Pluripotent stem cells can be classified into two distinct states, naïve and primed, which show different degrees of potency. One difficulty in stem cell research is the inability to distinguish these states in live cells. Studies on... more
Pluripotent stem cells can be classified into two distinct states, naïve and primed, which show different degrees of potency. One difficulty in stem cell research is the inability to distinguish these states in live cells. Studies on female mice have shown that reactivation of inactive X chromosomes occurs in the naïve state, while one of the X chromosomes is inactivated in the primed state. Therefore, we aimed to distinguish the two states by monitoring X chromosome reactivation. Thus far, X chromosome reactivation has been analysed using fixed cells; here, we inserted different fluorescent reporter gene cassettes (mCherry and eGFP) into each X chromosome. Using these knock-in 'Momiji' mice, we detected X chromosome reactivation accurately in live embryos, and confirmed that the pluripotent states of embryos were stable ex vivo, as represented by embryonic and epiblast stem cells in terms of X chromosome reactivation. Thus, Momiji mice provide a simple and accurate method f...
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Zfp318, a mouse gene with a Cys2/His2 zinc finger motif, is mainly expressed in germ cells in the testis. It encodes two alternative transcripts, which regulate androgen receptor-mediated transcriptional activation or repression by... more
Zfp318, a mouse gene with a Cys2/His2 zinc finger motif, is mainly expressed in germ cells in the testis. It encodes two alternative transcripts, which regulate androgen receptor-mediated transcriptional activation or repression by overexpression of them. However, the role of Zfp318 is still obscure in vivo, especially in spermatogenesis. To elucidate the role of Zfp318 during gamete production, we established a knockout mouse line. Zfp318-null male mice exhibited infertility, whereas Zfp318-null female mice displayed normal fertility. ZFP318 was expressed during multiple stages of spermatogenesis, from spermatocytes to round spermatids. The nuclei of secondary spermatocytes showed high levels of expression. Histological analysis and quantitative analysis of DNA content showed decreased numbers of both spermatids in the seminiferous tubules and mature spermatozoa in the epididymides of Zfp318-null mice. These results suggest that Zfp318 is expressed as a functional protein in testic...
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Loss of the endoplasmic reticulum resident chaperone calmegin leads to the production of sterile sperm that do not bind to the egg zona pellucida (M. Ikawa et al., 1997, Nature 387, 607–611). In the present study, we demonstrate that... more
Loss of the endoplasmic reticulum resident chaperone calmegin leads to the production of sterile sperm that do not bind to the egg zona pellucida (M. Ikawa et al., 1997, Nature 387, 607–611). In the present study, we demonstrate that calmegin −/− sperm were defective in migrating into the oviducts and in binding to the egg plasma membrane. Despite the impaired adhesive function, calmegin −/− sperm could fertilize eggs when zonae pellucidae were partially dissected, and eggs fertilized in this manner could develop normally to term. Since these sperm characteristics were similar to those found in fertilin β −/− sperm, we investigated the interaction of calmegin with fertilin β. Using immunoprecipitation techniques, calmegin was found to bind to sperm membrane proteins, fertilin α and β, during spermatogenesis. The binding was specific to calmegin: another endoplasmic reticulum chaperone calnexin, a calmegin homologue, was not able to bind to fertilin α and β. In the calmegin −/− mice, a loss of heterodimerization of fertilin α and β was observed and fertilin β was not detectable in mature sperm. The data not only explain why the calmegin and fertilin β knockout mouse lines share a common infertile phenotype, but also reveal the importance of the maturation of sperm membrane proteins in the endoplasmic reticulum.
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Genetics, Cellular Biology, Kinetics, DNA damage, DNA repair, and 20 moreTranscription Factors, Biological Sciences, Longevity, Molecular and cellular biology, Humans, Mutagenesis, Cell fusion, Skin Cancer, Mice, Female, Animals, Cockayne syndrome, Male, Nucleotide Excision Repair, Protein Function, In Vitro Studies, Autosomal Recessive, Life Span, DNA binding proteins, and Xeroderma Pigmentosum
Glycosylphosphatidylinositol (GPI)-anchored proteins are widely distributed on plasma membranes of eukaryotes. More than 50 GPI-anchored proteins have been shown to be spatiotemporally expressed in mice with a deficiency of GPI-anchor... more
Glycosylphosphatidylinositol (GPI)-anchored proteins are widely distributed on plasma membranes of eukaryotes. More than 50 GPI-anchored proteins have been shown to be spatiotemporally expressed in mice with a deficiency of GPI-anchor biosynthesis that causes embryonic lethality. Here, we examine the functional roles of GPI-anchored proteins in mouse skin using the Cre-loxP recombination system. We disrupted the Pig-a gene, an X-linked gene essential for GPI-anchor biosynthesis, in skin. The Cre-mediated Pig-a disruption occurred in skin at almost 100% efficiency in male mice bearing two identically orientated loxP sites within the Pig-a gene. Expression of GPI-anchored proteins was completely absent in the skin of these mice. The skin of such mutants looked wrinkled and more scaly than that of wild-type mice. Furthermore, histological examination of mutant mice showed that the epidermal horny layer was tightly packed and thickened. Electron microscopy showed that the intercellular space was narrow and there were many small vesicles embedded in the intercellular space that were not observed in equivalent wild-type mouse skin preparations. Mutant mice died within a few days after birth, suggesting that Pig-a function is essential for proper skin differentiation and maintenance.
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Human serum immunoglobulin G light chain (Fr.I-L), which was reduced and carboxamide-methylated, showed no effect on formyl-methionyl-leucyl-phenylalanine (FMLP)-induced chemotaxis nor on phagocytosis of yeasts when directly added to... more
Human serum immunoglobulin G light chain (Fr.I-L), which was reduced and carboxamide-methylated, showed no effect on formyl-methionyl-leucyl-phenylalanine (FMLP)-induced chemotaxis nor on phagocytosis of yeasts when directly added to guinea pig polymorphonuclear leukocytes (PMNs). However, intravenously administered Fr.I-L inhibited emigration of leukocytes into the peritoneal cavity and promoted phagocytosis of yeasts in a yeast-induced peritonitis model in mice. Moreover, Fr.I-L reduced FMLP-induced chemiluminescence (CL) from PMNs. These facts indicated that the anti-inflammatory action of Fr.I-L was caused by inhibiting emigration of leukocytes into the injured site and scavenging superoxide radicals from the cells.
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Developmental Biology, Biological Sciences, Molecular chaperones, Mice, Female, and 16 moreAnimals, Male, Membrane Fusion, Sterilization, Green Fluorescent Protein, Developmental, Fertilization, Egg, Acrosome, Spermatozoa, Calcium Binding Proteins, Zona Pellucida, Recombinant Proteins, Plasma Membrane, Chaperone, and Cell Membrane
The stratum corneum of the skin serves as an effective barrier for maintenance of the internal milieu against the external environment. At the cell periphery of the stratum corneum is the cell envelope, a highly insoluble membranous... more
The stratum corneum of the skin serves as an effective barrier for maintenance of the internal milieu against the external environment. At the cell periphery of the stratum corneum is the cell envelope, a highly insoluble membranous structure composed of precursor proteins cross-linked by ε-(gamma-glutamyl)lysine bonds. Transglutaminase 1 (TGase 1; keratinocyte TGase), a membrane-bound isozyme of the TGase family, has been proposed to catalyze this process of assembly. Deficient cross-linking of the cell envelope in some patients with the autosomal recessive skin disorder lamellar ichthyosis (LI) and several mutations of the TGase 1 gene that have been identified in families with LI suggest the importance of this gene in production of the cell envelope. In this study, we generated mice lacking the TGase 1 gene, and we report that they have erythrodermic skin with abnormal keratinization. In their stratum corneum, degradation of nuclei and keratohyalin F-granules was incomplete and cell envelope assembly was defective. The skin barrier function of TGase 1-null mice was markedly impaired, and these mice died within 4-5 h after birth. These results clearly demonstrate that the TGase 1 gene is essential to the development and maturation of the stratum corneum and to adaptation to the environment after birth. Thus, these TGase 1 knockout mice may be a useful model for severe cases of LI.
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Myeloid dendritic cells (mDCs) recognize and respond to polyI:C, an analog of dsRNA, by endosomal Toll-like receptor (TLR) 3 and cytoplasmic receptors. Natural killer (NK) cells are activated in vivo by the administration of polyI:C to... more
Myeloid dendritic cells (mDCs) recognize and respond to polyI:C, an analog of dsRNA, by endosomal Toll-like receptor (TLR) 3 and cytoplasmic receptors. Natural killer (NK) cells are activated in vivo by the administration of polyI:C to mice and in vivo are reciprocally activated by mDCs, although the molecular mechanisms are as yet undetermined. Here, we show that the TLR adaptor TICAM-1 (TRIF) participates in mDC-derived antitumor NK activation. In a syngeneic mouse tumor implant model (C57BL/6 vs. B16 melanoma with low H-2 expresser), i.p. administration of polyI:C led to the retardation of tumor growth, an effect relied on by NK activation. This NK-dependent tumor regression did not occur in TICAM-1-/- or IFNAR-/- mice, whereas a normal NK antitumor response was induced in PKR-/-, MyD88-/-, IFN-β-/-, and wild-type mice. IFNAR was a prerequisite for the induction of IFN-α/β and TLR3. The lack of TICAM-1 did not affect IFN production but resulted in unresponsiveness to IL-12 production, mDC maturation, and polyI:C-mediated NK-antitumor activity. This NK activation required NK-mDC contact but not IL-12 function in in vivo transwell analysis. Implanted tumor growth in IFNAR-/- mice was retarded by adoptively transferring polyI:C-treated TICACM-1-positive mDCs but not TICAM-1-/- mDCs. Thus, TICAM-1 in mDCs critically facilitated mDC-NK contact and activation of antitumor NK, resulting in the regression of low MHC-expressing tumors.
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Bcl-2 interacting cell death suppressor (Bis), also known as Bag3 or CAIR-1, is involved in antistress and antiapoptotic pathways. In addition to Bcl-2, Bis binds to several proteins, suggesting it has diverse functions in normal and... more
Bcl-2 interacting cell death suppressor (Bis), also known as Bag3 or CAIR-1, is involved in antistress and antiapoptotic pathways. In addition to Bcl-2, Bis binds to several proteins, suggesting it has diverse functions in normal and pathological conditions. To better define the physiological function of Bis in vivo, we developed bis-deficient mice with a cre-loxP system. Targeted disruption of exon 4 of the bis gene was demonstrated by Southern blotting and PCR, and Western blotting showed that no intact or truncated Bis protein was synthesized in bis(-/-) mice. While heterozygotes were fertile and appeared normal, Bis-deficient mice showed growth retardation and died by 3 wk after birth. The relative weight of the thymus and spleen was reduced and the total numbers of white blood cells, splenocytes, and thymocytes were significantly reduced compared with wild-type littermates. Serum profiles indicated significant hypoglycemia as well as decrease in triglyceride and cholesterol levels. Expression profiles of metabolic genes indicated that gluconeogenesis and beta-oxidation are activated in the liver of bis(-/-) mice. This activation, as well as a decrease in peripheral fat and an induction of fatty liver, appears to be an adaptive response to hypoglycemia. Our study reveals that the absence of Bis has considerable influences on postnatal growth and survival, possibly due to a nutritional impairment.
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Advances in assisted reproduction techniques such as in vitro fertilization and intracytoplasmic sperm injection have made paternity possible for many patients with male infertility. However, at least some sperm or spermatids are required... more
Advances in assisted reproduction techniques such as in vitro fertilization and intracytoplasmic sperm injection have made paternity possible for many patients with male infertility. However, at least some sperm or spermatids are required for these techniques to be successful, and patients incapable of producing spermatids cannot be helped. Male mice homozygous for the mutant juvenile spermatogonial depletion (jsd) gene show spermatogonial arrest and an elevated intratesticular testosterone level like many other experimental infertility models such as those with iradiation- or chemotherapy-induced testicular damage. In this category of infertile males, suppression of the testosterone level induces spermatogonial differentiation to the stage of spermatocytes but no further. In the present study with jsd mutant mice, we induced spermatogenesis first to spermatocytes and then to elongated spermatids by suppression of testosterone levels with a GnRH antagonist, Nal-Glu, at a dose of 2500 microg kg(-1) day(-1) for 4 wk and then withdrawal of Nal-Glu. Spermatids were seen in the cross-sections of seminiferous tubules in all mice treated by administration and subsequent withdrawal of Nal-Glu. Four weeks after withdrawal of Nal-Glu, some of the germ cells differentiated into elongated spermatids. Supplementation with testosterone and Nal-Glu after 4 wk of treatment with Nal-Glu alone also induced spermatogenesis similar to the induction by withdrawal of Nal-Glu. Thus, we ascribe the restoration of the differentiation of spermatocytes to spermatids to reelevation of the testosterone level. Furthermore, we successfully rescued male sterility in jsd mice by subsequent intracytoplasmic sperm injection using the elongated spermatids induced by the programmed hormone therapy.
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Cellular Biology, Biology, Transgenic Mice, Signal Transduction, Biological Sciences, and 17 moreMolecular, Growth Hormone, Erythropoietin, Cell Differentiation, Molecular and cellular biology, Prolactin, Natural Killer cells, Phenotype, Transcription Factor, And, Mammary gland, Body Weight, Knockout Mice, DNA binding proteins, Leukemia Inhibitory Factor, NK Cell, and Tyrosine Phosphorylation
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Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by MYH9 is associated with autosomal dominantly inherited diseases called MYH9 disorders. MYH9 disorders are characterized by macrothrombocytopenia and very characteristic inclusion... more
Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by MYH9 is associated with autosomal dominantly inherited diseases called MYH9 disorders. MYH9 disorders are characterized by macrothrombocytopenia and very characteristic inclusion bodies in granulocytes. MYH9 disorders frequently cause nephritis, sensorineural hearing disability and cataracts. One of the most common and deleterious mutations causing these disorders is the R702C missense mutation. We generated knock-in mice expressing the Myh9 R702C mutation. R702C knock-in hetero mice (R702C+/- mice) showed macrothrombocytopenia. We studied megakaryopoiesis of cultured fetal liver cells of R702C+/- mice and found that proplatelet formation was impaired: the number of proplatelet tips was decreased, proplatelet size was increased, and proplatelet shafts were short and enlarged. Although granulocyte inclusion bodies were not visible by May-Grünwald Giemsa staining, immunofluorescence analysis indicated that NMMHCIIA proteins aggregated and accumulated in the granulocyte cytoplasm. In other organs, R702C+/- mice displayed albuminuria which increased with age. Renal pathology examination revealed glomerulosclerosis. Sensory hearing loss was indicated by lowered auditory brainstem response. These findings indicate that Myh9 R702C knock-in mice mirror features of human MYH9 disorders arising from the R702C mutation.
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An anti-sperm monoclonal antibody was developed from female C57BL/6 mice immunized with epididymal sperm from a syngeneic male mouse. Immunofluorescence staining revealed that the antibody did not react to fresh epididymal sperm but... more
An anti-sperm monoclonal antibody was developed from female C57BL/6 mice immunized with epididymal sperm from a syngeneic male mouse. Immunofluorescence staining revealed that the antibody did not react to fresh epididymal sperm but attached to the capacitated sperm found in the peri-vitelline spaces of the ova. When the antibody was applied to sperm incubated in vitro, variously stained sperm were observed. It was presumed that the antigenic site detected by the antibody was hidden in the fresh epididymal sperm and was spread from the acrosomal cap region to the entire head during the capacitation process.
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Angiotensin-converting enzyme (ACE) inhibitors were excised from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) preparations of tuna and porcine muscles by heating at 120 degrees C for 5 min in 1 M AcOH-20 mM HCl. The inhibitors were... more
Angiotensin-converting enzyme (ACE) inhibitors were excised from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) preparations of tuna and porcine muscles by heating at 120 degrees C for 5 min in 1 M AcOH-20 mM HCl. The inhibitors were then purified by successive chromatographies. The final product from tuna was identified as Pro-Thr-His-Ile-Lys-Trp-Gly-Asp, which was the ACE inhibitor obtained from tuna muscle [Kohama et al. (1988) Biochem. Biophys. Res. Commun. 155, 332-337]. The porcine ACE inhibitor was found to be Pro-Ala-Asn-Ile-Lys-Trp-Gly-Asp, which was identical to the porcine muscle GAPDH peptide 79-86. These results strongly suggested that the ACE inhibitory octapeptides derived from GAPDH proteins by acid-limited proteolysis at Asp-Pro and Asp-Ala peptide bonds.
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It is intriguing that the disruption of a number of genes, e.g., Clgn, Ace, Adamla, Adam2 and Adam3 results in a similar sperm phenotype, i.e., failure of sperm to bind to the zona pellucida (ZP). Because calmegin functions as a... more
It is intriguing that the disruption of a number of genes, e.g., Clgn, Ace, Adamla, Adam2 and Adam3 results in a similar sperm phenotype, i.e., failure of sperm to bind to the zona pellucida (ZP). Because calmegin functions as a testis-specific molecular chaperone, there is a possibility of misfolding of ADAMs in sperm from Clgn-/- mice. In the first half of this review we describe the interaction of ADAMs with calmegin and try to elucidate the relationship of these proteins to establish the zona binding ability. In the second half of this review we describe other gene manipulated animals that lead to a defect in sperm-egg fusion. The first factor, CD9, was found serendipitously on the egg side, while the second factor, Izumo, on the sperm side, was discovered after 20 years of pursuit. Here we describe various gene manipulated animals that are useful to elucidate the mechanism of fertilisation.
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RIG-I-mediated type I interferon (IFN) production and nuclease-mediated viral RNA degradation are essential for antiviral innate immune responses. DDX60 is an IFN-inducible cytoplasmic helicase. Here, we report that DDX60 is a sentinel... more
RIG-I-mediated type I interferon (IFN) production and nuclease-mediated viral RNA degradation are essential for antiviral innate immune responses. DDX60 is an IFN-inducible cytoplasmic helicase. Here, we report that DDX60 is a sentinel for both RIG-I activation and viral RNA degradation. We show that DDX60 is an upstream factor of RIG-I that activates RIG-I signaling in a ligand-specific manner. DDX60 knockout attenuates RIG-I signaling and significantly reduces virus-induced type I IFN production in vivo. In addition, we show that DDX60 is involved in RIG-I-independent viral RNA degradation. DDX60 and RIG-I adaptor MAVS double-knockout mice reveal a role for DDX60-dependent RNA degradation in antiviral responses. Several viruses induced DDX60 phosphorylation via epidermal growth factor receptor (EGFR), leading to attenuation of the DDX60 antiviral activities. Our results define DDX60 as a sentinel for cytoplasmic antiviral response, which is counteracted by virus-mediated EGF recep...
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Polyinosinic-polycytidylic acid strongly promotes the antitumor activity of NK cells via TLR3/Toll/IL-1R domain-containing adaptor molecule 1 and melanoma differentiation-associated protein-5/mitochondrial antiviral signaling protein... more
Polyinosinic-polycytidylic acid strongly promotes the antitumor activity of NK cells via TLR3/Toll/IL-1R domain-containing adaptor molecule 1 and melanoma differentiation-associated protein-5/mitochondrial antiviral signaling protein pathways. Polyinosinic-polycytidylic acid acts on accessory cells such as dendritic cells (DCs) and macrophages (Mφs) to secondarily activate NK cells. In a previous study in this context, we identified a novel NK-activating molecule, named IFN regulatory factor 3-dependent NK-activating molecule (INAM), a tetraspanin-like membrane glycoprotein (also called Fam26F). In the current study, we generated INAM-deficient mice and investigated the in vivo function of INAM. We found that cytotoxicity against NK cell-sensitive tumor cell lines was barely decreased in Inam(-/-) mice, whereas the number of IFN-γ-producing cells was markedly decreased in the early phase. Notably, deficiency of INAM in NK and accessory cells, such as CD8α(+) conventional DCs and Mφs...
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It is intriguing that the disruption of a number of genes, e.g., Clgn, Ace, Adamla, Adam2 and Adam3 results in a similar sperm phenotype, i.e., failure of sperm to bind to the zona pellucida (ZP). Because calmegin functions as a... more
It is intriguing that the disruption of a number of genes, e.g., Clgn, Ace, Adamla, Adam2 and Adam3 results in a similar sperm phenotype, i.e., failure of sperm to bind to the zona pellucida (ZP). Because calmegin functions as a testis-specific molecular chaperone, there is a possibility of misfolding of ADAMs in sperm from Clgn-/- mice. In the first half of this review we describe the interaction of ADAMs with calmegin and try to elucidate the relationship of these proteins to establish the zona binding ability. In the second half of this review we describe other gene manipulated animals that lead to a defect in sperm-egg fusion. The first factor, CD9, was found serendipitously on the egg side, while the second factor, Izumo, on the sperm side, was discovered after 20 years of pursuit. Here we describe various gene manipulated animals that are useful to elucidate the mechanism of fertilisation.
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Although pronuclear DNA micro-injection has long been the most reliable method to produce transgenic pigs, the efficiency of production of transgenic offspring is generally plagued by 1% of the DNA-injected embryos. Therefore, a problem... more
Although pronuclear DNA micro-injection has long been the most reliable method to produce transgenic pigs, the efficiency of production of transgenic offspring is generally plagued by 1% of the DNA-injected embryos. Therefore, a problem with this method is the need for large numbers of pronuclear stage embryos. One great advancement would be the use of in vitro-matured (IVM) oocytes for the purpose of transgenic pig production. High developmental competence of IVM oocytes was proven by transfer of parthenogenetic IVM oocytes. A combined method of sperm vectors with the IVM of oocytes would make the production of transgenic pigs remarkably feasible. Rate of blastocyst formation following intracytoplasmic sperm injection (ICSI) by frozen sperm was over 20%, and transgene was expressed in approximately 50% of blastocysts generated. Somatic cell nuclear transfer would enable more efficient and sophisticated genetic modification of the pig. Simultaneous comparison between two nuclear tra...
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Cell Cycle, Cryopreservation, Stem Cell, Theriogenology, Biological Sciences, and 15 moreDNA, Cell line, Female, Intracytoplasmic Sperm Injection, Animals, Male, Genetic modification, Genetically Modified, Swine, Spermatozoa, Oocytes, Nuclear Transfer, Embryos, Chemical Treatment, and Somatic Cell Nuclear Transfer
Bone marrow stem cells (BMC) develop into hematopoietic and mesenchymal lineages but have not been known to differentiate into glomerular cells. To investigate whether such differentiation is possible, a search was made for donor... more
Bone marrow stem cells (BMC) develop into hematopoietic and mesenchymal lineages but have not been known to differentiate into glomerular cells. To investigate whether such differentiation is possible, a search was made for donor glomerular cells in lethally irradiated C57BL/6j (B6) mice given transplants of BMC from syngeneic mice transgenic for green fluorescence protein (GFP) ([GFP-->B6] mice). After the recipients of donor BMC manifested GFP-positive cells in their glomeruli, the numbers of such cells increased markedly, in a time-dependent manner, from 2 wk to 24 wk after bone marrow transplantation. Immunohistochemical analyses revealed that most GFP-positive cells in the glomeruli were neither macrophages nor T cells. With the use of a laser-scanning confocal microscope, GFP-positive cells were observed within the mesangium of [GFP-->B6] mice. Furthermore, indirect immunofluorescence assays demonstrated that desmin-positive cells in the glomeruli of these chimeric mice ...
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Both lymphotoxin-alpha (LTalpha)-deficient mice and alymphoplasia (aly) mice, a natural mutant strain, manifest a quite similar phenotype: lack of lymph nodes (LN) and Peyer's patches (PP), with disturbed spleen architecture. The... more
Both lymphotoxin-alpha (LTalpha)-deficient mice and alymphoplasia (aly) mice, a natural mutant strain, manifest a quite similar phenotype: lack of lymph nodes (LN) and Peyer's patches (PP), with disturbed spleen architecture. The mechanisms underlying the defective lymphoid organogenesis in these mice were investigated by generating aggregation chimeras; ex vivo fused morulae were implanted into pseudo-pregnant host females and allowed to develop to term. Chimeric mice between LTalpha-deficient mice and wild-type mice restored LN and PP almost completely, suggesting that LTalpha expressed by circulating bone marrow-derived cells is essential for lymphoid organogenesis as well as for organization of spleen architecture. By contrast, chimeric mice between aly mice and wild-type mice showed only limited restoration of LN and PP. This suggests that the putative aly gene product does not act as a circulating ligand for lymphoid organogenesis, like LTalpha. Rather, abnormal developmen...
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Notch family genes encode transmembrane proteins involved in cell-fate determination. Using gene targeting procedures, we disrupted the mouse Notch2 gene by replacing all but one of the ankyrin repeat sequences in the cytoplasmic domain... more
Notch family genes encode transmembrane proteins involved in cell-fate determination. Using gene targeting procedures, we disrupted the mouse Notch2 gene by replacing all but one of the ankyrin repeat sequences in the cytoplasmic domain with the E. coli (beta)-galactosidase gene. The mutant Notch2 gene encodes a 380 kDa Notch2-(beta)-gal fusion protein with (beta)-galactosidase activity. Notch2 homozygous mutant mice die prior to embryonic day 11.5, whereas heterozygotes show no apparent abnormalities and are fully viable. Analysis of Notch2 expression patterns, revealed by X-gal staining, demonstrated that the Notch2 gene is expressed in a wide variety of tissues including neuroepithelia, somites, optic vesicles, otic vesicles, and branchial arches, but not heart. Histological studies, including in situ nick end labeling procedures, showed earlier onset and higher incidence of apoptosis in homozygous mutant mice than in heterozygotes or wild type mice. Dying cells were particularly...
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The xeroderma pigmentosum group G (XP-G) gene (XPG) encodes a structure-specific DNA endonuclease that functions in nucleotide excision repair (NER). XP-G patients show various symptoms, ranging from mild cutaneous abnormalities to severe... more
The xeroderma pigmentosum group G (XP-G) gene (XPG) encodes a structure-specific DNA endonuclease that functions in nucleotide excision repair (NER). XP-G patients show various symptoms, ranging from mild cutaneous abnormalities to severe dermatological impairments. In some cases, patients exhibit growth failure and life-shortening and neurological dysfunctions, which are characteristics of Cockayne syndrome (CS). The known XPG protein function as the 3' nuclease in NER, however, cannot explain the development of CS in certain XP-G patients. To gain an insight into the functions of the XPG protein, we have generated and examined mice lacking xpg (the mouse counterpart of the human XPG gene) alleles. The xpg-deficient mice exhibited postnatal growth failure and underwent premature death. Since XPA-deficient mice, which are totally defective in NER, do not show such symptoms, our data indicate that XPG performs an additional function(s) besides its role in NER. Our in vitro studie...
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Genetics, Cellular Biology, Kinetics, DNA damage, DNA repair, and 20 moreTranscription Factors, Biological Sciences, Longevity, Molecular and cellular biology, Humans, Mutagenesis, Cell fusion, Skin Cancer, Mice, Female, Animals, Cockayne syndrome, Male, Nucleotide Excision Repair, Protein Function, In Vitro Studies, Autosomal Recessive, Life Span, DNA binding proteins, and Xeroderma Pigmentosum
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Carboxymethylated sperm-specific lactate dehydrogenase isozyme C4 (LDH-C4) proteins from mouse and rat testes were cleaved with cyanogen bromide and trypsin. Proteins were also citraconylated and digested with trypsin. In the case of... more
Carboxymethylated sperm-specific lactate dehydrogenase isozyme C4 (LDH-C4) proteins from mouse and rat testes were cleaved with cyanogen bromide and trypsin. Proteins were also citraconylated and digested with trypsin. In the case of mouse LDH-C4 isozyme, all 7 CNBr and 11 limited tryptic (arginine) peptides were isolated and sequenced. Some of the CNBr peptides were further fragmented with trypsin and chymotrypsin and their compositions and/or sequences characterized. Also, 34 of the 36 expected tryptic peptides were purified, and their compositions and sequences determined. Amino acid sequences of these peptides purified from mouse LDH-C4 were overlapped into a complete covalent structure of the 330 residues. For rat LDH-C4, 5 of 6 expected CNBr peptides, 5 of 8 expected arginine peptides, and 28 of the 34 expected tryptic peptides were isolated, and their compositions and sequences were determined. Some of the CNBr and arginine peptides were further fragmented with chymotrypsin, ...
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With the use of a computer graphics system, spacefilling models of mouse and rat testis lactate dehydrogenase C4 (LDH-C4) isozymes were constructed from amino acid sequence and published x-ray diffraction data. Thirty-two residues that... more
With the use of a computer graphics system, spacefilling models of mouse and rat testis lactate dehydrogenase C4 (LDH-C4) isozymes were constructed from amino acid sequence and published x-ray diffraction data. Thirty-two residues that differ between the mouse and rat LDH-C4 sequences are also displayed on the monomeric and tetrameric lactate dehydrogenase molecules. Immunological properties of mouse and rat LDH-C4 isozymes are compared and related to these 32 differences. Possible relationships between the structure, especially the amino acid sequence, and the unique enzymatic properties of LDH-C4 isozymes are also discussed.
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1. The LDH-X polypeptide was specifically immunoprecipitated from the cell-free translation products of poly A-containing mRNA from mouse testes, and it represents 1-2% of the total proteins synethesized in vitro. 2. The in-vitro... more
1. The LDH-X polypeptide was specifically immunoprecipitated from the cell-free translation products of poly A-containing mRNA from mouse testes, and it represents 1-2% of the total proteins synethesized in vitro. 2. The in-vitro synthesized LDH-X polypeptide appears to have the same mol. wt of 36,000 as mouse authentic LDH-X and, thus, any presequence of LDH-X must be very short, if present at all. 3. The LDH-X was not found in the mouse liver mRNA translation products immunoprecipitated by anti-LDH-X antibodies.
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A sensitive and specific radioimmunoassay for antiarrhythmic peptide (AAP) has been developed, utilizing 3-(4-hydroxy, 5-[125I]iodophenyl)propionyl AAP and guinea pig anti-AAP serum. Synthetic AAP was used as a standard and the... more
A sensitive and specific radioimmunoassay for antiarrhythmic peptide (AAP) has been developed, utilizing 3-(4-hydroxy, 5-[125I]iodophenyl)propionyl AAP and guinea pig anti-AAP serum. Synthetic AAP was used as a standard and the polyethylene glycol method was employed to separate free AAP from antibody-bound AAP. The minimum detectable dose of AAP was 0.2 pmol. In the assay system, immunoreactivity was shown not to be attributable to fragments derived from AAP, gelatin or collagen peptides, which sequences differ only by one amino acid from those of AAP. Immunoreactive (IR) AAP extracted from rat tissues and serum gave parallel dose-response curves to those of AAP. Thus, the AAP equivalents per g or ml of tissues measured in adult male rats were 203 pmol in heart, 166 pmol in kidney, 4 pmol in serum, and 2 to 6 pmol in the other tissues. IR-AAP was separated into two fractions by Sephadex G-25 chromatography. Fr. I was considered to have a larger molecular mass than AAP, while Fr. II...