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Biomedical subjects

Y Kitamura

Publications and source records attributed to Y Kitamura.

At least 415 records · Page 23Linked to original sources
PubMed1994

1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells.

Insulin stimulation drives the formation of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and 1-phosphatidylinositol 3-kinase (PI 3-kinase; ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase, EC 2.7.1.137), a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits. This interaction takes place via the phosphorylated YMXM motifs of IRS-1 and the Src homology region 2 (SH2) domains of p85. In this study, the stable overexpression in a Chinese hamster ovary (CHO) cell line of a mutant p85 alpha (delta p85) protein, which lacks a binding site for p110, disrupted the complex formation between IRS-1 and the catalytic subunit of PI 3-kinase in intact cells during insulin stimulation. Activation of insulin receptor kinase and the tyrosine phosphorylation of IRS-1 remained unaffected. In this cell line, both insulin-stimulated accumulation of phosphatidylinositol 3,4,5-trisphosphate and the insulin-stimulated glucose uptake due to the translocation of GLUT1 glucose transporters were markedly impaired, whereas neither phorbol 12-myristate 13-acetate-stimulated glucose uptake nor the insulin-stimulated activation of RAS was impaired. These results suggest that PI 3-kinase is required for glucose transport in insulin signaling in CHO cells.

Animals↗
PubMed1994

A complex of GRB2-dynamin binds to tyrosine-phosphorylated insulin receptor substrate-1 after insulin treatment.

Insulin drives the formation of a complex between tyrosine-phosphorylated IRS-1 and SH2-containing proteins. The SH2-containing protein Grb2 also possesses adjacent SH3 domains, which bind the Ras guanine nucleotide exchange factor Sos. In this report, we examined the involvement of another SH3 binding protein, dynamin, in insulin signal transduction. SH3 domains of Grb2 as GST fusion proteins bound dynamin from lysates of CHO cells expressing wild-type insulin receptor (IR) (CHO-IR cells) in a cell-free system (in vitro). Immunoprecipitation studies using specific antibodies against Grb2 revealed that Grb2 was co-immunoprecipitated with dynamin from unstimulated CHO-IR cells. After insulin treatment of CHO-IR cells, anti-dynamin antibodies co-immunoprecipitated the IR beta-subunit and IRS-1, as tyrosine-phosphorylated proteins and PI 3-kinase activity. However, purified rat brain dynamin did not bind directly to either the IR, IRS-1 or the p85 subunit of PI 3-kinase in vitro. Together, these results suggest that in CHO-IR cells, insulin stimulates the binding of dynamin to tyrosine-phosphorylated IRS-1 via Grb2 and that IRS-1 also associates with PI 3-kinase in response to insulin. This complex formation was reconstituted in vitro using recombinant baculovirus-expressed IRS-1, GST-Grb2 fusion proteins and dynamin peptides containing proline-rich sequences. Furthermore, dynamin GTPase activity was found to be stimulated when an IRS-1-derived phosphopeptide, containing the Grb2 binding site, was added to the dynamin-Grb2 complex in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptor Proteins, Signal Transducing↗
PubMed1994

Distribution of targets for avian retrovirus DNA integration in vivo.

The targeting of DNA integration in retrovirus-infected cells is a central yet very poorly understood aspect of the biology of the virus. To investigate this problem, we have assessed the use of specific sites for integration targets of avian leukosis virus (ALV) DNA within defined regions of turkey embryo fibroblast (TEF) cellular DNA. For this purpose, we developed an assay of sufficient sensitivity and specificity to allow detection and location of single integration events in a population of 5 million cells. Targets selected for study were either regions cloned by virtue of a previous integration event or clones chosen at random from cellular DNA. By use of this approach, we found that all genomic regions tested contained integration targets, with a frequency that varied from approximately 0.2 to 4 times that expected for random integration. Within regions, the frequency of use of specific sites varied considerably, with some sites used up to 280 times random frequency. When one region was introduced into cells at moderately high copy number by transfection, it provided integration targets in a pattern very much like that seen with the same sequence in vitro. On the basis of our sampling, we conclude that most or all regions of the TEF genome are accessible to ALV retroviral integration. As with integration in vitro, integration specificity seems to be determined largely by local structural features rather than accessibility of specific regions.

Animals↗
PubMed1994

C-kit gene is expressed by skin mast cells in embryos but not in puppies of Wsh/Wsh mice: age-dependent abolishment of c-kit gene expression.

The Wsh is a mutant allele at the W (c-kit) locus of mice, but no significant abnormalities are found at the coding region of the Wsh allele. Since cultured mast cells derived from the spleen of Wsh/Wsh mice do not express messenger RNA (mRNA) of c-kit, we studied the interrelation between the number of mast cells and the magnitude of c-kit mRNA expression in the skin of Wsh/Wsh mice of various ages. The number of mast cells in the skin of Wsh/Wsh embryos of 18 days postcoitum (pc) was approximately 40% that of normal control (+/+) embryos, but the number of mast cells decreased exponentially after birth; the number dropped to 0.6% that of +/+ mice at day 150 after birth. A weak but apparent signal of c-kit mRNA was detectable in the skin of 18-day pc Wsh/Wsh embryos by RNase protection assay but not in the skin of 5-day-old Wsh/Wsh mice. The number of c-kit protein-containing cells was significantly greater in the skin of 18-day pc Wsh/Wsh embryos than in the skin of 5-day-old Wsh/Wsh mice. The abolishment of c-kit mRNA expression appeared to be specific, because the expression of mast cell carboxypeptidase A mRNA but not of c-kit mRNA was detectable by in situ hybridization in skin mast cells of 5-day-old Wsh/Wsh mice. Taken together, the expression of c-kit mRNA was abolished first, then the content of c-kit protein dropped to undetectable levels, and then the disappearance of Wsh/Wsh mast cells themselves followed.

Age Factors↗
PubMed1994

Expression of neurotrophin genes in the brain of senescence-accelerated mouse (SAM) during postnatal development.

We compared the expression patterns of neurotrophin genes in the brain of senescence-accelerated mouse (SAMP8) which shows age-related impairment of learning behavior, with SAMR1 control which shows normal aging. By Northern blot analysis, NT-3 mRNA levels in the cortex were higher in SAMP8 than in SAMR1 mice during development, whereas in the midbrain, hippocampus and forebrain, NT-3 expression levels in SAMP8 were lower than those in SAMR1. At early stages, although NGF mRNA levels in SAMP8 were lower than those in SAMR1, BDNF mRNA levels were almost equivalent in both strains. By in situ hybridization analysis, NT-3 mRNA signals in the CA1 and CA2 regions in SAMP8 were shown to be reduced at early stages. However, BDNF mRNA signals were almost equivalent in both SAMR1 and SAMP8.

Aging↗
PubMed1994

Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling.

Insulin, IGF-1 or EGF induce membrane ruffling through their respective tyrosine kinase receptors. To elucidate the molecular link between receptor activation and membrane ruffling, we microinjected phosphorylated peptides containing YMXM motifs or a mutant 85 kDa subunit of phosphoinositide (PI) 3-kinase (delta p85) which lacks a binding site for the catalytic 110 kDa subunit of PI 3-kinase into the cytoplasm of human epidermoid carcinoma KB cells. Both inhibited the association of insulin receptor substrate-1 (IRS-1) with PI 3-kinase in a cell-free system and also inhibited insulin- or IGF-1-induced, but not EGF-induced, membrane ruffling in KB cells. Microinjection of nonphosphorylated analogues, phosphorylated peptides containing the EYYE motif or wild-type 85 kDa subunit (Wp85), all of which did not inhibit the association of IRS-1 with PI 3-kinase in a cell-free system, did not inhibit membrane ruffling in KB cells. In addition, wortmannin, an inhibitor of PI 3-kinase activity, inhibited insulin- or IGF-1-induced membrane ruffling. These results suggest that the association of IRS-1 with PI 3-kinase followed by the activation of PI 3-kinase are required for insulin- or IGF-1-induced, but not for EGF-induced, membrane ruffling.

Amino Acid Sequence↗
PubMed1994

The glycinin box: a soybean embryo factor binding motif within the quantitative regulatory region of the 11S seed storage globulin promoter.

The soybean embryo factor binding sequence in the glycinin A2B1a gene promoter was delimited to an A/T-rich 9 bp sequence, 5'-TAATAATTT-3', designated as the glycinin box, by DNA footprinting and gel mobility shift assay using synthetic oligonucleotides. It was shown that the interaction with the factor takes place at a defined DNA sequence rather than at random A/T-rich sequence blocks in the glycinin 5' flanking region. There are four glycinin boxes in the quantitative regulatory region between positions -545 and -378 of the glycinin A2B1a promoter. Multiple nonamer motifs similar to the glycinin box were also found in the equivalent regions of other glycinin and legumin promoters, suggesting that they must be conserved as a binding site for the embryo factor that activates the differential and stage-specific expression of seed 11S globulin genes in leguminous plants.

Base Sequence↗
PubMed1994

Ligand-independent activation of c-kit receptor tyrosine kinase in a murine mastocytoma cell line P-815 generated by a point mutation.

The c-kit proto-oncogene encodes a receptor tyrosine kinase that is known to play a crucial role in hematopoiesis, especially in mast cell growth and differentiation. Although a number of dominant loss-of-function mutations of c-kit gene have been well characterized in mice, rats, and humans, little is known about the c-kit mutations contributing to ligand-independent activation of the c-kit receptor tyrosine kinase (KIT). In a murine mastocytoma cell line, P-815, KIT has been found to be constitutively phosphorylated on tyrosine and activated in a ligand-independent manner. Sequencing of the whole coding region of c-kit cDNA showed that c-kit cDNA of P-815 cells carries a point mutation in codon 814, resulting in amino acid substitution of Tyr for Asp. Murine wild-type c-kit cDNA and mutant-type c-kit cDNA encoding Tyr in codon 814 were expressed in cells of a human embryonic kidney cell line, 293T. In the transfected cells, mutant-form KITTyr814 was strikingly phosphorylated on tyrosine and activated in immune complex kinase reaction regardless of stimulation with a ligand for KIT (stem cell factor), whereas tyrosine phosphorylation and activation was barely detectable in wild-form KIT. The data presented here provide evidence for a novel activating mutation of c-kit gene that might be involved in neoplastic growth or oncogenesis of some cell types, including mast cells.

Amino Acid Sequence↗
PubMed1994

Buspirone enhances head twitch behavior in mice.

We studied the effects of buspirone, a 5-HT1A receptor agonist, on head twitch behavior induced by 5-hydroxy-L-tryptophan (5-HTP) administered together with pargyline in mice. Buspirone dose dependently (0.1-10 mg/kg i.p.) enhanced head twitch behavior. This effect was blocked by (-)-propranolol and NAN-190 (1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]-piperazine hydrobromide). The enhancing effect of buspirone was also observed when mice were pretreated with p-chlorophenylalanine. These findings suggest that the enhancing effect of buspirone on head twitch behavior may be exerted through the activation of post-synaptic 5-HT1A receptors.

5-Hydroxytryptophan↗
PubMed1994

Demonstration of retinal cells expressing messenger RNAs of the c-kit receptor and its ligand.

We found cells expressing c-kit receptor and its ligand (Sl factor, SLF) in the retina of rats and mice. c-kit messenger RNA (mRNA) was detected in a limited number of cells in the inner nuclear layer of rats and mice by in situ hybridization. The c-kit-expressing cells were assumed to be a subpopulation of the amacrine cells on the basis of their size and distribution pattern. c-kit protein-containing cells were demonstrated in the mouse retina by using ACK2 monoclonal antibody against the extracellular domain of the mouse c-kit receptor. The c-kit protein was detected in cell bodies and processes of the presumed amacrine cells. Hybridization signals for SLF mRNA were observed in the retinal ganglion cells. The results suggest that the c-kit receptor and its ligand may play some roles in the formation of junctions between the amacrine and retinal ganglion cells.

Animals↗
PubMed1994

Transient and localized expression of bone morphogenetic protein 4 messenger RNA during fracture healing.

Temporal and spatial distribution of a gene encoding murine bone morphogenetic protein 4 (mBMP-4) during fracture repair were investigated in mice by RT-PCR and in situ hybridization. For in situ hybridization, fractured ribs and surrounding tissues were decalcified and hybridized with a mBMP-4-specific complementary RNA probe labeled with digoxigenin-11 UTP. mBMP-4 messenger RNA (mRNA) was not detected in ribs without fracture, whereas it was detected only in the early phase of fracture from 12 to 72 h after the onset of fracture before new cartilage or bone formation. The mBMP-4 mRNAs were present in cells distributed in three distinct regions, namely, the proliferating periosteum, the medullary cavity, and the muscles near the fracture site. These BMP-4-positive cells did not express bone gla protein mRNA, which is a marker of the mature osteogenic cell. RT-PCR also showed a transient increase in the level of BMP-4 mRNA in the early phase of fracture repair. The findings provide us with some new information. (1) The BMP-4 gene is produced by less differentiated osteoprogenitor cells, not by differentiated osteoblasts. (2) The BMP-4 gene is enhanced by the impact of fracture and localized in callus-forming tissue before callus formation. Together with the activities of BMP-4, as was previously described, our results suggest that newly produced BMP-4 gene product is one of the local contributing factors in callus formation in the early phase of fracture healing.

Animals↗
PubMed1994

Localization of the mRNA for bone matrix proteins during fracture healing as determined by in situ hybridization.

The expression of the mRNAs for osteonectin (ON), osteopontin (OPN), osteocalcin (OC), and matrix Gla protein (MGP) was studied by in situ hybridization during the healing process of an experimental fracture in adult rat femora. At day 1 postoperatively, ON mRNA was detected in the proliferating periosteum. At day 3, ON, OPN, and OC mRNAs were detected in woven bone. From day 5, MGP and ON mRNAs were detected in the immature chondrocytes. From day 7, ON, OPN, and OC mRNAs were detected in the osteoblastic cells in newly formed endosteal trabecular bone. OPN mRNA was also detected in some of the osteocytes in trabecular bone. From day 14, OPN and MGP mRNAs were detected in newly formed periosteal hypertrophic chondrocytes, and the ON, OPN, and OC mRNAs were detected in osteoblastic cells in newly formed periosteal trabecular bone. Although the cell types that expressed each mRNA in fractured bones were similar to those in embryonic bones, the time course of these mRNA expression in fractured bones was different from that in embryonic bones. We considered that this system is useful to investigate the phenotypic change in osteogenic and chondrogenic lineage cells that appears during fracture healing at the molecular level.

Animals↗
PubMed1994

Expression of mRNA of murine bone-related proteins in ectopic bone induced by murine bone morphogenetic protein-4.

To determine whether a system of ectopic bone formation induced by osteosarcoma-derived bone-inducing substance (bone morphogenetic protein-4) can be used as a model of developing bone at the molecular level, we studied the expression of bone-related protein mRNAs in the process of ectopic bone formation using non-radioisotopic in situ hybridization. Osteonectin mRNA was detected in fibroblast-like cells, which are similar to periosteal cells from the early to middle stages of bone development. The proportion of osteonectin mRNA-expressing cells was greater than that of osteopontin mRNA-expressing cells in hypertrophic chondrocytes and osteoblast-like cells. In contrast, osteopontin mRNA was localized in a limited population of hypertrophic chondrocytes, a single layer of osteoblast-like cells adjacent to the bone trabeculae in the middle stage of bone formation, and in a limited subset of osteocytes in the late stage. A strong osteocalcin mRNA signal was detected in osteoblast-like cells from the middle to late stages and in a limited subset of osteocytes in the late stage of bone development. Since the sequential gene expression pattern of bone-related proteins in the present system is comparable to that in embryonic osteogenesis, this system may be useful as a model for studying gene expression in osteogenesis.

Animals↗
PubMed1994

The enucleation of thyroid tumors indeterminate before surgery as papillary thyroid carcinoma: should immediate reoperation be performed?

The management of patients with thyroid nodules which are indeterminate as papillary thyroid carcinoma (PTC) before surgery but identified as such after surgery still poses great difficulty for the surgeon. Immediate reoperation is often not performed due to the indolent biological characteristics of this disease and the increased risk of associated surgical complications. We conducted a study to determine whether or not immediate reoperation should be performed. The prognosis of 91 patients with PTC who underwent enucleation only was compared with that of 321 control patients with non-advanced PTC who underwent radical surgery during the same period. There were no deaths due to PTC in either group. Distant metastasis was observed in 1.1% and 0.6% of the enucleation and control patients, respectively, while local recurrence was observed in 3.3% and 8.1%, respectively. These results suggest that immediate reoperation is not necessary for patients who have undergone enucleation only.

Adolescent↗
PubMed1994

Hemiaplasia of the thyroid associated with Graves' disease: report of three cases and a review of the literature.

We recently treated three additional patients with hemiaplasia of the thyroid associated with Graves' disease, making a total of eight such cases. All eight of these patients were women and their chief complaints were goiter in five cases, whereas exophthalmos or palpebral edema were noted in six cases. All eight patients underwent surgery for Graves' disease. The left lobe was absent in six cases and the right lobe in two, whereas the isthmus was absent in six cases and the pyramidal lobe in four. A total of 102 cases of hemiaplasia of the thyroid, including our present three cases, have been reported in the world literature since 1970, with 32 of them consisting of hemiaplasia associated with hyperthyroidism. Of these, the cause of hyperthyroidism was Graves' disease in 22 cases, an autonomously functioning thyroid nodule in 7, and thyrotoxic multinodular goiter in 3. The left lobe was absent in 19 cases while the right lobe was missing in 12, and laterality was unknown in 1 case.

Adolescent↗
PubMed1994

Suppressive effect of FK-506, a novel immunosuppressant, against MPTP-induced dopamine depletion in the striatum of young C57BL/6 mice.

Systemic injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to damage the dopaminergic nigrostriatal system in C57BL/6 mice. We have investigated the effects of immunosuppressants, FK-506 and cyclosporin A (CsA), on MPTP-induced dopamine (DA) depletion in the striatum of young C57BL/6 mice. 10 days after MPTP treatment (25 mg/kg i.p. given daily, 5 days), DA in the striatum was depleted by 80%. However, pretreatment with FK-506, a novel immunosuppressant, significantly protected MPTP-induced DA depletion in the striatum, but FK-506 itself did not affect the DA content. CsA, another immunosuppressant, also protected MPTP-induced DA depletion. From these results can be seen that immunosuppressants seem to inhibit MPTP neurotoxicity toward nigrostriatal dopaminergic neurons of young C57BL/6 mice.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1994

Expression and localization of gamma-aminobutyric acid A (GABAA) receptor alpha 1 subunit and L-glutamate decarboxylase (GAD) mRNAs in rat retina: an analysis by in situ hybridization.

The localization of the mRNAs encoding gamma-aminobutyric acidA receptor alpha 1 subunit (GABAA alpha 1) and L-glutamate decarboxylase (GAD) was elucidated in the rat retina by in situ hybridization. Soma diameter analysis of signal positive cells in the ganglion cell layer demonstrated that a subpopulation including alpha-cells of retinal ganglion cells expressed GABAA alpha 1 mRNA and a subpopulation of ganglion cells smaller than alpha-cells expressed GAD mRNA.

Animals↗