Biomedical subjects

K Fukasawa

Publications and source records attributed to K Fukasawa.

At least 55 records · Page 3Linked to original sources

PubMed1997

Genomic instability and apoptosis are frequent in p53 deficient young mice.

The loss of p53 tumor suppressor functions results in genetic instability, characteristically associated with changes in chromosome ploidy and gene amplification. In vivo, we find that cells from various organs of 4 to 6-week old p53-nullizygous (p53-/-) mice display aneuploidy and frequent gene amplification as well as evidence for apoptosis. Regardless of tissue types, many p53-/- cells contain multiple centrosomes and abnormally formed mitotic spindles. Thus, chromosome instability in vivo may be associated with abnormal centrosome amplification. Moreover, we observed a significant increase in the number of cells overexpressing c-Myc in p53-/- mice. Consistent with previous studies showing that c-Myc overexpression is associated with gene amplification in vitro, many of the p53-/- cells exhibited, in the same cell, c-Myc overexpression and amplified c-myc, dihydrofolate reductase (DHFR), and carbamoyl-phosphate synthetase-aspartate transcarbamoyl-dihydroorotase (CAD) genes. Furthermore, apoptosis was frequently observed in cells isolated from p53-/- mice. The apoptotic cells contained abnormally amplified centrosomes, displayed aneuploidy, high levels of c-Myc expression, as well as gene amplification. These results indicate that a high number of aberrant cells is eliminated by p53-independent pathways in vivo.

Amino Acid Sequence↗

PubMed1997

Synergy between the Mos/mitogen-activated protein kinase pathway and loss of p53 function in transformation and chromosome instability.

Constitutive activation of mitogen-activated protein kinase (MAPK) is a property common to many oncoproteins, including Mos, Ras, and Raf, and is essential for their transforming activities. We have shown that high levels of expression of the Mos/MAPK pathway in Swiss 3T3 fibroblast cause cells in S phase to undergo apoptosis, while cells in G1 irreversibly growth arrest. Interestingly, cells in G2 and M phases also arrest at a G1-like checkpoint after proceeding through mitosis. These cells fail to undergo cytokinesis and are binucleated. Thus, constitutive overexpression of Mos and MAPK cannot be tolerated, and fibroblasts transformed by Mos express only low levels of the mos oncogene product. Here, we show that p53 plays a key role in preventing oncogene-mediated activation of MAPK. In the absence of p53 (p53-/-), the growth arrest normally observed in wild-type p53 (p53+/+) mouse embryo fibroblasts (MEFs) is markedly reduced. The mos transformation efficiency in p53-/- MEFs is two to three orders of magnitude higher than that in p53+/+ cells, and p53-/- cells tolerate > 10-fold higher levels of both Mos and activated MAPK. Moreover, we show that, like Mos, both v-ras and v-raf oncogene products induce apoptosis in p53+/+ MEFs. These oncogenes also display a high transforming activity in p53-/- MEFs, as does a gain-of-function MAPK kinase mutant (MEK*). Thus, the p53-dependent checkpoint pathway is responsive to oncogene-mediated MAPK activation in inducing irreversible G1 growth arrest and apoptosis. Moreover, we show that the chromosome instability induced by the loss of p53 is greatly enhanced by the constitutive activation of the Mos/MAPK pathway.

3T3 Cells↗

PubMed1997

Thoracic myelopathy due to intraspinal rheumatoid nodules.

A fifty-six-year-old woman with classical rheumatoid arthritis had subacute onset of paraparesis due to thoracic epidural rheumatoid nodules. Although plain radiograms and computed tomograms of the thoracic spine were negative except for old compression fractures, magnetic resonance imaging revealed thoracic spinal cord compression due to masses at multiple levels. There was a steady recovery after excision surgery.

Female↗

PubMed1996

Molecular cloning and expression of rat liver aminopeptidase B.

We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNAs, a cDNA clone with 2212 base pairs encoding aminopeptidase B (EC 3.4.11.6). The open reading frame encodes a 649-amino acid protein with a theoretical molecular mass of 72,545 Da and bears the consensus sequence of the zinc metalloexopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase A, aminopeptidase N, and leukotriene-A4 hydrolase. Escherichia coli SOLR cells infected with the pBluescript phagemid excised from the Uni-ZAP XR vector containing the aminopeptidase B cDNA had a high L-arginyl-beta-naphthylamidase activity. The recombinant protein was purified to homogeneity from the recombinant E. coli extracts. The enzyme had Cl--dependent aminopeptidase activity specifically restricted to the Arg and Lys derivatives and contained 1 mol of zinc per mol of the enzyme.

Amino Acid Sequence↗

PubMed1996

The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway.

Animals↗

PubMed1996

Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I.

Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block. Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturation-promoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturation-promoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These studies define important new functions for the Mos/MAPK cascade in mouse oocyte maturation and, under these conditions, reveal novel detail of the early stages of oocyte meiosis I.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Abnormal centrosome amplification in the absence of p53.

The centrosome plays a vital role in mitotic fidelity, ensuring establishment of bipolar spindles and balanced chromosome segregation. Centrosome duplication occurs only once during the cell cycle and is therefore highly regulated. Here, it is shown that in mouse embryonic fibroblasts (MEFs) lacking the p53 tumor suppressor protein, multiple copies of functionally competent centrosomes are generated during a single cell cycle. In contrast, MEFs prepared from normal mice or mice deficient in the retinoblastoma tumor suppressor gene product do not display these abnormalities. The abnormally amplified centrosomes profoundly affect mitotic fidelity, resulting in unequal segregation of chromosomes. These observations implicate p53 in the regulation of centrosome duplication and suggest one possible mechanism by which the loss of p53 may cause genetic instability.

Animals↗

PubMed1996

[A basic and clinical evaluation of a new immunoradiometric assay kit for human serum tissue polypeptide antigen (TPA)].

A new immunoradiometric assay kit (IRMA) of human serum tissue polypeptide antigen (TPA) based on combination monoclonal antibodies was evaluated. Using a new TPA-IRMA, the procedure of TPA measurement was faster and the range of measurement was more wide than a conventional TPA-IRMA. 89% (76/85 cases) of patients with malignant tumor and 96.4% (27/28) of patients with metastatic malignant tumor were positively detected. This assay of new TPA-M kit is sensitive to the level of serum TPA which is corresponding to a therapy. It is concluded that a new TPA-IRMA is very useful in monitoring and assessing malignant tumors.

Adult↗

PubMed1995

Mutagenic analysis of functional domains of the mos proto-oncogene and identification of the sites important for MAPK activation and DNA binding.

We constructed in-frame deletion/replacement mutations in the Xenopus mos proto-oncogene that lie within conserved Mos-specific codons, but outside of the regions that are conserved among the src kinase family of genes. All gene products were assayed in vitro for kinase activity and in vivo for their ability to induce oocyte maturation, embryonic cleavage arrest and cellular transformation. Most mutations in Mos eliminated both kinase and biological activity. However, a mutation in Mos that removed two basic amino acid residues (R94 and K97) downstream from the lysine at the ATP binding site (K90) markedly enhanced autophosphorylation activity. Moreover, this mutant displayed markedly reduced biological activity, lacked transforming activity, and failed to activate mitogen activated protein kinase (MAPK). A second mutant Mos product, lacking amino acids R45-A54, displayed a five-fold increase in cellular transforming activity. This Mos mutant specifically localized to the cytoplasm; in contrast to wild-type (wt) Mos that localized to both the nucleus and the cytoplasm. These data indicate that Mos transforming activity is mediated via signalling exerted in the cytoplasm, presumably through MAPK, and that nuclear localization of the oncogene product interferes with transforming activity. We also show that amino acids R45-A54 are important for Mos DNA binding activity.

3T3 Cells↗

PubMed1995

Mos overexpression in Swiss 3T3 cells induces meiotic-like alterations of the mitotic spindle.

High levels of mos protooncogene product are expressed during oocyte meiotic maturation and Mos has been implicated in formation of the spindle and spindle pole. Here, we show that in Swiss 3T3 cells with 4N DNA content, high levels of Mos lead to the production of binucleated cells. The Swiss 3T3 cells in mitosis, before binucleation occurs, are anastral and the spindle poles are juxtaposed to the cell membrane. These phenotypes may be related to the meiotic process of attachment of the spindle pole to the oocyte membrane during polar body formation. The production of binucleated somatic cells could result from attachment of the altered mitotic spindle pole to the cell membrane that interferes with cytokinesis but not karyokinesis. This can explain at least one form of genetic instability that leads to altered DNA content in tumor cells.

3T3 Cells↗

PubMed1995

Determination of v-Mos-catalyzed phosphorylation sites and autophosphorylation sites on MAP kinase kinase by ESI/MS.

MAP kinase kinase (MAPKK), a key component of the MAP kinase cascade, is activated through phosphorylation by several protein kinases, including the oncogene v-Mos and its cellular counterpart, c-Mos. The v-Mos-catalyzed phosphorylation sites on recombinant MAPKK1 were identified by electrospray ionization mass spectrometry as S218 and S222, located within a sequence that aligns with the T loop structure of cAMP-dependent protein kinase; these are the same as the Raf-1 phosphorylation site identified previously [Alessi, D. R., et al. (1994) EMBO J. 13, 1610-1619]. Phosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of MAPKK occurred at several residues and was increased upon the stimulation of MAPKK activity by v-Mos. Major autophosphorylation sites were residues S298 and Y300. Minor autophosphorylation sites included T23, S299, S218, and either S24 or S25. Sequence similarities were noted between MAPKK autophosphorylation sites and exogenous phosphorylation sites on MAP kinase. Phosphorylation of either S218 or S222 was sufficient for partial MAPKK activation by Mos, and phosphorylation of S222 alone was sufficient for autophosphorylation at S298 and Y300. Mass spectral analysis was also performed on MAPKK1 purified from rabbit skeletal muscle. The peptide containing S218 and S222 was observed in only a singly phosphorylated form, and the peptide containing S298, S299, and Y300 was observed in multiply phosphorylated forms, suggesting that MAPKK is only partially phosphorylated within the T loop but significantly modified in the autophosphorylation loop under physiological conditions.

3T3 Cells↗

PubMed1995

Overexpression of mos oncogene product in Swiss 3T3 cells induces apoptosis preferentially during S-phase.

When Swiss 3T3 cells are acutely infected with Moloney murine sarcoma virus containing the v-mos oncogene, 90% of the cells round up and detach from the monolayer (floating cells) and express high levels of v-Mos. The majority of the floating cells are generated between 30 and 70 h post infection when the cellular level of Mos reaches approximately 0.1% of the total protein. Seventy percent of the floating cells exclude trypan blue but are growth arrested with 2C or 4C DNA content, whereas the remaining floating cells with < 2C DNA content, are dead or dying, and show characteristic apoptotic phenotypes. The apoptotic cells are most likely generated from cells in S-phase since these cells are absent from the viable floating cell population and the percentage of cells with < 2C DNA approximated the expected S-phase fraction of logarithmically growing cells. In addition, 5'-bromo-2'-deoxyuridine-labeling studies showed that approximately 50% of the floating cells with typical apoptotic phenotypes were metabolically-labelled with the drug. These analyses show that cell populations in different stages of the cell cycle are differently affected by high levels of v-Mos expression and cells in S-phase appear to be uniquely sensitive and undergo apoptosis.

3T3 Cells↗

PubMed1994

Transformation of mammalian cells by constitutively active MAP kinase kinase.

Mitogen-activated protein (MAP) kinase kinase (MAPKK) activates MAP kinase in a signal transduction pathway that mediates cellular responses to growth and differentiation factors. Oncogenes such as ras, src, raf, and mos have been proposed to transform cells by prolonging the activated state of MAPKK and of components downstream in the signaling pathway. To test this hypothesis, constitutively active MAPKK mutants were designed that had basal activities up to 400 times greater than that of the unphosphorylated wild-type kinase. Expression of these mutants in mammalian cells activated AP-1-regulated transcription. The cells formed transformed foci, grew efficiently in soft agar, and were highly tumorigenic in nude mice. These findings indicate that constitutive activation of MAPKK is sufficient to promote cell transformation.

3T3 Cells↗

PubMed1994

Chromophobe renal cell carcinoma: a report of two cases.

Chromophobe renal cell carcinoma (RCC) is a recently established subtype of RCC, which has rarely been reported in Japan. In this communication, the authors report two Japanese cases of chromophobe RCC together with the immunohistochemical findings. The tumors were composed of sheets and cribriform glands formed by tumor cells with cloudy and reticular cytoplasm. Ultrastructurally, the cytoplasm was filled with numerous microvesicles. The tumor cells were positive for cytokeratin, epithelial membrane antigen, and Tamm-Horsfall protein. Occasionally, LeuM1-positive cells were also noted. Vimentin was negative, unlike the usual RCC. Reactivity for peanut agglutinin was more frequent than that to Lotus tetragonolobus agglutinin. The results of this study suggest that the tumor cells possessed phenotypes similar to the distal nephron rather than to the proximal tubular cells.

Adult↗

PubMed1994

Similarities between somatic cells overexpressing the mos oncogene and oocytes during meiotic interphase.

The mos protooncogene encodes a serine/threonine kinase and is a key regulator of oocyte meiotic maturation. After acute infection of Swiss 3T3 cells with virus containing the v-mos oncogene, cells expressing high levels of v-Mos round up and detach from the monolayer (floating cells), while cells that remain attached express 10-fold lower levels of v-Mos and are transformed. The floating cells are growth arrested with their chromosomes partially condensed in the absence of histone H1 kinase activity, while mitogen-activated protein kinase activity is very high. Collectively, these properties are similar to properties observed in maturing oocytes between meiosis I and II. In v-mos-transformed cell populations, mitogen-activated protein kinase activity is also elevated, correlating with the degree of morphological transformation and the level of Mos expression. Moreover, phosphoprotein modifications specific for M are found in both the floating cells and in v-mos-transformed cells, regardless of their cell cycle stage. One explanation for both morphological transformation and the phenotypes of the floating cells is that Mos imposes a meiotic program on different stages of the somatic cell cycle. The extent of this meiotic phenotype is proportional to the level of v-Mos expression. These results suggest that both morphological transformation and the phenotypes of the floating cells induced by Mos in Swiss 3T3 cells are related to its normal activities during oocyte maturation.

3T3 Cells↗

PubMed1993

Ubiquitous nuclear proteins bind to 5' upstream region of major Kunitz chymotrypsin inhibitor gene in winged bean.

Winged bean Kunitz chymotrypsin inhibitor (WCI) accumulates abundantly in seeds and tuberous roots of winged bean plant. In seeds, the WCI mRNA is observed transiently during seed maturation period. The WCI is encoded by a multigene family and the major WCI (WCI-3) is encoded by two nearly identical genes (WCI-3a and WCI-3b genes), in which nucleotide sequences in the 1.1 kb 5' flanking regions are about 99% homologous to each other and the transcribed regions are completely identical. Here we report the detection of two types of nuclear proteins which bind to the multiple sites in the 5' upstream region of the WCI-3a gene. One of the proteins, band 1-forming protein, also bound to cauliflower mosaic virus 35S (CaMV35S) promoter, but another protein, band 3-forming protein, did not. DNaseI footprinting analysis showed that these proteins bound to AT-rich upstream regions in the WCI-3a gene. Addition of poly(dA-dT)-poly(dA-dT) to the binding reaction inhibited the formation of the retarded bands, while poly(dI-dC)-poly(dI-dC) did not. In various organs and throughout seed maturation period, proteins with invariable binding specificities were detected, and these binding proteins met some operational criteria for high-mobility-group (HMG) proteins. These results suggest that leguminous seed AT-binding proteins reported on several seed storage protein genes may be HMG-like proteins which are present ubiquitously in plant organs.

Base Sequence↗

PubMed1993

[Characteristics of day service center clients and their families based on their medical conditions].

Clients utilizing day service centers in Hokkaido, and their families were surveyed for the purpose of analyzing present conditions of health, ADL level, family structure, motivation, activities, and changes in physical and psychosocial status. The average age of subjects was 76 years old with women generally being older than men. Most were living with their families. As to disease, cerebrovascular diseases were most common followed by hypertension and spinal-articulation diseases. Women had more hypertension problems compared to men who had more cerebrovascular conditions. Effectiveness of these day service programs were seen in the improvement of psychological functions. Persons with cerebrovascular disease had greater participation in functional rehabilitation training programs, and their ADL skills were improved.

Activities of Daily Living↗

PubMed1992

Hypertension in unilateral atrophic kidney secondary to ureteropelvic junction obstruction.

A patient with atrophic unilateral hydronephrosis due to ureteropelvic junction obstruction associated with hypertension was successfully treated by nephrectomy. Preoperatively, plasma renin activity was elevated in both the peripheral vein and affected renal vein. Renin concentration in the resected kidney was high, and immunohistochemical localization of renin was observed along the afferent arterioles of the juxtaglomerular apparatus and in arterioles at some distance from the glomeruli.

Adult↗