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Aim To examine the contribution of the FUT2 gene and ABO blood type to the development of Type 1 diabetes in Japanese children. Methods We analysed FUT2 variants and ABO genotypes in a total of 531 Japanese children diagnosed with Type 1 diabetes and 448 control subjects. The possible association of FUT2 variants and ABO genotypes with the onset of Type 1 diabetes was statistically examined. Results The se2 genotype (c.385A>T) of the FUT2 gene was found to confer susceptibility to Type 1A diabetes in a recessive effects model [odds ratio for se2/se2, 1.68 (95% CI 1.20-2.35); corrected P value = 0.0075]. Conclusions The FUT2 gene contributed to the development of Type 1 diabetes in the present cohort of Japanese children. [ABSTRACT FROM AUTHOR]

Aims The aim of this study was to clarify the significance of previously reported susceptibility variants in the development of autoimmune Type 1 diabetes in non-white children. Tested variants included rs2290400, which has been linked to Type 1 diabetes only in one study on white people. Haplotypes at 17q12-q21 encompassing rs2290400 are known to determine the susceptibility of early-onset asthma by affecting the expression of flanking genes. Methods We genotyped 63 variants in 428 Japanese people with childhood-onset autoimmune Type 1 diabetes and 457 individuals without diabetes. Possible association between variants and age at diabetes onset was examined using age-specific quantitative trait locus analysis and ordered-subset regression analysis. Results Ten variants, including rs2290400 in GSDMB, were more frequent among the people with Type 1 diabetes than those without diabetes. Of these, rs689 in INS and rs231775 in CTLA4 yielded particularly high odds ratios of 5.58 (corrected P value 0.001; 95% CI 2.15-14.47) and 1.64 (corrected P value 5.3 × 10-5; 95% CI 1.34-2.01), respectively. Age-specific effects on diabetes susceptibility were suggested for rs2290400; heterozygosity of the risk alleles was associated with relatively early onset of diabetes, and the allele was linked to the phenotype exclusively in the subgroup of age at onset ≤ 5.0 years. Conclusions The results indicate that rs2290400 in GSDMB and polymorphisms in INS and CTLA4 are associated with the risk of Type 1 diabetes in Japanese children. Importantly, cis-regulatory haplotypes at 17q12-q21 encompassing rs2290400 probably determine the risk of autoimmune Type 1 diabetes predominantly in early childhood. [ABSTRACT FROM AUTHOR]

Self-catalyzed GaAs nanowires (NWs) were grown on a Si(111) substrate via molecular beam epitaxy (MBE) using a vapor-liquid-solid mechanism under an alternating supply of Ga and As. Compared to GaAs NWs grown using the conventional MBE method, the length and diameter of the GaAs NWs grown with an alternating supply of Ga and As were longer and narrower. In addition, the formation of polycrystals on the substrate was decreased, and it was possible to grow the GaAs NWs at a lower temperature using the alternating method. Furthermore, an increase in the formation of twin boundaries (TBs) in the GaAs NWs was observed. The effect of using an alternating supply of Ga and As on the shape and formation of TBs is also discussed. (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

The pharmacokinetics of (1R,2R-diaminocyclohexane)oxalatoplatinum(II) (1-OHP, NSC-266046), a second-generation antitumor platinum complex, was studied in rabbits and compared with that of cisplatin. The rabbits were given a single i.v. dose of 1-OHP or cisplatin (10 mumol/kg). A comparison of tissue platinum levels at 24 h postinjection showed that platinum levels were lower in the eight organs examined, which included the kidney and liver, after the injection of 1-OHP than following cisplatin administration. Plasma-decay profiles of three platinum species, that is, the unchanged species, filterable platinum, and total platinum, were examined. Plasma levels of the unchanged species and filterable platinum for 1-OHP declined more rapidly than those for cisplatin. The ratio of plasma filterable-to-total platinum indicated that the protein-binding ability of 1-OHP was greater than that of cisplatin. As for urinary excretion, amounts of the unchanged species and total platinum excreted during the 24 h period postinjection were 28% and 76% of the dose for 1-OHP and 23% and 57% of the dose for cisplatin, respectively. The renal clearance of both the unchanged species and filterable platinum in plasma for 1-OHP was about 2-fold that for cisplatin. 1-OHP is reported to be much less nephrotoxic than cisplatin. This may be due in part to its pharmacokinetic behavior or to pharmacokinetic differences resulting from chemical reactions that make 1-OHP less toxic than cisplatin. [ABSTRACT FROM AUTHOR]

Unlabelled: To determine how long the measles virus genome was detected in a patient with congenital measles, the peripheral blood mononuclear cells were tested for 203 d. The measles virus genome was detected up to 140 d.Conclusion: The period for which the measles virus genome was detected in this patient with congenital measles was much longer than in normal children with measles. [ABSTRACT FROM AUTHOR]

We evaluated the usable specimen thickness of Si for lattice imaging on a transmission electron microscopy (TEM) instrument operating at 300 kV and equipped with a complementary metal-oxide-semiconductor camera by using an original reference material (RM) and comparing the lattice images obtained from Si patterns of the RM with various thicknesses. Lattice images of the {111} planes of crystalline Si are successfully observed for patterns with thicknesses of up to 508 nm. However, the contrast of these lattice fringes at a thickness of 508 nm is not distinct, even when recorded using a longer exposure time (5.0 s) than that required to obtain lattice images of patterns with thicknesses of 316 nm or less (0.5 s). Based on these results, we conclude that the practical thickness of crystalline Si specimens for accurate structural analysis and TEM magnification calibration via lattice imaging is less than approximately 500 nm under the experimental conditions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is activated by environmental contaminants such as dioxins and polycyclic aromatic hydrocarbons. Following ligand binding, AhR binds to xenobiotic responsive elements and modulates the transcription of AhR target genes. Multiple studies have shown that AhR plays important roles in a range of cancer cells and is attracting attention as a therapeutic target for cancer treatment. We have previously reported that AhR agonists inhibit tumorsphere formation in an AhR-dependent manner in the MCF-7 breast cancer cell line. In the present study, we found that FDI-6, an inhibitor of the transcription factor Forkhead Box M1 (FOXM1) induced the mRNA expression of AhR target genes, nuclear translocation of AhR, and transcriptional activity of AhR. In addition, FDI-6 dose-dependently reduced the mRNA expression of FOXM1-regulated genes in AhR-expressing MCF-7 cells, although not in AhR-deficient MCF-7 cells. Furthermore, FDI-6 was found to suppress tumorsphere formation via the AhR in MCF-7 cells and HepG2 human liver cancer cell line. On the basis of the findings of this study, we show that FDI-6, a FOXM1 inhibitor, functions as an AhR agonist, and suppresses tumorsphere formation via the AhR., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Poly(ADP-ribosyl)ation is a post-translational modification of proteins by transferring poly(ADP-ribose) (PAR) to acceptor proteins by the action of poly(ADP-ribose) polymerase (PARP). Two tankyrase (TNKS) isoforms, TNK1 and TNK2 (TNKS1/2), are ubiquitously expressed in mammalian cells and participate in diverse cellular functions, including wnt/β-catenin signaling, telomere maintenance, glucose metabolism and mitosis regulation. For wnt/β-catenin signaling, TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin, a key component of the β-catenin degradation complex, which allows Axin's ubiquitination and subsequent degradation, thereby activating β-catenin signaling. In the present study, we focused on the functions of TNKS1/2 in neuronal development. In primary hippocampal neurons, TNKS1/2 were detected in the soma and neurites, where they co-localized with PAR signals. Treatment with XAV939, a selective TNKS1/2 inhibitor, suppressed neurite outgrowth and synapse formation. In addition, XAV939 also suppressed norepinephrine uptake in PC12 cells, a rat pheochromocytoma cell line. These effects likely resulted from the inhibition of β-catenin signaling through the stabilization of Axin, which suggests TNKS1/2 enhance Axin degradation by modifying its poly(ADP-ribosyl)ation, thereby stabilizing wnt/β-catenin signaling and, in turn, promoting neurite outgrowth and synapse formation.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxins and polycyclic aromatic hydrocarbons. Recent studies have suggested that AhR is involved in cancer immunity. In the present study, we examined whether AhR regulates the expression of immune checkpoint genes in breast cancer cells. We discovered that the mRNA expression of V-set domain containing T cell activation inhibitor 1 (VTCN1) that negatively regulates T cell immunity was upregulated by AhR agonists in breast cancer cell lines, MCF-7 and T47D. Furthermore, AhR knockout or knockdown experiments clearly demonstrated that upregulation of VTCN1 gene expression by 3-methylcholanthrene was AhR dependent. Luciferase reporter and chromatin immunoprecipitation assays revealed that this upregulation of VTCN1 gene expression was induced by the recruitment of AhR to the AhR responsive element in the VTCN1 gene promoter in MCF-7 cells. Taken together, AhR directly regulates VTCN1 gene expression in MCF-7 cells.

Breast cancer is the most commonly diagnosed cancer among women worldwide. Despite a variety of drugs available for the treatment of patients with breast cancer, drug resistance remains a significant clinical problem. Therefore, there is an urgent need to develop drugs with new mechanisms of action. Camalexin is the main indole phytoalexin in Arabidopsis thaliana and other crucifers. Camalexin inhibits the proliferation of various cancer cells. However, the mechanism by which camalexin inhibits cell proliferation remains unclear. In this study, we found that camalexin inhibited cell proliferation and migration of breast cancer cell lines. Furthermore, camalexin also suppressed breast cancer stem cell-derived mammosphere formation. We previously reported that the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) agonist suppresses mammosphere formation. Several compounds with indole structures are known to act as AhR agonists. Therefore, we hypothesized that the inhibition of mammosphere formation by camalexin may involve AhR activation. We found that camalexin increased the nuclear translocation of AhR, AhR-mediated transcriptional activation, and expression of AhR target genes. In addition, camalexin suppressed mammosphere formation in AhR-expressing breast cancer cells more than in the breast cancer cells that lacked AhR expression. Taken together, the data demonstrate that camalexin is a novel AhR agonist and that the inhibition of cell proliferation, migration, and mammosphere formation by camalexin involves the activation of AhR. Our findings suggest that camalexin, an AhR agonist, may be a novel therapeutic agent for breast cancer., (© 2021. The Japanese Society of Pharmacognosy.)

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates various toxicological and biological functions. We reported previously that 3-methylcholanthrene (3MC), an exogenous AhR agonist, inhibited tumorsphere (mammosphere) formation from breast cancer cell lines, while the endogenous AhR agonist, indirubin, very weakly inhibited this process. However, the difference in inhibition mechanism of mammosphere formation by 3MC or indirubin is still unknown. In this study, we established AhR-re-expressing (KOTR-AhR) cells from AhR knockout MCF-7 cells using the tetracycline (Tet)-inducible gene expression systems. To identify any difference in inhibition of mammosphere formation by 3MC or indirubin, RNA-sequencing (RNA-seq) experiments were performed using KOTR-AhR cells. RNA-seq experiments revealed that cell division cycle 20 (CDC20), which regulates the cell cycle and mitosis, was decreased by 3MC, but not by indirubin, in the presence of AhR expression. Furthermore, the mRNA and protein levels of CDC20 were decreased by 3MC in MCF-7 cells via the AhR. In addition, mammosphere formation was suppressed by small interfering RNA-mediated CDC20 knockdown compared to the negative control in MCF-7 cells. These results suggest that AhR activation by 3MC suppresses mammosphere formation via downregulation of CDC20 expression in breast cancer cells. This study provides useful information for the development of AhR-targeted anti-cancer drugs., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The aryl hydrocarbon receptor (AhR) regulates expression of genes encoding drug/xenobiotic metabolizing enzymes. Cytochrome P450 (CYP) 3A5 is involved in drug metabolism. However, regulation of CYP3A5 gene expression is not yet well understood. In this study, we aimed to investigate the effect of the ligands of AhR on CYP3A5 gene expression. CYP3A5 mRNA expression was induced by the polycyclic aromatic hydrocarbons (PAHs) such as 3-methylcholanthrene (3MC) and benzo[a]pyrene in HepG2 cells. To determine whether the PAHs induced CYP3A5 gene expression via AhR, we generated AhR knockout (AhR KO) HepG2 cells. CYP3A5 mRNA expression was not induced by 3MC treatment in AhR KO cells. In addition, we generated AhR rescue cells from AhR KO cells and evaluated CYP3A5 mRNA expression. We found that CYP3A5 mRNA expression was induced by 3MC treatment in AhR rescue cells. Taken together, these results demonstrated that CYP3A5 mRNA expression was induced by PAHs via AhR in HepG2 cells. Our findings suggest that ligand-activated AhR affects CYP3A5-mediated drug metabolism.

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear protein that is activated by binding to DNA lesions and catalyzes poly(ADP-ribosyl)ation of nuclear acceptor proteins, including PARP1 itself, to recruit DNA repair machinery to DNA lesions. When excessive DNA damage occurs, poly(ADP-ribose) (PAR) produced by PARP1 is translocated to the cytoplasm, changing the activity and localization of cytoplasmic proteins, e.g., apoptosis-inducing factor (AIF), hexokinase, and resulting in cell death. This cascade, termed parthanatos, is a caspase-independent programmed cell death distinct from necrosis and apoptosis. In contrast, PARP1 is a substrate of activated caspases 3 and 7 in caspase-dependent apoptosis. Once cleaved, PARP1 loses its activity, thereby suppressing DNA repair. Caspase cleavage of PARP1 occurs within a nuclear localization signal near the DNA-binding domain, resulting in the formation of 24-kDa and 89-kDa fragments. In the present study, we found that caspase activation by staurosporine- and actinomycin D-induced PARP1 autopoly(ADP-ribosyl)ation and fragmentation, generating poly(ADP-ribosyl)ated 89-kDa and 24-kDa PARP1 fragments. The 89-kDa PARP1 fragments with covalently attached PAR polymers were translocated to the cytoplasm, whereas 24-kDa fragments remained associated with DNA lesions. In the cytoplasm, AIF binding to PAR attached to the 89-kDa PARP1 fragment facilitated its translocation to the nucleus. Thus, the 89-kDa PARP1 fragment is a PAR carrier to the cytoplasm, inducing AIF release from mitochondria. Elucidation of the caspase-mediated interaction between apoptosis and parthanatos pathways extend the current knowledge on mechanisms underlying programmed cell death and may lead to new therapeutic targets., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Aryl hydrocarbon receptor (AhR) and androgen receptor (AR) are ligand-activated transcription factors with profound cross-talk between their signal transduction pathways. Previous studies have shown that AhR agonists activate the transcription of AR-regulated genes in an androgen-independent manner; however, the underlying mechanism remains unclear. To decipher this mechanism, we evaluated the effects of 3-methylcholanthrene (3MC), a potent AhR agonist, on the transcription of AR-regulated genes in three AR-expressing cell lines. 3MC induced the expression of not only three representative AR-regulated chromosomal genes but also the exogenous AR-responsive luciferase reporter gene. No significant difference in the 3MC-induced luciferase activity was detected in the presence of SKF-525A, a non-specific inhibitor of CYP enzymes. The androgenic effects of 3MC were diminished by AhR and AR knockdown. Following 3MC treatment, the amount of nuclear AhR and AR increased synchronously. Co-immunoprecipitation revealed that AhR and AR formed a complex in the nucleus of cells treated with 3MC. AR was recruited to the proximal promoter and distal enhancer regions of the PSA gene upon the addition of 3MC. We propose that AhR activated by 3MC forms a complex with unliganded AR which translocates from the cytoplasm to the nucleus. Nuclear AR now binds the transcriptional regulatory region of AR-regulated genes and activates the transcription., (© The Author(s) 2020. Published by Oxford University Press.)

Triple-negative breast cancer (TNBC) is associated with poor prognosis, because of no effective targeted therapy. In the present study, we demonstrated the crucial role of the aryl hydrocarbon receptor (AhR) in mediating the effects of the chemotherapeutic agent doxorubicin (DOX) in the chemotherapeutic sensitivity of TNBC. Firstly, we established AhR knockout (KO) MDA-MB 231 TNBC cells. The cytotoxic effects of DOX were more pronounced in AhR KO cells than in parental cells. In addition, our results indicated that AhR KO cells showed downregulated expression of DOX-metabolism enzyme, aldo-keto reductase (AKR) 1C3, relative to those of parental cells. Furthermore, AhR was found to enhance AKR1C3 promoter reporter activity, suggesting that AKR1C3 mRNA transcription is activated by AhR. Additionally, our findings confirmed that the downregulation of AKR1C3 expression enhanced DOX sensitivity in MDA-MB 231 cells. Finally, AhR and AKR1C3 expression were positively correlated in human breast cancer. Taken together, our results suggested that AhR is involved in DOX sensitivity by regulating AKR1C3 expression in TNBC cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The most important risk factor for severe respiratory syncytial virus (RSV) infection is considered young age due to the immature immune system. The risk at young age is reported greater for RSV than for other respiratory infectious agents. Based on the strong association between young age and severity of RSV infection due to immature immunity, we aimed to assess whether there were any age-related differences in fever responses, as one clinical aspect of the immune response. In our observational study over two seasons (2014-2015 and 2015-2016), daily body temperatures of children under 3 years old with RSV infection were recorded from the first medical visit during the acute phase to defervescence. The body temperature records were analyzed among 171 children of four age groups (< 6, < 12, < 24 and ≥ 24 months), in terms of fever development, degrees of fever onset, the highest fever during the period, and fever duration. There were 54 patients in the group of < 6 months, 41 in the group of < 12 months, 58 in the group of < 24 months, and 18 in the group of ≥ 24 months. We thus found the correlation between age and fever responses under 24 months old; namely, the more the age advanced, the more frequently high and prolonged fever was experienced. Importantly, infants under 6 months old tend to show the suppressed fever responses. In conclusion, young infants with reduced fever response during RSV infection do not implicate less severity and needs attentive management.

Background: Diazoxide is the first-line treatment for pediatric hyperinsulinemic hypoglycemia (HI). This study aimed to elucidate the pharmacokinetics of diazoxide in children with HI., Methods: We obtained 81 blood samples from 22 children with HI. Measured serum diazoxide concentrations were used for population pharmacokinetic analysis. Patient factors influencing pharmacokinetics were estimated using nonlinear mixed-effects model analysis. Relationships between drug exposure and adverse drug reactions were also investigated., Results: Diazoxide disposition in the body was described by a 1-compartment model. Oral clearance (CL/F) and the volume of distribution were proportional to body weight (WT), as expressed by CL/F in males (liters/h) = 0.0358 + 0.00374 × WT (kg). CL/F in females was 39% greater than that in males. Steady-state concentrations of diazoxide were similar following twice- and 3 times-daily dosing when the total daily doses were comparable. A patient whose serum diazoxide concentration exceeded 100 μg/mL over a 4-month period developed hyperglycemia. No significant correlation was observed between severity of hirsutism and diazoxide concentration., Conclusion: We have proposed for the first time a population pharmacokinetic model for diazoxide in children with HI. The potential risk of diabetes mellitus and/or hyperglycemia increases when serum concentrations of diazoxide exceed 100 μg/mL., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Background: Lysinuric protein intolerance (LPI) is a rare autosomal recessive disorder affecting the transport of cationic amino acid caused by mutations in solute carrier family 7 amino acid transporter light chain, y + L system, member 7 (SLC7A7). This disorder occurs worldwide, especially in Finland and Japan, where founder effect mutations have been reported. Detailed features of the clinical symptoms and mutation types in Japanese LPI, however, remain unclear to date., Methods: An epidemiological nationwide survey of LPI patients was carried out via mail to all domestic university and general hospitals in Japan. Next, the clinical information for each LPI patient was obtained, in the form of a questionnaire, from the attending physicians who replied to the letters., Results: We received answered questionnaires for 43 LPI patients in 19 hospitals. We selected 35 patients who were genetically diagnosed with LPI. The most common clinical manifestations were with protein aversion, ferritinemia, increased serum lactate dehydrogenase, and hyperammonemia. The most frequent SLC7A7 mutation in Japanese LPI patients is p.R410*, which is a founder effect mutation in northern Japan. In total, nine types of mutation were detected in this survey, six of which (p.R410*, p.S238F, c.1630delC, p.S489P, c.1673delG, and IVS3-IVS5del9.7 kb) have not been reported in other countries., Conclusion: The clinical and genetic features of 35 Japanese patients with LPI were characterized, and no correlation between genotype and phenotype was observed. The importance of early diagnosis for better prognosis of LPI is emphasized., (© 2016 Japan Pediatric Society.)

The store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel is activated by diminished luminal Ca(2+) levels in the endoplasmic reticulum and sarcoplasmic reticulum (SR), and constitutes one of the major Ca(2+) entry pathways in various tissues. Tubular aggregates (TAs) are abnormal structures in the skeletal muscle, and although their mechanism of formation has not been clarified, altered Ca(2+) homeostasis related to a disordered SR is suggested to be one of the main contributing factors. TA myopathy is a hereditary muscle disorder that is pathologically characterized by the presence of TAs. Recently, dominant mutations in the STIM1 gene, encoding a Ca(2+) sensor that controls CRAC channels, have been identified to cause tubular aggregate myopathy (TAM). Here, we identified heterozygous missense mutations in the ORAI1 gene, encoding the CRAC channel itself, in three families affected by dominantly inherited TAM with hypocalcemia. Skeletal myotubes from an affected individual and HEK293 cells expressing mutated ORAI1 proteins displayed spontaneous extracellular Ca(2+) entry into cells without diminishment of luminal Ca(2+) or the association with STIM1. Our results indicate that STIM1-independent activation of CRAC channels induced by dominant mutations in ORAI1 cause altered Ca(2+) homeostasis, resulting in TAM with hypocalcemia., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Primary ciliary dyskinesia (PCD) is a genetic disease that causes abnormalities in ciliary structure and/or function. Ciliated cells line the upper and lower respiratory tracts and the Eustachian tube. Impairment of mucus clearance at these sites leads to sinusitis, repeated pulmonary infections, bronchiectasis, and chronic otitis media. Situs inversus occurs randomly in approximately 50% of subjects with PCD. The triad of situs inversus, bronchiectasis and sinusitis is known as Kartagener syndrome. PCD is usually an autosomal recessive disease, but occasional instances of X-linked transmission have been reported. Specific diagnosis requires examination of ciliary function or structure on light and electron microscopy. Early diagnosis and respiratory management are important in order to prevent the development of bronchiectasis and deterioration in lung function. We report early diagnosis of PCD on nasal mucosal biopsy in two newborns who presented with prolonged respiratory distress and rhinorrhea., (© 2014 The Authors. Pediatrics International © 2014 Japan Pediatric Society.)

Background: This multicenter observational study was conducted to investigate the efficacy and safety of insulin detemir (detemir) for diabetes management in Japanese children and adolescents., Methods: Data from the Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes database were analyzed. Ninety children (32 boys, 58 girls; mean age, 11.9 ± 3.8 years) who transferred from a neutral protamine Hagedorn insulin or insulin glargine basal-bolus regimen to detemir basal-bolus therapy and who were observed for at least 12 months were identified. Clinical data obtained at 0, 3, 6, and 12 months were analyzed to determine the type of bolus insulin used, number and timing of detemir injections, detemir dose as a proportion of the total insulin dose, hemoglobin A1c (HbA1c), fasting blood glucose (FBG) and frequency of severe hypoglycemia., Results: Twelve months after switching to detemir, the detemir dose represented 39.8% of the total insulin dose, and 37.8% of patients were being treated with twice-daily injections. HbA1c and FBG were significantly reduced from baseline at 3 and 6 months but not at 12 months. Considering the seasonal HbA1c variation in the Japanese population, a separate analysis was performed using data for 65 children (21 boys, 44 girls; mean age, 11.6 ± 2.9 years) who switched to detemir during the winter. Subset analysis showed significant HbA1c reductions from baseline at all specified times. The incidence of severe hypoglycemia during detemir treatment was 4.4 episodes per 100 patient-years., Conclusions: Detemir is an effective and safe basal insulin for diabetes management in Japanese children and adolescents., (© 2012 The Authors. Pediatrics International © 2012 Japan Pediatric Society.)

Objective: To determine the HLA-DRB1, DQB1, DPB1, A, C, and B genotypes among Japanese children with autoimmune type 1 diabetes., Methods: Four hundred and thirty patients who were GADAb and/or IA-2Ab-positive (Type 1A) were recruited from 37 medical centers as part of a nationwide multicenter collaborative study. DNA samples from 83 siblings of the children with Type 1A diabetes and 149 parent-child trios were also analyzed. A case-control study and a transmission disequilibrium test (TDT) were then performed., Results: The susceptible and protective DRB1 and DQB1 alleles and haplotypes were confirmed. DPB1 alleles unique to the Japanese population and those common to multiple ethnic groups were also present. A linkage disequilibrium (LD) analysis showed both susceptible and protective haplotypes. The TDT did not reveal any alleles that were transmitted preferentially from the mother or father to children with Type 1A. Homozygosity for DRB1-09:01-DQB1-03:03 and heterozygosity for DRB1-04:05-DQB1-04:01 and DRB1-08:02-DQB1-03:02 were associated with an extremely high risk of Type 1A. A comparison of children with Type 1A and their parents and siblings suggested a dose effect of susceptible DRB1-DQB1 haplotypes and an effect of protective alleles on immunological pathogenesis. DRB1-09:01 appeared to be strongly associated with an early onset in preschool children with Type 1A diabetes., Conclusions: This study demonstrated the characteristic association of HLA-class II and class I genes with Type 1A diabetes among Japanese children. A TDT did not reveal the genomic imprinting of HLA-class II and class I genes in Type 1A diabetes., (© 2011 John Wiley & Sons A/S.)

Cdc25 dual specificity phosphatases positively regulate the cell cycle by activating cyclin-dependent kinase/cyclin complexes. Of the three mammalian Cdc25 isoforms, Cdc25A is phosphorylated by genotoxic stress-activated Chk1 or Chk2, which triggers its SCFbeta-TrCP-mediated degradation. However, the roles of Cdc25B and Cdc25C in cell stress checkpoints remain inconclusive. We herein report that c-Jun NH2-terminal kinase (JNK) induces the degradation of Cdc25B. Nongenotoxic stress induced by anisomycin caused rapid degradation of Cdc25B as well as Cdc25A. Cdc25B degradation was dependent mainly on JNK and partially on p38 mitogen-activated protein kinase (p38). Accordingly, cotransfection with JNK1, JNK2, or p38 destabilized Cdc25B. In vitro kinase assays and site-directed mutagenesis experiments revealed that the critical JNK and p38 phosphorylation site in Cdc25B was Ser101. Cdc25B with Ser101 mutated to alanine was refractory to anisomycin-induced degradation, and cells expressing such mutant Cdc25B proteins were able to override the anisomycin-induced G2 arrest. These results highlight the importance of a novel JNK/p38-Cdc25B axis for a nongenotoxic stress-induced cell cycle checkpoint.

Polycyclic aromatic hydrocarbons (PAHs) and dioxins are ubiquitous environmental pollutants and activate the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. It has been reported that testosterone represses 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced transcription of the cytochrome P450 (CYP) 1A1 gene in LNCaP cells. In this study, we investigated the mechanism for the repression of 3-methylcholanthrene (3MC)-induced transcription of AhR-regulated genes, CYP1A1, CYP1A2, CYP1B1, and AhR repressor (AhRR), by 5alpha-dihydroteststerone (DHT) in LNCaP and T47D cells, which are androgen receptor (AR)- and AhR-positive. Real-time PCR analysis showed that DHT repressed 3MC-induced mRNA expression of the CYP1 family and AhRR genes. DHT repressed 3MC-induced luciferase activity in an AhR response element-driven luciferase reporter assay in LNCaP and T47D cells. The inhibitory effect of DHT was abolished by knockdown of AR protein with siRNA. The protein levels of AhR and AhR nuclear translocator (Arnt), the AhR-dimerizing partner, were not affected by DHT. Co-immunoprecipitation assay showed that DHT significantly facilitated the complex formation between AR and AhR in 3MC-treated cells. These results suggest that complex formation between activated AR and AhR plays an important role in the suppression of 3MC-induced transcription of CYP1 family genes by DHT.

Quinoid polycyclic aromatic hydrocarbons are potent redox-active compounds that undergo enzymatic and nonenzymatic redox cycling with their semiquinone radical. We previously reported that acenaphthenequinone (AcQ) can damage human lung epithelial A549 cells through the formation of reactive species (RS). However, the biochemical mechanisms by which RS-generating enzymes cause oxidative burst during AcQ exposure remain elusive. Here we examined the biochemical mechanism of AcQ-induced RS generation by using selective metabolic inhibitors in A549 cells. We found that AA861, a 5-lipoxygenase (5-LO)-specific inhibitor significantly decreases RS generation. This inhibition of RS seems to be 5-LO specific because other inhibitors did not suppress AcQ-induced RS generation by nicotinamide adenine nucleotide phosphate (reduced) oxidase and/or xanthine oxidase. In addition, the inhibition of 5-LO by AA861 markedly reduced AcQ-induced nuclear factor kappa B (NF-kappa B) activation. We further found the activation of 5-LO pathway by exposing cells to AcQ mediates the secretion of inflammatory leukotriene B4, which can be significantly suppressed by a potent RS scavenger, N-acetylcysteine. Thus, based on our findings, we propose that AcQ-induced damage is likely due to increased RS generation and NF-kappa B activity through 5-LO activation.

Diesel exhaust particulate extract (DEPE) was obtained from diesel exhaust particulates with Soxhlet extraction using dichloromethane. After separating DEPE into 11 fractions by liquid-liquid extraction, the neutral fraction (N) showed anti-estrogenic activity and the weak acid (phenol) fraction (WA(P)) showed estrogenic and anti-estrogenic activities by a yeast two-hybrid assay system expressing human estrogen receptor alpha. Both fractions were thoroughly fractionated by silica gel column chromatography and reversed-phase HPLC. In the WA(P) fraction, 3-methyl-4-nitrophenol and 2,6-dimethyl-4-nitrophenol were identified by LC-MS/MS as estrogenic compounds. This is the first study to identify 2,6-dimethyl-4-nitrophenol in DEPE and the first study to show that it is an estrogenic compound. In the N fraction, 1-hydroxypyrene was also identified by LC-MS/MS as an anti-estrogenic compound., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Diesel exhaust particles (DEPs) contain oxygen-containing polycyclic aromatic hydrocarbons (PAHs) called quinoid PAHs. Some quinoid PAHs generate free radicals as they undergo enzymatic and nonenzymatic redox cycling with their corresponding semiquinone radicals. Reactive oxygen species (ROS) produced by these reactions can cause severe oxidative stress connected with inflammatory processing. Although humans and animals are continuously exposed to these chemicals in the environment, little is known about which quinoid PAHs are active. In this study, we estimated the intracellular ROS production and nuclear factor kappa B (NF-kappaB) translocation in A549 cells exposed to isomers of quinoid PAHs having two to four rings. We found that both acenaphthenequinone (AcQ) and 9,10-phenanthrenequinone (PQ) enhanced ROS generation and that AcQ translocated NF-kappaB from the cytosol to the nucleus. However, PQ, which has been reported to induce apoptosis, did not influence NF-kappaB activation. In addition, AcQ induced cyclooxygenase-2 (COX-2) expression which is a key enzyme in the inflammatory processing involved in the activation of NF-kappaB. Upregulation of NF-kappaB and COX-2 expression by AcQ treatment was suppressed by the antioxidant N-acetylcysteine (NAC). These results provide that AcQ might play an important role in human lung inflammatory diseases as an air pollutant.

Natural and artificial substances present in the environment can affect our health. Testicular toxicants in particular are troublesome, because they disturb gonadal function of males. Translocation of substances into the seminiferous epithelium where sperm production proceeds is restricted due to the blood-testis barrier, but this permeability barrier temporarily disappears under physiological and sub-physiological conditions. This means that any substance could enter the seminiferous epithelium and disturb sperm production. To reduce the risk posed by such toxins, it is important to accurately determine which substances possess the toxicity. However, existing assay systems are not satisfactory in terms of both accuracy and sensitivity. Here, we report the establishment of such a system. We injected the androgen antagonists, flutamide and vinclozolin, directly into seminiferous tubules of live mice, which had been treated with busulfan for a temporal arrest of spermatogenesis, and the testes were histologically examined to see the effect of the injected materials on spermatogenesis that was in the process of recovery. The injection of either substance brought about a severe impairment of spermatogenesis at an amount over a million times smaller than that used in the previous assay systems where animals are administered with test substances outside of the testis. In contrast, these androgen antagonists at the same doses showed lesser effects when intratubularly or intraperitoneally administered into mice that had not been pretreated with busulfan. We propose that the method adopted in this study is a novel assay system to identify potential testicular toxicants.

Estrogen receptor alpha (ERalpha) is a ligand-inducible transcription factor that mediates the biological effects of estrogens and antiestrogens. Many point mutations in the human ERalpha gene have been reported to be associated with breast cancer, endometrial cancer, and psychiatric diseases. However, functional analyses for most mutants with amino acid changes are still lacking. In the present study, to investigate the effects of point mutations on the function, gel-shift assays and luciferase assays were performed for eight kinds of mutated ERalpha proteins, including a single nucleotide change of C207G (N69K), G478T (G160C), T887C (L296P), A908G (K303R), C926T (S309F), A1058T (E353V), A1186G (M396V), and G1231deletion (411fsX7). The mutated ERalpha expression plasmids were constructed by site-directed mutagenesis. With gel-shift assays using in vitro translated ERalpha proteins, binding to the consensus estrogen response element (ERE) was observed for the mutated ERalpha proteins except ERalpha (G160C) and ERalpha (411fsX7), the binding of which was comparable with that of the wild type. Western blot analyses showed that ERalpha (G160C) could not be efficiently translated with the in vitro transcription/translation system and that ERalpha (411fsX7) produced a truncated protein. To investigate the transactivation potency, wild-type or mutated ERalpha expression plasmids were co-transfected with pGL3-3EREc38 reporter plasmid into human breast adenocarcinoma MDA-MB-435 cells. The concentration-response curves (10pM-100nM E2) of the mutant ERalpha proteins except ERalpha (E353V) and ERalpha (411fsX7) were similar to that of wild-type ERalpha. However, at a low level of E2 (100pM), the mutants ERalpha (N69K), ERalpha (L296P), ERalpha (S309F), and ERalpha (M396V) showed a significant decrease of transactivation compared with that of the wild-type ERalpha. The mutants ERalpha (E353V) and ERalpha (411fsX7) did not show responsiveness to E2 and antiestrogens, 4-hydroxytamoxifen (4OHT) and ICI 182,780. The mutant ERalpha (S309F) showed decreased responsiveness for the antiestrogenicity of 4OHT. In conclusion, we found that some of the naturally occurring human ERalpha mutants with amino acid changes may have an altered responsiveness to estrogen and antiestrogens.

Water samples were collected at 17 sites in Kahokugata Water Basin, a closed water basin in central Japan. We determined the concentration of dioxins of the water samples. Linear relationships between toxic equivalent (TEQ) concentrations of dioxin and concentrations of suspended solid (SS) were obtained at sites in Kahokugata Lagoon and in the rivers flowing into the lagoon. Homologue composition of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) indicated that all the water samples were still strongly influenced by chlorinated herbicides, such as chloronitrofen (CNP) and pentachlorophenol (PCP) that had been widely used in rice fields. The main isomer distributions of the PCDD homologues were not significantly different among the sampling sites, while the main isomer distributions of the PCDF homologues were considerably different among the sampling sites. At a few sampling points in the downstream part of one of the rivers, high concentrations of 1,3,6,7,8-pentachloro dibenzofuran (1,3,6,7,8-PeCDF) and its related isomers (1,3,6,8-chlorine-substituted PCDFs) were traced to a dye manufacturing plant. These non-toxic isomers are usually only minor constituents in environmental water samples and are not indicators of any known dioxin sources. The dyeing discharge was found to make a contribution only in the water samples collected near the plant and the seasonal variation of the contribution might depend on the flow rate of the river.

The phospho-site adapter protein 14-3-3 binds to target proteins at amino acid sequences matching the consensus motif Arg-X-X-Ser/Thr-X-Pro, where the serine or threonine residue is phosphorylated and X is any amino acid. The dual-specificity phosphatase CDC25B, which is involved in cell cycle regulation, contains five 14-3-3 binding motifs, but 14-3-3 preferentially binds to the motif at Ser309 in CDC25B1 (or Ser323 in CDC25B3). In the present study, we demonstrate that amino acid residues C-terminal to the 14-3-3 binding motif strongly affect the efficiency of 14-3-3 binding. Alanine substitutions at residues downstream of the Ser309 motif dramatically reduced 14-3-3 binding, although phosphorylation of Ser309 was unaffected. We also observed that binding of endogenous 14-3-3 to mutant CDC25B occurred less efficiently than to the wild type. Mutants to which 14-3-3 cannot bind efficiently tend to be located in the nucleus, although not as specifically as the alanine substitution mutant of Ser309. These results indicate that amino acid sequences C-terminal to the consensus binding site have an important role in the efficient binding of 14-3-3 to at least CDC25B, which may partly explain why some consensus sequences are inactive as 14-3-3 binding sites.

The damage to and recovery of the Japanese coastline from Suzu, Ishikawa Prefecture to Mikuni, Fukui Prefecture was investigated visually over three years after a C-heavy oil spill from the Russian tanker "Nakhodka" in the Japan Sea on January 2, 1997. The beached C-heavy oil tended to remain for a long time on coasts of bedrock and boulder/cobble/pebble but it was removed rapidly from coasts of gravel/sand and man-made structures such as concrete tetrapods. On the coasts of the latter type, wave energy appeared to be the main force removing the oil. One year after the spill, C-heavy oil tended to remain strongly on the sheltered coasts of bedrock and boulder/cobble/pebble. Even on coasts of this type, the contamination was remarkably absent by 2 years after the spill. The concentration levels of polycyclic aromatic hydrocarbons (PAHs) in oil lumps, sand and seawater were monitored during 3 years following the spill. The concentrations of PAHs having 2 or 3 rings decreased more quickly than did those of PAHs having 4 or more rings, suggesting that volatilization was the main cause of the decrease. On the other hand, the concentrations of PAHs having 4 to 6 rings did not start to decrease until 7 months after the spill. The main cause of the decrease seemed to be photolysis. The concentration of BaP in seawater off the polluted coasts was high 1 month after the spill and then decreased. Three years after the spill, the level fell to the sub ng/L level, which was as low as the level in seawater along unpolluted clean coasts in Japan. The concentration of BaP in greenling was higher than the normal level only during the first two months after the spill. These results suggest that the coastlines in Ishikawa and Fukui Prefectures that were polluted with C-heavy oil recovered in 3 years.

Estrogenic activities of cigarette smoke condensates obtained from the extraction of particulate matters from mainstream and sidestream cigarette smoke with benzene/ethanol were evaluated by using a yeast two-hybrid assay system expressing human estrogen receptor alpha (hERalpha). To identify the constituents of the cigarette smoke condensate which are responsible for the estrogenic activity, the condensate was fractionated into eleven fractions by liquid-liquid extractions. Among these fractions, the neutral fractions of mainstream and sidestream smoke showed the strongest estrogen receptor-mediated activity by the yeast two-hybrid assay. Then the neutral fractions were fractionated by medium-pressure liquid chromatography with silica gel column. In the fractions that showed strong estrogenic activity, 2-hydroxyfluorene (2-OHFle), 2- and 3-hydroxyphenanthrene (2- and 3-OHPhe), 1-hydroxypyrene (1-OHPyr) and n-propyl-p-hydroxybenzoate (n-PHB) were identified by LC- and GC-MS and HPLC with fluorescence detection. 2-OHFle, 2-OHPhe and n-PHB exhibited estrogenic activity, whereas weak activity was observed with 3-OHPhe and 1-OHPyr. Several other hydroxylated polycyclic aromatic hydrocarbons having no activity were also identified. This is a first study to identify estrogenic hydroxylated PAHs in cigarette smoke condensate. The present findings points out the necessity for detailed investigation of exposure to aerosols containing apparently estrogenic compounds.

A new dinuclear docking Pt(II) complex, (cis-diammine) (l-1,2-cyclohexanediamine)(mu-dichloro)-diplatinum(II) oxalate was synthesized by reacting oxaliplatin(l-OHP, [Pt(oxalato)(L-dach)]), L-dach = 1R, 2R-cyclohexanediamine), with cisplatin (CDDP). Elemental analysis of the compound indicated that it was 1:1 molar ratio complex of oxaliplatin and cisplatin. A plausible chemical structure has been proposed as Cl(-) bridged dinuclear complex, judged from its yellow coloration and NMR spectral analysis. This complex can be denoted as, i.e. [Pt(2)Cl(2)(NH(3))(2)(L-dach)](COO)(2) (L-OHP/CDDP). The complex showed higher cytotoxicity against L1210 than the parent complexes and low cross-resistance against L1210/CDDP and L1210/DACH. Its antitumor activity was also tested in vivo against murine leukemia L1210 cell lines. The complex showed much higher activity than the mixture(1:1 molar ratio) of oxaliplatin and cisplatin. The antitumor effect against L1210/CDDP was very high, showing collateral sensitivity, being similar to that of oxaliplatin, and against L1210/DACH it showed no cross-resistance.