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1.  A common missense variant of monocarboxylate transporter 9 (MCT9/SLC16A9) gene is associated with renal overload gout, but not with all gout susceptibility 
Human Cell  2013;26:133-136.
Gout is a common disease caused by hyperuricemia, which shows elevated serum uric acid (SUA) levels. From a viewpoint of urate handling in humans, gout patients can be divided into those with renal overload (ROL) gout with intestinal urate underexcretion, and those with renal underexcretion (RUE) gout. Recent genome-wide association studies (GWAS) revealed an association between SUA and a variant in human monocarboxylate transporter 9 (MCT9/SLC16A9) gene. Although the function of MCT9 remains unclear, urate is mostly excreted via intestine and kidney where MCT9 expression is observed. In this study, we investigated the relationship between a variant of MCT9 and gout in 545 patients and 1,115 healthy volunteers. A missense variant of MCT9 (K258T), rs2242206, significantly increased the risk of ROL gout (p = 0.012), with odds ratio (OR) of 1.28, although it revealed no significant association with all gout cases (p = 0.10), non-ROL gout cases (p = 0.83), and RUE gout cases (p = 0.34). In any case groups and the control group, minor allele frequencies of rs2242206 were >0.40. Therefore, rs2242206 is a common missense variant and is revealed to have an association with ROL gout, indicating that rs2242206 relates to decreased intestinal urate excretion rather than decreased renal urate excretion. Our study provides clues to better understand the pathophysiology of gout as well as the physiological roles of MCT9.
PMCID: PMC3844819  PMID: 23990105
Gouty arthritis; Single nucleotide polymorphism (SNP); Gut urate excretion; Carnitine; Solute carrier (SLC) family transporter
2.  ABCG2 dysfunction causes hyperuricemia due to both renal urate underexcretion and renal urate overload 
Scientific Reports  2014;4:3755.
Gout is a common disease which results from hyperuricemia. We have reported that the dysfunction of urate exporter ABCG2 is the major cause of renal overload (ROL) hyperuricemia, but its involvement in renal underexcretion (RUE) hyperuricemia, the most prevalent subtype, is not clearly explained so far. In this study, the association analysis with 644 hyperuricemia patients and 1,623 controls in male Japanese revealed that ABCG2 dysfunction significantly increased the risk of RUE hyperuricemia as well as overall and ROL hyperuricemia, according to the severity of impairment. ABCG2 dysfunction caused renal urate underexcretion and induced hyperuricemia even if the renal urate overload was not remarkable. These results show that ABCG2 plays physiologically important roles in both renal and extra-renal urate excretion mechanisms. Our findings indicate the importance of ABCG2 as a promising therapeutic and screening target of hyperuricemia and gout.
PMCID: PMC3895923  PMID: 24441388
3.  Genome-wide quantification of homeolog expression ratio revealed nonstochastic gene regulation in synthetic allopolyploid Arabidopsis 
Nucleic Acids Research  2014;42(6):e46.
Genome duplication with hybridization, or allopolyploidization, occurs commonly in plants, and is considered to be a strong force for generating new species. However, genome-wide quantification of homeolog expression ratios was technically hindered because of the high homology between homeologous gene pairs. To quantify the homeolog expression ratio using RNA-seq obtained from polyploids, a new method named HomeoRoq was developed, in which the genomic origin of sequencing reads was estimated using mismatches between the read and each parental genome. To verify this method, we first assembled the two diploid parental genomes of Arabidopsis halleri subsp. gemmifera and Arabidopsis lyrata subsp. petraea (Arabidopsis petraea subsp. umbrosa), then generated a synthetic allotetraploid, mimicking the natural allopolyploid Arabidopsis kamchatica. The quantified ratios corresponded well to those obtained by Pyrosequencing. We found that the ratios of homeologs before and after cold stress treatment were highly correlated (r = 0.870). This highlights the presence of nonstochastic polyploid gene regulation despite previous research identifying stochastic variation in expression. Moreover, our new statistical test incorporating overdispersion identified 226 homeologs (1.11% of 20 369 expressed homeologs) with significant ratio changes, many of which were related to stress responses. HomeoRoq would contribute to the study of the genes responsible for polyploid-specific environmental responses.
PMCID: PMC3973336  PMID: 24423873
4.  Common variants of a urate-associated gene LRP2 are not associated with gout susceptibility 
Rheumatology International  2013;34:473-476.
A recent genome-wide association study revealed that there is an association between serum uric acid (SUA) levels and rs2544390, a common variant in low-density lipoprotein-related protein 2 (LRP2/Megalin) gene. Two other variants of LRP2, rs2229268 and rs3755166, are also found to have associations with dyslipidemia and Alzheimer’s disease, respectively, which also could have a relationship with SUA in human. Although no studies report that LRP2 transports urate, LRP2 is a multi-ligand receptor and expresses in many tissues including kidney, suggesting a direct and/or indirect relationship with gout. In the present study, we investigated the association between gout and these variants of LRP2 with 741 clinically diagnosed male gout patients and 1,302 controls. As a result, the three common LRP2 variants, rs2544390, rs2229268 and rs3755166, showed no association with gout (P = 0.76, 0.55, and 0.22, respectively). Our study is the first to reveal that an SUA-related gene LRP2 is not involved in gout susceptibility.
PMCID: PMC3953547  PMID: 24366390
Gouty arthritis; Hyperuricemia; Hyperlipidemia; Urate exporter; Low-density lipoprotein receptor (LDLR); LDLR gene family
5.  A common variant of leucine-rich repeat-containing 16A (LRRC16A) gene is associated with gout susceptibility 
Human Cell  2013;27:1-4.
Gout is a common disease resulting from hyperuricemia which causes acute arthritis. Recently, genome-wide association studies revealed an association between serum uric acid levels and a common variant of leucine-rich repeat-containing 16A (LRRC16A) gene. However, it remains to be clarified whether LRRC16A contributes to the susceptibility to gout. In this study, we investigated the relationship between rs742132 in LRRC16A and gout. A total of 545 Japanese male gout cases and 1,115 male individuals as a control group were genotyped. rs742132 A/A genotype significantly increased the risk of gout, conferring an odds ratio of 1.30 (95 % CI 1.05–1.60; p = 0.015). LRRC16A encodes a protein called capping protein ARP2/3 and myosin-I linker (CARMIL), which serves as an inhibitor of the actin capping protein (CP). CP is an essential element of the actin cytoskeleton, which binds to the barbed end of the actin filament and regulates its polymerization. In the apical membrane of proximal tubular cells in the human kidney, the urate-transporting multimolecular complex (urate transportsome) is proposed to consist of several urate transporters and scaffolding proteins, which interact with the actin cytoskeleton. Thus, if there is a CARMIL dysfunction and regulatory disability in actin polymerization, urate transportsome may be unable to operate appropriately. We have shown for the first time that CARMIL/LRRC16A was associated with gout, which could be due to urate transportsome failure.
Electronic supplementary material
The online version of this article (doi:10.1007/s13577-013-0081-8) contains supplementary material, which is available to authorized users.
PMCID: PMC3889988  PMID: 24318514
Gouty arthritis; Single nucleotide polymorphism (SNP); Urate transport; PDZ domain-containing 1 (PDZK1); Sodium–proton exchanger regulatory factor 1 (NHERF1)
6.  Development of an online UV–visible microspectrophotometer for a macromolecular crystallography beamline 
Journal of Synchrotron Radiation  2013;20(Pt 6):948-952.
An online UV–visible microspectrophotometer has been developed for the macromolecular crystallography beamline at SPring-8. Details of this spectrophotometer are reported.
Measurement of the UV–visible absorption spectrum is a convenient technique for detecting chemical changes of proteins, and it is therefore useful to combine spectroscopy and diffraction studies. An online microspectrophotometer for the UV–visible region was developed and installed on the macromolecular crystallography beamline, BL38B1, at SPring-8. This spectrophotometer is equipped with a difference dispersive double monochromator, a mercury–xenon lamp as the light source, and a photomultiplier as the detector. The optical path is mostly constructed using mirrors, in order to obtain high brightness in the UV region, and the confocal optics are assembled using a cross-slit diaphragm like an iris to eliminate stray light. This system can measure optical densities up to a maximum of 4.0. To study the effect of radiation damage, preliminary measurements of glucose isomerase and thaumatin crystals were conducted in the UV region. Spectral changes dependent on X-ray dose were observed at around 280 nm, suggesting that structural changes involving Trp or Tyr residues occurred in the protein crystal. In the case of the thaumatin crystal, a broad peak around 400 nm was also generated after X-ray irradiation, suggesting the cleavage of a disulfide bond. Dose-dependent spectral changes were also observed in cryo-solutions alone, and these changes differed with the composition of the cryo-solution. These responses in the UV region are informative regarding the state of the sample; consequently, this device might be useful for X-ray crystallography.
PMCID: PMC3795562  PMID: 24121346
UV–visible spectroscopy; protein crystallography; radiation damage; microspectroscopy; SPring-8
7.  Common dysfunctional variants in ABCG2 are a major cause of early-onset gout 
Scientific Reports  2013;3:2014.
Gout is a common disease which mostly occurs after middle age, but more people nowadays develop it before the age of thirty. We investigated whether common dysfunction of ABCG2, a high-capacity urate transporter which regulates serum uric acid levels, causes early-onset gout. 705 Japanese male gout cases with onset age data and 1,887 male controls were genotyped, and the ABCG2 functions which are estimated by its genotype combination were determined. The onset age was 6.5 years earlier with severe ABCG2 dysfunction than with normal ABCG2 function (P = 6.14 × 10−3). Patients with mild to severe ABCG2 dysfunction accounted for 88.2% of early-onset cases (twenties or younger). Severe ABCG2 dysfunction particularly increased the risk of early-onset gout (odds ratio 22.2, P = 4.66 × 10−6). Our finding that common dysfunction of ABCG2 is a major cause of early-onset gout will serve to improve earlier prevention and therapy for high-risk individuals.
PMCID: PMC3684804  PMID: 23774753
8.  Hypoxia Stimulates the EMT of Gastric Cancer Cells through Autocrine TGFβ Signaling 
PLoS ONE  2013;8(5):e62310.
Epithelial mesenchymal transition (EMT) is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. We therefore investigated the effect of hypoxic condition on EMT of gastric cancer cells. Gastric cancer cells were cultured in normoxia (21% O2) or hypoxia (1% O2) for 24 h. EMT was evaluated as the percentage of spindle-shaped cells in total cells. Effect of transforming growth factor β1 (TGFβ1) or tyrosine kinase inhibitors on the EMT was evaluated. The expression level of TGFβ1 and TGFβR was evaluated by real time RT-PCR. The TGFβ1 production from cancer cells was measured by ELISA. Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of OCUM-2M cells. The expression level of TGFβ1 mRNA under hypoxia was significantly higher than that under normoxia in all of three cell lines. The expression level of TGFβR mRNA was significantly increased by hypoxia in OCUM-2MD3 cells, but not in OCUM-2M cells. TGFβR inhibitor, SB431542 or Ki26894, significantly suppressed EMT of OCUM-2MD3 and OCUM-12. TGFβ1 production from OCUM-2MD3 and OCUM-12 cells was significantly increased under hypoxia in comparison with that under normoxia. These findings might suggest that hypoxia stimulates the EMT of gastric cancer cells via autocrine TGFβ/TGFβR signaling.
PMCID: PMC3656884  PMID: 23690936
9.  Isolation of Human Monoclonal Antibodies to the Envelope E2 Protein of Hepatitis C Virus and Their Characterization 
PLoS ONE  2013;8(2):e55874.
We isolated and characterized two human monoclonal antibodies to the envelope E2 protein of hepatitis C virus (HCV). Lymphoblastoid cell lines stably producing antibodies were obtained by immortalizing peripheral blood mononuclear cells of a patient with chronic hepatitis C using Epstein-Barr virus. Screening for antibody-positive clones was carried out by immunofluorescence with Huh7 cells expressing the E2 protein of HCV strain H (genotype 1a) isolated from the same patient. Isotype of resulting antibodies, #37 and #55, was IgG1/kappa and IgG1/lambda, respectively. Epitope mapping revealed that #37 and #55 recognize conformational epitopes spanning amino acids 429 to 652 and 508 to 607, respectively. By immunofluorescence using virus-infected Huh7.5 cells as targets both antibodies were reactive with all of the nine different HCV genotypes/subtypes tested. The antibodies showed a different pattern of immuno-staining; while #37 gave granular reactions mostly located in the periphery of the nucleus, #55 gave diffuse staining throughout the cytoplasm. Both antibodies were shown by immuno-gold electron microscopy to bind to intact viral particles. In a neutralization assay (focus-forming unit reduction using chimeric infectious HCV containing structural proteins derived from genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, 6a, and 7a), #55 inhibited the infection of all HCV genotypes tested but genotype 7a to a lesser extent. #37 did not neutralize any of these viruses. As a broadly cross-neutralizing human antibody, #55 may be useful for passive immunotherapy of HCV infection.
PMCID: PMC3567033  PMID: 23409074
10.  Loss of Myeloid Related Protein-8/14 Exacerbates Cardiac Allograft Rejection 
Circulation  2011;124(25):2920-2932.
The calcium-binding proteins myeloid-related protein (MRP)-8 (S100A8) and MRP-14 (S100A9) form MRP-8/14 heterodimers (S100A8/A9, calprotectin) that regulate myeloid cell function and inflammatory responses, and serve as early serum markers for monitoring acute allograft rejection. Despite functioning as a pro-inflammatory mediator, the pathophysiological role of MRP-8/14 complexes in cardiovascular disease is incompletely defined. This study investigated the role of MRP-8/14 in cardiac allograft rejection using MRP-14-deficient mice (MRP14-/-) that lack MRP-8/14 complexes.
Methods and Results
We examined parenchymal rejection (PR) after major histocompatibility complex (MHC) class II allomismatched cardiac transplantation (bm12 donor heart and B6 recipients) in wild-type (WT) and MRP14-/- recipients. Allograft survival averaged 5.9 ± 2.9 weeks (n=10) in MRP14-/- recipients, compared to > 12 weeks (n = 15, p < 0.0001) in WT recipients. Two weeks after transplantation, allografts in MRP14-/- recipients had significantly higher PR scores (2.8 ± 0.8, n=8) than did WT recipients (0.8 ± 0.8, n=12, p<0.0001). Compared to WT recipients, allografts in MRP14-/- recipients had significantly increased T-cell and macrophage infiltration, as well as increased mRNA levels of IFN-γ and IFN-γ–associated chemokines (CXCL9, CXCL10, and CXCL11), IL-6, and IL-17, with significantly higher levels of Th17 cells. MRP14-/- recipients also had significantly more lymphocytes in the adjacent paraaortic lymph nodes than did WT recipients (cell number per lymph node: 23.7 ± 0.7 × 105 for MRP14-/- vs. 6.0 ± 0.2 × 105 for WT, p < 0.0001). The dendritic cells (DCs) of the MRP14-/- recipients of bm12 hearts expressed significantly higher levels of the co-stimulatory molecules CD80 and CD86 than did those of WT recipients 2 weeks after transplantation. Mixed leukocyte reactions using allo-EC-primed MRP14-/- DCs resulted in significantly higher antigen-presenting function than reactions using WT DCs. Ovalbumin-primed MRP14-/- DCs augmented proliferation of OT-II CD4+ T cells with increased IL-2 and IFN-γ production. Cardiac allografts of B6 MHC class II-/- hosts and of B6 WT hosts receiving MRP14-/- DCs had significantly augmented inflammatory cell infiltration and accelerated allograft rejection, compared to WT DCs from transferred recipient allografts. Bone marrow–derived MRP14-/- DCs infected with MRP-8 and MRP-14 retroviral vectors showed significantly decreased CD80 and CD86 expression compared to controls, indicating that MRP-8/14 regulates B7-costimulatory molecule expression.
Our results indicate that MRP-14 regulates B7 molecule expression and reduces antigen presentation by DCs, and subsequent T-cell priming. The absence of MRP-14 markedly increased T-cell activation and exacerbated allograft rejection, indicating a previously unrecognized role for MRP-14 in immune cell biology.
PMCID: PMC3277828  PMID: 22144572
MRP-8 (S100A8); MRP-14 (S100A9); T-lymphocytes; macrophages; dendritic cells; antigen-presenting cells; cytokine; heart transplantation; pathogenesis
11.  Breast cancer manifested by hematologic disorders 
Journal of Thoracic Disease  2012;4(6):650-654.
Breast cancer is the most common type of cancer in women. However, it is very rarely manifested as hematologic disorders. A 35-year-old woman was admitted because of disseminated intravascular coagulation. Examinations revealed the presence of breast cancer in her left breast; therefore, paclitaxel was administered weekly. Although disseminated intravascular coagulation was controlled, pulmonary dysfunction due to lymphangitis carcinomatosa suddenly occurred 10 weeks after treatment. Pulmonary dysfunction was effectively treated with epirubicin and cyclophosphamide. Twenty-three weeks after treatment, the patient developed liver dysfunction accompanied with jaundice due to progressive metastatic lesions in the liver; liver dysfunction improved after the administration of vinorelbine. Subsequently, because of the recurrence of pulmonary dysfunction, rechallenge with epirubicin and cyclophosphamide was performed and was effective; however, this therapy was discontinued because of its adverse effects. She expired of liver failure 33 weeks after the occurrence of disseminated intravascular coagulation. Metastatic tumors in the bone marrow, lung, and liver showed different sensitivities to different anti-cancer agents. We report a case of breast cancer manifested by hematologic disorders which was treated by a sequential chemotherapy.
PMCID: PMC3506789  PMID: 23205295
Breast cancer; disseminated intravascular coagulation; multiple organ metastases
12.  1,8-cineole, a TRPM8 agonist, is a novel natural antagonist of human TRPA1 
Molecular Pain  2012;8:86.
Essential oils are often used in alternative medicine as analgesic and anti-inflammatory remedies. However, the specific compounds that confer the effects of essential oils and the molecular mechanisms are largely unknown. TRPM8 is a thermosensitive receptor that detects cool temperatures and menthol whereas TRPA1 is a sensor of noxious cold. Ideally, an effective analgesic compound would activate TRPM8 and inhibit TRPA1.
We screened essential oils and fragrance chemicals showing a high ratio of human TRPM8-activating ability versus human TRPA1-activating ability using a Ca2+-imaging method, and identified 1,8-cineole in eucalyptus oil as particularly effective. Patch-clamp experiments confirmed that 1,8-cineole evoked inward currents in HEK293T cells expressing human TRPM8, but not human TRPA1. In addition, 1,8-cineole inhibited human TRPA1 currents activated by allyl isothiocyanate, menthol, fulfenamic acid or octanol in a dose-dependent manner. Furthermore, in vivo sensory irritation tests showed that 1,8-cineole conferred an analgesic effect on sensory irritation produced by TRPA1 agonists octanol and menthol. Surprisingly, 1,4-cineole, which is structurally similar and also present in eucalyptus oil, activated both human TRPM8 and human TRPA1.
1,8-cineole is a rare natural antagonist of human TRPA1 that has analgesic and anti-inflammatory effects possibly due to its inhibition of TRPA1.
PMCID: PMC3567430  PMID: 23192000
1,8-cineole; Pain relief; TRP channels; TRPA1
Interactions between CXCL12 and its receptors CXCR4 or CXCR7 are involved in tumor growth and metastasis in various types of human cancer. However, CXCL12 expression and its role in lung cancer are not fully elucidated. Here we examined the expression of CXCL12 in 54 lung cancer cell lines consisting of 23 small cell lung cancers (SCLCs) and 31 non-small cell lung cancers (NSCLCs). CXCL12 was overexpressed in lung cancer cell lines compared to non-malignant human bronchial epithelial cell lines (N = 6). CXCL12 expression was positively but weakly correlated with the expression of CXCR4 or CXCR7. We also examined CXCL12 expression in 89 NSCLC specimens and found that CXCL12 expression was significantly higher in tumor specimens from female patients, non-smokers and adenocarcinoma patients. Small interfering RNAs targeting CXCL12 inhibited cellular proliferation, colony formation and migration of CXCL12-overexpressing lung cancer cells; however, this inhibition did not occur in lung cancer cells that lacked CXCL12. Furthermore, the anti-CXCL12 neutralizing antibody mediated inhibitory effects in three lung cancer cell lines that overexpressed CXCL12, but not in two CXCL12 non-expressing lung cancer cell lines nor two non-malignant bronchial epithelial cell lines. The present study demonstrates that: CXCL12 is concomitantly overexpressed with CXCR4 or CXCR7 in lung cancers; CXCL12 is highly expressed in NSCLCs from females, non-smokers and adenocarcinoma patients; and disruption of CXCL12 inhibits the growth and migration of lung cancer cells. Our findings indicate that CXCL12 is required for tumor growth and provide a rationale for the anti-CXCL12 treatment strategy in lung cancer.
PMCID: PMC3368436  PMID: 20378003
CXCL12; CXCR4; CXCR7; overexpression; lung cancer
14.  Azithromycin Inhibits Mucus Hypersecretion from Airway Epithelial Cells 
Mediators of Inflammation  2012;2012:265714.
To examine the in vivo effects of the 15-member macrolide, azithromycin (AZM), on mucus hypersecretion, we induced hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium by intranasal instillation of ovalbumin (OVA) in OVA-sensitized rats, or by intranasal lipopolysaccharides (LPS) instillation. Oral administration of AZM (5–10 mg/kg) or clarithromycin (CAM, 5–10 mg/kg) significantly inhibited OVA- and LPS-induced mucus production, whereas josamycin (JM) or ampicillin (ABPC) showed no effect. In vitro effects of AZM on airway epithelial cells were examined using NCI-H292 cells and human nasal epithelial cells cultured in air-liquid interface. Mucus secretion was evaluated by enzyme-linked immunosorbent assay using an anti-MUC5AC monoclonal antibody. AZM or CAM significantly inhibited tumor necrosis factor-α (TNF-α) (20 ng/mL)-induced MUC5AC secretion from NCI-H292 cells at 10−6–10−7 M, whereas JM or ABPC showed no effect. AZM significantly inhibited TNF-α (20 ng/mL)-induced MUC5AC secretion from human nasal epithelial cells at 10−4 M. MUC5AC mRNA expression was also significantly inhibited. These results indicate that the 15-member macrolide, AZM, exerts direct inhibitory effects on mucus secretion from airway epithelial cells and that it may be useful for the treatment of mucus hypersecretion caused by allergic inflammation and LPS stimulation.
PMCID: PMC3347724  PMID: 22577246
15.  Ubiquitylation of an ERAD substrate occurs on multiple types of amino acids 
Molecular cell  2010;40(6):917-926.
Any protein synthesized in the secretory pathway has the potential to misfold and would need to be recognized and ubiquitylated for degradation. This is astounding since only a few ERAD-specific E3 ligases have been identified. To begin to understand substrate recognition, we wished to map the ubiquitylation sites on the NS-1 non-secreted immunoglobulin light chain, which is an ERAD substrate. Ubiquitin is usually attached to lysine residues and less frequently to the N-terminus of proteins. In addition, several viral E3s have been identified that attach ubiquitin to cysteine or serine/threonine residues. Mutation of lysines, serines, and threonines in the NS-1 variable region was necessary to significantly reduce ubiquitylation and stabilize the protein. The Hrd1 E3 ligase was required to modify all three amino acids. Our studies argue that ubiquitylation of ER proteins relies on very different mechanisms of recognition and modification than those used to regulate biological processes.
PMCID: PMC3031134  PMID: 21172657
16.  In Situ Enzyme Activity in the Dissolved and Particulate Fraction of the Fluid from Four Pitcher Plant Species of the Genus Nepenthes 
PLoS ONE  2011;6(9):e25144.
The genus Nepenthes, a carnivorous plant, has a pitcher to trap insects and digest them in the contained fluid to gain nutrient. A distinctive character of the pitcher fluid is the digestive enzyme activity that may be derived from plants and dwelling microbes. However, little is known about in situ digestive enzymes in the fluid. Here we examined the pitcher fluid from four species of Nepenthes. High bacterial density was observed within the fluids, ranging from 7×106 to 2.2×108 cells ml−1. We measured the activity of three common enzymes in the fluid: acid phosphatases, β-d-glucosidases, and β-d-glucosaminidases. All the tested enzymes detected in the liquid of all the pitcher species showed activity that considerably exceeded that observed in aquatic environments such as freshwater, seawater, and sediment. Our results indicate that high enzyme activity within a pitcher could assist in the rapid decomposition of prey to maximize efficient nutrient use. In addition, we filtered the fluid to distinguish between dissolved enzyme activity and particle-bound activity. As a result, filtration treatment significantly decreased the activity in all enzymes, while pH value and Nepenthes species did not affect the enzyme activity. It suggested that enzymes bound to bacteria and other organic particles also would significantly contribute to the total enzyme activity of the fluid. Since organic particles are themselves usually colonized by attached and highly active bacteria, it is possible that microbe-derived enzymes also play an important role in nutrient recycling within the fluid and affect the metabolism of the Nepenthes pitcher plant.
PMCID: PMC3174996  PMID: 21949872
17.  Inhibitory effect of a TGFβ receptor type-I inhibitor, Ki26894, on invasiveness of scirrhous gastric cancer cells 
British Journal of Cancer  2010;102(5):844-851.
Gastric cancer cells frequently metastasise, partly because of their highly invasive nature. Transforming growth factor-β (TGF-β) receptor signalling is closely associated with the invasion of cancer cells. The aim of this study was to clarify the effect of a TGF-β receptor (TβR) phosphorylation inhibitor on the invasiveness of gastric cancer cells.
Four gastric cancer cell lines, including two scirrhous-type cell lines and two non-scirrhous-type cell lines, were used. A TβR type I (TβR-I) kinase inhibitor, Ki26894, inhibits the phosphorylation of Smad2 at an ATP-binding site of TβR-I. We investigated the expression levels of TβR and phospho-Smad2, and the effects of TGF-β in the presence or absence of Ki26894 on Smad2 phosphorylation, invasion, migration, epithelial-to-mesenchymal transition (EMT), Ras homologue gene family member A (RhoA), ZO-2, myosin, and E-cadherin expression of gastric cancer cells.
TβR-I, TβR-II, and phospho-Smad2 expressions were found in scirrhous gastric cancer cells, but not in non-scirrhous gastric cancer cells. Ki26894 decreased Smad2 phosphorylation induced by TGF-β1 in scirrhous gastric cancer cells. Transforming growth factor-β1 upregulated the invasion, migration, and EMT ability of scirrhous gastric cancer cells. Transforming growth factor-β1 significantly upregulated the activity of RhoA and myosin phosphorylation, whereas TGF-β1 decreased ZO-2 and E-cadherin expression in scirrhous gastric cancer cells. Interestingly, Ki26894 inhibited these characteristics in scirrhous gastric cancer cells. In contrast, non-scirrhous gastric cancer cells were not affected by TGF-β1 or Ki26894 treatment.
A TβR-I kinase inhibitor decreases the invasiveness and EMT of scirrhous gastric cancer cells. Ki26894 is therefore considered to be a promising therapeutic compound for the metastasis of scirrhous gastric carcinoma.
PMCID: PMC2833252  PMID: 20145621
scirrhous gastric cancer; TGF-β; epithelial-to-mesenchymal transition; Smad2; phosphorylation inhibitor
18.  Genetic variants in mannose receptor gene (MRC1) confer susceptibility to increased risk of sarcoidosis 
BMC Medical Genetics  2010;11:151.
Mannose receptor (MR) is a member of the C-type lectin receptor family involved in pathogen molecular-pattern recognition and thought to be critical in shaping host immune response. The aim of this study was to investigate potential associations of genetic variants in the MRC1 gene with sarcoidosis.
Nine single nucleotide polymorphisms (SNPs), encompassing the MRC1 gene, were genotyped in a total of 605 Japanese consisting of 181 sarcoidosis patients and 424 healthy controls.
Suggestive evidence of association between rs691005 SNP and risk of sarcoidosis was observed independent of sex and age in a recessive model (P = 0.001).
These results suggest that MRC1 is an important candidate gene for sarcoidosis. This is the first study to imply that genetic variants in MRC1, a major member of the C-type lectin, contribute to the development of sarcoidosis.
PMCID: PMC2987761  PMID: 21029423
19.  Hyper-expansion of large DNA segments in the genome of kuruma shrimp, Marsupenaeus japonicus 
BMC Genomics  2010;11:141.
Higher crustaceans (class Malacostraca) represent the most species-rich and morphologically diverse group of non-insect arthropods and many of its members are commercially important. Although the crustacean DNA sequence information is growing exponentially, little is known about the genome organization of Malacostraca. Here, we constructed a bacterial artificial chromosome (BAC) library and performed BAC-end sequencing to provide genomic information for kuruma shrimp (Marsupenaeus japonicus), one of the most widely cultured species among crustaceans, and found the presence of a redundant sequence in the BAC library. We examined the BAC clone that includes the redundant sequence to further analyze its length, copy number and location in the kuruma shrimp genome.
Mj024A04 BAC clone, which includes one redundant sequence, contained 27 putative genes and seemed to display a normal genomic DNA structure. Notably, of the putative genes, 3 genes encode homologous proteins to the inhibitor of apoptosis protein and 7 genes encode homologous proteins to white spot syndrome virus, a virulent pathogen known to affect crustaceans. Colony hybridization and PCR analysis of 381 BAC clones showed that almost half of the BAC clones maintain DNA segments whose sequences are homologous to the representative BAC clone Mj024A04. The Mj024A04 partial sequence was detected multiple times in the kuruma shrimp nuclear genome with a calculated copy number of at least 100. Microsatellites based BAC genotyping clearly showed that Mj024A04 homologous sequences were cloned from at least 48 different chromosomal loci. The absence of micro-syntenic relationships with the available genomic sequences of Daphnia and Drosophila suggests the uniqueness of these fragments in kuruma shrimp from current arthropod genome sequences.
Our results demonstrate that hyper-expansion of large DNA segments took place in the kuruma shrimp genome. Although we analyzed only a part of the duplicated DNA segments, our result suggested that it is difficult to analyze the shrimp genome following normal analytical methodology. Hence, it is necessary to avoid repetitive sequence (such as segmental duplications) when studying the other unique structures in the shrimp genome.
PMCID: PMC2838849  PMID: 20187930
20.  Development of a shutterless continuous rotation method using an X-ray CMOS detector for protein crystallography 
Journal of Applied Crystallography  2009;42(Pt 6):1165-1175.
A shutterless continuous rotation method using an X-ray complementary metal-oxide semiconductor (CMOS) detector has been developed for high-speed, precise data collection in protein crystallography. The new method and detector were applied to the structure determination of three proteins by multi- and single-wavelength anomalous diffraction phasing and have thereby been proved to be applicable in protein crystallography.
A new shutterless continuous rotation method using an X-ray complementary metal-oxide semiconductor (CMOS) detector has been developed for high-speed, precise data collection in protein crystallography. The principle of operation and the basic performance of the X-ray CMOS detector (Hamamatsu Photonics KK C10158DK) have been shown to be appropriate to the shutterless continuous rotation method. The data quality of the continuous rotation method is comparable to that of the conventional oscillation method using a CCD detector and, furthermore, the combination with fine ϕ slicing improves the data accuracy without increasing the data-collection time. The new method is more sensitive to diffraction intensity because of the narrow dynamic range of the CMOS detector. However, the strong diffraction spots were found to be precisely measured by recording them on successive multiple images by selecting an adequate rotation step. The new method has been used to successfully determine three protein structures by multi- and single-wavelength anomalous diffraction phasing and has thereby been proved applicable in protein crystallography. The apparatus and method may become a powerful tool at synchrotron protein crystallography beamlines with important potential across a wide range of X-ray wavelengths.
PMCID: PMC3246825  PMID: 22477775
protein crystallography; shutterless continuous rotation method; X-ray CMOS detectors; X-ray wavelength capabilities
22.  Comparison of Human T Cell Repertoire Generated in Xenogeneic Porcine and Human Thymus Grafts1 
Transplantation  2008;86(4):601-610.
Xenogeneic thymus transplantation is an effective approach to achieving T cell tolerance across highly disparate xenogeneic species barriers. We have previously demonstrated that phenotypically normal, specifically tolerant human T cells are generated in porcine thymic grafts. In this study, we assessed the diversity of the human T cell repertoire generated in porcine thymic xenografts. We also examined the ability of porcine thymus grafts to coexist with human thymus grafts.
Fetal swine (SW) or human (HU) thymus with human fetal liver (FL) fragments were transplanted under the kidney capsule of 3Gy irradiated NOD/SCID mouse recipients. Thymus tissue was harvested approximately 16 weeks post-transplant for analysis of mixed lymphocyte reactions and spectratyping of human CD4 and CD8 single positive (SP) thymocytes.
TCR β genes of human CD4 and CD8 SP cells developing in HU and SW thymus grafts showed similar, normal CDR3 length distributions. Human T cells developing in SW thymus grafts showed specific unresponsiveness to the MHC of the donor swine, in MLR assays. In 2 of 3 animals receiving SW and HU thymus grafts under opposite kidney capsules, both grafts functioned. In animals with surviving SW grafts, thymocytes from the SW but not the HU grafts showed specific unresponsiveness to the SW donor.
SW thymus grafts support generation of human T cells with a diverse TCR repertoire. Human thymocytes in human thymus grafts are not tolerized by the presence of an additional porcine thymus, but tolerance might be achieved by post-thymic encounter with porcine antigens.
PMCID: PMC2680689  PMID: 18724231
Xenotransplantation; thymus; thymopoiesis; T cell repertoire; Spectratyping
23.  Establishment and characterization of pleomorphic adenoma cell systems: an in-vitro demonstration of carcinomas arising secondarily from adenomas in the salivary gland 
BMC Cancer  2009;9:247.
Among the salivary gland carcinomas, carcinoma in pleomorphic adenoma has been regarded as a representative carcinoma type which arises secondarily in the background of a pre-existent benign pleomorphic adenoma. It is still poorly understood how and which benign pleomorphic adenoma cells transform into its malignant form, carcinoma ex pleomorphic adenoma.
We have established five cell systems from a benign pleomorphic adenoma of the parotid gland of a 61-year-old woman. They were characterized by immunofluorescence, classical cytogenetics, p53 gene mutational analysis, fluorescence in-situ hybridization, and histopathological and immunohistochemical examinations of their xenografts, to demonstrate their potency of secondary transformation.
We established and characterized five cell systems (designated as SM-AP1 to SM-AP5) from a benign pleomorphic adenoma of the parotid gland. SM-AP1 to SM-AP3 showed polygonal cell shapes while SM-AP4 and SM-AP5 were spindle-shaped. SM-AP1-3 cells were immunopositive for keratin only, indicating their duct-epithelial or squamous cell differentiation, while SM-AP4/5 cells were positive for both keratin and S-100 protein, indicating their myoepithelial cell differentiation. Chromosome analyses showed numeral abnormalities such as 5n ploidies and various kinds of structural abnormalities, such as deletions, translocations, derivatives and isodicentric chromosomes. Among them, der(9)t(9;13)(p13.3;q12.3) was shared by all five of the cell systems. In addition, they all had a common deletion of the last base G of codon 249 (AGG to AG_) of the p53 gene, which resulted in generation of its nonsense gene product. Transplanted cells in nude mice formed subcutaneous tumors, which had histological features of squamous cell carcinoma with apparent keratinizing tendencies. In addition, they had ductal arrangements or plasmacytoid appearances of tumor cells and myxoid or hyaline stromata, indicating some characteristics of pleomorphic adenoma.
This study demonstrates in vitro that certain cell types from pleomorphic adenoma are able to clone and survive over a long term and develop subcutaneous tumors in nude mice. The histological features of squamous cell carcinoma from the transplanted cell systems in nude mice might suggest a secondary onset of malignancy from a pre-existing benign adenoma.
PMCID: PMC2722671  PMID: 19622142
24.  Incidental MALT Type Lymphoma Exhibiting Prominent Plasma Cell Differentiation Associated with Hashimoto’s Thyroiditis. A Two Case Report 
Head and Neck Pathology  2008;3(1):27-30.
We present here two cases of incidental extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) showing prominent plasma cell differentiation associated with Hashimoto’s thyroiditis (HT). Histological examination demonstrated that both lesions exhibited HT including lymphoplasmacytic infiltration with the formation of germinal centers, destruction of the normal thyroid follicular architecture, Hürthle cell changes, and squamous metaplasia. The dominant tumor nodules of both cases contained large, well-circumscribed but unencapsulated aggregation of mature plasma cells and scattered centrocyte-like cells (CCL-cells). Both lesions contained a few lymphoepithelial lesions. Moreover, immunohistochemical study demonstrated that plasma cells and CCL-cells of these two lesions contained monotypic intracytoplasmic kappa light chain. Other small B-cell lymphomas, plasmacytoma and plasmablastic lymphoma were excluded using stains for CD5, CD10, CD23, CD43, CD56. Cyclin D1, human herpes virus type-8.
PMCID: PMC2807536  PMID: 20596985
Thyroid gland; MALT type lymphoma; Hashimoto’s thyroiditis; Plasma cell granuloma; Immunohistochemistry
25.  Role of NF-κB-dependent Caveolin-1 Expression in the Mechanism of Increased Endothelial Permeability Induced by Lipopolysaccharide*S 
The Journal of biological chemistry  2007;283(7):4210-4218.
We investigated the role of NF-κB activation by the bacterial product lipopolysaccharide (LPS) in inducing caveolin-1 (Cav-1) expression and its consequence in contributing to the leakiness of the endothelial barrier. We observed that LPS challenge of human lung microvascular endothelial cells induced concentration- and time-dependent increases in expression of Cav-1 mRNA and protein. The NEMO (NF-κB essential modifier binding domain)-binding domain peptide (IkB kinase (IKK)-NEMO-binding domain (NBD) peptide), which prevents NF-κB activation by inhibiting the interaction of IKKγ with the IKK complex, blocked LPS-induced Cav-1 mRNA and protein expression. Knockdown of NF-κB subunit p65/RelA expression with small interfering RNA also prevented LPS-induced Cav-1 expression. Caveolae open to the apical and basal plasmalemma of endothelial cells increased 2–4-fold within 4 h of LPS exposure. IKK-NBD peptide markedly reduced the LPS-induced increase in the number of caveolae as well as transendothelial albumin permeability. These observations were recapitulated in mouse studies in which IKK-NBD peptide prevented Cav-1 expression and interfered with the increase in lung microvessel permeability induced by LPS. Thus, LPS mediates NF-κB-dependent Cav-1 expression that results in increased caveolae number and thereby contributes to the mechanism of increased transendothelial albumin permeability.
PMCID: PMC2435298  PMID: 18077459

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