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Tohru Kimura

Publications and source records attributed to Tohru Kimura.

At least 19 recordsLinked to original sources
PubMed2006

PGC7/Stella protects against DNA demethylation in early embryogenesis.

DNA methylation is an important means of epigenetic gene regulation and must be carefully controlled as a prerequisite for normal early embryogenesis. Although global demethylation occurs soon after fertilization, it is not evenly distributed throughout the genome. Genomic imprinting and epigenetic asymmetry between parental genomes, that is, delayed demethylation of the maternal genome after fertilization, are clear examples of the functional importance of DNA methylation. Here, we show that PGC7/Stella, a maternal factor essential for early development, protects the DNA methylation state of several imprinted loci and epigenetic asymmetry. After determining that PGC7/Stella binds to Ran binding protein 5 (RanBP5; a nuclear transport shuttle protein), mutant versions of the two proteins were used to examine exactly when and where PGC7/Stella functions within the cell. It is likely that PGC7/Stella protects the maternal genome from demethylation only after localizing to the nucleus, where it maintains the methylation of several imprinted genes. These results demonstrate that PGC7/Stella is indispensable for the maintenance of methylation involved in epigenetic reprogramming after fertilization.

Animals↗
PubMed2006

The stabilization of beta-catenin leads to impaired primordial germ cell development via aberrant cell cycle progression.

Primordial germ cells (PGCs) are germ cell precursors that are committed to sperm or oocytes. Dramatic proliferation during PGC development determines the number of founder spermatogonia and oocytes. Although specified to a germ lineage, PGCs produce pluripotent embryonic germ (EG) cells in vitro and testicular teratomas in vivo. Wnt/beta-catenin signaling regulates pluripotency and differentiation in various stem cell systems, and dysregulation of this signaling causes various human cancers. Here, we examined the role of Wnt/beta-catenin signaling in PGC development. In normal PGC development, Wnt/beta-catenin signaling is suppressed by the GSK3beta-mediated active degradation of beta-catenin and the low expression of canonical Wnt molecules. The effects of aberrant activation of Wnt/beta-catenin signaling in PGCs were analyzed using mice carrying a deletion of the exon that encodes the GSK3beta phosphorylation sites in the beta-catenin locus. Despite the potential activity of Wnt/beta-catenin signaling in stem cell maintenance and carcinogenesis in various cell lineages, teratomas were not induced in the mice expressing the nuclear-localized beta-catenin in PGCs. Instead, the mutant mice showed germ cell deficiency caused by the delayed cell cycle progression of the proliferative phase PGCs. Our results show that the suppression of Wnt/beta-catenin signaling is a prerequisite for the normal development of PGCs.

Animals↗
PubMed2005

Differential effects of GATA-1 on proliferation and differentiation of erythroid lineage cells.

The zinc finger transcription factor GATA-1 is essential for both primitive (embryonic) and definitive (adult) erythropoiesis. To define the roles of GATA-1 in the production and differentiation of primitive and definitive erythrocytes, we established GATA-1-null embryonic stem cell lines in which GATA-1 was able to be conditionally expressed by using the tetracycline conditional gene expression system. The cells were subjected to hematopoietic differentiation by coculturing on OP9 stroma cells. We expressed GATA-1 in the course of primitive and definitive erythropoiesis and analyzed the ability of GATA-1 to rescue the defective erythropoiesis caused by the GATA-1 null mutation. Our results show that GATA-1 functions in the proliferation and maturation of erythrocytes in a distinctive manner. The early-stage expression of GATA-1 during both primitive and definitive erythropoiesis was sufficient to promote the proliferation of red blood cells. In contrast, the late-stage expression of GATA-1 was indispensable to the terminal differentiation of primitive and definitive erythrocytes. Thus, GATA-1 affects the proliferation and differentiation of erythrocytes by different mechanisms.

Anti-Bacterial Agents↗
PubMed2004

Mili, a mammalian member of piwi family gene, is essential for spermatogenesis.

The piwi family genes, which are defined by conserved PAZ and Piwi domains, play important roles in stem cell self-renewal, RNA silencing, and translational regulation in various organisms. To reveal the function of the mammalian homolog of piwi, we produced and analyzed mice with targeted mutations in the Mili gene, which is one of three mouse homologs of piwi. Spermatogenesis in the MILI-null mice was blocked completely at the early prophase of the first meiosis, from the zygotene to early pachytene, and the mice were sterile. However, primordial germ cell development and female germ cell production were not disturbed. Furthermore, MILI bound to MVH, which is an essential factor during the early spermatocyte stage. The similarities in the phenotypes of the MILI- and MVH-deficient mice and in the physical binding properties of MILI and MVH indicate a functional association of these proteins in post-transcriptional regulation. These data indicate that MILI is essential for the differentiation of spermatocytes.

Animals↗
PubMed2004

Contribution of oligosaccharides to protection of the H,K-ATPase beta-subunit against trypsinolysis.

The proton-pumping H+,K+-adenosinetriphosphatase (H,K-ATPase), responsible for acid secretion by the gastric parietal cell, faces a harshly acidic environment, with some pepsin from neighboring chief cells, at its luminal surface. Its large catalytic alpha-subunit is mostly oriented cytoplasmically. The smaller beta-subunit (HKbeta), is mainly extracellular, with one transmembrane domain and a small cytoplasmic domain. Seven N-linked oligosaccharides in the extracellular domain of HKbeta are thought to contribute to protection of the H,K-ATPase, since previous work has shown that their complete removal, by peptide N-glycosidase F (PNGase F), greatly increased susceptibility of HKbeta to proteolysis. The possibility of graded protection by different numbers of oligosaccharides was investigated here with the use of mutant HKbeta cDNA, having various N-glycosylation sites mutated (Asn to Gln), transfected into HEK-293 cells. Membrane preparations, two days after transfection, were solubilized in 1% Triton X-100 and subjected to trypsinolysis (pH 8, 37 degrees C, trypsin:protein 1:10-1:25). Relative amounts of HKbeta remaining after 20 min trypsin were determined, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probing of Western blots with an antibody to the HKbeta extracellular domain, by chemiluminescent development of blots and densitometry of resulting films. Maturely glycosylated HKbeta was made significantly more susceptible to trypsin than wild type when at least five oligosaccharides were deleted, while the high-mannose form (pre-beta), from the endoplasmic reticulum, became significantly more susceptible than wild-type pre-beta with removal of only two or more oligosaccharides. For each mutant, and wild type, pre-beta was consistently more susceptible than the mature form. While the number, and kind, of oligosaccharides seem to affect protection for HKbeta against trypsinolysis, other aspects of protein maturation, including proper folding of peptide domains and possible subtle alterations of conformation during Golgi processing, are also likely to contribute to this protection.

Adenosine Triphosphatases↗
PubMed2004

Q-switched ruby laser irradiation on spotty pigmentation in the skin of the hairless dog.

OBJECTIVE: To investigate macroscopically and histopathologically the dermatological changes after Q-switched ruby laser (QRL) irradiation with different exposure doses in UVB-induced pigmentation in hairless dogs. METHODS: QRL irradiation with 3.0, 5.0 and 7.0 J/cm(2) was carried out on the UVB-induced spotty pigmentation in the skin of the hairless dog. Gross appearance was observed daily throughout this study. Histopathological examination was performed 1 day before QRL irradiation and 1 and 3 days and 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14 and 16 weeks after QRL irradiation. RESULTS: Immediately after QRL irradiation, spotty pigmentation was removed. One week after QRL irradiation, re-epithelialization started from the margin of the irradiated sites. between 5 and 10 weeks after QRL irradiation, the skin color returned to normal and some portions showed recurrence of hyperpigmentation. Histopathologically, spotty pigmentation had a heavy deposition of melanin granules in the stratum basale, spinosum and corneum. One day after QRL irradiation, the skin showed destruction of melanin granules. Seven days after QRL irradiation, re-epithelialization began from the surroundings of the QRL-irradiated sites and the pilosebaceous units. The delayed process of re-epithelialization was dependent on the incident exposure dose with QRL. The repaired epidermis was devoid of melanin granules. By 5 weeks after QRL irradiation with 3.0 and 5.0 J/cm(2), the stratum basale and spinosum revealed a redistribution of melanin granules. In the sites of recurrent hyperpigmentation, the bases of the remaining hair follicles showed a notable increase in the reproduction of melanin granules. Melanin granules abundantly aggregated in the bottom portion of the nucleus in each epidermal cell. CONCLUSION: These results revealed that hairless dogs were invaluable laboratory animals, which developed spotty pigmentation after successive UVB irradiation. In addition, UVB-induced spotty pigmentation in hairless dogs is useful for investigating the process of depigmentory treatment with QRL irradiation and recurrence of this lesion.

Animals↗
PubMed2004

Depigmentation and rejuvenation effects of kinetin on the aged skin of hairless descendants of Mexican hairless dogs.

The depigmenting and anti-aging effects of kinetin (KN) solutions on the aged skin of hairless dogs were clinically and histologically investigated. Grossly, all KN-treated sites became mildly depigmented. At 50 days of topical treatment with KN solutions, apparent improvement was observed in the skin texture, wrinkling, and pigmentation. At 100 days of KN treatment, both the skin rejuvenation and depigmentation effects became more prominent. Throughout the experimental period of KN treatment, no adverse effects were found in any sites treated with KN solutions. In the colorimetric system, at 100 days of topical treatment with KN solutions, the L* and b* values in the sites treated with KN solutions significantly increased. Histologically, at 50 days of topical treatment with KN solutions, the KN-treated sites showed a decrease in the thickness of the corneal layers. Melanin granules decreased throughout all epidermal layers. In the dermis, the large number of fine collagen and elastic fibers were densely aligned. At the end of the treatment, this agent was equally effective against pigmented lesions irrespective of the concentration of KN solution. The distribution of melanin granules returned to normal in the skin of adult hairless dogs. Throughout the present study, there were no histologic abnormalities in the epidermis and the dermis. These results revealed that topical treatment with lower concentrations of KN solutions normalized hyperpigmentation and improved the aged skin structure of hairless dogs. In addition, it was clarified that KN solutions had no adverse effects on the skin of hairless dogs and that this agent was a safe chemical for long-term application.

Adenine↗
PubMed2004

Clinical and histopathological findings in pustular psoriaform dermatitis (pityriasis rosea) in pigs.

Three cases of pustular psoriaform dermatitis (pityriasis rosea) in pigs were clinically and histopathologically examined. Grossly, the affected skin was characterized by multiple, circumscribed lesions. Three pigs were the descendants derived from the same Landrace boar. Skin lesions expanded centrifugally to became ring-shaped plaques. There were no abnormal values in hematological and serum biochemical profiles. Histopathologically, the epidermis showed remarkable thickening. The dermal lesions were characterized by a prominent component of superficial and deep perivascular infiltration of eosinophils. Dilatation of microvasculature was accompanied with congested vessels. These results revealed that the etiology of pustular psoriaform dermatitis in pigs was associated with a hereditary predisposition derived from the specific boars. This dermatosis is histopathologically characterized by microcirculatory disturbances with infiltration of abundant eosinophils.

Animals↗
PubMed2004

Stage-specific expression of mouse germ cell-less-1 (mGCL-1), and multiple deformations during mgcl-1 deficient spermatogenesis leading to reduced fertility.

A mouse homologue of Drosophila germ cell-less, mouse germ cell-less-1 (mgcl-1), is highly expressed in the testis. Previous report revealed that the fertility of the mgcl-1(-/-) male mice is reduced significantly as a result of various morphological abnormalities in the sperm (Kimura et al., 2003). To elucidate the function of mgcl-1 in spermatogenesis, the expression of mGCL-1 in the wild-type testis was examined. Immunohistochemical studies demonstrated that mGCL-1 first appeared in the nuclei of the pachytene spermatocytes at stage VI of the seminiferous epithelium, and existed in those of spermatids until step 8 during spermatogenesis. mGCL-1 was not detectable after step 9 spermatids. The testicular cells and epididymal sperm were further analyzed morphologically using mgcl-1(-/-) mice. In the testis, deformed nuclei first occurred in the pachytene spermatocytes at stage VI, which is consistent with the time of the first appearance of the mGCL-1 protein in the wild-type testis. Abnormal nuclei and acrosomes were found in spermatids after step 5, and nuclei of the spermatids and epididymal sperm were frequently invaginated. In addition, variously deformed sperm such as bent-neck, multi-headed or multi-nucleated sperm were observed in the mgcl-1(-/-) cauda epididymidis. However, several key structures such as the acroplaxome marginal ring (Kierszenbaum et al., 2003), postacrosomal sheath, and posterior ring apparently formed. In addition, MN7 and MN13, essential substances for fertilization that are located in sperm heads, were detectable in the mgcl-1 null sperm. These observations provide important insights into the mechanisms regulating the nuclear architecture and causes of human infertility.

Animals↗
PubMed2003

Enhanced degradation of MDM2 by a nuclear envelope component, mouse germ cell-less.

A mouse homologue of Drosophila germ cell less, mouse germ cell less-1 (mgcl-1), encodes a nuclear envelope component essential for nuclear integrity. To analyze the molecular function of mGCL-1, we carried out two hybrid screening and found that mGCL-1 bound to the gene product of tumor susceptibility gene 101 (tsg101). Effects of mGCL-1 on the expression of MDM2-p53 axis were examined, since TSG101 has been shown to elevate the amount of MDM2 by inhibiting the ubiquitination. mGCL-1 significantly reduced the amount of MDM2 probably by changing the sub-cellular localization of the MDM2 and facilitating the ubiquitination of MDM2. In addition, the amount of p53 was increased and transactivation by p53 was enhanced by mGCL-1. Thus, mGCL-1 turned out to be a factor modulating MDM2-p53 axis by enhanced degradation of MDM2.

Animals↗
PubMed2003

Quantity and quality control of gastric proton pump in the endoplasmic reticulum by ubiquitin/proteasome system.

The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the noncatalytic beta-subunit. These subunits are assembled in the endoplasmic reticulum (ER) and leave the ER to reach to the cell surface as a functional holoenzyme. We studied the quantity control mechanism of the H(+),K(+)-ATPase in the ER by using a heterologous expression system in human embryonic kidney 293 cells. The alpha-subunit in the alpha-expressing cells was degraded more rapidly than in the alpha+beta-expressing cells. It was stabilized, however, in the presence of a proteasome inhibitor, lactacystin. Polyubiquitination of the alpha-subunit was observed in the alpha-expressing cells as well as in the alpha+beta-expressing cells. The extent of polyubiquitination was higher in the former alpha-expressing cells especially in the presence of lactacystin. On the other hand, polyubiquitination of the beta-subunit was not observed in the absence and presence of lactacystin. When the alpha-subunit was coexpressed with a mutant beta-subunit that lacks alpha/beta assembly capacity, degradation of the alpha-subunit was accelerated in parallel with increased polyubiquitination of the alpha-subunit. These results indicate that the ubiquitin/proteasome system is involved in degradation of the unassembled alpha-subunits in the ER to control the cell surface expression of the functional alpha/beta holoenzymes.

Cell Line↗
PubMed2003

pH of TGN and recycling endosomes of H+/K+-ATPase-transfected HEK-293 cells: implications for pH regulation in the secretory pathway.

The influences of the gastric H+/K+ pump on organelle pH during trafficking to and from the plasma membrane were investigated using HEK-293 cells stably expressing the alpha- and beta-subunits of human H+/K+-ATPase (H+/K+-alpha,beta cells). The pH values of trans-Golgi network (pHTGN) and recycling endosomes (pHRE) were measured by transfecting H+/K+-alpha,beta cells with the pH-sensitive GFP pHluorin fused to targeting sequences of either TGN38 or synaptobrevin, respectively. Immunofluorescence showed that H+/K+-ATPase was present in the plasma membrane, TGN, and RE. The pHTGN was similar in both H+/K+-alpha,beta cells (pHTGN 6.36) and vector-transfected ("mock") cells (pHTGN 6.34); pHRE was also similar in H+/K+-alpha,beta (pHRE 6.40) and mock cells (pHRE 6.37). SCH28080 (inhibits H+/K+-ATPase) caused TGN to alkalinize by 0.12 pH units; subsequent addition of bafilomycin (inhibits H+ v-ATPase) caused TGN to alkalinize from pH 6.4 up to a new steady-state pHTGN of 7.0-7.5, close to pHcytosol. Similar results were observed in RE. Thus H+/K+-ATPases that trafficked to the plasma membrane were active but had small effects to acidify the TGN and RE compared with H+ v-ATPase. Mathematical modeling predicted a large number of H+ v-ATPases (8000) active in the TGN to balance a large, passive H+ leak (with PH approximately 10-3 cm/s) via unidentified pathways out of the TGN. We propose that in the presence of this effective, though inefficient, buffer system in the Golgi and TGN, H+/K+-ATPases (estimated to be approximately 4000 active in the TGN) and other transporters have little effect on luminal pH as they traffic to the plasma membrane.

Acid-Base Equilibrium↗
PubMed2003

Ion pump-interacting proteins: promising new partners.

The sorting and regulation of the Na,K and H,K-ATPases requires that the pump proteins must associate, at least transiently, with kinases, phosphatases, scaffolding molecules, and components of the cellular trafficking machinery. The identities of these interacting proteins and the nature of their associations with the pump polypeptides have yet to be elucidated. We have begun a series of yeast two-hybrid screens employing structurally defined segments of pump polypeptides as baits in order to gain insight into the nature and function of these interacting proteins.

Animals↗
PubMed2003

Mouse germ cell-less as an essential component for nuclear integrity.

A mouse homologue of the Drosophila melanogaster germ cell-less (mgcl-1) gene is expressed ubiquitously, and its gene product is localized to the nuclear envelope based on its binding to LAP2 beta (lamina-associated polypeptide 2 beta). To elucidate the role of mgcl-1, we analyzed two mutant mouse lines that lacked mgcl-1 gene expression. Abnormal nuclear morphologies that were probably due to impaired nuclear envelope integrity were observed in the liver, exocrine pancreas, and testis. In particular, functional abnormalities were observed in testis in which the highest expression of mgcl-1 was detected. Fertility was significantly impaired in mgcl-1-null male mice, probably as a result of severe morphological abnormalities in the sperm. Electron microscopic observations showed insufficient chromatin condensation and abnormal acrosome structures in mgcl-1-null sperm. In addition, the expression patterns of transition proteins and protamines, both of which are essential for chromatin remodeling during spermatogenesis, were aberrant. Considering that the first abnormality during the process of spermatogenesis was abnormal nuclear envelope structure in spermatocytes, the mgcl-1 gene product appears to be essential for appropriate nuclear-lamina organization, which in turn is essential for normal sperm morphogenesis and chromatin remodeling.

Animals↗
PubMed2003

Conditional loss of PTEN leads to testicular teratoma and enhances embryonic germ cell production.

The tumor suppressor gene PTEN, which is frequently mutated in human cancers, encodes a lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3] and antagonizes phosphatidylinositol 3 kinase. Primordial germ cells (PGCs), which are the embryonic precursors of gametes, are the source of testicular teratoma. To elucidate the intracellular signaling mechanisms that underlie germ cell differentiation and proliferation, we have generated mice with a PGC-specific deletion of the Pten gene. Male mice that lacked PTEN exhibited bilateral testicular teratoma, which resulted from impaired mitotic arrest and outgrowth of cells with immature characters. Experiments with PTEN-null PGCs in culture revealed that these cells had greater proliferative capacity and enhanced pluripotent embryonic germ (EG) cell colony formation. PTEN appears to be essential for germ cell differentiation and an important factor in testicular germ cell tumor formation.

Animals↗
PubMed2002

Mutational study on the roles of disulfide bonds in the beta-subunit of gastric H+,K+-ATPase.

The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the non-catalytic beta-subunit. Correct assembly between the alpha- and beta-subunits is essential for the functional expression of H(+),K(+)-ATPase. The beta-subunit contains nine conserved cysteine residues; two are in the cytoplasmic domain, one in the transmembrane domain, and six in the ectodomain. The six cysteine residues in the ectodomain form three disulfide bonds. In this study, we replaced each of the cysteine residues of the beta-subunit with serine individually and in several combinations. The mutant beta-subunits were co-expressed with the alpha-subunit in human embryonic kidney 293 cells, and the role of each cysteine residue or disulfide bond in the alpha/beta assembly, stability, and cell surface delivery of the alpha- and beta-subunits and H(+),K(+)-ATPase activity was studied. Mutant beta-subunits with a replacement of the cytoplasmic and transmembrane cysteines preserved H(+),K(+)-ATPase activity. All the mutant beta-subunits with replacement(s) of the extracellular cysteines did not assemble with the alpha-subunit, resulting in loss of H(+),K(+)-ATPase activity. These mutants did not permit delivery of the alpha-subunit to the cell surface. Therefore, each of these disulfide bonds of the beta-subunit is essential for assembly with the alpha-subunit and expression of H(+),K(+)-ATPase activity as well as for cell surface delivery of the alpha-subunit.

Cell Line↗