Naoko Yamagishi, Ken Kurokawa, Keisuke Kajita, Toshihito Tanahashi and Kazuhito Rokutan : High-throughput screening of immunomodulators identifies VEGF as a potential biomarker for trait anxiety and depressive mood in healthy Japanese university students., The 7th World Congress on Stress, Leiden, Netherland, Aug. 2010.
32.
Sakurako Katuura, Yoshiko Kanmezaki, Yuki Kuwano, Naoko Yamagishi, Ken kurokawa, Yuzuru Satake, Toshihito Tanahashi and Kazuhito Rokutan : High-throughput screening of brief naturalistic stress-responsive cytokines in Japanese university students taking examinations., The 7th World Congress on Stress, Netherland, Aug. 2010.
33.
Ken Kurokawa, Toshihito Tanahashi, Tutomu Iima, Yuta Yamamoto, Kensei Nishida, Kiyoshi Masuda, Yuki Kuwano, Shigetada Kondo, M Fukushima and Kazuhito Rokutan : microRNAs regulate 5-fluorouracil resistance in human colon cancer cells, Digestive Disease Week 2010, New Orleans, May 2010.
34.
Yuki Kuwano, Yuzuru Satake, Kensei Nishida, Kiyoshi Masuda, Ken Kurokawa, Shigetada Kondo, Toshihito Tanahashi and Kazuhito Rokutan : The mechanism of transfomer 2-beta gene expression in response to oxidative stress in human colon epithelial cells, Digestive Disease Week 2010, New Orleans, May 2010.
35.
Zhi-Rong Qian, Toshihito Tanahashi, Katsuhiko Yoshimoto, Shozo Yamada, Sakurako Katsuura, Wang EL, Kazuhito Rokutan and Toshiaki Sano : MicroRNA Expression Abnormalities in Pituitary Adenomas Are Associated with Distinctive Pathologic Features and May Contribute to Tumorigenesis., 99th Annual Meeting of United States and Canadian Academy of Pathology, Washington, D.C., Mar. 2010.
36.
Hiroyoshi Sei, Sachiko Chikahisa, Kumiko Tominaga, Tomoko Kawai, Katsutaka Oishi, Norio Ishida and Kazuhito Rokutan : Bezafibrate, a PPARs agonist, enhances EEG delta oscillation during NREM sleep in mice, Neuroscience 2008, Washington DC, USA, Nov. 2008.
37.
Junichi Iga, Shu-ichi Ueno, Ken Yamauchi, Shusuke Numata, I Motoki, Shinya Tayoshi, S Kinouchi, K Ohta, H Song, Kyoko Morita, Kazuhito Rokutan, H Tanabe, A Dano and Tetsuro Ohmori : Gene expression and association analysis of LIM (PDLIM5) in major depression, Neuroscience, Atlanta, Oct. 2006.
38.
Kazunori Sekine, Kyoko Morita, Masuda Kiyoshi, Gou Satou, Kazuhito Rokutan and Noriaki Takeda : Microarray analysis of stress-related gene expression in patients with Ménière's disease, 24TH Barany Society Meeting, Uppsala, Sweden, Jun. 2006.
39.
Kazuhito Rokutan : Role of heat shock proteins in mastric mucosal protection, Shandon Digestive Disease Week(DDW)Plenary Lecture, Shandon, Aug. 2004.
40.
Kazuhito Rokutan : Pathophysiology of Nox1 in the gastrointestinal tract, Second Internatinal Conference on NAD(P)H oxidase, Atlanta, Mar. 2004.
41.
Kazuhito Rokutan : Roles of Nox1 in innate immune response and cancer development in the gastrointestinal tract, 1st Internatinal Conference on NAD(P)H oxidase, Frankfurt, May 2002.
42.
Takeshi Nikawa, Madoka Ikemoto, Takako Kitano, Mihoko Kano, Shigetada Teshima, Kazuhito Rokutan, Kyoichi Kishi, Shinichi Takeda, Ikuya Nonaka, Kenneth M. Baldwin, Ryutaro Izumi and Takae Towatari : Space Shuttle flight enhances degradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway, 2nd iInternational Society of Proteolysis, Munich, Geramany, Oct. 2001.
43.
Takeshi Nikawa, Madoka Ikemoto, Takako Kitano, Mihoko Kano, Shigetada Teshima, Kazuhito Rokutan, Kyoichi Kishi, Shinichi Takeda, Ikuya Nonaka, Kenneth M. Baldwin, Ryutaro Izumi and Takae Towatari : Speaceflight and unweighting enhance degradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway and antiozidative nutrient prevents muscle protein degradation in tail-suspended rats, 17th International Congress of Nutrition, Wien, Aug. 2001.
44.
Takeshi Nikawa, Madoka Ikemoto, Chiho Watanabe, Takako Kitano, Shigetada Teshima, Kazuhito Rokutan, Kyoichi Kishi, Shinichi Takeda, Ikuya Nonaka, Kenneth M. Baldwin and Ryutaro Izumi : Tail-suspension stimulates expression of muscle proteases and cysteine supplementation prevents loss of muscle weight in tail-suspended rats, 21th Annual Gravitational Physiology Meeting, Nagoya, Apr. 2000.
45.
Takeshi Nikawa, K Tokuoka, Shigetada Teshima, Madoka Ikemoto, Takae Towatari, David H. Alpers, Kazuhito Rokutan and Kyoichi Kishi : Synergistic effects of retinoic acid and IL-1 on expression of the bone-type alkaline phosphatase and retinoid receptors in small intestinal epithelial IEC-6 cells, Annual Meeting of the American Association of the American Gastroenterological Association, Orlando, May 1999.
46.
Takeshi Nikawa, Kazuhito Rokutan, Shigetada Teshima, David H. Alpers and Kyoichi Kishi : Inflammatory cytokines are involved in retinoic acid-mediated maturation of small intestinal epithelial cells, 12th Federation of Asian and Oseanian Biochemists and Molecular Biologists, Tokushima, Oct. 1996.
47.
Takeshi Nikawa, Shigetada Teshima, David H. Alpers, Kyoichi Kishi and Kazuhito Rokutan : Involvement of inflammatory cytokines in retinoic acid-mediated maturation of small intestinal epithelial cells, Annual Meeting of the American Gastroenterological Association, San Francisco, May 1996.
48.
Takeshi Nikawa, Atsuko Sakai, M Emgle, David H. Alpers, Kazuhito Rokutan and Kyoichi Kishi : Stimulatory action of retinoic acid on differentiation of rat small intestinal cells, Annual Meeting of the American Gastroenterological Association, San Diego, May 1995.
S Kano, 西田 憲生, 桑野 由紀, 増田 清士, K Kurokawa, 六反 一仁 : Ultraconserved exon-containing SRSF3 mRNA isoform is specifically translated to the truncated SRSF3protein under oxidative stress, 第35回日本分子生物学会, 2012年12月.
銭 志栄, 棚橋 俊仁, 吉本 勝彦, 山田 正三, 勝浦 桜子, 六反 一仁, 佐野 壽昭 : MicroRNA Expression Abnormalities in Pituitary Adenomas are Associated with Distinctive Pathologic Features and May Contribute to Tumorigenesis, 第55回日本病理学会秋期特別総会, 2009年11月.
86.
銭 志栄, 棚橋 俊仁, 吉本 勝彦, 山田 正三, 勝浦 桜子, 六反 一仁, 佐野 壽昭 : MicroRNA expression abnormalities in pituitary adenomas are associated with distinctive pathologic features and may contribute to tumorigenesis, 第13回日本内分泌病理学会学術総会, 2009年10月.
Yuki Kuwano, Manami Honda, Kinuyo Fujita, Yoko Akaike, Shizuka Kano, Yuzuru Satake, Kensei Nishida and Kazuhito Rokutan : Chronic academic stress increases a group of microRNAs in peripheral blood in healthy Japanese students., The international conference on social stratification and health 2013, Sep. 2013.
Takeshi Nikawa, Madoka Ikemoto, Chiho Watanabe, Takako Kitano, Keeneth M. Baldwin, Ryutaro Izumi, Ikuya Nonaka, Shigetada Teshima, Kazuhito Rokutan, Shinichi Takeda and Kyoichi Kishi : Development of effective space food against weightlessness-induced muscle atrophy, The Progress Report of Ground Research Announcement for Space Utilization, Dec. 2001.