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Purpose: Testicular germ cell tumors (TGCT) have a unique epigenetic profile distinct from that of other types of cancer. Elucidation of these properties has a potential to identify novel markers for TGCTs.Experimental Design: We conducted comprehensive analysis of DNA methyltransferase (DNMT) gene expression in TGCTs. Based on the expression profiles of DNMT genes in TGCTs, we generated a rabbit polyclonal anti-human DNMT3L antibody. We then studied the role of DNMT3L in TGCTs by the treatment of two embryonal carcinoma (EC) cell lines with a small interfering RNA system. Finally, we evaluated the immunohistochemical detection of DNMT3L in TGCT tissues. We also compared the patterns of DNMT3L immunohistochemistry with those of CD30 and SOX2.Results: Among the DNMT genes, we found that mRNA for DNMT3L was specifically expressed in TGCTs, but neither in normal testicular tissues nor in cancer cells of somatic tissue origin. DNMT3L protein was strongly expressed in two EC cell lines, but not in the cell lines of somatic tissue origin. Transfection of small interfering RNA for DNMT3L significantly reduced DNMT3L expression and resulted in growth suppression and apoptosis in EC cells. Immunohistochemical analysis showed that DNMT3L protein was present only in EC cells, but not in the other types of TGCT components and cancer cells of somatic tissue origin. DNMT3L staining was more prominent and specific than CD30 or SOX2 staining for detecting EC cells.Conclusion: DNMT3L is a novel marker and is essential for the growth of human embryonal carcinoma. Clin Cancer Res; 16(10); 2751–9. ©2010 AACR.

Supplementary Data from DNMT3L Is a Novel Marker and Is Essential for the Growth of Human Embryonal Carcinoma

Background Rearranged during transfection (RET) rearrangements occur in 1-2% of non-small cell lung cancers (NSCLCs). Alectinib administered at doses of 300 mg and 600 mg twice daily (BID) is approved for. Alk rearranged NSCLC in Japan and other countries, respectively. Since alectinib has activity against RET, we conducted a phase (P) 1/2 study of alectinib to determine its activity in Japanese patients with. Ret rearranged NSCLC. Methods This study was a single-arm, open-label, multi-institutional P1/2 trial. Previously treated patients with RET-rearranged NSCLC, screened by nation-wide network (LC-SCRUM-Japan), were recruited. In P1, alectinib (600 or 450 mg BID) was administered following a 3+3 design and its safety was assessed. During P2, alectinib was administered at the recommended dose (RD) determined in P1. The primary endpoint was the objective response rate (ORR) in RET inhibitor-naive patients treated with the RD of alectinib. Results Thirty-four patients were administered alectinib. In cohort 1 (600 mg BID) of P1, we observed 5 dose-limiting toxicities (DLTs), including grade 3 rash and thromboembolic event, in 3 of 6 patients. In cohort 2 (450 mg BID), we observed no DLTs in 3 patients. Pharmacokinetic analysis revealed that AUC0-10 to 600 mg BID was higher than that previously reported in global trials. We determined 450 mg BID as the RD for P2. In 25 RET inhibitor-naive patients, one achieved an objective response (4%) and 13 achieved disease control at 8 weeks (52%). The median progression-free survival (PFS) was 3.4 months (95% CI, 2.0-5.4), while the median overall survival was 19.0 months (5.4-NE). We observed grade 3 adverse events (AEs) (4%) including pneumonitis in P2. Conclusions Alectinib exerts limited activity against RET-rearranged NSCLC. Further investigation to elucidate the mechanisms underlying sensitivity and resistance of RET inhibitors is required to improve outcomes for these patients.

The human foot provides numerous functions that let humans deal with various environments. Recently, study of the structure of the human foot and adjustment of an appropriate reaction force and vertical free moment during bipedal locomotion has gained attention. However, little is known about the mechanical (morphological) contribution of the foot structure to the reaction force and free moment. It is difficult to conduct a comparative experiment to investigate the contribution systematically by using conventional methods with human and cadaver foot experiments. This study focuses on the oblique transverse tarsal joint (TTJ) of the human foot, whose mechanical structure can generate appropriate free moments. We conduct comparative experiments with a rigid foot, a non-oblique joint foot (i.e. mimicking only the flexion/extension of the midfoot), and an oblique joint foot. Axial loading and walking experiments were conducted with these feet. The axial loading experiment demonstrated that the oblique foot generated free moment in the direction of internal rotation, as observed in the human foot. The walking experiment showed that the magnitude of the free moment generated with the oblique foot is significantly lower than that with the rigid foot during the stance phase. Using this constructive approach, the present study demonstrated that the oblique axis of the TTJ can mechanically generate free moments. This capacity might affect the transverse motion of bipedal walking.

Submitting data compliant with the Clinical Data Interchange Standards Consortium (CDISC) standards is mandatory for new drug applications (NDAs). The standards set by CDISC are widely adopted in the pharmaceutical business world. Introduction of CDISC standards in academia can be necessary to reduce labor, resolve the shortage of data managers in academia, and gain new knowledge through standardized data accumulation. However, the introduction of CDISC standards has not progressed in communities within the academia that do not apply for NDAs. Therefore, herein, we created study data tabulation model (SDTM)-compliant datasets within the academia, without outsourcing, to reduce costs associated with investigator-initiated clinical trials. First, we input data from paper case report forms (CRFs) into an electronic data capture system with minimal function for paper CRFs, "Ptosh," which is compatible with SDTM. Then, we developed a generic program to convert data exported from Ptosh into fully SDTM-compliant datasets. The consistency was then verified with an SDTM validator, Pinnacle21 Community V3.0.1 (P21C). This resulted in generation of SDTM datasets, resolving all "Rejects" in P21C, thereby achieving the required quality level. Although Ptosh directly exports data in SDTM format, manual mapping of items on CRFs to SDTM variables prepared in Ptosh is necessary. SDTM mapping requires extensive knowledge and skills, and it was assumed that mapping is challenging for the staff without in-depth knowledge of CDISC standards and datasets. Therefore, for CDISC dissemination in academia, it is crucial to secure the staff, time, and funding to acquire the knowledge.

Background This study aimed to investigate the contribution of a proliferation-inducing ligand (APRIL), a member of the tumor necrosis factor (TNF) superfamily implicated in plasma cell survival, to the development of plasma cell–rich lesions in immunoglobulin G4–related disease (IgG4-RD). Methods We performed immunohistochemical staining for APRIL with Stalk-1 and Aprily-8 antibodies specifically recognizing APRIL-producing cells and secreted APRIL, respectively, in renal and submandibular lesions of IgG4-RD in comparison with those of Sjögren’s syndrome and sialolithiasis. Results Numerous Stalk-1-positive APRIL-producing cells were detectable in lesions of IgG4-RD. These cells, identified as CD163-positive M2 macrophages, secreted APRIL that distributed close to and even on infiltrating plasma cells. In contrast, APRIL-producing cells and the secreted form of APRIL were rarely detectable in lesions of Sjögren’s syndrome or sialolithiasis. Notably, APRIL expression decreased concomitantly with the level of plasma cell infiltration after successful glucocorticoid treatment. Conclusions Abundant infiltration into tissue lesions of APRIL-producing M2 macrophages and retention of secreted APRIL in plasma–cell–rich areas support a role for APRIL in the pathogenesis of plasma cell–rich lesions in IgG4-RD.

Background The rearranged during transfection (RET) fusion gene was discovered as a driver oncogene in 1-2% of non-small cell lung cancers (NSCLCs). Alectinib is an approved anaplastic lymphoma kinase (ALK) inhibitor that may also be effective for RET fusion-positive NSCLC. Methods/design RET fusion-positive NSCLC patients treated with at least one regimen of chemotherapy are being recruited. In step 1, alectinib (600 or 450 mg, twice daily) will be administered following a 3+3 design. The primary endpoint is safety. In step 2, alectinib will be administered at the recommended dose (RD) defined by step 1. The primary endpoint is the response rate of RET inhibitor treatment-naive patients. Conclusion This is the first study to investigate the safety and preliminary efficacy of alectinib in RET fusion-positive NSCLC patients. If successful, alectinib treatment may lead to substantial and important changes in the management of NSCLC with RET fusion genes. J. Med. Invest. 64: 317-320, August, 2017.

The LCuBr-catalyzed C–H imidation of arenes by N-fluorobenzenesulfonimide (NFSI), previously reported by us, utilizes an inexpensive catalyst and is applicable to a broad scope of complex arenes. The computational and experimental study reported here shows that the mechanism of the reaction is comprised of two parts: (1) generation of the active dinuclear CuII–CuII catalyst; and (2) the catalytic cycle for the C–H bond imidation of arenes. Computations show that the LCuIBr complex used in experiments is not an active catalyst. Instead, upon reacting with NFSI it converts to an active dinuclear CuII–CuII catalyst that is detected using HRMS techniques. The catalytic cycle starting from the CuII–CuII dinuclear complex proceeds via (a) one-electron oxidation of the active catalyst by NFSI to generate an imidyl radical and dinuclear CuII–CuIII intermediate, (b) turnover-limiting single-electron-transfer (SET1) from the arene to the imidyl radical, (c) fast C–N bond formation with an imidyl anion and an aryl radical cation, (d) reduction of the CuII–CuIII dinuclear intermediate by the aryl radical to regenerate the active catalyst and produce an aryl-cation intermediate, and (e) deprotonation and rearomatization of the arene ring to form the imidated product. The calculated KIE for the turnover-limiting SET1 step reproduces its experimentally observed value. A simple predictive tool was developed and experimentally validated to determine the regiochemical outcome for a given substrate. We demonstrated that the pre-reaction LCuX complexes, where X = Cl, Br and I, show a similar reactivity pattern as these complexes convert to the same catalytically active dinuclear CuII–CuII species.

Aromatic C–H amination chemistry has attracted significant attentions as an alternative of conventional amination of aryl halides. Among C–H amination reactions, dehydrogenative coupling of arenes and N–H reagents represents an ideal transformation because prefunctionalization steps of arenes or aminating reagents are not required. Herein, we report direct C–H/N–H coupling between naphthalene and sulfonimides mediated by 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) as a photosensitizer under irradiation of blue light.

The structures and stabilities of 1,8-anthrylene cyclic oligomers having acetylene or diacetylene linkers were investigated by DFT calculations with M05 and M06 series functionals. The structures o...

The rearranged during transfection (RET) fusion gene was discovered as a driver oncogene in 1-2% of non-small cell lung cancers (NSCLCs). Alectinib is an approved anaplastic lymphoma kinase (ALK) inhibitor that may also be effective for RET fusion-positive NSCLC.RET fusion-positive NSCLC patients treated with at least one regimen of chemotherapy are being recruited. In step 1, alectinib (600 or 450 mg, twice daily) will be administered following a 3+3 design. The primary endpoint is safety. In step 2, alectinib will be administered at the recommended dose (RD) defined by step 1. The primary endpoint is the response rate of RET inhibitor treatment-naïve patients.This is the first study to investigate the safety and preliminary efficacy of alectinib in RET fusion-positive NSCLC patients. If successful, alectinib treatment may lead to substantial and important changes in the management of NSCLC with RET fusion genes. J. Med. Invest. 64: 317-320, August, 2017.

The present study investigated the antiallergic and anti-inflammatory effects of 10-hydroxy-cis-12-octadecenoic acid (HYA), a novel gut microbial metabolite of linoleic acid, in NC/Nga mice, a model of atopic dermatitis (AD). Feeding HYA decreased the plasma immunoglobulin E level and skin infiltration of mast cells with a concomitant decrease in dermatitis score. HYA feeding decreased TNF-α and increased claudin-1, a tight junction protein, levels in the mouse skin. Cytokine expression levels in the skin and intestinal Peyer's patches cells suggested that HYA improved the Th1/Th2 balance in mice. Immunoglobulin A concentration in the feces of the HYA-fed mice was approximately four times higher than that in the control mice. Finally, denaturing gradient gel electrophoresis of the PCR-amplified 16 S rRNA gene of fecal microbes indicated the modification of microbiota by HYA. Taken together, the alterations in the intestinal microbiota might be, at least in part, associated with the antiallergic effect of HYA.

New fluorescent 2,9-diethynylanthracene derivatives ethynylated at unsymmetrical positions were synthesized. The addition of a (trialkylsilyl)ethynyllithium to 2-chloro-9-anthrone gave the corresponding 2-chloro-9-ethynylanthracene, and subsequent nickel-catalyzed coupling with a (trialkylsilyl)ethynyl Grignard reagent furnished the disilylated derivatives. These compounds were used for the synthesis of various 2,9-bis(phenylethynyl)anthracenes and dianthrylbutadiynes. UV–vis and fluorescence spectra were measured to evaluate the substituent effects on the absorption and emission bands.

This study investigated the longitudinal changes in brain activation balance in motor-related areas after Constraint-Induced Movement Therapy (CIMT).The subjects included seven ischemic stroke patients with mild right hemiparesis. Eight normal subjects were also included. The patients underwent functional MRI and motor function tests (Fugl-Meyer Assessment; FMA, modified Wolf Motor Function Test; mWMFT) both before and immediately after CIMT and also after a 3-month follow-up.The motor function test scores improved immediately after CIMT; moreover, these scores were either maintained or improved even at the 3-month follow-up. In a comparison of the chronological data of the contralaterality index of the affected hand movement, the cerebellar activity changed significantly to ipsilateral activation immediately after CIMT and thereafter the cerebellar activity further changed to ipsilateral activation at the 3-month follow-up. A correlation was observed among the contralateral activation, FMA and mWMFT scores in SM1 and the ipsilateral activation and in the mWMFT scores in the cerebellum at the 3-month follow-up examinations.The participation of the contralateral SM1 and the ipsilateral cerebellum is thus considered to play an important role in the satisfactory recovery of the motor function after CIMT intervention.

Testicular germ cell tumors (TGCTs) have a unique epigenetic profile distinct from that of other types of cancer. To further evaluate epigenetics of TGCTs, this study examines DNA methylation patterns of DNA repetitive elements in TGCTs. Bisulfite genomic sequencing and combined bisulfite restriction analysis (COBRA) were used to analyze the methylation patterns of DNA repetitive elements (LINE1 and Alu repeats) in embryonal carcinoma (EC) derived cell lines, primary TGCT tissues, noncancerous testicular tissues adjacent to TGCTs and cancer cells derived from somatic tissues (testicular malignant lymphoma tissues and renal cell carcinoma cell lines). Through both bisulfite genomic sequencing and COBRA, LINE1 was extensively hypomethylated in both seminomatous and nonseminomatous TGCT tissues as well as EC cell lines. We studied two Alu repeats locating in the 5' end of E-cadherin and XIST by bisulfite genomic sequencing. These two Alu elements were extensively hypomethylated in seminomatous TGCTs, but methylated in nonseminomatous TGCTs, including two EC derived cell lines. This increased unmethylated profile in seminomatous TGCTs was observed also by COBRA for Alu repeats. Although partial demethylation of DNA repetitive elements was observed in cancer cells of somatic tissue origin, the degree of demethylation was more pronounced in TGCTs than in cancer cells of somatic tissue origin. We observed abnormal demethylation of DNA repetitive elements in some of the tissues adjacent to TGCTs. The results indicate that the underlying mechanisms to undergo or maintain demethylation of DNA repetitive sequences differ between TGCTs and cancer cells of somatic tissue origin.

Purpose: Testicular germ cell tumors (TGCT) have a unique epigenetic profile distinct from that of other types of cancer. Elucidation of these properties has a potential to identify novel markers for TGCTs. Experimental Design: We conducted comprehensive analysis of DNA methyltransferase (DNMT) gene expression in TGCTs. Based on the expression profiles of DNMT genes in TGCTs, we generated a rabbit polyclonal anti-human DNMT3L antibody. We then studied the role of DNMT3L in TGCTs by the treatment of two embryonal carcinoma (EC) cell lines with a small interfering RNA system. Finally, we evaluated the immunohistochemical detection of DNMT3L in TGCT tissues. We also compared the patterns of DNMT3L immunohistochemistry with those of CD30 and SOX2. Results: Among the DNMT genes, we found that mRNA for DNMT3L was specifically expressed in TGCTs, but neither in normal testicular tissues nor in cancer cells of somatic tissue origin. DNMT3L protein was strongly expressed in two EC cell lines, but not in the cell lines of somatic tissue origin. Transfection of small interfering RNA for DNMT3L significantly reduced DNMT3L expression and resulted in growth suppression and apoptosis in EC cells. Immunohistochemical analysis showed that DNMT3L protein was present only in EC cells, but not in the other types of TGCT components and cancer cells of somatic tissue origin. DNMT3L staining was more prominent and specific than CD30 or SOX2 staining for detecting EC cells. Conclusion: DNMT3L is a novel marker and is essential for the growth of human embryonal carcinoma. Clin Cancer Res; 16(10); 2751–9. ©2010 AACR.

Introduction: Dehydrated hereditary stomatocytosis (DHSt) or hereditary xerocytosis (HX) is a form of congenital hemolytic anemia characterized by red blood cell (RBC) dehydration. Heterozygous mutations in PIEZO1, a mechanically-activated ion channel, cause DHSt. Recently, KCNN4, which encodes the Gardos channel, has been found to be the second pathogenic gene for DHSt. DHSt is characterized by an alteration in the RBC morphology in target cells, stomatocytes, and/or echinocytes, and RBC deformability assessments by ektacytometry as well as RBC ion flux measurements are currently the standard laboratory tests for DHSt, but their use in laboratories is limited. The flow cytometric osmotic fragility (FCM-OF) test is a useful diagnostic test for hereditary spherocytosis (HS) and also for hereditary elliptocytosis (HE). In this study, we showed that the FCM-OF test could also successfully diagnose DHSt. Subjects: A total of 46 cases of RBC membrane disorders were examined, and tentative diagnoses were made based on the RBC morphology, acid glycerol lysis time, and eosin 5'-maleimide binding tests, resulting in HS (n=31), HE (n=6), and DHSt (n=9). Methods: The number of RBCs in isotonic and hypotonic buffers were measured by flow cytometry. The degree of osmotic fragility was expressed as the "percentage residual RBCs (%RRC)". We confirmed the DHSt diagnosis by the massively paralleled sequencing using our custom panels targeting 68 hemolytic anemia-related genes with the next-generation sequencer. Results: Both HS and HE patients showed a decrease in %RRC; HS (18.0±8.9%, p Discussion: We examined 77 patients with congenital hemolytic anemia recently, and 59 cases were confirmed by diagnostic tests (76.6%). The results were as follows: 48 cases of RBC membrane abnormality (62.3%), 6 cases of RBC enzymopathy (7.8%), and 5 cases of hemoglobinopathy (6.5%). Of the cases of RBC membrane disorders, 31 cases of HS, 9 cases of DHSt, and 8 cases of HE were identified. These observations suggest that DHSt is the second-most common RBC membranopathy in Japan, and that the FCM-OF test and targeted sequencing efficiently discriminate DHSt from other RBC membrane disorders. Disclosures No relevant conflicts of interest to declare.

DNA methylation is the best known and most thoroughly studied epigenetic mechanism. Hypermethylation of CpG islands associated with silencing of tumour suppressor genes or tumour-related genes is a common hallmark of human cancer. The list of tumour-related genes with aberrant hypermethylation in their CpG islands has been increasing. There is also the potential for using DNA methylation profile data as markers for various types of human cancer. In this paper, we review the methylation profile of testicular germ cell tumours (TGCTs). We show that TGCTs have distinctive DNA methylation profiles that differ from those of somatic tissue-derived cancers or somatic tissues. We also discuss the methylation profile of TGCTs in terms of the DNA reprogramming that occurs in primordial germ cells or pre-implantation embryos. Finally, we describe the potential clinical utility of this unique methylation phenotype in TGCTs with regard to developing a novel tumour marker. These data suggest that unmethylated DNA fragments in TGCTs may have diagnostic implications. Further elucidation of epigenetic profiles in TGCTs is expected to provide a new insight into the biology of this disease.

The direct C—H imidation of a variety of arenes and heteroarenes is possible with N-fluorobenzenesulfonamide in the presence of CuBr and 6,6′-dimethyl-2,2′-bipyridine.

Versatile imidation of aromatic C–H bonds was accomplished. In the presence of copper bromide and 6,6′-dimethyl-2,2′-bipyridyl, a range of aromatics, such as polycyclic aromatic hydrocarbons, aromatic bowls, porphyrins, heteroaromatics, and natural products, can be imidated by N-fluorobenzenesulfonimide. A dramatic ligand-accelerated copper catalysis and an interesting kinetic profile were uncovered.

A common deletion at chromosomal arm 14q32 in human renal cell carcinoma (RCC) prompted us to explore a tumor suppressor gene (TSG) in this region. We report that imprinted DLK1 at 14q32, a regulator of adipocyte differentiation, is a candidate TSG in RCCs. DLK1 expression was lost in 39 out of 50 (78%) primary RCC tissues, whereas expression of DLK1 was maintained in every normal kidney tissue examined. DLK1 was expressed in only one of 15 (7%) RCC-derived cell lines. In order to see the biological significance of DLK1 inactivation in RCCs, we tested the effect of restoration of DLK1 in RCC cell lines, using a recombinant retrovirus containing the gene. Reintroduction of DLK1 into DLK1-null RCC cell lines markedly increased anchorage-independent cell death, anoikis and suppressed tumor growth in nude mice. We then investigated the underlying mechanisms for DLK1 inactivation in RCCs. We found loss of heterozygosity at this region in 12 out of 50 RCC tissues (24%). To explore the role of epigenetic regulation of DLK1 inactivation in RCCs, we conducted methylation analysis of the upstream region and the gene body of DLK1. We could not find a differentially methylated region in either the upstream region or the gene body of DLK1. However, we found that gain of methylation upstream of GTL2, a reciprocal imprinted gene for DLK1, is a critical epigenetic alteration for the inactivation of DLK1 in RCCs. The present data have shown that gain of methylation upstream of the untranslated GTL2 leads to pathological downregulation of DLK1 in RCCs.

Genome-wide epigenetic modification plays a crucial role in regulating genome functions at critical stages of development. In particular, DNA methylation is known to be reprogrammed on a genome-wide level in germ cells and in preimplantation embryos, although it is relatively stable in somatic cells. In this reprogramming process, the genome becomes demethylated, and methylated de novo during later stages of development. Reprogramming of DNA methylation in male germ cells has not been fully investigated. Testicular germ cell tumors (TGCTs) possess a pluripotential nature and display protean histology from germ cells to embryonal and somatic cell differentiation. These properties make TGCT a unique model for studying germ cell development and gametogenesis in respect of DNA reprogramming. In order to obtain an insight into the epigenetic dynamics of TGCTs, we conducted a comprehensive analysis of differential methylated regions (DMRs) on H19 and IGF2 in TGCTs compared with testicular malignant lymphomas. In the present study, we show that methylation imprint at the promoter and CTCF-binding site upstream of H19 was completely erased in both seiminomatous and non-seminomatous TGCTs, whereas differential methylation was observed in testicular lymphomas. The erasure of methylation imprint was also observed in TGCTs with malignant transformation. We found biallelic unmethylation at the promoter and the CTCF-binding site upstream of H19 is required, but not sufficient for the biallelic expression of H19 in TGCTs. These data suggest that factors other than methylation contribute to transcriptional regulation of imprinted genes in TGCTs. The present data have shown that TGCTs carry distinctive epigenetic profiles at the core-imprinting domain of H19/IGF2 from other neoplasms of somatic cell origin. The data also suggest that both seminomatous and non-seminomatous TGCTs carry methylation profiles similar to fetal germ cells, but not adult germ cells, indicating the origin of TGCTs as fetal germ cells.

Fluorine-free and hydrophobic room-temperature ionic liquids (RTILs) composed of the bis(2-ethylhexyl)sulfosuccinate (BEHSS) ion, which is known as the surface-active anion constituting Aerosol OT®, and symmetric tetraalkylammonium ions ((CnH2n+1)4N+; n = 4–8), have been prepared. Physicochemical properties of the water-saturated RTILs such as density, conductivity, viscosity and mutual solubility with water (W) have been measured. The RTIL|W interface is polarizable for the RTILs with n = 5–8 in spite of the high water content, 3.6–8.9 wt% in the water-saturated RTILs. The width of the polarized potential window of the RTIL|W interfaces is quantitatively correlated with the solubility of the RTIL in W. The RTIL–W two-phase systems are not spontaneously emulsified and no reverse micelles are formed in the water-saturated RTILs, although BEHSS− is known to form stable water-in-oil emulsions and reverse micelles in oil–water two-phase systems.

Testicular germ-cell tumors (TGCTs) are pluripotent and display protean histology from the germ-cell stage until embryonal and somatic-cell differentiation. These properties make TGCT a fascinating model for studying germ-cell development and gametogenesis. Methylation patterns specific to cell type (stem cells, germ cells, and somatic tissues) occur throughout the normal development of mice. To shed light on the epigenetic phenotypes among histological subtypes of TGCTs, we investigated the methylation and expression of several cancer testis antigen (CTA) genes (MAGEA1, MAGEA3, and SYCP1) in TGCTs. In the current study, we showed that the 5' ends of MAGEA1 and MAGEA3 on the X chromosome are unmethylated in seminomatous TGCTs, regardless of whether MAGEA1 and MAGEA3 are expressed and are methylated in nonseminomatous TGCTs when expression is absent. These distinctive epigenetic phenotypes of MAGEA1 and MAGEA3 also were observed in pure seminomas and in the seminomatous elements of mixed-type TGCTs. In contrast, the 5' end of SYCP1, on chromosome 1, remained predominantly unmethylated, regardless of expression, in both seminomatous and nonseminomatous TGCTs. This pattern of transcriptional regulation of SYCP1 is similar to that observed for XIST in TGCTs. On the basis of the epigenetic phenotypes of CTA genes, we concluded that, first, consistent unmethylated DNA profiles in seminomatous TGCTs imply that methylation may not be the primary control mechanism of programmed gene expression in seminomatous TGCTs, and, second, that nonseminomatous TGCTs might be midway between seminomatous TGCTs and somatic tissues because gene expression in nonseminomatous TGCTs is regulated by methylation in some genes (MAGEA1 and MAGEA3) but not others (SYCP1 and XIST).

Photoluminescence and surface plasmon emission light have been investigated in the attenuated total reflection (ATR) Kretschmann configuration. Surface plasmons (SPs), two-dimensional optical waves, were excited when rhodamine-B (RB) dye Langmuir-Blodgett (LB) films on metal thin film were directly irradiated by laser beams in the Kretschmann configuration. Emission light, that is, three-dimensional optical waves through the prism were observed corresponding to the resonant conditions of SP excitations in the configuration. The intensity of the SP emission light was small for the RB LB film with 4 layers, but the intensities increased in proportion to the thickness for the LB films having above 8 layers. Photoluminescence intensities also strongly depended upon the nanostructured separation between the RB LB films and metal thin films. It is thought that the phenomena are very useful for applications utilizing dye molecules on metal and conversion between three- and two-dimensional optical waves.

Polystyrene submicron and micron sphere films were fabricated and the optical properties were investigated using surface plasmon spectroscopy (SPS) and reverse irradiation utilizing the SPS Kretschmann configuration. The diameters of the spheres were 0.1 and 1.0 µm. For the 0.1-µm-sphere films, the dip angles of the SPS curves shifted to higher angles with the amount of spheres in the films. However, for the 1.0-µm-sphere films, the SPS curves did not shift with the amount of spheres which is considered to be due to the change of the effective dielectric constant in the decay length of the evanescent field intensity of surface plasmon. Furthermore, emitted light due to reverse irradiation was also observed as a function of the emitted angle in the Kretschmann configuration. The peak angles of the emitted light almost corresponded to the dip angles of the SPS curves and the emitted light was considered to be due to surface plasmon excitation.

An overabundance of X chromosomes in testicular germ cell tumors and the identification of the candidate testicular germ cell tumor susceptibility gene TGCT1 on Xq27 highlight the potential involvement of X chromosomes in testicular germ cell tumor pathogenesis. The current study was designed to shed light on the question whether the multiple X chromosomes in testicular germ cell tumor are active or inactive through a complex mechanism of X chromosomal gain and XIST expression.We analyzed 4 testicular germ cell tumor derived cell lines and 20 primary testicular germ cell tumor tissues. The number of X chromosomes was determined by fluorescence in situ hybridization using the X chromosome specific probe. The expression patterns of XIST and the 3 X-linked genes androgen receptor (AR), fragile X mental retardation (FMR1 ) and Glypican 3 (GPC3 ) were studied by reverse transcriptase-polymerase chain reaction. Bisulfite genomic sequencing was used to analyze the methylation patterns of the AR, FMR1 and GPC3 genes. The relative expression levels of the 2 X-linked proto-oncogenes ARAF1 and ELK1 were assayed by quantitative reverse transcriptase-polymerase chain reaction.XIST expression was common in seminomatous testicular germ cell tumors (2 of 2 or 100% of seminoma derived cell lines and 10 of 12 or 83% of seminomatous testicular germ cell tumor tissues) but not in nonseminomatous testicular germ cell tumors (0 of 2 or 0% nonseminoma derived cell lines and 2 of 8 or 25% of nonseminomatous testicular germ cell tumor tissues). However, X chromosomal gain was consistently observed in the 2 types of tumors. XIST expression in testicular germ cell tumors and normal testicular parenchyma was not associated with methylation of the AR, FMR1 or GPC3 genes. After determining the expression patterns of AR, FMR1 and GPC3 in testicular germ cell tumor samples we concluded that multiple X chromosomes in testicular germ cell tumors were predominantly hypomethylated and active regardless of XIST expression. The biological significance of excess active X chromosomes in testicular germ cell tumors was suggested by enhanced expression of the 2 X-linked oncogenes ARAF1 and ELK1 in the testicular germ cell tumor derived cell lines.The current data may suggest the potential oncogenic implications of X chromosomal gain in testicular germ cell tumors.

Emission light through the prism in the Kretschmann configuration of prism/Ag thin film/rhodamine-B (RB) Langmuir–Blodgett (LB) film has been observed by means of direct irradiation of a laser beam from air to the RB LB film, that is, the reverse irradiation method against the conventional attenuated total reflection (ATR) method. The emission light intensities and the spectra strongly depended upon the emission angle at the prism. The relation between the peak wavelengths of the spectra and the emission angles corresponded to the dispersion property of the resonant excitations of surface plasmons (SPs) in the ATR configuration. It was concluded that the emission light was caused by multiple excitations of SPs in the ATR configuration. Emission light through the prism was also observed below the reflection angle of the resonant one when the SP was resonantly excited in the conventional ATR measurement. This emission light also showed the same property as the one in the reverse irradiation method. It is thought that the phenomenon will be used as a new sensing device.

The family of neurotrophins, encompassing nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5), is important in the regulation of neuronal development and function. We examined the expression of neurotrophin messenger RNAs (mRNAs) in the rat urinary bladder during pre- and postnatal development using competitive reverse transcription-polymerase chain reaction. The mRNA levels showed a biphasic pattern of expression; one peak was at prenatal ages (embryonic day (E)15-E18) and the other peak was at postnatal ages (postnatal day (P)14-P28). NT-4/5, BDNF and NGF mRNA levels were greatest at E15, E16 and E18, respectively. In contrast, NT-3 mRNA levels were significantly highest at P14. These data suggest that neurotrophins are involved in the mechanisms of bladder nerve growth for the prenatal period and reorganization of bladder reflex pathways during the second to the fourth postnatal week.

Little is known about how the prenatal innervation of the urinary bladder develops. The objective of this study was to define the timing and pattern of innervation of the developing rat bladder using growth-associated protein-43 (GAP-43) immunohistochemistry. In addition, we examined the development of two different classes of transmembrane receptor proteins, the Trk family of tyrosine kinases and neurotrophin receptor p75 immunoreactivities. GAP-43-positive nerves were first detected in the bladder at embryonic day (E) 16. They were growing rapidly to the bladder dome and covered the whole bladder by E18. The density of GAP-43-containing nerves increased in the muscle layer by postnatal day (P) 0. Both Trk receptor-positive and p75 neurotrophin receptor-positive fibers were also first seen at E16. The number of Trk immunoreactive nerves reached a peak at E18, and then decreased over the following days. In contrast, p75-labeled fibers were abundant at E18-P14. There were few GAP-43 or neurotrophin receptor-positive fibers in the adult. GAP-43 immunohistochemistry provided us with a picture of innervation in the developing rat bladder. Furthermore, the demonstration of neurotrophin receptors positive fibers in the prenatal and early postnatal bladders suggests that neurotrophins may contribute to the development of the peripheral nervous system in the urinary bladder.

Electroluminescent (EL) devices of metal-free phthalocyanines (H 2 Pc) were fabricated and electrical and luminescent properties due to NO 2 gas adsorption were studied. The device had an ITO bottom electrode (anode)/H 2 Pc (100 nm)/Al top electrode (cathode) structure. The H 2 Pc and the Al films were vacuum evaporated. Especially, the Al films were deposited using an oblique deposition method in order to form porous structure to enable NO 2 gas adsorption into the H 2 Pc layer through the Al film. The conduction currents and the EL intensities of the device increased with NO 2 adsorption. Open-circuit voltage generations due to NO 2 gas adsorption were also observed. It was estimated that the adsorbed NO 2 molecules in H 2 Pc generated holes by capturing negative electrons and charge separation of the negative charged NO 2 and positive holes induced the open circuit voltage. It was concluded that the increase in the conduction currents due to the gas adsorption was caused by the modification of the depletion layer, and that efficient injection of electrons and recombination of carriers produced the improvement in the EL efficiency.

Sequential reaction of substrate (I) with ethynyllithium reagents (II) and Grignard reagents (IV) provides silylated 2,9-diethynylanthracenes (V).

A novel and fundamental method was reported to judge states of lithium ion batteries (LIBs) using the capacitance and the voltage of the cells that were estimated from the real-time currents and voltage characteristics of the cells. We measured the differential capacitance, that is, dQ/dV or delta Q/ delta V that is equal to the currents (I) divided by differential voltages (dV/dt) calculated from the current and the voltage characteristics of the cell during the charging/ discharging, where Q is the charge that flows through the cell, V is the voltage of the cell and t is time. It is thought that the capacitance decrease with the degradation of the cell because the effective area of the electrodes is decreasing due to formation of undesirable compounds. The differential capacitance in some specific voltage range for the LIBs was approximately directly proportional to the state of the degradation of the cell. Therefore, it is concluded that the novel method is very useful to judge the state of the LIBs.

Spectra of emission light from Ag/ Rhodamine-B (RB) LB films due to surface plasmon (SP) excitations have been investigated using an attenuated total reflection (ATR) method and a reversed ATR (RATR) method. In the R-ATR configuration, emission light was observed through the prism, and the intensities and spectra strongly depended upon the emission angle where the light was observed. The relation between the wavelengths and the emission angles agreed with the resonant conditions of excitations of SPs in the ATR configuration. It was concluded that the emission light was caused by excitations of multiple SPs.