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The potential liability to hypercatabolism in lipodystrophy remains to be fully elucidated. Here we report a 28‐year‐old Japanese woman with acquired generalized lipodystrophy, who presented with recurrence of panniculitis and anemia. After corticosteroid treatment was started, she showed rapid reductions in body weight and lean mass by 15% at maximum, accompanied by an elevated urea nitrogen/creatinine ratio, which recovered almost fully as the corticosteroid treatment was tapered and discontinued. She had multiple risk factors for hypercatabolism: lack of metabolic reserves, insulin resistance, and hyperglycemia due to lipodystrophy, lowered daily activity due to anemia, persistent inflammation, and wasting associated with panniculitis, and relatively insufficient energy and protein intake during hospitalization. More attention should be paid to the potential liability to hypercatabolism in patients with lipodystrophy, and to skeletal muscle loss as an adverse effect of corticosteroid treatment in patients at high risk, such as those with diabetes or decreased metabolic reserves. [ABSTRACT FROM AUTHOR]

In this study, intermittent creep‐fatigue tests were applied on Ni‐based Alloy 617 and Alloy 625. Scanning electron microscope (SEM) observation and electron back‐scatter diffraction (EBSD) analysis were employed to the evaluation of the degradation of the crystallinity under the creep‐fatigue loads. It was found that the initial damage under creep‐fatigue load basically appeared as intergranular cracks. In addition, the lifetimes of Alloy 625 samples fluctuated significantly due to the growth of NbC precipitates. The initial damage of these two alloys was dominated by the growth and accumulation of dislocations and vacancies around the interface that consisted of large lattice mismatch. Local atomic diffusion was activated when the summation of the nominal stress and local stress caused by the large lattice mismatch exceeded a critical value. The stress‐induced acceleration of the degradation of the crystallinity of the alloys was analyzed by applying the modified Arrhenius equation. Instead of the most popular inversion methods according to the final failure mode, the quantitative description in life prediction was developed based on the dynamic progress for acceleration mechanism of the degradation. It is of importance to perfecting the frontiers of damage mechanics approach. Highlights: The degradation mechanism was obtained by analyzing the damage behavior of two alloys.IQ value was used to characterize the degree of degradation under creep‐fatigue load.The degree of degradation and life assessment was expressed quantitatively. [ABSTRACT FROM AUTHOR]

Excess deposition of extracellular matrix in the myocardium is a predictor of reduced left ventricular function. Although reducing the hemodynamic load is known to improve myocardial fibrosis, the mechanisms underlying the reversal of the fibrosis have not been elucidated. We focused on the elasticity of myocardial tissue, which is assumed to influence the fibroblast phenotype. Normal and fibrotic myocardium were cultured in 16 kPa and 64 kPa silicone gel‐coated dishes supplemented with recombinant TGFβ protein, respectively. Matrix‐degrading myocardium was cultured in 64 kPa silicone gel‐coated dishes with recombinant TGFβ protein and then in 16 kPa silicone gel‐coated dishes. Cardiac fibroblasts were cultured in this three‐part in vitro pathological models and compared. Fibroblasts differentiated into activated or matrix‐degrading types in response to the pericellular environment. Comprehensive gene expression analysis of fibroblasts in each in vitro condition showed Selenbp1 to be one of the genes responsible for regulating differentiation of fibroblasts. In vitro knockdown of Selenbp1 enhanced fibroblast activation and inhibited conversion to the matrix‐degrading form. In vivo knockdown of Selenbp1 resulted in structural changes in the left ventricle associated with progressive tissue fibrosis and left ventricular diastolic failure. Selenbp1 is involved in regulating fibroblast differentiation and appears to be one of the major molecules regulating collagen turnover in cardiac fibrosis. [ABSTRACT FROM AUTHOR]

Background: Despite evidence for the role of human leukocyte antigen (HLA) in the genetic predisposition to Parkinson's disease (PD), the complex haplotype structure and highly polymorphic feature of the major histocompatibility complex (MHC) region has hampered a unified insight on the genetic risk of PD. In addition, a majority of the reports focused on Europeans, lacking evidence on other populations. Objectives: The aim of this study is to elucidate the genetic features of the MHC region associated with PD risk in trans‐ethnic cohorts. Methods: We conducted trans‐ethnic fine‐mapping of the MHC region for European populations (14,650 cases and 1,288,625 controls) and East Asian populations (7712 cases and 27,372 controls). We adopted a hybrid fine‐mapping approach including both HLA genotype imputation of genome‐wide association study (GWAS) data and direct imputation of HLA variant risk from the GWAS summary statistics. Results: Through trans‐ethnic MHC fine‐mapping, we identified the strongest associations at amino acid position 13 of HLA‐DRβ1 (P = 6.0 × 10−15), which explains the majority of the risk in HLA‐DRB1. In silico prediction revealed that HLA‐DRB1 alleles with histidine at amino acid position 13 (His13) had significantly weaker binding affinity to an α‐synuclein epitope than other alleles (P = 9.6 × 10−4). Stepwise conditional analysis suggested additional independent associations at Ala69 in HLA‐B (P = 1.0 × 10−7). A subanalysis in Europeans suggested additional independent associations at non‐HLA genes in the class III MHC region (EHMT2; P = 2.5 × 10−7). Conclusions: Our study highlights the shared and distinct genetic features of the MHC region in patients with PD across ethnicities. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society [ABSTRACT FROM AUTHOR]

Founder animals carrying high proportions of somatic mutation induced by CRISPR–Cas9 enable a rapid and scalable strategy for the functional screening of numerous target genes in vivo. In this functional screening, genotyping using pooled amplicons with next‐generation sequencing is the most suitable approach for large‐scale management of multiple samples and accurate evaluation of the efficiency of Cas9‐induced somatic mutations at target sites. Here, we present a simple workflow for genotyping of multiple CRISPR–Cas9‐based knockout founders by pooled amplicon sequencing. Using custom barcoded primers, pooled amplicons from multiple individuals can be run in a single‐indexed library on the Illumina MiSeq platform. Additionally, a user‐friendly web tool, CLiCKAR, is available to simultaneously perform demultiplexing of pooled sequence data and evaluation of somatic mutation in each phenotype. CLiCKAR provides users with practical reports regarding the positions of insertions/deletions, as well as the frameshift ratio and tables containing mutation sequences, and read counts of each phenotype, with just a few clicks by the implementation of demultiplexing for pooled sample data and calculation of the frameshift ratio. This genotyping workflow can be harnessed to evaluate genotype–phenotype correlations in CRISPR–Cas9‐based loss‐of‐function screening of numerous target genes in various organisms. [ABSTRACT FROM AUTHOR]

Aim: To elucidate the incidences and circumstances of falls and fall‐related injuries, and to explore the physical characteristics of community‐dwelling ambulatory stroke survivors who experienced falls. Methods: A total of 144 community‐dwelling ambulatory survivors of hemiparetic stroke (mean age 68.0 years [SD 10.4 years]) who were undergoing rehabilitation in an adult daycare center participated in this prospective study. The mean duration from stroke onset was 5.21 years (SD 3.15 years). The occurrence of falls was collected for 1 year with a fall diary. The incidence rates of falls and fall‐related injuries, and the detailed circumstances of falls were descriptively analyzed. The characteristics of fallers were explored by comparing background information, motor impairments and results of physical function tests, including the 10‐m walk test, Timed Up and Go test and five‐times‐sit‐to‐stand test, between fallers and non‐fallers. Results: The incidence rates of falls and fall‐related fractures were 0.88 per person‐year and 2.8 per 100 person‐years, respectively. Falls occurred more frequently during daytime and in winter. Falls were caused most often by losing balance while walking indoors, especially on the way to the toilet. After falling, 34.1% of individuals who fell could not stand up by themselves. The time of the five‐times‐sit‐to‐stand test was significantly longer in fallers than in non‐fallers (P < 0.05). Conclusions: The incidence rate of falls was high among community‐dwelling ambulatory survivors of hemiparetic stroke. Appropriate approaches, including mastering the skills to cope with falling, are required, especially for individuals with reduced lower limb muscle strength. Geriatr Gerontol Int 2019; 19: 240–244. [ABSTRACT FROM AUTHOR]

Abstract: Background: Telomeres are known to provide genomic stability and telomere length has been associated with cardiovascular diseases. Moreover, a higher telomerase activity has been shown to be associated with ventricular arrhythmias (VA) in ischemic cardiomyopathy. Increasing evidence suggests that genetic variation in key telomere genes has an impact on telomerase activity. Each copy of the minor allele of SNP rs12696304, at a locus including TERC (telomerase), has been associated with ∼75 base pairs reduction in mean telomere length likely mediated by an effect on TERC expression. We investigated the impact of genetic variation of this SNP on telomerase and its association with VA in ischemic cardiomyopathy patients. Methods and results: Ninety ischemic cardiomyopathy patients with primary prevention implantable cardioverter defibrillators (ICDs) were recruited. Thirty‐five received appropriate ICD therapy for potentially fatal VA (cases), while the remaining 55 patients did not (controls). No significant differences in baseline demographics were seen between the groups. TS was measured by qPCR, telomerase activity by TRAP assay, and SNP genotyping with Taqman probes. Telomerase was highest in C homozygous allele and had a significant association with VA in this group only (C/C,C/G,G/G; P‐value 0.04, 0.33, 0.43). Conclusion: The present study is the first to examine the association between telomerase, a SNP at a locus including TERC, and VA in ischemic cardiomyopathy patients. Homozygosity for C‐allele significantly effects telomerase expression and its association with VA in this cohort. Large‐scale prospective studies are required to determine if this genetic variation predisposes patients to greater arrhythmic tendency post‐MI. [ABSTRACT FROM AUTHOR]

Recent advances in genome editing using programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, have facilitated reverse genetics in Xenopus tropicalis. To establish a practical workflow for analyzing genes of interest using CRISPR-Cas9, we examined various experimental procedures and conditions. We first compared the efficiency of gene disruption between Cas9 protein and mRNA injection by analyzing genotype and phenotype frequency, and toxicity. Injection of X. tropicalis embryos with Cas9 mRNA resulted in high gene-disrupting efficiency comparable with that produced by Cas9 protein injection. To exactly evaluate the somatic mutation rates of on-target sites, amplicon sequencing and restriction fragment length polymorphism analysis using a restriction enzyme or recombinant Cas9 were performed. Mutation rates of two target genes (slc45a2 and ltk) required for pigmentation were estimated to be over 90% by both methods in animals exhibiting severe phenotypes, suggesting that targeted somatic mutations were biallelically introduced in almost all somatic cells of founder animals. Using a heteroduplex mobility assay, we also showed that off-target mutations were induced at a low rate. Based on our results, we propose a CRISPR-Cas9-mediated gene disruption workflow for a rapid and efficient analysis of gene function using X. tropicalis founders. [ABSTRACT FROM AUTHOR]

Left-right asymmetry of bilaterian animals is established during early development. In mice, frogs and fishes, the ciliated left-right organizer plays an essential role in establishing bilateral asymmetry, and leftward flow of extracellular fluid generated by ciliary motion results in Nodal activity on the left side. However, H+/K+- ATPase activity is also involved in the determination of left-right asymmetry in a variety of animals, and it has been thought to be an ancestral mechanism in deuterostomes. In sea urchin, the determination of the left-right asymmetry based on H+/K+- ATPase activity was already clarified, but it remains to be uncovered whether ciliary motion is involved in the left-right asymmetry of the embryo. Here, we show evidence that ciliary motion is involved in the establishment of left-right asymmetry of sea urchin embryo. Furthermore, we show that the initial cilia generated on small micromeres during the early stage of embryogenesis may be involved in this process. These results suggest that the cilia-mediated mechanism for the determination of left-right asymmetry may be acquired at the base of the deuterostomes. [ABSTRACT FROM AUTHOR]

Xenopus laevis tadpoles can completely regenerate their appendages, such as tail and limbs, and therefore provide a unique model to decipher the molecular mechanisms of organ regeneration in vertebrates. Epigenetic modifications are likely to be involved in this remarkable regeneration capacity, but they remain largely unknown. To examine the involvement of histone modification during organ regeneration, we generated transgenic X. laevis ubiquitously expressing a fluorescent modification-specific intracellular antibody (Mintbody) that is able to track histone H3 lysine 9 acetylation (H3K9ac) in vivo through nuclear enhanced green fluorescent protein (EGFP) fluorescence. In embryos ubiquitously expressing H3K9ac-Mintbody, robust fluorescence was observed in the nuclei of somites. Interestingly, H3K9ac-Mintbody signals predominantly accumulated in nuclei of regenerating notochord at 24 h postamputation following activation of reactive oxygen species ( ROS). Moreover, apocynin (APO), an inhibitor of ROS production, attenuated H3K9ac-Mintbody signals in regenerating notochord. Our results suggest that ROS production is involved in acetylation of H3K9 in regenerating notochord at the onset of tail regeneration. We also show this transgenic Xenopus to be a useful tool to investigate epigenetic modification, not only in organogenesis but also in organ regeneration. [ABSTRACT FROM AUTHOR]

Background: Embryonic stem (ES) cells are pluripotent cells with the ability to differentiate to any cell type of the resident organism. In recent years, significant advances have been made in using these cells to obtain large numbers of cardiomyocyte (CM) -like cells for scientific research and clinical application. A vast number of protocols have emerged describing differentiation methods without the use of animal serum or extracts restrictive for use in a human clinical setting. These techniques follow a complicated procedure, which although successful, show a relatively varied yield among cell batches. Results: We have designed a three-step differentiation protocol using defined reagents and a monolayer culture without feeder cells, avoiding embryoid body formation and multiple trypsin treatment, in which beating foci appeared as early as day 6 in in vitro differentiating conditions. Our results show a high yield of CM reaching approximately 60% of the differentiated cells after 13 days in vitro. Conclusions: We provide a fast, simple, reliable and reproducible protocol for inducing murine ES cells toward a CM-like phenotype comparable to available high-yield protocols, without the use of intermediate trypsinization/passage steps. Developmental Dynamics 245:157-165, 2016. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: After chemotherapy for metastatic testicular tumors, masses may remain, often in the metastatic sites. This study analyses the role of resections for the residual masses. Methods: Seventy-seven patients with advanced (stage II, III) testicular tumors were treated. Of these, 38 patients, including eight with seminoma and 30 patients with non-seminomatous germ cell tumors, underwent resection of residual masses after chemotherapy and have been followed for a median of 41.5 months (range 2–138) after the resection. Results: Residual masses were necrosis/fibrosis in 19 patients, mature teratoma in 11 and cancer in eight. The ratio of cancer in stage III (41.2%) was significantly higher than that in stage II (4.8%). Ten of 38 (26.3%) patients experienced recurrences in sites other than the resected sites, and five of 10 patients have died of cancer. Most recurrences (80%) occurred within two years. Recurrences after resection were detected in 4.8% of stage II patients, 52.9% of stage III, 16.7% of necrosis/fibrosis and mature teratoma, and 62.5% of cancer. The survival rate of patients with cancer was significantly lower in spite of adjuvant chemotherapy after surgery. Conclusions: Resection for residual masses after chemotherapy in metastatic testicular tumors was useful in confirming the tissue and in controlling the metastatic sites. Recurrences were often found in patients with cancer in the residual mass and the prognosis of patients with cancer was poor, therefore the development of more effective therapy for patients with cancer is required to improve the prognosis. [ABSTRACT FROM AUTHOR]

Biaxial melt-drawing produces a high-performance poly(tetrafluoroethylene) (PTFE) membrane with excellent mechanical properties and superior transparency. Unique two-step molding gives the better adhesion between the PTFE powder materials even with ultra-high molecular weight of 107. Such void-free film can be successfully biaxially drawn in the molten state. The resultant membrane exhibits high tensile strength of 120MPa and high transparency of 8.5% haze. [ABSTRACT FROM AUTHOR]

Toll-like receptor 9 ( TLR9) has a key role in the recognition of pathogen DNA in the context of infection and cellular DNA that is released from damaged cells. Pro-inflammatory TLR9 signalling pathways in immune cells have been well investigated, but we have recently discovered an alternative pathway in which TLR9 temporarily reduces energy substrates to induce cellular protection from stress in cardiomyocytes and neurons. However, the mechanism by which TLR9 stimulation reduces energy substrates remained unknown. Here, we identify the calcium-transporting ATPase, SERCA2 (also known as Atp2a2), as a key molecule for the alternative TLR9 signalling pathway. TLR9 stimulation reduces SERCA2 activity, modulating Ca2+ handling between the SR/ ER and mitochondria, which leads to a decrease in mitochondrial ATP levels and the activation of cellular protective machinery. These findings reveal how distinct innate responses can be elicited in immune and non-immune cells-including cardiomyocytes-using the same ligand-receptor system. [ABSTRACT FROM AUTHOR]

Transcription activator-like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X. laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues ( tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40-80%. Next, we show early introduction of TALEN-mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X. laevis. [ABSTRACT FROM AUTHOR]

Transcription activator-like effector nucleases (TALENs) have recently arisen as effective tools for targeted genome engineering. Here, we report streamlined methods for the construction and evaluation of TALENs based on the 'Golden Gate TALEN and TAL Effector Kit' (Addgene). We diminished array vector requirements and increased assembly rates using six-module concatemerization. We altered the architecture of the native TALEN protein to increase nuclease activity and replaced the final destination vector with a mammalian expression/ in vitro transcription vector bearing both CMV and T7 promoters. Using our methods, the whole process, from initiating construction to completing evaluation directly in mammalian cells, requires only 1 week. Furthermore, TALENs constructed in this manner may be directly applied to transfection of cultured cells or mRNA synthesis for use in animals and embryos. In this article, we show genomic modification of HEK293T cells, human induced pluripotent stem cells, Drosophila melanogaster, Danio rerio and Xenopus laevis, using custom-made TALENs constructed and evaluated with our protocol. Our methods are more time efficient compared with conventional yeast-based evaluation methods and provide a more accessible and effective protocol for the application of TALENs in various model organisms. [ABSTRACT FROM AUTHOR]

Background: Deep sequencing of single cell-derived cDNAs offers novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA-seq analysis requires multiple steps with consequent sample loss and stochastic variation at each step significantly affecting output. Thus, a simpler and better protocol is desirable. The recently developed hyperactive Tn5-mediated library preparation, which brings high quality libraries, is likely one of the solutions. Results and Conclusions: Here, we tested the applicability of hyperactive Tn5-mediated library preparation to deep sequencing of single cell cDNA, optimized the protocol, and compared it with the conventional method based on sonication. This new technique does not require any expensive or special equipment, which secures wider availability. A library was constructed from only 100 ng of cDNA, which enables the saving of precious specimens. Only a few steps of robust enzymatic reaction resulted in saved time, enabling more specimens to be prepared at once, and with a more reproducible size distribution among the different specimens. The obtained RNA-seq results were comparable to the conventional method. Thus, this Tn5-mediated preparation is applicable for anyone who aims to carry out deep sequencing for single cell cDNAs. Developmental Dynamics 241:1584-1590, 2012. © 2012 Wiley Periodicals Inc. [ABSTRACT FROM AUTHOR]

Background: Measurement of urinary sulfated bile acid (USBA) is a non-invasive method to detect bile congestion. Our aim was to evaluate the feasibility of USBA analysis for the early detection of biliary atresia (BA). Methods: We determined the USBA-to-creatinine ratio (USBA/cr) in 1148 infants at 10-40 days after birth. All infants were followed until the 3- to 4-month postnatal routine health check. The cutoff value for USBA/cr was 55.0 µmol/g creatinine. Results: Among the infants tested, 47 (4.10%) had USBA/cr ratios that exceeded the cutoff value. Two of these 47 infants had liver disease; one was diagnosed with neonatal hepatitis syndrome, and the other was diagnosed with BA. The BA patient underwent USBA analysis for the first time on day 18 after birth and hepatoportoenterostomy on day 49. No other infants were diagnosed with hepatobiliary disease during the follow-up period. Conclusion: This USBA analysis provided the correct assessment without fail and identified a case of BA. This approach could be used for the screening and early detection of BA when the false-positive rate is decreased by improving the methods for sample collection and urine storage. [ABSTRACT FROM AUTHOR]

We investigated the characteristics of a novel type I keratin gene in Xenopus laevis during ontogenesis. The transcript was first detected in the posterior region at the late neurula stage, and then restricted to the fin and external gill during embryogenesis. To examine the transcriptional regulation of the keratin gene in vivo, we generated transgenic lines with fluorescent reporter genes driven by its 4.2-kb upstream sequence. The promoter/enhancer activity recapitulated the endogenous gene expression during embryogenesis. Sequential deletion analyses revealed that the regions proximal to the promoter were essential for fin-specific expression. Reporter expression was detected in various organs, including the fin and gill. In particular, robust expression was observed in the developing limbs and gill. The reporter fluorescence rapidly decreased with internal gill resorption during metamorphosis. The transgenic lines carrying the promoter/enhancer should represent valuable tools for elucidating the formation, development and resorption of various organs, especially the gill. Developmental Dynamics 239:3172-3181, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Amphibian body skin provides an opportunity to investigate the molecular mechanism of thyroid hormone (TH)-dependent organ remodeling during metamorphosis. Global gene expression changes in the TH-dependent body skin remodeling were studied with microarray analysis. We identified 401 genes that were differentially expressed more than fourfold for 7 days after TH-treatment. As expected, larval- and adult-type keratin genes were significantly inactivated and activated, respectively. The expression changes of the Gene Ontology annotated genes demonstrated significant correlation with the morphological and physiological changes in body skin metamorphosis. The ‘transcription and proteolysis’ category genes were first upregulated 1 day after TH-treatment. Subsequently, the ‘cell cycle’ category genes were activated at 3 days. The ‘defense response’ and ‘immune response’ category genes were the late TH-response genes, which were downregulated and upregulated at 5 and 7 days, respectively. From these genes, adult-type keratin-c (xak-c) gene was selected as a suitable gene to visually monitor the emergence of adult-type epidermal cells during skin remodeling, because the gene is specifically expressed in adult epidermal basal cells. We generated enhanced green fluorescent protein (EGFP)-transgenic Xenopus laevis driven by the promoter of xak-c gene. The keratin promoter faithfully expressed the EGFP gene in adult-type basal cells. Spatial and temporal EGFP-fluorescence patterns of filial 1 (F1)-offspring tadpoles visually demonstrated an event of sequential replacement of larval keratinocytes with the newly generated adult counterparts. [ABSTRACT FROM AUTHOR]

Thrombopoietin (TPO) is the principal physiologic regulator of platelet production. We have searched for small molecule compounds that mimic the action of TPO by using human TPO receptor-expressed in Ba/F3 cells, resulting in the discovery of AKR-501 (YM477). AKR-501 specifically targeted the TPO receptor and stimulated megakaryocytopoiesis throughout the development and maturation of megakaryocytes just as rhTPO did. AKR-501, however, was shown to be effective only in humans and chimpanzees with high species specificity. Therefore, we examined the in vivo platelet-increasing effect of AKR-501 in human platelet producing non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice transplanted with human fetal liver CD34+ cells. Daily oral administration of AKR-501 dose-dependently increased the number of human platelets in these mice, with significance achieved at doses of 1 mg/kg and above. The peak unbound plasma concentrations of AKR-501 after administration at 1 mg/kg in NOD/SCID mice were similar to those observed following administration of an active oral dose in human subjects. These results suggest that AKR-501 is an orally-active TPO receptor agonist that may be useful in the treatment of patients with thrombocytopenia. [ABSTRACT FROM AUTHOR]

Embryonic stem (ES) cells are thought to have unique chromatin structures responsible for their capacity for self-renewal and pluripotency. To examine this possibility, we sought nuclear proteins in mouse ES cells that specifically bind to histones using a pull-down assay with synthetic peptides of histone H3 and H4 tail domain as baits. Nuclear proteins preferentially bound to the latter. We identified 45 proteins associated with the histone H4 tail and grouped them into four categories: 10 chromatin remodeling proteins, five histone chaperones, two histone modification-related proteins, and 28 other proteins. mRNA expression levels of 20 proteins selected from these 45 proteins were compared between undifferentiated and retinoic acid (RA)-induced differentiated ES cells. All of the genes were similarly expressed in both states of ES cells, except nucleoplasmin 3 (NPM3) that was expressed at a higher level in the undifferentiated cells. NPM3 proteins were localized in the nucleoli and nuclei of the cells and expression was decreased during RA-induced differentiation. When transfected with NPM3 gene, ES cells significantly increased their proliferation compared with control cells. The present study strongly suggests that NPM3 is a chromatin remodeling protein responsible for the unique chromatin structure and replicative capacity of ES cells. [ABSTRACT FROM AUTHOR]

Background: The rates of acquisition and spontaneous eradication of Helicobacter pylori infection in children has yet to be established. To determine these rates in children living in an urban region of Japan, the levels of urine H. pylori antibodies in children of three different age groups were measured. Methods: A urine-based enzyme-linked immunosorbent assay (ELISA) was used to detect H. pylori antibodies twice within a 12 month interval over 2 years in 452 healthy children living in Tokyo. The subjects were divided into three groups: ages 4, 7, and 10 years. Results: The prevalence of H. pylori infection was not different among the groups, being between 4.0% and 6.7%. The rate of turn to positivity for H. pylori infection was 1.5% per year and the rate of turn to negativity was 1.1%, but in the 10 year age group the rates were markedly lower than in the younger children. Conclusion: The prevalence of H. pylori infection in Tokyo was 4.0–6.7% and was not different among 4, 7, and 10 year age groups. [ABSTRACT FROM AUTHOR]

The thyroid hormone receptor (THR) is a member of the nuclear transcription factor and plays a central role in regulating anuran metamorphosis. Previous studies with mammalian cells have suggested that THR is involved in chromatin remodeling through histone methylation. In the present study, we cloned cDNA of lysine-specific demethylase gene, xLSD1, from Xenopus laevis and examined its expression in relation to metamorphosis. Overexpression of xLSD1 in A6 cells, a Xenopus laevis cell line, resulted in the decrease of methylation status of lysine residues of histone H3, indicating that the protein of cloned xLSD1 was functionally active. The expression of LSD1 at mRNA levels was up-regulated in the body skin and the intestine during natural and thyroid hormone-induced metamorphosis. Larval epidermal basal cells and intestinal epithelial cells at the premetamorphic stage were identified as the xLSD1-expressing cells. At the metamorphic climax stage the progenitor cells of adult epidermal basal cells also expressed xLSD1, whereas those of the adult intestinal epithelial cells did not. We propose that LSD1 participates in the regulation of metamorphosis through THR- or another transcriptional factor-induced chromatin remodeling. [ABSTRACT FROM AUTHOR]

A suitable model for the preclinical study of human platelet production in vivo has not been available. NOD/SCID mice were characterized as representing an efficient engraftment model for human hematopoietic stem cells, which resulted in the production of human platelets. Here, we evaluated in vivo human thrombopoiesis and ex vivo human platelet functions in NOD/SCID mice transplanted with human cord blood (CB) CD34+ cells. Human platelets and human CD45+ cells appeared in peripheral blood of NOD/SCID mice from 4 wk after transplantation. Human platelets produced in these mice showed CD62P expression and the activation of GPIIb/IIIa on human platelets on stimulation with an agonist. PEG-rHuMGDF (0, 0.5 and 5 μg/kg/d s.c.) was injected for 14 d into mice that had been confirmed to produce human platelets stably. The number of human platelets increased about twofold at 0.5 μg/kg/d and about fivefold at 5 μg/kg/d after 14 d. Withdrawal of PEG-rHuMGDF administration caused the human platelet count to return to the pretreatment level. Further, re-administration of PEG-rHuMGDF induced a similar human thrombopoietic response as it did on initial administration. These results suggest that NOD/SCID mice engrafted with human CB CD34+ cells will be useful for the study of human platelet production in vivo. [ABSTRACT FROM AUTHOR]

A complete spontaneous regression of diffuse large B cell lymphoma involving the right breast, confirmed by aspiration cytology, is reported. The patient visited a hospital due to the rapid growth of a tumor in the right breast. Five years previously she underwent a craniotomy for a brain tumor, diagnosed as B-cell malignant lymphoma, and received several courses of irradiation to the brain. Analysis of the breast tumor cells obtained by aspiration revealed lymphoma cells morphologically, which were similar to the tumor cells in the brain expressing CD20. While waiting for further examination, the tumor regressed rapidly and was not palpable after 20 days. An excisional biopsy of the breast exhibited no definite malignant lymphoma cells among a diffuse population of CD45RO and CD8-positive small lymphocytes. Nucleotide sequencing of HCDR3s of the brain tumor and breast tumor cells showed a completely matched sequence, revealing the breast mass to be a metastatic lesion from the tumor of the brain. Although there was no tumorous lesion, the patient received additional chemotherapy and has shown no sign of recurrence in the breast for 7 years. We were able to confirm that the breast lymphoma shown in the aspiration cytology was a metastatic one, which was not proven histologically prior to chemotherapy, and regard the present case as a malignant lymphoma of the breast showing spontaneous regression. The present case shows a rare occurrence of spontaneous regression of diffuse large B cell malignant lymphoma after aspiration and suggests that CD8-positive T cells might be related to the regression. [ABSTRACT FROM AUTHOR]

Anuran larval skin undergoes a process of metamorphosis into preadult and adult skin. Basal skein, larval basal and adult basal cells are basement membrane-attaching cells in the larval, preadult and adult epidermis, respectively, and are identified as cells expressing genes of RLK ( Rana larval keratin), both RLK and RAK ( Rana adult keratin), and RAK. Larval to preadult skin conversion takes place in the histological entity called the skin transformation center (STC). The present study performed a cDNA subtractive gene screening on cDNA of the larval and the preadult skin, and cloned the secreted protein acidic and rich in cysteine (SPARC) gene as an upregulated gene in the larva to preadult skin conversion. RAK gene-positive basal skein cells and fibroblasts in and around the STC were weakly and strongly sparc-positive, respectively. Using sparc and rak, we redefined the STC and visualized it on a histological section as an approximately 150 µm-long region that contained about 20 rak-negative and weakly sparc-positive basal cells. Intense sparc expression was observed in basal skein cells, but not in larval basal cells, suggesting that SPARC acts as a suppressor of rak during epidermal differentiation. This suggestion was tested by investigating the effect of SPARC on cultured larval basal cells. We observed that SPARC suppressed the expression of rak, but not rlk. [ABSTRACT FROM AUTHOR]

A cellulase [endo-β-1,4-d-glucanase (EC 3.2.1.4)] was isolated from the hepatopancreas of abalone Haliotis discus hannai by successive chromatographies on TOYOPEARL CM-650M, hydroxyapatite and Sephacryl S-200 HR. The molecular mass of the cellulase was estimated to be 66 000 Da by SDS/PAGE, thus the enzyme was named HdEG66. The hydrolytic activity of HdEG66 toward carboxymethylcellulose showed optimal temperature and pH at 38 °C and 6.3, respectively. cDNAs encoding HdEG66 were amplified by the polymerase chain reaction from an abalone hepatopancreas cDNA library with primers synthesized on the basis of partial amino-acid sequences of HdEG66. By overlapping the nucleotide sequences of the cDNAs, a sequence of 1898 bp in total was determined. The coding region of 1785 bp located at nucleotide position 56–1840 gave an amino-acid sequence of 594 residues including the initiation methionine. The N-terminal region of 14 residues in the deduced sequence was regarded as the signal peptide as it was absent in HdEG66 protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Thus, matured HdEG66 was thought to consist of 579 residues. The C-terminal region of 453 residues in HdEG66, i.e. approximately the C–terminal three quarters of the protein, showed 42–44% identity to the catalytic domains of glycoside hydrolase family 9 (GHF9)-cellulases from arthropods and Thermomonospora fusca . While the N-terminal first quarter of HdEG66 showed 27% identity to the carbohydrate-binding module (CBM) of a Cellulomonas fimi cellulase, CenA. Thus, the HdEG66 was regarded as the GHF9-cellulase possessing a family II CBM in the N-terminal region. By genomic PCR using specific primers to the 3′-terminal coding sequences of HdEG66-cDNA, a DNA of 2186 bp including three introns was amplified. This strongly suggests that the origin of HdEG66 is not from symbiotic bacteria but abalone itself. [ABSTRACT FROM AUTHOR]

AbstractBackground: Simultaneous determinations of human chorionic gonadotropin hormone (hCG) and hCG-β frequently produce discrepancies, that is when hCG or hCG-β is normal, the other is elevated. Accordingly, we examined the significance of simultaneous determination of serum hCG and hCG-β in testicular tumors. Methods: Simultaneous determination of hCG and hCG-β was performed in 54 patients with testicular seminoma and 74 with non-seminomatous testicular tumors. Results: For detection of seminoma patients, hCG-β was more effective than hCG because hCG-β was positive in 83% (45/54) of the patients and hCG was positive in 50% (27/54). In non-seminomatous testicular tumor cases, hCG-β was positive in 74% (55/74) and hCG was positive in 82% (61/74). The cases of hCG <1.0 mIU/mL and HCG-β > 0.1 ng/mL were significantly more frequently seen in patients with seminoma than in those with non-seminomatous testicular tumor (P < 0.001). Fourteen patients had recurrent tumor. At recurrence, only hCG was elevated in nine cases, only hCG-β was elevated in two cases and both in one case. For diagnosis of falsely positive hCG, testosterone administration was effective because after testosterone administration, serum hCG levels became undetectable (< 1.0 mIU/mL) within one week in three examined cases. Conclusion: Human chorionic gonadotropin-β was a better marker of seminoma than hCG. For earlier detection of recurrence, both markers should be examined. For diagnosis of falsely positive hCG, testosterone administration was effective. [ABSTRACT FROM AUTHOR]

This article proposes the TLB-unified cache, which is one of the indirect tagged cache implementation methods. In the indirect tagged cache, the cache tag functions as a pointer to another address. It requires less hardware than the conventional method. In order to maintain the consistency between the indirect tag and the cache in the indirect tagged cache, however, there must be a high-speed selective cache invalidating mechanism. From such a viewpoint, this article proposes time stamp invalidation as one of the invalidating mechanisms. We present an implementation of time stamp invalidation for the TLB-unified cache, where the TLB and the cache share a tag. As the next step, the amount of hardware resources that can be saved by using the indirect tag is evaluated. It is then shown that the saved hardware resources can be transferred to other on-chip units in order to improve their performance. Lastly, the performance of the TLB-unified cache is evaluated by trace-driven simulation, and it is shown that the performance can be improved with less hardware complexity than the conventional method. © 1999 Scripta Technica, Syst Comp Jpn, 30(11): 94–106, 1999 [ABSTRACT FROM AUTHOR]

Abstract Purpose: Reverse transcriptase–polymerase chain reaction (RT–PCR) offers a potentially more sensitive assay for detecting cells expressing prostate-specific antigen (PSA) mRNA in peripheral circulation. But the sensitivity and specificity are variable depending on the position of the PSA amplification. To increase sensitivity and specificity, the whole PSA cDNA (1466 bp) was separated into eight different parts. Methods: We examined RT–PCR on 12 urogenital cell lines, including three prostate cancer (LNCaP, PC3, DU145), five human renal cell carcinoma (SMKT-R3, TOS-1, TOS-2, R4, ACHN), two urinary bladder cancer (YTS-1, KK-47) and two testicular cancer (NEC8, NEC14) cell lines. The sizes of the eight fragmented PSA used in the experiment were PSA-1 (1–257 bp), PSA-2 (1–322 bp), PSA-3 ( 172–507 bp), PSA-4 (172–851 bp), PSA-5 (595–1347 bp), PSA-6 (682 967 bp), PSA-7 (682–1347 bp) and PSA-8 (863–1466 bp). Results: All cell lines had positive signals from PSA-6, PSA-7 and PSA-8. The positive signals from PSA-1, PSA-2 and PSA-3 were detected in some other cell lines in addition to the three prostate cancer cell lines. Only LNCaP which produces the PSA protein had a positive signal from PSA-5. PC3 and DU145 (which do not produce PSA) and LNCaP had a positive signal from PSA-4. Therefore, the inner primer PSA-4′ (578–782 bp) used to increase sensitivity and specificity. Nested RT–PCR on the 12 cell lines, using the PSA-4 and 4′ primers, detected more clear bands in the three prostate cancer cells. Conclusion: Nested RT–PCR using PSA-4 (outer primer) and PSA-4′ (inner primer) may be useful for detecting prostate cancer cells in the peripheral blood. [ABSTRACT FROM AUTHOR]

AbstractBackground and Methods: Bipedal lymphography and percutaneous fine needle aspiration biopsy (FNAB) of pelvic lymph node was done in 200 patients with bladder cancer. Results: A diagnosis of metastasis to the pelvic lymph node was obtained by this method in 34 patients. Of these 34 patients, only 12 (35%) were positive or suspected of having pelvic lymph node metastasis by computed tomography. Sixteen patients (47%) had unequivocally positive or highly suspicious lymphogram and 18 patients (53%) had normal lymphogram. Seventy-eight cases, including eight FNAB-positive cases, were treated by radical cystectomy and regional lymph node dissection. Sensitivity, specificity, positive predictive value and negative predictive value of FNAB were 57, 100, 100 and 91%, respectively. Thirteen FNAB-positive M0 patients could be treated by multimodality therapy and evaluated after neoadjuvant therapy by repeated lymphography and FNAB. All were changed to N0 by FNAB after neoadjuvant therapy. In eight patients treated by total cystectomy and lymph node dissection, four had down staged to pN0 but four had one positive node. In five follow-up cases without surgery, one had recurrent pelvic lymph node metastasis 35 months later. Cause-specific survival of these patients was 76% at 5 years and 57% at 8 years. Conclusions: Pretreatment diagnosis of pelvic lymph node metastasis and multimodality therapy may improve the prognosis of regional lymph node-positive bladder cancer. Because it is difficult to diagnose pN1 using FNAB, radical operation should be performed even if neoadjuvant therapy is effective and down staging is obtained. [ABSTRACT FROM AUTHOR]

Examines the application of neobladder after neoadjuvant therapy and total cystectomy as a treatment for invasive bladder cancer. Application of whole-layer core biopsy (WLCB) of the bladder tumor; Presentation of the indications for total cystectomy and urinary diversion; Evaluation of muscle invasion in invasive bladder cancer.