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1.  Time-Dependent Expression Profiles of microRNAs and mRNAs in Rat Milk Whey 
PLoS ONE  2014;9(2):e88843.
Functional RNAs, such as microRNA (miRNA) and mRNA, are present in milk, but their roles are unknown. To clarify the roles of milk RNAs, further studies using experimental animals such as rats are needed. However, it is unclear whether rat milk also contains functional RNAs and what their time dependent expression profiles are. Thus, we prepared total RNA from whey isolated from rat milk collected on days 2, 9, and 16 postpartum and analyzed using microarrays and quantitative PCR. The concentration of RNA in colostrum whey (day 2) was markedly higher than that in mature milk whey (days 9 and 16). Microarray analysis detected 161 miRNAs and 10,948 mRNA transcripts. Most of the miRNAs and mRNA transcripts were common to all tested milks. Finally, we selected some immune- and development-related miRNAs and mRNAs, and analysed them by quantitative PCR (in equal sample volumes) to determine their time-dependent changes in expression in detail. Some were significantly more highly expressed in colostrum whey than in mature milk whey, but some were expressed equally. And mRNA expression levels of some cytokines and hormones did not reflect the protein levels. It is still unknown whether RNAs in milk play biological roles in neonates. However, our data will help guide future in vivo studies using experimental animals such as rats.
PMCID: PMC3923055  PMID: 24533154
2.  Regulation of Innate Immune Function in Bovine Oviduct Epithelial Cells in Culture: The Homeostatic Role of Epithelial Cells in Balancing Th1/Th2 Response 
This study aimed to investigate the role of epithelial cells in regulating innate immunity in bovine oviduct epithelial cell (BOEC) culture. We studied the effect of Escherichia coli lipopolysaccharide (LPS) and its interaction with ovarian steroids, estradiol (E2) and progesterone (P4), and luteinizing hormone (LH) at concentrations observed during the preovulatory period on immune responses in BOEC culture. Immunohistochemistry of oviduct tissue showed intensive expression of Toll-like receptor-4 (TLR-4) and TLR-2 in epithelial cells. A dose of 10 ng/ml LPS stimulated TLR-4, cyclooxygenase-2 (COX-2), nuclear factor kappa B inhibitor A (NFKBIA), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) expression, indicating an early pro-inflammatory response. A dose of 100 ng/ml LPS did not induce expression of these genes but stimulated TLR-2, IL-10,IL-4 and microsomal prostaglandin E synthase-1 (mPGES-1) expression and PGE2 secretion, indicating an anti-inflammatory response. Ovarian steroids and LH completely block LPS (10 ng/ml)-induced TLR-4, IL-1β and TNF-α expression as well as LPS (100 ng/ml)-induced TLR-2 expression. Taken together, this study suggests the existence of an early signaling system to respond to infection in the BOEC. In addition, ovarian steroids and LH may play a critical role in inducing homeostasis and in controlling hyperactive pro-inflammatory responses detrimental to epithelial cells, sperm and the embryo.
PMCID: PMC3934114  PMID: 23800958
Bovine oviduct epithelial cell; Ovarian steroids; TLR-4; Th1/Th2 response
3.  Characterization of Legionella pneumophila Isolated from Environmental Water and Ashiyu Foot Spa 
BioMed Research International  2013;2013:514395.
Hot springs are the most common infectious source of Legionella pneumophila in Japan. However, little is known about the association between L. pneumophila and environmental waters other than hot springs. In this study, water samples from 22 environmental water sites were surveyed; of the 22 samples, five were L. pneumophila positive (23%). L. pneumophila was mainly isolated from ashiyu foot spas, a type of hot spring for the feet (3/8, 38%). These isolates had genetic loci or genes that encoded the virulence factors of L. pneumophila. Moreover, these isolates showed higher intracellular growth and stronger cytotoxicity compared with the reference strain. These results suggest that ashiyu foot spa can be the original source for L. pneumophila infection.
PMCID: PMC3728520  PMID: 23956987
4.  Downregulation of Lymphatic Vessel Formation Factors in PGF2α-induced Luteolysis in the Cow 
Prostaglandin F2α (PGF2α) induces luteolysis in cows and causes infiltration of immune cells, which resembles inflammatory immune response. Since the general immune response is mediated by the lymphatic system, we hypothesized that luteolysis is associated with generation of an immune response that involves lymphatic vessels in the bovine corpus luteum (CL). The CL was obtained from Holstein cows at the mid-luteal phase (days 10–12, ovulation = day 0) by ovariectomy at various time points after PGF2α injection. Lymphatic endothelial cell (LyEC) marker, endothelial hyaluronan receptor 1 (LYVE1), levels decreased significantly 12 h after PGF2α injection. Podoplanin, another LyEC marker, decreased from 15 min after PGF2α injection. PGF2α also diminished mRNA expression of lymphangiogenic factors, such as vascular endothelial growth factor (VEGF) C, VEGFD and VEGF receptor 3 (VEGFR3). During PGF2α-induced luteolysis, the levels of mRNA expression of tumor necrosis factor α (TNFα; the major pro-inflammatory cytokine) and chemokine (C-X-C motif) ligand 1 (neutrophil chemokine) were increased. On the other hand, chemokine (C-C motif) ligand 21, which regulates outflow of immune cells from tissues via the lymphatic vessels during an immune response, was decreased. This study demonstrated that the lymphatic network in the CL is disrupted during luteolysis and suggests that during luteolysis, immune cells can induce a local immune response in the CL without using the lymphatic vessels.
PMCID: PMC3934130  PMID: 23524297
Corpus luteum; Cow; Luteolysis; Lymphatic vessel; Tumor necrosis factor α
5.  Early diagnosis of asparaginase-associated pancreatitis based on elevated serum elastase-1 levels: Case reports 
Biomedical Reports  2013;1(4):651-653.
L-asparaginase (L-asp) is a well-known anticancer agent used in the treatment of acute lymphoblastic leukemia (ALL) in children. However, it is also known to induce several acute complications, such as acute pancreatitis. This is a presentation of two pediatric acute lymphoblastic leukemia (ALL) cases of asparaginase-associated pancreatitis (AAP) diagnosed at an early stage based on elevated serum elastase-1 levels, in the presence of normal serum amylase levels. Early diagnosis and treatment of AAP, although imperative, is occasionally difficult if only standard diagnostic procedures are followed. Elastase-1 is a potentially useful marker for the early diagnosis of AAP. Therefore, the measurement of elastase-1 levels, in addition to amylase and lipase levels, is recommended in L-asp-treated patients.
PMCID: PMC3917048  PMID: 24649003
asparaginase; pancreatitis; elastase-1; acute lymphoblastic leukemia; chemotherapy
6.  Rapid Accumulation of Polymorphonuclear Neutrophils in the Corpus luteum during Prostaglandin F2α-Induced Luteolysis in the Cow 
PLoS ONE  2012;7(1):e29054.
Prostaglandin F2α (PGF2α) induces luteolysis within a few days in cows, and immune cells increase in number in the regressing corpus luteum (CL), implying that luteolysis is an inflammatory-like immune response. We investigated the rapid change in polymorphonuclear neutrophil (PMN) numbers in response to PGF2α administration as the first cells recruited to inflammatory sites, together with mRNA of interleukin-8 (IL-8: neutrophil chemoattractant) and P-selectin (leukocyte adhesion molecule) in the bovine CL. CLs were collected by ovariectomy at various times after PGF2α injection. The number of PMNs was increased at 5 min after PGF2α administration, whereas IL-8 and P-selectin mRNA increased at 30 min and 2 h, respectively. PGF2α directly stimulated P-selectin protein expression at 5–30 min in luteal endothelial cells (LECs). Moreover, PGF2α enhanced PMN adhesion to LECs, and this enhancement by PGF2α was inhibited by anti-P-selectin antibody, suggesting that P-selectin expression by PGF2α is crucial in PMN migration. In conclusion, PGF2α rapidly induces the accumulation of PMNs into the bovine CL at 5 min and enhances PMN adhesion via P-selectin expression in LECs. It is suggested that luteolytic cascade by PGF2α may involve an acute inflammatory-like response due to rapidly infiltrated PMNs.
PMCID: PMC3250407  PMID: 22235260
7.  Protective Role of Heme Oxygenase-1 in Listeria monocytogenes-Induced Abortion 
PLoS ONE  2011;6(9):e25046.
It is well-known fact that various pathogens, including bacteria, virus, and protozoa, induce abortion in humans and animals. However the mechanisms of infectious abortion are little known. In this study, we demonstrated that Listeria monocytogenes infection in trophoblast giant cells decreased heme oxygenase (HO)-1 and B-cell lymphoma-extra large (Bcl-XL) expression, and that their overexpression inhibited cell death induced by the infection. Furthermore, HO-1 and Bcl-XL expression levels were also decreased by L. monocytogenes in pregnant mice. Treatment with cobalt protoporphyrin, which is known to induce HO-1, inhibited infectious abortion. Taken together, our study indicates that L. monocytogenes infection decreases HO-1 and Bcl-XL expression and induces cell death in placenta, leading to infectious abortion.
PMCID: PMC3174987  PMID: 21949846
8.  Cooperation between LepA and PlcH Contributes to the In Vivo Virulence and Growth of Pseudomonas aeruginosa in Mice▿  
Infection and Immunity  2010;79(1):211-219.
Pseudomonas aeruginosa-derived large extracellular protease (LepA) and hemolytic phospholipase C (PlcH) are considered to play an important role in the pathogenicity of this organism. Although bacterial growth appears to be closely related to virulence, little is known about whether LepA and PlcH participate in the growth and virulence of P. aeruginosa. In this study, we investigated whether LepA and PlcH contribute to the virulence and growth of P. aeruginosa using a wild-type strain and mutants. The growth rate of the isogenic lepA single mutant was lower than that of the wild-type strain in a minimal medium containing serum albumin or hemoglobin as the sole carbon and nitrogen source. Furthermore, the growth rate of the lepA plcH double mutant decreased greatly compared with that of the wild-type strain in a minimal medium containing erythrocytes as a sole nutrient source for growth. Thus, these results indicate that cooperation between LepA and PlcH would contribute to the utilization of erythrocytes as a sole nutrient source for the growth of P. aeruginosa. In addition, mouse infection experiments demonstrated that the virulence of the lepA and plcH single mutants was attenuated, and the numbers of the mutants were lower than the numbers of the wild-type strain in peritoneal lavage fluid and whole-blood specimens. In particular, the virulence and growth rate of the lepA plcH double mutant were markedly lower than those of the wild-type strain. Collectively, these results suggest that LepA and PlcH contribute to the in vivo virulence and growth of P. aeruginosa.
PMCID: PMC3019895  PMID: 21041488
9.  Evidence that polymorphonuclear neutrophils infiltrate into the developing corpus luteum and promote angiogenesis with interleukin-8 in the cow 
After ovulation in the cow, the corpus luteum (CL) rapidly develops within a few days with angiogenesis and progesterone production. CL formation resembles an inflammatory response due to the influx of immune cells. Neutrophils play a role in host defense and inflammation, and secrete chemoattractants to stimulate angiogenesis. We therefore hypothesized that neutrophils infiltrate in the developing CL from just after ovulation and may play a role in angiogenesis of the CL.
Methods and Results
Polymorphonuclear neutrophils (PMN) were detected in CL tissue by Pas-staining, and interleukin-8 (IL-8, a neutrophil-specific chemoattractant) was measured in supernatant of the CL tissue culture: considerable amounts of PMNs and the high level of IL-8 were observed during the early luteal phase (days 1-4 of the estrous cycle). PMNs and IL-8 were low levels in the mid and late luteal phases, but IL-8 was increased during luteal regression. The PMN migration in vitro was stimulated by the supernatant from the early CL but not from the mid CL, and this activity was inhibited by neutralizing with an anti-IL-8 antibody, indicating the major role of IL-8 in inducing active PMN migration in the early CL. Moreover, IL-8 stimulated proliferation of CL-derived endothelial cells (LECs), and both the supernatant of activated PMNs and IL-8 stimulated formation of capillary-like structures of LECs.
PMNs migrate into the early CL partially due to its major chemoattractant IL-8 produced at high levels in the CL, and PMNs is a potential regulator of angiogenesis together with IL-8 in developing CL in the cow.
PMCID: PMC3129584  PMID: 21651784
10.  A Triacylated Lipoprotein from Mycoplasma genitalium Activates NF-κB through Toll-Like Receptor 1 (TLR1) and TLR2▿  
Infection and Immunity  2008;76(8):3672-3678.
Mycoplasma genitalium is a sexually transmitted bacterial pathogen that causes nongonococcal chlamydia-negative urethritis, mucopurulent cervicitis, endometritis, pelvic inflammatory disease, and tubal factor infertility in humans. However, pathogenic agents that induce inflammatory responses have not been identified in M. genitalium. In this study, we examined the involvement of Toll-like receptors (TLRs) in activation of the immune response by a lipoprotein from M. genitalium and their active component responsible for NF-κB activation. The Triton X-114 detergent phase of M. genitalium was found to induce NF-κB through TLR2. The active component of the Triton X-114 detergent phase was a lipoprotein precursor, MG149. The activation of NF-κB by MG149 was inhibited by a dominant negative (DN) construct of TLR1 but not by a DN construct of TLR6. These results indicate that the activation of NF-κB by MG149 is dependent on TLR1 and TLR2. A synthetic lipopeptide derived from MG149 containing three acyl chains also induced NF-κB through TLR1 and TLR2. Thus, the results show that MG149, a triacylated lipoprotein from M. genitalium, activates NF-κB through TLR1 and TLR2.
PMCID: PMC2493199  PMID: 18474641
11.  Growth and Endocrine Function in Long-term Adult Survivors of Childhood Stem Cell Transplant 
The number of long-term surviving stem cell transplant (SCT) recipients has increased steadily, and attention has now extended to the late complications of this procedure. The objective of this study was to investigate relationship among growth and endocrine functions in long-term adult survivors of childhood SCT. The inclusion criteria of this study were survival at least 5 yr after SCT and achievement of adult height. Fifty-four patients (39 males) fulfilled these criteria and were included in this study. Growth was mainly evaluated by height standard deviation score (SDS) and individual longitudinal growth curves. Among the 54 patients, those that received SCT before 10 yr of age showed significantly greater reductions in changes in height SDS (mean –1.75, range –4.80 to –0.10) compared with those that received SCT at or after 10 yr of age (mean –0.50, range –1.74 to 1.20; P<0.001). The mean loss of height for all patients who received SCT during childhood was estimated to be approximately 1 SDS/6.5 yr (r=0.517). Individual longitudinal growth curves indicated that a significant growth spurt was absent in severe short stature patients during the pubertal period without severe endocrine dysfunctions including GH deficiency. The incidence of growth disorder in long-term adult survivors depends on the age at SCT and whether they received radiation therapy. Life-long follow-up is necessary for survivors to detect, prevent and treat the late endocrine complications in SCT survivors.
PMCID: PMC4004878  PMID: 24790374
growth disorder; endocrine function; long-term adult survivors
12.  Mycoplasma pneumoniae-Derived Lipopeptides Induce Acute Inflammatory Responses in the Lungs of Mice▿  
Infection and Immunity  2007;76(1):270-277.
The pathogenesis of Mycoplasma pneumoniae infection is considered to be in part attributable to excessive immune responses. In this study, we investigated whether synthetic lipopeptides of subunit b of F0F1-type ATPase (F0F1-ATPase), NF-κB-activating lipoprotein 1 (N-ALP1), and N-ALP2 (named FAM20, sN-ALP1, and sN-ALP2, respectively) derived from M. pneumoniae induce cytokine and chemokine production and leukocyte infiltration in vivo. Intranasal administration of FAM20 and sN-ALP2 induced infiltration of leukocyte cells and production of chemokines and cytokines in bronchoalveolar lavage fluid, but sN-ALP1 failed to do so. The activity of FAM20 was notably higher than that of sN-ALP2. FAM20 and sN-ALP2 induced tumor necrosis factor alpha (TNF-α) through Toll-like receptor 2 in mouse peritoneal macrophages. Moreover, in the range of low concentrations of lipopeptides, FAM20 showed relatively high activity of inducing TNF-α in mouse peritoneal macrophages compared to synthetic lipopeptides such as MALP-2 and FSL-1, derived from Mycoplasma fermentans and Mycoplasma salivarium, respectively. These findings indicate that the F0F1-ATPase might be a key molecule in inducing cytokines and chemokines contributing to inflammatory responses during M. pneumoniae infection in vivo.
PMCID: PMC2223659  PMID: 17954722
13.  Serratia marcescens Serralysin Induces Inflammatory Responses through Protease-Activated Receptor 2▿  
Infection and Immunity  2006;75(1):164-174.
The Serratia marcescens-derived protease serralysin is considered to play an important role in the pathogenesis of infection. Protease-activated receptor 2 (PAR-2) is activated by trypsin and also several other trypsin-like serine proteases, leading to the modulation of inflammatory and immune responses. However, little is known about the activation of PAR-2 by bacterial proteases and its roles in bacterial infection. In this study, we investigated whether S. marcescens serralysin activates host inflammatory responses through PAR-2. Our results demonstrated that serralysin induces interleukin-6 (IL-6) and IL-8 mRNA expression in a human lung squamous cell carcinoma, EBC-l cells. In addition, serralysin activated activator protein 1 (AP-1)-, CCAAT/enhancer-binding protein (C/EBP)-, and nuclear factor-κB (NF-κB)-driven promoters in EBC-1 cells. An electrophoretic mobility shift assay showed that serralysin activates the binding of AP-1, C/EBPβ, and NF-κB in the cells. Inactivation of serralysin resulted in the failure of transactivation of AP-1-, C/EBP-, and NF-κB-driven promoters in the cells. Furthermore, serralysin activated AP-1-, C/EBP-, and NF-κB-driven promoters via PAR-2 in HeLa cells. PAR-2 antagonist peptides decreased serralysin-induced transactivation of AP-1-, C/EBP-, and NF-κB-driven promoters in EBC-1 cells. Considered together, these results suggest that serralysin requires PAR-2 to activate the critical transcription factors AP-1, C/EBPβ, and NF-κB for host inflammatory responses.
PMCID: PMC1828393  PMID: 17043106
14.  Relationship between job stress and self-rated health among Japanese full-time occupational physicians 
We investigated relationship between job stress and self-rated health among Japanese nese full-time occupational physicians (OPs).
In 2000, we mailed self-administrated questionnaires to 716 OPs. Of these OPs, 349 (49%) returned sufficiently completed questionnaires for analyses. oblique-rotated principal factor analysis of the job stress questionnaire extracted three components; low understanding of occupational health services in companies (low understanding), conflicts between occupational physicians and their coworkers (conflicts), and discrepancies between occupational physicians’ routine work and occupational health services (discrepancies).
The model, in which low understanding contributed to self-rated health through job satisfaction and self-rated health was influenced by job satisfaction and discrepancies, provided a good fit to the data.
We found that a potential relationship between job stress and self-rated health among Japanese full-time OPs. The present results implied that among full-time OPs, low understanding contributed negatively to self-rated health through job satisfaction, and that self-rated health was influenced positively by job satisfaction and negatively by discrepancies.
PMCID: PMC2723404  PMID: 21432124
job stress; job satisfaction; self-rated health; occupational physicians; structural equation model
15.  Levels and Concentration Ratios of Polychlorinated Biphenyls and Polybrominated Diphenyl Ethers in Serum and Breast Milk in Japanese Mothers 
Environmental Health Perspectives  2006;114(8):1179-1185.
Blood and/or breast milk have been used to assess human exposure to various environmental contaminants. Few studies have been available to compare the concentrations in one matrix with those in another. The goals of this study were to determine the current levels of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in Japanese women, with analysis of the effects of lifestyle and dietary habits on these levels, and to develop a quantitative structure–activity relationship (QSAR) with which to predict the ratio of serum concentration to breast milk concentration. We measured PBDEs and PCBs in 89 paired samples of serum and breast milk collected in four regions of Japan in 2005. The geometric means of the total concentrations of PBDE (13 congeners) in milk and serum were 1.56 and 2.89 ng/g lipid, respectively, whereas those of total PCBs (15 congeners) were 63.9 and 37.5 ng/g lipid, respectively. The major determinant of total PBDE concentration in serum and milk was the geographic area within Japan, whereas nursing duration was the major determinant of PCB concentration. BDE-209 was the most predominant PBDE congener in serum but not in milk. The excretion of BDE 209 in milk was lower than that of BDE 47 and BDE 153. QSAR analysis revealed that two parameters, calculated octanol/water partition and number of hydrogen-bond acceptors, were significant descriptors. During the first weeks of lactation, the predicted partitioning of PBDE and PCB congeners from serum to milk agreed with the observed values. However, the prediction became weaker after 10 weeks of nursing.
PMCID: PMC1552037  PMID: 16882522
breast milk; partition coefficient; polybrominated diphenyl ethers; polychlorinated biphenyls; quantitative structure—activity relationship; serum
16.  Relationship between coping skills and job satisfaction among Japanese full-time occupational physicians 
The present study investigated the relationship between coping skills and job satisfaction among Japanese full-time occupational physicians (OPs).
In 2000 we mailed self-administered questionnaires to 716 full-time OPs who were members of “Sanyu-kai”, the only Japanese association of full-time OPs. The questionnaires included age, gender, marital status, main type of company’s work, the number of full-time OPs, the number of employees, working years as an OP, tenure in the present company, job stress, and coping skills question. The coping skills questions consisted of 11 items which were decided after discussion among several experienced full-time OPs. In total, 351 (49%) of the OPs returned suitable questionnaires for analyses.
Considering age, gender, marital status, and coping skills, multiple regression analysis (stepwise method) found that age, simplification of work, obvious roles for staff, consultations, and communication in the community and company were factors which contributed significantly to job satisfaction. Structural equation modeling showed that age and coping skills such as work system improvements, consultations, and communication in the community and company influenced job satisfaction.
Our results indicated that the age and coping skills influenced job satisfaction among full-time OPs. Our results are also considered to support the training of OPs in the future.
PMCID: PMC2723387  PMID: 21432099
full-time occupational physician; coping skills; job satisfaction; multiple regression analysis; structural equation modeling
17.  In Vitro Activities of Moxifloxacin and Other Fluoroquinolones against Mycoplasma pneumoniae 
A total of 105 isolates of Mycoplasma pneumoniae were evaluated for susceptibility to moxifloxacin, sparfloxacin, levofloxacin, and ciprofloxacin. Moxifloxacin, a newly synthesized compound, showed the greatest activity. The MICs and MBCs at which 50 and 90% of isolates were affected were 0.15 (MIC50 and MBC50) and 0.3 μg/ml (MIC90 and MBC90) respectively. The results indicate that moxifloxacin might be promising an antimycoplasmal agent.
PMCID: PMC90571  PMID: 11353651

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