Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Company of Biologists  

<title>ABSTRACT</title>
Heterochromatin-related epigenetic mechanisms, such as DNA methylation, facilitate pairing of homologous chromosomes during the meiotic prophase of mammalian spermatogenesis. In pro-spermatogonia, de novo DNA methylation plays a key role in completing meiotic prophase and initiating meiotic division. However, the role of maintenance DNA methylation in the regulation of meiosis, especially in the adult, is not well understood. Here, we reveal that NP95 (also known as UHRF1) and DNMT1 – two essential proteins for maintenance DNA methylation – are co-expressed in spermatogonia and are necessary for meiosis in male germ cells. We find that Np95- or Dnmt1-deficient spermatocytes exhibit spermatogenic defects characterized by synaptic failure during meiotic prophase. In addition, assembly of pericentric heterochromatin clusters in early meiotic prophase, a phenomenon that is required for subsequent pairing of homologous chromosomes, is disrupted in both mutants. Based on these observations, we propose that DNA methylation, established in pre-meiotic spermatogonia, regulates synapsis of homologous chromosomes and, in turn, quality control of male germ cells. Maintenance DNA methylation, therefore, plays a role in ensuring faithful transmission of both genetic and epigenetic information to offspring.

DOI： 10.1242/dev.194605

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

During spermatogenesis, intricate gene expression is coordinately regulated by epigenetic modifiers, which are required for differentiation of spermatogonial stem cells (SSCs) contained among undifferentiated spermatogonia. We previously found that KMT2B conveys H3K4me3 at bivalent and monovalent promoters in undifferentiated spermatogonia. Because these genes are expressed late in spermatogenesis or during embryogenesis, we expect that many of them are potentially programmed by KMT2B for future expression. Here, we show that one of the genes targeted by KMT2B, Tsga8, plays an essential role in spermatid morphogenesis. Loss of Tsga8 in mice leads to male infertility associated with abnormal chromosomal distribution in round spermatids, malformation of elongating spermatid heads and spermiation failure. Tsga8 depletion leads to dysregulation of thousands of genes, including the X chromosome genes that are reactivated in spermatids, and insufficient nuclear condensation accompanied by reductions of TNP1 and PRM1, key factors for histone-to-protamine transition. Intracytoplasmic sperm injection (ICSI) of spermatids rescued the infertility phenotype, suggesting competency of the spermatid genome for fertilization. Thus, Tsga8 is a KMT2B target that is vitally necessary for spermiogenesis and fertility.

DOI： 10.1242/dev.196212

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BACKGROUND: The clinical course and prognosis of progressive fibrosing interstitial lung diseases (PF-ILDs) vary between individuals. Notably, predictive serum biomarkers for disease management are needed. Serum human epididymis protein 4 (HE4) is reportedly elevated in patients with idiopathic pulmonary fibrosis (IPF); however, its clinical utility remains unknown. We evaluated the potential of serum HE4 as a biomarker for patients with PF-ILD. METHODS: Serum HE4 was measured in a retrospective study consisting of 34 patients with PF-ILD and 40 healthy volunteers. The relationship between serum HE4 levels and clinical parameters or prognosis was investigated. To validate the significance of results obtained, a prospective observational study was performed in 37 patients presenting PF-ILD and 40 control patients without PF-ILD. RESULTS: Serum HE4 levels were higher in patients with PF-ILD than in healthy volunteers (P < 0.01). Moreover, serum HE4 levels correlated with the extent of honeycombing on chest high-resolution computed tomography (r = 0.41, P = 0.015). In multivariate analysis using the Cox proportional hazard model, higher HE4 levels (>238 pmol/L) were associated with an elevated mortality risk; hazard ratio (HR) 7.27, 95% CI 1.56-34.0, P = 0.01 in the derivation cohort; HR 44.3, 95% CI 4.19-468, P < 0.01 in validation cohort. CONCLUSIONS: Serum HE4 levels may serve as a new diagnostic and prognostic biomarker for patients with PF-ILD.

DOI： 10.1016/j.resinv.2020.08.002

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MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An in vitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.

DOI： 10.1016/j.ajhg.2019.11.011

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Respiratory failure is a life-threatening problem for pre-term and term infants, yet many causes remain unknown. Here, we present evidence that whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (Wfdc2), a protease inhibitor previously unrecognized in respiratory disease, may be a causal factor in infant respiratory failure. Wfdc2 transcripts are detected in the embryonic lung and analysis of a Wfdc2-GFP knock-in mouse line shows that both basal and club cells, and type II alveolar epithelial cells (AECIIs), express Wfdc2 neonatally. Wfdc2-null-mutant mice display progressive atelectasis after birth with a lethal phenotype. Mutant lungs have multiple defects, including impaired cilia and the absence of mature club cells from the tracheo-bronchial airways, and malformed lamellar bodies in AECIIs. RNA sequencing shows significant activation of a pro-inflammatory pathway, but with low-quantity infiltration of mononuclear cells in the lung. These data demonstrate that Wfdc2 function is vitally important for lung aeration at birth and that gene deficiency likely causes failure of the lung mucosal barrier.

DOI： 10.1242/dmm.040139

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The mammalian male germline is sustained by a pool of spermatogonial stem cells (SSCs) that can transmit both genetic and epigenetic information to offspring. However, the mechanisms underlying epigenetic transmission remain unclear. The histone methyltransferase Kmt2b is highly expressed in SSCs and is required for the SSC-to-progenitor transition. At the stem-cell stage, Kmt2b catalyzes H3K4me3 at bivalent H3K27me3-marked promoters as well as at promoters of a new class of genes lacking H3K27me3, which we call monovalent. Monovalent genes are mainly activated in late spermatogenesis, whereas most bivalent genes are mainly not expressed until embryonic development. These data suggest that SSCs are epigenetically primed by Kmt2b in two distinguishable ways for the upregulation of gene expression both during the spermatogenic program and through the male germline into the embryo. Because Kmt2b is also the major H3K4 methyltransferase for bivalent promoters in embryonic stem cells, we also propose that Kmt2b has the capacity to prime stem cells epigenetically.

DOI： 10.1242/dev.169102

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Global histone hyperacetylation is suggested to play a critical role for replacement of histones by transition proteins and protamines to compact the genome during spermiogenesis. However, the underlying mechanisms for hyperacetylation-mediated histone replacement remains poorly understood. Here, we report that EPC1 and TIP60, two critical components of the mammalian nucleosome acetyltransferase of H4 (NuA4) complexes, are coexpressed in male germ cells. Strikingly, genetic ablation of either Epc1 or Tip60 disrupts hyperacetylation and impairs histone replacement, in turn causing aberrant spermatid development. Taking these observations together, we reveal an essential role of the NuA4 complexes for histone hyperacetylation and subsequent compaction of the spermatid genome.

DOI： 10.1128/MCB.00082-17

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Background: In the male germline, neonatal prospermatogonia give rise to spermatogonia, which include stem cell population (undifferentiated spermatogonia) that supports continuous spermatogenesis in adults. Although the levels of DNA methyltransferases change dynamically in the neonatal and early postnatal male germ cells, detailed genome-wide DNA methylation profiles of these cells during the stem cell formation and differentiation have not been reported.
Results: To understand the regulation of spermatogonial stem cell formation and differentiation, we examined the DNA methylation and gene expression dynamics of male mouse germ cells at the critical stages: neonatal prospermatogonia, and early postntal (day 7) undifferentiated and differentiating spermatogonia. We found large partially methylated domains similar to those found in cancer cells and placenta in all these germ cells, and high levels of non-CG methylation and 5-hydroxymethylcytosines in neonatal prospermatogonia. Although the global CG methylation levels were stable in early postnatal male germ cells, and despite the reported scarcity of differential methylation in the adult spermatogonial stem cells, we identified many regions showing stage-specific differential methylation in and around genes important for stem cell function and spermatogenesis. These regions contained binding sites for specific transcription factors including the SOX family members.
Conclusions: Our findings show a distinctive and dynamic regulation of DNA methylation during spermatogonial stem cell formation and differentiation in the neonatal and early postnatal testes. Furthermore, we revealed a unique accumulation and distribution of non-CG methylation and 5hmC marks in neonatal prospermatogonia. These findings contrast with the reported scarcity of differential methylation in adult spermatogonial stem cell differentiation and represent a unique phase of male germ cell development.

DOI： 10.1186/s12864-015-1833-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

In many adult tissues, homeostasis relies on self-renewing stem cells that are primed for differentiation. The reconciliation mechanisms of these characteristics remain a fundamental question in stem cell biology. We propose that regulation at the post-transcriptional level is essential for homeostasis in murine spermatogonial stem cells (SSCs). Here, we show that Nanos2, an evolutionarily conserved RNA-binding protein, works with other cellular messenger ribonucleoprotein (mRNP) components to ensure the primitive status of SSCs through a dual mechanism that involves (1) direct recruitment and translational repression of genes that promote spermatogonial differentiation and (2) repression of the target of rapamycin complex 1 (mTORC1), a well-known negative pathway for SSC self-renewal, by sequestration of the core factor mTOR in mRNPs. This mechanism links mRNA turnover to mTORC1 signaling through Nanos2-containing mRNPs and establishes a post-transcriptional buffering system to facilitate SSC homeostasis in the fluctuating environment within the seminiferous tubule.

DOI： 10.1016/j.devcel.2015.05.014

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：De Gruyter Mouton  

Stem cells are identified classically by an in vivo transplantation assay plus additional characterization, such as marker analysis, linage-tracing and in vitro/ex vivo differentiation assays. Stem cell lines have been derived, in vitro, from adult tissues, the inner cell mass (ICM), epiblast, and male germ stem cells, providing intriguing insight into stem cell biology, plasticity, heterogeneity, metastable state, and the pivotal point at which stem cells irreversibly differentiate to non-stem cells. During the past decade, strategies for manipulating cell fate have revolutionized our understanding about the basic concept of cell differentiation: stem cell lines can be established by introducing transcription factors, as with the case for iPSCs, revealing some of the molecular interplay of key factors during the course of phenotypic changes. In addition to de-differentiation approaches for establishing stem cells, another method has been developed whereby induced expression of certain transcription factors and/or micro RNAs artificially converts differentiated cells from one committed lineage to another
notably, these cells need not transit through a stem/progenitor state. The molecular cues guiding such cell fate conversion and reprogramming remain largely unknown. As differentiation and de-differentiation are directly linked to epigenetic changes, we overview cell fate decisions, and associated gene and epigenetic regulations.

DOI： 10.1515/bmc-2014-0036

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Epigenetic modifications influence gene expression and chromatin remodeling. In embryonic pluripotent stem cells, these epigenetic modifications have been extensively characterized
by contrast, the epigenetic events of tissue-specific stem cells are poorly understood. Here, we define a new epigenetic shift that is crucial for differentiation of murine spermatogonia toward meiosis. We have exploited a property of incomplete cytokinesis, which causes male germ cells to form aligned chains of characteristic lengths, as they divide and differentiate. These chains revealed the stage of spermatogenesis, so the epigenetic differences of various stages could be characterized. Single, paired and medium chain-length spermatogonia not expressing Kit (a marker of differentiating spermatogonia) showed no expression of Dnmt3a2 and Dnmt3b (two de novo DNA methyltransferases)
they also lacked the transcriptionally repressive histone modification H3K9me2. By contrast, spermatogonia consisting of ~8-16 chained cells with Kit expression dramatically upregulated Dnmt3a2/3b expression and also displayed increased H3K9me2 modification. To explore the function of these epigenetic changes in spermatogonia in vivo, the DNA methylation machinery was destabilized by ectopic Dnmt3b expression or Np95 ablation. Forced Dnmt3b expression induced expression of Kit
whereas ablation of Np95, which is essential for maintaining DNA methylation, interfered with differentiation and viability only after spermatogonia become Kit positive. These data suggest that the epigenetic status of spermatogonia shifts dramatically during the Kit-negative to Kit-positive transition. This shift might serve as a switch that determines whether spermatogonia self-renew or differentiate. © 2013. Published by The Company of Biologists Ltd.

DOI： 10.1242/dev.094045

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The human epididymis 4 (HE4) protein is expressed in the epididymis and respiratory tract. We previously reported that HE4 is also expressed in pulmonary adenocarcinoma. The purpose of this study was to investigate serum levels of HE4 as a biological marker in pulmonary adenocarcinoma. As the trained set, 102 patients with pulmonary adenocarcinoma who underwent surgery in our institute from 2008 to 2011 were evaluated. They were compared with 58 healthy controls and 16 cases of benign lung disease. In the validation, we used 104 patients with pulmonary adenocarcinoma operated on between 2000 and 2007. Postoperative changes of serum HE4 levels were investigated in 35 patients. The level of HE4 was determined by enzyme immunometric assay and compared with clinicopathological factors. In the trained set, HE4 levels in sera in pulmonary adenocarcinoma were significantly higher than in healthy controls and benign lung disease. Receiver operating characteristic curve showed that HE4 was a good discriminator of pulmonary adenocarcinoma (cut-off point, 50.3 pM; area under curve, 0.825; 95 % confidence interval, 0.76-0.89, p &lt; 0.001). In the validation set, serum HE4 levels were significantly correlated with age, nodal status, and carcinoembryonic antigen. Furthermore, postoperative increase of HE4 serum levels showed a significant correlation with recurrence (p = 0.032). The 5-year overall survival rate was 52.6 % in the HE4-positive group compared with 97.1 % in the HE4-negative group (p = 0.001). These data showed that HE4 expression in sera is associated with progression of pulmonary adenocarcinoma and a possible biomarker.

DOI： 10.1007/s13277-012-0499-8

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The human epididymis 4 (HE4) gene product, also known as whey-acidic-protein four-disulfide core domain protein 2, was identified as the transcript expressed in the epididymis and respiratory tract. HE4 is also expressed in lung adenocarcinoma. We investigated mRNA expressions of full-length HE4 and splice variants in lung adenocarcinoma, and the clinical impact of these genes was evaluated. One hundred and fifty-two patients with pulmonary adenocarcinoma underwent surgery in our institute from 2000 to 2008. We employed immunohistochemical analysis to determine the expression of HE4 and molecular analysis to evaluate full-length HE4 or splice variant gene expression in pulmonary adenocarcinoma. All of the 152 cases were full-length HE4 mRNA-positive; 88 of the 152 (57.9%) were HE4-V1-positive, and 140 of the 152 (92.1%) were HE4-V3-positive. Regarding the relationship between the clinicopathological characteristics of patients and these gene expressions, the histological subtype, tumor size, and vascular invasion were significantly associated with HE4-V3 expression. HE4-V3 expression was also closely correlated with the prognosis. The 5-year disease-free survival in the HE4-V3 high expression group showed a significantly favorable prognosis compared with the low expression group (p = 0.02). The 5-year overall survival rate in the HE4-V3 high expression group was significantly higher than in the HE4-V3 low expression group (p = 0.028). These data showed that high-level HE4-V3 expression is associated with a favorable prognosis in lung adenocarcinoma. Further investigation of HE4 splice variants may offer a new insight into this possibility.

DOI： 10.1007/s13277-011-0252-8

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During meiosis, specific histone modifications at pericentric heterochromatin (PCH), especially histone H3 tri- and dimethylation at lysine 9 (H3K9me3 and H3K9me2, respectively), are required for proper chromosome interactions. However, the molecular mechanism by which H3K9 methylation mediates the synapsis is not yet understood. We have generated a Cbx3-deficient mouse line and performed comparative analysis on Suv39h1/h2-, G9a- and Cbx3-deficient spermatocytes. This study revealed that H3K9me2 at PCH depended on Suv39h1/h2- mediated H3K9me3 and its recognition by the Cbx3 gene product HP1 gamma. We further found that centromere clustering and synapsis were commonly affected in G9a- and Cbx3-deficient spermatocytes. These genetic observations suggest that HP1 gamma/G9a-dependent PCH-mediated centromere clustering is an axis for proper chromosome interactions during meiotic prophase. We propose that the role of the HP1 gamma/G9a axis is to retain centromeric regions of unpaired homologous chromosomes in close alignment and facilitate progression of their pairing in early meiotic prophase. This study also reveals considerable plasticity in the interplay between different histone modifications and suggests that such stepwise and dynamic epigenetic modifications may play a pivotal role in meiosis.

DOI： 10.1242/dev.064444

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DOI： 10.1016/j.diff.2010.09.045

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Background: Histone methylation is thought to be central to the epigenetic mechanisms that maintain and confine cellular identity in multi-cellular organisms. To examine epigenetic roles in cellular homeostasis, we conditionally mutated the histone 3 lysine 4 methyltransferase, Mll2, in embryonic stem (ES) cells, during development and in adult mice using tamoxifen-induced Cre recombination.
Results: In ES cells, expression profiling unexpectedly revealed that only one gene, Magoh2, is dependent upon Mll2 and few other genes were affected. Loss of Mll2 caused loss of H3K4me3 at the Magoh2 promoter and concomitant gain of H3K27me3 and DNA methylation. Hence Mll2, which is orthologous to Drosophila Trithorax, is required to prevent Polycomb-Group repression of the Magoh2 promoter, and repression is further accompanied by DNA methylation. Early loss of Mll2 in utero recapitulated the embryonic lethality found in Mll2-/- embryos. However, loss of Mll2 after E11.5 produced mice without notable pathologies. Hence Mll2 is not required for late development, stem cells or homeostasis in somatic cell types. However it is required in the germ cell lineage. Spermatogenesis was lost upon removal of Mll2, although spermatogonia A persisted.
Conclusion: These data suggest a bimodal recruit and maintain model whereby Mll2 is required to establish certain epigenetic decisions during differentiation, which are then maintained by redundant mechanisms. We also suggest that these mechanisms relate to the epigenetic maintenance of CpG island promoters.

DOI： 10.1186/1756-8935-2-5

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Language：Japanese   Publisher：JAPANESE SOCIETY OF OVA RESEARCH  

Mammalian spermatogenesis is a classic adult stem cell system. Identification of a crucial self renewal factor, glial cell line derived neurotrophic factor (GDNF), has provided new opportunities in cell culture systems of testicular cells. Spermatogonia can be maintained for years in culture with GDNF. Transplantation experiments of cultured spermatogonia have shown that they are able to reconstitute all the germ cells with proper programming in the testes, suggesting that the stem cell population of spermatogonia is maintained <i>in vitro</i> for at least, a couple years. Recently, it has also been reported that cultured spermatogonia have the potency to convert to multi-potent stem cells. The rapid progress of <i>in vitro</i> culture systems of spermatogonia makes it possible to apply the culture systems not only to the study of male infertility but also to regenerative medicine.<br>

DOI： 10.1274/jmor.26.178

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Stem cells are maintained in an undifferentiated state by interacting with a microenvironment known as the "niche," which is comprised of various secreted and membrane proteins. Our goal was to identify niche molecules participating in stem cell-stem cell and/or stem cell-supporting cell interactions. Here, we isolated genes encoding secreted and membrane proteins from purified male germ stem cells using a signal sequence trap approach. Among the genes identified, we focused on the junctional adhesion molecule 4 (JAM4), an immunoglobulin type cell adhesion molecule. JAM4 protein was actually localized to the plasma membrane in male germ cells. JAM4 expression was downregulated as cells differentiated in both germ cell and hematopoietic cell lineages. To analyze function in vivo, we generated JAM4-deficient mice. Histological analysis of testes from homozygous nulls did not show obvious abnormalities, nor did liver and kidney tissues, both of which strongly express JAM4. The numbers of hematopoietic stem cells in bone marrow were indistinguishable between wild-type and mutant mice, as was male germ cell development. These results suggest that JAM4 is expressed in stem cells and progenitor cells but that other cell adhesion molecules may substitute for JAM4 function in JAM4-deficient mice both in male germ cell and hematopoietic lineages.

DOI： 10.1128/MCB.01502-06

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Mammalian spermatogenesis is maintained by a continuous supply of differentiating cells from self-renewing stem cells. The stem cell activity resides in a small subset of primitive germ cells, the undifferentiated spermatogonia. However, the relationship between the establishment of this population and the initiation of differentiation in the developing testes remains unclear. In this study, we have investigated this issue by using the unique expression of Ngn3, which is expressed speci.cally in the undifferentiated spermatogonia, but not in the differentiating spermatogonia or their progenitors, thegonocytes. Our lineage analyses demonstrate that the first round of mouse spermatogenesis initiates directly from gonocytes, without passing through the Ngn3-expressing stage (Ngn3(-) lineage). By contrast, the subsequent rounds of spermatogenesis are derived from Ngn3(-) positive undifferentiated spermatogonia, which are also immediate descendents of the gonocytes and represent the stem cell function (Ngn3(+) lineage). Thus, in mouse spermatogenesis, the state of the undifferentiated spermatogonia is not an inevitable step but is a developmental option that ensures continuous sperm production. In addition, the segregation of gonocytes into undifferentiated spermatogonia (Ngn3(-) lineage) or differentiating spermatogonia (Ngn3(-) lineage) is topographically related to the establishment of the seminiferous epithelial cycle, thus suggesting a role of somatic components in the establishment of stem cells.

DOI： 10.1242/dev.02316

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Tie2 is a receptor-type tyrosine kinase expressed on hematopoietic stem cells and endothelial cells. We used cultured embryonic stem (ES) cells to determine the function of Tie2 during early vascular development and hematopoiesis. Upon differentiation, the ES cell-derived Tie2(+)FIk1(+) fraction was enriched for hematopoietic and endothelial progenitor cells. To investigate lymphatic differentiation, we used a monoclonal antibody against LYVE-11 and found that LYVE-1(+) cells derived from Tie2(+)FIk1(+) cells possessed various characteristics of lymphatic endothelial cells. To determine whether Tie2 played a role in this process, we analyzed differentiation of Tie2(-/-) ES cells. Although the initial numbers of LYVE-1(+) and PECAM-1(+) cells derived from Tie2(-/-) cells did not vary significantly, the number of both decreased dramatically upon extended culturing. Such decreases were rescued by treatment with a caspase inhibitor, suggesting that reductions were due to apoptosis as a consequence of a lack of Tie2 signaling. Interestingly, Tie2(-/-) ES cells did not show measurable defects in development of the hematopoletic system, suggesting that Tie2 is not essential for hematopoletic cell development.

DOI： 10.1182/blood-2005-05-1823

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

The theory of the &apos;&apos; stem cell niche &apos;&apos; was originally proposed for the hematopoietic system, and the existence of the niche as an actual entity was proved in the Drosophila germ cell system. Historically, mammalian spermatogenesis has been studied extensively as a prime example of a stem cell system, and studies have established a stem-progenitor hierarchical order of spermatogonia. In the niche on the basal lamina of seminiferous tubules, spermatogonial stem cells (SSCs) are secluded from the outside world and divide constantly to self-renew and differentiate. During the last 10 years, the development and exploitation of the germ cell transplantation method has expanded our understanding of the nature of SSCs and their niches. The ability to maintain and expand SSCs in vitro, which recently became possible, has further reinforced this research area as a mecca of stem cell biology Nonetheless, the mammalian germ stem cell and its niche remain to be defined more strictly and precisely. We are still on a journey in search of the real stem cell and its true niche.

DOI： 10.1532/IJH97.05088

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

Spermatogonial stem cells (SSCs), having yet to possess decisive markers, can only be detected retrospectively by transplantation assay. It was reported recently that mouse gonocytes collected from DBA/2 and ICR neonates propagated in vitro. This cultured germ cell, named the germline stem cell (GS cell), produced functional sperm to make progeny when transplanted into recipient mouse testes. Here we show that GS cell lines can be established not only front neonatal testes but also from the testis of adult mice. We also confirmed that GS cells once transplanted into a host testis can be recovered to resume in vitro expansion, indicating that they are convertible mutually with SSCs in adult testes. Confocal laser microscopic examination showed GS cells resemble undifferentiated spermatogonia in the adult testis. This unique cell line could be useful for research in germ cell biology and applicable as a new tool for the genetic engineering of animals.

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Questions persist regarding male germ stem cells and how they mature during the prespermatogenic period of testicular development. We successfully labeled the prespermatogonia with green fluorescence protein (GFP) by using Oct-4 enhancer/promoter. This study shows that GFP was specifically expressed in prespermatogonia, spermatogonia and spermatids that faithfully reproduce the endogenous expression of Oct-4. Histochemical analysis revealed that most of the TRA98-positive gonocytes are also positive for GFP. However, the frequency of GFP expressing cells out of TRA98 expressing cells decreased together with the maturation of gonocytes in the first week after birth. To compare the stem cell activity between GFP-positive and -negative populations, we performed a transplantation of sorted cells into testes from an individual population. Colonization efficiency of germ cells from a GFP-positive population resulted in a 30-fold increase in colonization compared with a GFP-negative population. Since the expression of Oct-4 in prespermtogonia correlates is-ell with the stem activity, Oct-4 might be a crucial molecule in the stem cell property of spermatogonia but not in cell survival.

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Cell to cell interaction in bone marrow is crucial for differentiation of hematopoietic cells. We have shown that EphB4 receptor is expressed in erythroid progenitor and its activation accelerates erythroid differentiation. To elucidate the role of EphB4 activation in erythropoiesis, we analyzed effects of EphB4 on cell adhesive pathways. Cell adhesion with the extension of filopodial pseudopod was observed by EphB4 activation. EphB4 activation also enhanced an effect of fibronectin-mediated adhesive pathway along with formation of the c-Cbl/CrkL complex. The tyrosine kinase activity of EphB4 was dispensable for those phenomena. These results suggest that activation of EphB4 participates in adhesive but not repulsive signals independently of its tyrosine kinase activity in hematopoietic cells. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2004.07.016

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in mammalian testis, a typical stem cell system ensures continuous spermatozoa production. Lines of experiments have demonstrated that stem cell activity resides in the most primitive small subset of germ cells, that is, A(s) (A(single)), A(pr) (A(paired)), and A(al) (A(aligned)) spermatogonia, also collectively called undifferentiated spermatogonia. However, their cellular or molecular nature is largely to be elucidated because a gene that is specifically expressed in these cells has not yet been identified, which makes it difficult to study them. In this study, we demonstrate that a class B basic helix-loop-helix (bHLH) transcription factor neurogenin3 (ngn3) is expressed specifically in As, Apr, and A,, spermatogonia because ngn3 is expressed in c-Kit negative spermatogonia throughout the seminiferous cycle, and transgenic labeling with GFP revealed connection of 1, 2, 4, 8, 16, or 32 ngn3-positive cells via intercellular bridges. ngn3 is first expressed at the prepubertal stage in c-Kit negative prespermatogonia. Lineage tracing, using the Cre-loxP system, demonstrates that ngn3-positive germ cells give rise to eventually all the spermatogenesis in mature testis. To our knowledge, ngn3 is the first reported gene that delineates these earliest stages of spermatogenesis. Considering its molecular nature, ngn3 could be involved in their differentiation control. Moreover, visualization with GFP and targeting expression of exogenous genes are valuable tools to investigate the mammalian spermatogenic stem cell system. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2004.01.036

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The stem cell properties of gonocytes and prospermatogonia at prepubertal stages are still largely unknown: it is not clear whether gonocytes and prospermatogonia are a special cell type or similar to adult undifferentiated spermatogonia. To characterize these cells, we have established transgenic mice carrying EGFP (enhanced green fluorescence protein) cDNA under control of an Oct4 18-kb genomic fragment containing the minimal promoter and proximal and distal enhancers; Oct4 is reported to be expressed in undifferentiated spermatogonia at prepubertal stages. Generation of transgenic mice enabled us to purify gonocytes and prospermatogonia from the somatic cells of the testis. Transplantation studies of testicular cells so far have been done with a mixture of germ cells and somatic cells. This is the first report that establishes how to purify germ cells from total testicular cells, enabling evaluation of cell-autonomous repopulating activity of a subpopulation of prospermatogonia. We show that prospermatogonia differ markedly from adult spermatogonia in both the size of the KIT-negative population and cell cycle characteristics. The GFP(+)KIT(-) fraction of prospermatogonia has much higher repopulating activity than does the GFP(+)KIT(+) population in the adult environment. Interestingly, the GFP(+)KIT(+) population still exhibits repopulating activity, unlike adult KIT-positive spermatogonia. We also show that ALCAM, activated leukocyte cell adhesion molecule, is expressed transiently in gonocytes. Sertoli cells and myoid cells also express ALCAM at the same stage, suggesting that ALCAM may contribute to gonocyte-Sertoli cell adhesion and migration of gonoyctes toward the basement membrane. (C) 2003 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0012-1606(03)00111-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Immunostaining with NJ-1 monoclonal antibody (MoAb) revealed that NJ-1 is expressed on megakaryocytes (MKs). NJ-1-positive and lineage-negative progenitor cells have a higher potency to proliferate and differentiate into MKs. MKs were divided into NJ-1 (+)MKs and NJ-1(-)MKs. NJ-1 (+)MKs are immature MKs because of their low potential to generate proplatelets. When cultured CD41-positive MK cells were analyzed with RTPCR, we found that the expression of NJ-1 is down-regulated. NJ-1 (+)MKs have a high adherent potential to endothelial cells comparing with NJ-1 (-)MKs, and this binding ability could be inhibited by the NJ-1-Fc fusion protein. We hypothesize that NJ-1 +MKs are immature MKs and the NJ-1 molecule is involved in MK adhesion to endothelial cells. (C) 2002 Elsevier Science (USA).

DOI： 10.1006/bbrc.2002.6721

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The salivary system in mammals is comprised of three independently developed pairs of organs, the parotid, submaxillar, and sublingual glands. Each gland is composed of various ductal and acinar cell types that fulfill multiple roles. However, the molecular mechanisms regulating their biogenesis and functions are still largely unknown. In this paper, we report that two class B basic helix-loop-helix (bHLH) transcriptional regulators delineate the ductal and the acinar cells in salivary glands. Sgn1, a novel class B bHLH factor, is specifically expressed in the salivary duct cells, while the acinar cells are characterized by the expression of another class B bHLH factor, Mist1. The molecular nature of Sgn1 was also investigated: it binds to specific sequences of DNA as a dimer with a class A bHLH factor and acts as a negative transcriptional regulator against other bHLH factors. This study provides an important cue towards better understanding of the generation and function of multiple cell types in salivary glands. In addition, Sgn1 expression exhibits a reverse relationship with the development of male phenotypes, suggesting its role in gender dimorphism in the salivary glands. (C) 2001 Elsevier Science.

DOI： 10.1006/dbio.2001.0473

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE ASIA  

The Pic-1, Oct-1,2, Unc-86 (POU) transcription factor Oct-4 is specifically expressed in the germ cell line, and a previous study has indicated that the expression of the lacZ gene inserted into an 18 kb genomic fragment encompassing the Oct-4 gene can come close to mimicking the endogenous embryonic expression pattern of Oct-4 in transgenic mice. In the present study transgenic mice expressing green fluorescent protein (GFP) in the germ cell line were generated using the same Oct-4 genomic fragments and the expression pattern was analyzed in detail through all stages of germ cell development. The GFP expressing primordial germ cells were first detected as early as 8.0 days post-coitum (d.p.c.; early head fold stage) at the base of the allantois in living embryos. The GFP expression was thereafter found in both male and female germ cells at all developmental stages except in male germ cells after differentiating into type A spermatogonia in the postnatal testis. There was also a lower level of expression in female germ cells in the prophase of the first meiotic division. These transgenic mice therefore proved to be powerful tools for isolating living germ cells at various developmental stages to study their nature and to isolate new genes.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Activation of transcription by Oct-4 from remote binding sites requires a cofactor that is restricted to embryonal stem cells. The adenovirus E1A protein can mimic the activity of this stem cell-specific factor and stimulates Oct-4 activity in differentiated cells. Here we characterize the Oct-4-E1A interaction and show that the E1A 289R protein harbors two independent Oct-4 binding sites, both of which specifically interact with the POU domain of Oct-4, Furthermore, we demonstrate that, like E1A, the human papillomavirus E7 oncoprotein also specifically binds to the Oct-4 POU domain. E7 and Oct-4 can form a complex both in vitro and in vivo, Expression of E7 in differentiated cells stimulates Oct-ii-mediated transactivation from distal binding sites, Moreover, Oct-4, but not other Oct factors, is active when expressed in cells transformed by human papillomavirus, Our results suggest that different viruses have evolved oncoproteins that share the ability to target Oct-4 and to mimic a stem cell-specific activity.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We assayed IL-6 in 105 cerebrospinal fluid (CSF) samples from patients with ALS, MS, HTLV-1 associated myelopathy (HAM), and controls. There was considerable overlap in n-6 levels in all patient groups. The mean n-6 in 27 patients with ALS was significantly higher than in 21 patients in the other neurological disease (OND) group (P=0.0075). There were no significant differences in MS or HAM and the OND control group. Overall, CSF IL-6 correlated with protein concentration but not with percentage IgG or IgG-albumin index. Patients with CSF oligoclonal bands were no more likely to have detectable IL-6 than patients without oligoclonal bands. Similarly, IL-6 did not correlate with clinical disease activity in MS when subgroups of patients were compared or when an individual patient was followed over time. The elevated IL-6 in ALS may reflect an ongoing humoral immune response, or n-6 may be non-specifically expressed in these patients as a putative neurotrophic factor in response to nerve cell degeneration. (C) 1998 Elsevier Science B.V.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

Interleukin-2 (IL-2) receptor gamma chain-deficient mice with a truncated mutation showed the absence or severe reduction of natural killer cells, decreased numbers of T- and B-cells, marked hypoplasia of the thymus and peripheral lymphoid tissues, defective formation of lymphoid follicles and germinal centre in the peripheral lymphoid tissues, and the absence of Peyer's patches in the intestinal mucosa. In addition, marked splenomegaly with extramedullary haematopoiesis, increased level of IgM and decreased levels of IgG and IgE in serum, severe reduction of conventional B cells (B-2) in the peripheral lymphoid tissues, the presence of lgM-producing CD5(+) B cells (B-1) and their differentiation into plasma cells and Motto cells in the spleen, and increased production and differentiation of macrophages in various tissues were found in the mutant mice, However, the development of both marginal metallophilic macrophage populations in the spleen and of their related macrophages in the other tissues of the mutant mice was severely impaired. All these abnormalities seem to be induced by the loss-of-function of the IL-2 receptor gamma chain, From 8 weeks of age on, inflammatory changes occurred in the intestines, mesenteric lymph nodes, lungs, liver, and kidneys of the mutant mice. Besides the absence of Hassall's corpuscles, thymic cysts were frequently observed in the mutant mice. These pathological abnormalities suggest that the gamma chain is implicated not only in lymphoid and haematopoietic development but also in thymic epithelial cell ontogeny.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The POU transcription factor Oct-4 is expressed in totipotent and pluripotent cells of the early mouse embryo and the germ cell lineage, Transactivation capacities of regions flanking the DNA binding domain of Oct-4 were analyzed in undifferentiated and differentiated cell lines, The amino- and carboxy-terminal regions (N domain and C domain) fused to the Gall DNA binding domain both functioned as transactivation domains in all cell lines tested. However, the C domain failed to activate transcription in some cell lines in the context of the native protein. The underlying regulator, mechanism appears to involve the POU domain of Oct-4 and can discriminate between different POU domains, since constructs in,which the C domain was instead fused to the POU domain of Pit-1 were again equally active in all cell lines, These results indicate that the C domain is subject to cell-type-specific regulation mediated by the Oct-4 POU domain. Phosphopeptide analysis revealed that the cell-type-specific difference of C-domain activity correlates with a difference in Oct-4 phosphorylation status, Since Oct-4 is expressed in a variety of distinct cell types during murine embryogenesis, these results suggest an additional regulatory mechanism for determining Oct-4 function in rapidly changing cell types during development.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS UNITED KINGDOM  

We have examined phosphorylation mediated by cross-talk between growth signal pathways induced by IL-2 and IL-5. To analyze the phosphorylation process in the same cells, we established two sublines, T88-M beta 1, which is a subline of a murine IL-5-dependent cell line, T88-M, by introduction of the human IL-2 receptor beta chain (IL-2R beta), and secondly CTLL-5R alpha beta, which is a subline of a murine IL-2-dependent cell line, CTLL-2, by introduction of the murine IL-5 receptor a chain (IL-5R alpha) and IL-5 receptor beta chain (IL-5R beta, beta c) genes, Both T88-M beta 1 and CTLL-5R alpha beta expressed high-affinity receptors for IL-2 and IL-5, and proliferated in response to both factors, Tyrosine phosphorylation of IL-2R beta was induced by stimulation of T88-M beta 1 with not only IL-2 but also IL-5. Anti-IL-SRP-directed immune complexes from T88-M beta 1 stimulated with IL-5 as well as with IL-2 contained an activated tyrosine kinase, However, stimulation with IL-5 but not IL-2 induced the tyrosine phosphorylation of IL-5R beta, beta c, suggesting that IL-2 does not activate a tyrosine kinase which efficiently catalyzes the IL-5R beta molecule in response to IL-5, On the other hand, the detection of JAK1 and the other common set of phosphotyrosine-containing proteins after stimulation with either IL-5 or IL-2 suggests the existence of the same tyrosine phosphorylation pathways.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

The totipotential stem cells of the pregastrulation mouse embryo which give rise to all embryonic somatic tissues and germ cells express Oct-4. The expression is downregulated during gastrulation and is thereafter only maintained in the germline lineage, Oct-4/lacZ transgenes were used to determine how this pattern of expression was achieved, and resulted in the identification of two separate regulatory elements. The distal element drives Oct-4 expression in preimplantation embryos, in migratory and postmigratory primordial germ cells but is inactive in cells of the epiblast, In cell lines this element is specifically active in embryonic stem and embryonic germ cells. The proximal element directs the epiblast-specific expression pattern, including downregulation during gastrulation; in cell lines its activity is restricted to epiblast-derived cells. Thus, Oct-4 expression in the germline is regulated separately from epiblast expression, This provides the first marker for the identification of totipotent cells in the embryo, and suggests that expression of Oct-4 in the totipotent cycle is dependent on a set of factors unique to the germline.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS UNITED KINGDOM  

Oct3/4, a hallmark of the earliest stages of embryogenesis, is expressed in undifferentiated embryonal carcinoma (EC) and embryonic stem (ES) cells. Oct3/4 gene expression is dependent on the promoter region, the proximal enhancer and the newly identified distal enhancer. We have analysed in vivo occupancy of these elements. In undifferentiated EC and ES cells, strong footprints were detected at specific sites of all three regulatory elements. These were promptly lost upon RA treatment in ES cells and in P19 EC cells, in parallel with sharply reduced Oct3/4 mRNA levels, Thus, the occupancy of regulatory elements is coupled with Oct3/4 expression, and RA treatment causes coordinated factor displacement, leading to extinction of gene activity. In F9 EC cells, footprint was first abolished at the proximal enhancer. However, this loss of binding site occupancy did not result in a decrease in Oct3/4 mRNA levels. The partial factor displacement seen in F9 EC cells, combined with the observation that EC and ES cells utilize the proximal and distal enhancers in a differential manner, indicate the complex pattern of Oct3/4 gene regulation, which could reflect a cell type- and lineage-specific expression of the gene in vivo.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：W B SAUNDERS CO  

The interleukin-2 (IL-2) receptor gamma chain is indispensable for IL-2-, IL-4-, IL-7-, IL-9-, and IL-15-mediated signaling. Mutations of the human gamma chain cause the X-linked severe combined immunodeficiency (XSCID), showing that T and natural killer cells absolutely require the gamma chain for their development in humans, To elucidate the roles of the gamma chain in hematopoiesis, we have generated mice, by gene targeting, that express a form of the gamma chain lacking the cytoplasmic region. Male mice carrying the truncated gamma-chain mutant, which mimics mutations in patients with XSCID, showed a decrease in the number of lymphocytes and an increase in monocytes; the number of T cells was profoundly reduced and no natural killer cells were detected, which is similar to the characteristic of human XSCID. Unlike human XSCID, the levels of B cells were also reduced. In spite of the severe decrease in CD45R(+)/sIgM(+) B cells, the level of IgM in serum of the 8-week-old mutant mice was higher than that of control littermates. Interestingly, the stem cell population with surface phenotypes of CD34, c-kit, and Sca-1 was significantly increased. Furthermore, the colony-forming assay showed that the mutant mice had 15-fold higher numbers of hematopoietic progenitor cells in the spleen as compared with that of controls, These results indicate that functional loss of the gamma chain causes significant effects on the immunological system in mice. (C) 1996 by The American Society of Hematology.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The third component of the interleukin (IL) 2 receptor, gamma chain, is essential not only for IL-2- but also for IL-4-, IL-7-, IL-9-, and IL-15-induced proliferation of lymphocytes. To elucidate the mechanisms by which the gamma chain is expressed, we have analyzed the promoter region of the gamma chain gene. The 633-base pair fragment upstream of the initiation codon showed the promoter activity in human hematopoietic cell lines, Jurkat and THP-1, when linked to the luciferase gene. With a series of 5'-deletion mutants, the basal promoter activity was found in a fragment from nucleotide 80 to 58 upstream from the RNA start site, including an Ets binding sequence, Treatment of cells with either 12-O-tetradecanoylphorbol-13-acetate or phytohemagglutinin but not forskolin induced transcription from the gamma chain gene promoter. A viral trans acting transcriptional activator, Tax, of human T-cell leukemia virus type I elevated expression of the gamma chain gene. In contrast, IL-2 decreased transcription from the IL-2 receptor gamma chain promoter. These results suggest that expression of the gamma chain is regulated at the transcription level by extracellular stimuli and may be implicated in immune response.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The functional interleukin 2 (IL-2) receptors contain the beta and gamma chains which are necessary for the transduction of cell growth signals. Monoclonal antibodies specific for the beta chain and gamma chain coimmunoprecipitated JAK1 and 114-kDa JAK2 tyrosine kinases, respectively. Tyrosine phosphorylation of JAK1 and JAK2, was induced upon IL-2 stimulation, and IL-2 activated the JAK2 kinase. These results demonstrate that the JAK1 and JAK2 tyrosine kinases are physically associated with the beta chain and gamma chain, respectively, and suggest that regulation of the kinases may be linked to IL-2-induced signal transduction.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TOHOKU UNIV MEDICAL PRESS  

We previously demonstrated the existence of a third component, p64, of IL-2 receptor (IL-2R), tentatively named the gamma chain of IL-2R. Our recent studies provided evidence suggesting that the gamma chain endows the beta chain of IL-2R with IL-2 binding ability. The gamma chain was detected in lymphoid transfectants of IL-2Rbeta cDNA, which showed the intermediate-affinity IL-2R, but not in nonlymphoid transfectants of IL-2Rbeta cDNA, which showed no IL-2 binding activity. The comparative study between two subclones of lymphoid MOLT4 transfectant of IL-2Rbeta cDNA demonstrated that the amount of the gamma chain coprecipitated with IL-2Rbeta was proportional to numbers of the IL-2 binding sites. These results suggest the possibility that the gamma chain associates with IL-2Rbeta and has an important role in formation of the intermediate-affinity IL-2R complex. On the other hand, we have also demonstrated the association of IL-2Rbeta with a certain tyrosine kinase, of which activation by IL-2 could be indispensable process at the initial pathway of signal transduction.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have established and characterized five new monoclonal antibodies (mAbs) which specifically immunoprecipitate the human interleukin-2 receptor beta chain (IL-2R-beta). One of them, TU30, recognizes the intracytoplasmic 'serine-rich region' of IL-2R-beta that is critical for IL-2 signal transduction. The others, TU12, TU21, TU23 and TU25, completely inhibit IL-2 binding, as does the previously characterized TU27. However, reciprocal binding competition assays show that the epitopes recognized by the individual mAbs are different from each other. The mAbs inhibit the growth of IL-2-dependent cells. The magnitude of their inhibitory effects is dependent on not only the affinities of the mAbs for IL-2R-beta but also upon the number of IL-2R-alpha subunits expressed on IL-2-dependent cells. These mAbs should be useful in studying the structure and function of the IL-2R.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LITTLE BROWN CO  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

We have cloned and sequenced a cDNA encoding gp34, a novel glycoprotein expressed in cells bearing human T-cell leukemia virus type I (HTLV-I). HTLV-I has a trans-acting transcriptional activator, p40tax, that is thought to be implicated in leukemogenesis through the activation of cellular enhancers. With a subline (JPX-9) of the human T-cell line Jurkat, in which p40tax is inducible, gp34 was shown to be of cellular origin and to be transcriptionally activated by p40tax. It was also demonstrated that two species of mRNA are generated from one copy of the gp34 gene and that these mRNAs encode the identical gp34 product and differ in the 3' untranslated region. Analysis of the deduced amino acid sequence of gp34 showed that it lacks typical signal peptides; however, it has a hydrophobic stretch for membrane anchoring and four possible N-linked glycosylation sites at the carboxy-terminal portion, indicating that it belongs to the family of membrane proteins whose carboxy-terminal portion protrudes out of the cell. The gp34 gene displayed relatively delayed induction compared with other genes activated by p40tax. Taken together with the observation of the dependence of gp34 expression on HTLV-I p40tax, unlike other p40tax-dependent genes such as those for the interleukin-2 receptor alpha-chain and c-fos, which are expressed or induced under physiological conditions, we predict that the mechanism involved in the induction of gp34 expression by p40tax is distinct from and more intricate than those for the previously characterized genes.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

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Language：English   Publishing type：Research paper (international conference proceedings)  

We previously established a monoclonal antibody, TU11 mAb, which is specific for human IL-2 receptor (IL-2R) β chain (p75) and does not inhibit IL-2-binding to IL-2Rβ. Using TU11 mAb, we first demonstrated the existence of a third component, p64, of IL-2R, tentatively named the γ chain of IL-2R. TU11 mAb precipitated not only the β chain but also the α and γ chains in the lysates of cells bearing the high-affinity IL-2R in the presence of IL-2 without any chemical crosslinker. The γ chain was also detected in lymphoid MOLTαβ and MOLTβ cells, which were stably transfected with both α and β cDNA, and with β cDNA alone, respectively, but not in fibroblastoid COSαβ and COSβ cells, which were stably transfected with both α and β cDNA, and with β cDNA alone, respectively. Furthermore, IL-2-mediated growth signals were transduced in the lymphoid transfectant cells but not in the fibroblastoid transfectant cells, suggesting the possibility that the γ chain along with the β chain has an essential role in the transduction of IL-2-mediated growth signals. Using TU11 mAb, we secondly demonstrated that IL-2 rapidly induces tyrosine phosphorylation of both the β and γ chains in an IL-2-dose-dependent manner. The tyrosine phosphorylation of β and γ chains were also detected in the lymphoid transfectant cells but not in the fibroblastoid transfectant cells, indicating the correlation between tyrosine kinase activation and IL-2-mediated growth signaling. The β chain was phosphorylated in in vitro on serine, threonine and tyrosine residues, but the γ chain was phosphorylated in in vitro predominantly on tyrosine residues, suggesting the possibility that the γ chain itself is a tyrosine kinase molecule.

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Language：English   Publishing type：Research paper (scientific journal)  

A new monoclonal antibody (mAb), TU11 mAb, was found to be specific for the p75 subunit of human interleukln 2 receptor (IL-2R). TU11 mAb reacted with human and Gibbon ape cell lines positive for lL-2Rp75 but not with cell lines negative for lL-2Rp75 tested. TU11 mAb specifically detected a single cell surface molecule with a molecular weight of 75 kd. The 75 kd molecule was identical to p75 detected by TU27 mAb specific for human lL-2Rp75 in sequential immunoprecipitation. TU11 mAb did not block lL-2 binding to the high-affinity lL-2R at all and precipitated p75 bound with IL-2. Using TU11 mAb, we have demonstrated here that solubilized p75 has an IL-2 binding site. © 1989 The Japanese Society for Immunology.

DOI： 10.1093/intimm/1.4.373

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Language：English   Publisher：(公社)日本生化学会  

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Language：Japanese  

J-GLOBAL

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J-GLOBAL

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Language：Japanese  

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J-GLOBAL

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J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：Japanese   Publisher：日本発生生物学会  

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DOI： 10.1146/annurev.immunol.14.1.179

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：MARY ANN LIEBERT INC PUBL  

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Language：Japanese   Presentation type：Symposium, workshop panel (public)  

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Language：Japanese   Presentation type：Symposium, workshop panel (public)  

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Presentation type：Poster presentation  

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Language：English   Presentation type：Oral presentation (invited, special)  

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Language：Japanese   Presentation type：Symposium, workshop panel (public)  

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Language：Japanese   Presentation type：Symposium, workshop panel (public)  

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Language：Japanese   Presentation type：Symposium, workshop panel (public)  

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Language：Japanese   Presentation type：Symposium, workshop panel (public)  

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Language：Japanese   Presentation type：Symposium, workshop panel (public)  

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