Biomedical subjects

M Yanagida

Publications and source records attributed to M Yanagida.

At least 91 records · Page 5Linked to original sources

PubMed1997

Distinct subunit functions and cell cycle regulated phosphorylation of 20S APC/cyclosome required for anaphase in fission yeast.

We show here that the fission yeast gene products Cut9 and Nuc2 are the subunits of the 20S complex, the putative APC (anaphase promoting complex)/cyclosome which contains ubiquitin ligase activity required for cyclin and Cut2 destruction. The assembly of Cut9 into the 20S complex requires functional Nuc2, and vice versa. The size of fission yeast APC/cyclosome is similar to that of higher eukaryotes, but differs greatly from that (36S) of budding yeast. The 20S complex is present in cells arrested at different stages of the cell cycle, and becomes slightly heavier in mitosis than interphase. Cut9 in the 20S complex is hyperphosphorylated specifically at the time of metaphase. The truncated forms of Cut9 block entry into mitosis, however. The 20S assembly impaired in the cut9 mutant can be restored by elevating the level of a novel gene product Hcnl, similar to budding yeast Cdc26. Furthermore, deletion of protein kinase PKA (Pkal) suppresses the phenotype of the cut9 mutation and reduces phosphorylation of Cut9. In contrast, PP1 (Dis2) phosphatase mutation shows the reverse effect on the phenotype of cut9. The Cut9 subunit is likely to be a target for regulating APC/ cyclosome function through protein-protein interactions and phosphorylation.

Amino Acid Sequence↗

PubMed1997

Evidence for cell cycle-specific, spindle pole body-mediated, nuclear positioning in the fission yeast Schizosaccharomyces pombe.

Specific changes in spatial order occur during cell cycle progression in fission yeast. Growth of the rod-shaped cells is highly regulated and undergoes a cell cycle and size-regulated switch from monopolar to bipolar tip extension. During both phases of growth, the interphase nucleus is maintained in a central location. Following the separation of the genome to the cell tips in mitosis, the two nuclei migrate back towards the cell equator before stopping in two new positions that will become the middle of the two new cells. Here we use simultaneous labeling of microtubules, chromatin and spindle pole bodies in wild-type and cdc mutants, to show that nuclear positioning is achieved by regulation of spindle pole body-mediated nuclear migration. We show that the number and location of nuclear positioning signals is regulated in a cell cycle-specific manner and that spindle pole body-mediated forces are likely to be responsible for maintaining correct nuclear position once the nuclei have reached the appropriate position in the cell. Accentuating the movement of the nuclei back towards the cell equator after mitosis by artificially increasing cell length shows that the spindle pole body leads the nucleus during this migration. When multiple spindle pole bodies are associated with the same or different nuclei they all go to the same point indicating that the different spindle pole bodies are responding to the same positional cue. In a septation-defective mutant cell, which contains four nuclei, the spindle pole bodies on the four different nuclei initially group as two pairs in regions that would become the middle of the new cells, were the cell able to divide. In the subsequent interphase, the nuclei aggregate as a group of four in the centre of the cell. The presence of two or three clusters of spindle pole bodies in larger cells with eight nuclei suggests that the mechanisms specifying the normally central location for multiple nuclei may be unable to operate properly as the cells get larger. Perturbation of microtubules with the microtubule poison thiabendazole prevents the spindle pole body clustering in septation mutants, demonstrating that nuclear positioning requires a functional microtubule cytoskeleton.

Anaphase↗

PubMed1997

Ascorbic acid 2-O-alpha-glucoside-induced redox modulation in human keratinocyte cell line, SCC: mechanisms of photoprotective effect against ultraviolet light B.

We previously reported that the topical application of ascorbic acid 2-O-alpha-glucoside (AA-2G) suppressed the cutaneous inflammation by ultraviolet irradiation in human and guinea pigs (Miyai et al., Nishinihon J. Dermatol., 58, 439-443 (1996)). In this paper, the effect of AA-2G on the lethal damage induced by ultraviolet B (UVB) was studied using a human keratinocyte cell line, SCC, established from squamous cell carcinoma. The photoprotective effect of AA-2G on cytotoxicity of UVB in SCC cells was dose dependent (0.125-1 mM) and more effective than that of ascorbic acid (AsA) at 1 mM. This protection was completely abolished in the presence of an alpha-glucosidase inhibitor, castanospermine, indicating that release of AsA from this derivative was essential for reduction of the actinic injury. AA-2G significantly suppressed cytotoxicities of hydrogen peroxide and superoxide anion produced by xanthine and xanthine oxidase. AA-2G exhibited a preventive effect against the cytotoxicity produced by tert-butylhydroperoxide, an inducer of lipid peroxidation, in the presence of alpha-tocopherol, but not in the absence of alpha-tocopherol. Cytotoxicity of UVB was also effectively reduced by the combination of AA-2G and alpha-tocopherol. In addition, AA-2G reduced UVB-promoted formation of lipid peroxide and accumulation of lipofuscin, which is known to be a complex of cellular proteins and metabolites of lipid peroxide. These data suggest that AA-2G prevents the acute inflammation induced by UVB irradiation partly through scavenging reactive oxygen species and potentiating the antioxidative activity of alpha-tocopherol.

Ascorbic Acid↗

PubMed1996

Fission yeast Cut1 and Cut2 are essential for sister chromatid separation, concentrate along the metaphase spindle and form large complexes.

Fission yeast Schizosaccharomyces pombe temperature-sensitive (ts) cut1 mutants fail to separate sister chromatids in anaphase but the cells continue to divide, leading to bisection of the undivided nucleus (the cut phenotype). If cytokinesis is blocked, replication continues, forming a giant nucleus with polyploid chromosomes. We show here that the phenotype of ts cut2-364 is highly similar to that of cut1 and that the functions of the gene products of cut1+ and cut2+ are closely interrelated. The cut1+ and cut2+ genes are essential for viability and interact genetically. Cut1 protein concentrates along the short spindle in metaphase as does Cut2. Cut1 (approximately 200 kDa) and Cut2 (42 kDa) associate, as shown by immunoprecipitation, and co-sediment as large complexes (30 and 40S) in sucrose gradient centrifugation. Their behavior in the cell cycle is strikingly different, however: Cut2 is degraded in anaphase by the same proteolytic machinery used for the destruction of cyclin B, whereas Cut1 exists throughout the cell cycle. The essential function of the Cut1-Cut2 complex which ensures sister chromatid separation may be regulated by Cut2 proteolysis. The C-terminal region of Cut1 is evolutionarily conserved and similar to that of budding yeast Esp1, filamentous fungi BimB and a human protein.

Amino Acid Sequence↗

PubMed1996

Requirement for PP1 phosphatase and 20S cyclosome/APC for the onset of anaphase is lessened by the dosage increase of a novel gene sds23+.

Ubiquitin-dependent proteolysis is required for the onset of anaphase. We show that protein dephosphorylation by protein phosphatase 1 (PP1) is also essential for initiating anaphase in fission yeast. PP1 may directly or indirectly regulate the 20S cyclosome/APC (anaphase-promoting complex) required for anaphase-promoting proteolysis. Using anti-phosphopeptide antibodies, PP1 is shown to be dephosphorylated at the C-terminus, upon the onset of anaphase, for reactivation. sds23+, a novel gene, is a multicopy suppressor for mutations in PP1 and the 20S cyclosome/APC, implying that the gene dosage increase can relieve the requirement for PP1 and the cyclosome/APC for the onset of anaphase. The sds23+ gene is not essential for cell viability, but a mutant with the gene deleted cannot form colonies at 22 and 36 degrees C. In the sds23 deletion mutant, the progression of anaphase and cytokinesis is retarded and cell shape is aberrant. These defects are overcome by plasmids carrying the genes encoding subunits of the 20S cyclosome/APC or PP1. These results demonstrate functions other than promoting anaphase for the components of the 20S cyclosome/APC and also a close functional relationship of Sds23 with PP1 and 20S cyclosome/APC.

Amino Acid Sequence↗

PubMed1996

20S cyclosome complex formation and proteolytic activity inhibited by the cAMP/PKA pathway.

The 20S cyclosome complex (also known as the anaphase-promoting complex) has ubiquitin ligase activity and is required for mitotic cyclin destruction and sister chromatid separation. The formation and activation of the 20S cyclosome complex is regulated by an unknown mechanism. Here we show that Cut4 (ref. 6) is an essential component of the cyclosome in fission yeast. Cut4 shares sequence similarity with BimE, a protein that regulates mitosis in Aspergillus nidulans. Mutations in cut4 result in hypersensitivity to cyclic AMP and to stress-inducing heavy metals, inhibition of the onset of anaphase, disruption of the 20S complex, and inhibition of mitotic cyclin ubiquitination. These phenotypes are fully suppressed by cAMP phosphodiesterase and the protein kinase A (PKA) regulatory subunit and weakly suppressed by Sti1 (an activator of the Hsp70 and Hsp90 chaperones). Suppression correlates with the amount of 20S complex, indicating that cyclosome formation and activation is inhibited by the cAMP/PKA pathway.

Anaphase↗

PubMed1996

Dis3, implicated in mitotic control, binds directly to Ran and enhances the GEF activity of RCC1.

Using the two-hybrid method, we isolated a Saccharomyces cerevisiae cDNA encoding a protein homologous to Schizosaccharomyces pombe protein Dis3sp, using as bait, human GTPase Ran. The DIS3 gene is essential for viability and complements S.pombe mutant dis3-54 which is defective in mitosis. Although Dis3sc has no homology to RanBP1, it bound directly to Ran and the S.cerevisiae Ran homologue Cnr1, but not to the S.cerevisiae RCC1 homologue Srm1. Upon binding to Ran with a 1:1 molar ratio, Dis3sc enhanced a nucleotide-releasing activity of RCC1 on Ran. In the presence of Dis3sc, the K(m) of RCC1 on Ran decreased by half, while the kcat was unchanged. In vivo, Dis3sp was present as oligomers of M(r) 670-200 kDa as previously reported, and the 200 kDa oligomer of Dis3sp was found to include Spi1 and Pim1, the S.pombe homologues of Ran and RCC1, respectively. Although the biological function of the heterotrimeric oligomer consisting of Dis3, Spi1 and Pim1 is unknown, our results indicate that Dis3 is a component of the RCC1-Ran pathway.

Amino Acid Sequence↗

PubMed1996

Selective growth of human mast cells induced by Steel factor, IL-6, and prostaglandin E2 from cord blood mononuclear cells.

To establish the method for generating a large number of mature human mast cells, we cultured cord blood mononuclear cells (CBMC) in several conditions in the presence of Steel factor (SF). Among several cytokines tested, IL-6 enhanced SF-dependent mast cell growth from purified CD34+ cells for more than 8 wk in culture. When CBMC were cultured instead of CD34+ cells, IL-6 enhanced the mast cell development in the presence but not in the absence of PGE2. PGE2 enhanced the SF- and IL-6-dependent development of mast cells from CBMC probably by blocking granulocyte-macrophage CSF (GM-CSF) secretion from accessory cells, because 1) PGE2, or anti-GM-CSF enhanced the mast cell development induced by SF and IL-6 from CBMC, but not from CD34+ cells; 2) GM-CSF inhibited the enhancing effect of IL-6 on the mast cell development from CD34+ cells; and 3) PGE2 inhibited GM-CSF secretion from CBMC. The mast cells cultured in the presence of SF, IL-6, and PGE2 for >10 wk were 99% pure, and seemed to be functionally mature, because 1) they contained 5.62 micrograms of histamine and 3.46 micrograms of tryptase per 10(6) cells; and 2) when sensitized with human IgE and then challenged with anti-human IgE, the cells released a variety of mediators such as histamine, and an increase in intracellular Ca2+ was found in advance of the activation of membrane movement by using a confocal laser-scanning microscope. Electron-microscopic analysis revealed that some of the cultured mast cells are morphologically mature since they filled with scroll granules and contained crystal granules.

Base Sequence↗

PubMed1996

Cut2 proteolysis required for sister-chromatid seperation in fission yeast.

Although mitotic cyclins are well-known substrates for ubiquitin-mediated proteolysis at the metaphase-anaphase transition, their degradation is not essential for separation of sister chromatids; several lines of evidence suggest that proteolysis of other protein(s) is required, however. Here we report the anaphase-specific proteolysis of the Schizosaccharomyces pombe Cut2 protein, which is essential for sister-chromatid separation. Cut2 is located in the nucleus, where it is concentrated along the short metaphase spindle. The rapid degradation of Cut2 at anaphase requires its amino-terminal region and the activity of Cut9 (ref. 14), a component of the 20S cyclosome/anaphase-promoting complex (APC), which is necessary for cyclin destruction. Expression of non-degradable Cut2 blocks sister-chromatid separation but not cell-cycle progression. This defect can be overcome by grafting the N terminus of cyclin B onto the truncated Cut2, demonstrating that the regulated proteolysis of Cut2 is essential for sister-chromatid separation.

Amino Acid Sequence↗

PubMed1996

Adenomyoepithelioma of the Breast: A Case Reprot and a Review of Literature.

A 74-year-old woman complained of a small nodule in the outer lower quadrant of her left breast. On physical examination, a 0.9 x 0.8 cm, round-shaped and firmly elastic nodule was palpated. Excisional biopsy was performed. Histologically, the tumor was separated into, with a bicellular pattern, containing both numerous glandular structures and numerous spindle-shaped cells. Immunohistochemical staining for EMA and cytokeratin showed strongly positive immunoreactivity for epithelial cells. Staining for &aipha;-SMA showed strongly positive immunoreactivity for myoepithelial components. Staining for keratin and S-100 protein showed weakly positive immunoreactivity for myoepithelial cells. Microscopically, the tumor was diagnosed as adenomyoepithelioma of the breast. Immunohistochemical examination is needed to distinguish epithelial cell proliferation from myoepithelial cell proliferation. Immunohistochemical examination using antibodies against EMA, alpha-SMA, Keratin, cytokeratin and S-100 protein, is indispensable.

Journal Article↗

PubMed1996

Caffeine-resistance in fission yeast is caused by mutations in a single essential gene, crm1+.

Caffeine is a base analogue and is known to affect a wide variety of cellular processes. In order to dissect genetically molecules which mediate the biological effects of caffeine, temperature-sensitive (ts) and caffeine-resistant mutants were isolated from fission yeast, Schizosaccharomyces pombe. Surprisingly, all twelve ts isolates contained a mutation in the same locus, crm1. Cells of the ts crm1 mutant showed an abnormal chromosome structure at the restrictive temperature, an elevated expression of Pap1-dependent transcription, and cross-resistance to an unrelated drug such as staurosporine. Overproduction of pap1+ also conferred caffeine resistance, whilst the resistance of the crm1 mutant is abolished in the pap1- background. These results show that the crm1+ gene is a major locus for caffeine resistance, which arises from Pap1-dependent transcriptional activation.

Alkaloids↗

PubMed1996

Nerve growth factor enhances antigen-specific antibody production in ascorbate-stimulated murine splenocytes.

In murine splenocytes, a primary antigen-specific antibody response is stimulated by 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), a stable form of ascorbate, as described in our previous paper. We examined here the effect of nerve growth factor (NGF) on the antigen-specific antibody production in vitro augmented by AA-2G. NGF (> or = 10 ng/ml), which alone had no effect, enhanced the anti-sheep-red-blood-cell (SRBC) antibody response stimulated by AA-2G. The effect of NGF plus AA-2G or AA-2G alone was abrogated by the presence of castanospermine, an alpha-glucosidase inhibitor, suggesting that the active form of AA-2G is ascorbate. The repeated additions; but not one addition, of ascorbate also resulted in a synergism with NGF on the antibody production. These results suggest that NGF might be a cytokine which functions as a regulatory factor for ascorbate-dependent immune responses.

Animals↗

PubMed1996

Dissection of fission yeast microtubule associating protein p93Dis1: regions implicated in regulated localization and microtubule interaction.

BACKGROUND: Fission yeast microtubule associating protein (MAP) p93Dis1 functions for sister chromatid separation: dis1 mutants fail to separate chromosomes, while the spindle elongates but without cyclin destruction. p93Dis1 localizes along microtubules in interphase cytoplasm, but shifts to the spindle pole body (SPB) and spindle microtubules upon the entry into mitosis. In this study, regions of p93Dis1 were dissected to examine their role. RESULTS: Nitrocellulose filter blotting shows that recombinant Dis1 binds to bovine brain microtubules in vitro. A basic central region rich in S, T and P is essential for this association. However, the whole p93Dis1 with N- and C-termini containing a conserved repeat motif and heptad repeats, respectively, is necessary for normal microtubule association in vivo. The N-truncated region also binds to microtubules but only to the portions near the SPBs. Overproduction phenotypes indicate that p93Dis1 greatly affects spindle formation and cell morphogenesis. The central region is essential but, by itself, not sufficient for generating such effects. CONCLUSIONS: We propose that p93Dis1 consists of three regions which carry distinct properties for localization: the N-region for cell cycle dependent localization, the central region for direct microtubule association, and the C-region for SPB and nuclear localization. The essential role of p93Dis1 is carried out in the C-region, while the N-region acts as a regulator.

Amino Acid Sequence↗

PubMed1996

The regulatory subunits of fission yeast protein phosphatase 2A (PP2A) affect cell morphogenesis, cell wall synthesis and cytokinesis.

BACKGROUND: Protein phosphatase 2A (PP2A) holoenzymes have a trimeric structure, consisting of a catalytic subunit C and two regulatory subunits A (PR65) and B (PR55). In fission yeast the C subunits, being 80% identical to their mammalian counterparts, are essential for viability and negatively regulate the entry into mitosis. Genetic analyses in budding yeast and Drosophila show that the regulatory subunits are implicated in chromosome segregation, cell morphogenesis and/or cytokinesis. RESULTS: We isolated fission yeast genes paa1+ and pab1+ encoding the regulatory subunits PR65 and PR55, respectively. Gene disruption showed that the paa1+ gene was essential for viability while pab1+ was not required at 26-33 degrees C. Microtubule and actin distributions were anomalous in gene disrupted delta paa1 cells which were incapable of forming a polarized cell shape. Gene disrupted delta pab1 cells were pear- or round-shaped, and lost the polar distributions of actin and microtubules. In addition, delta pab1 cells were defective in cell wall synthesis and sporulation at permissive temperatures. At restrictive temperatures, delta pab1 cells showed an osmoremedial temperature-sensitive phenotype and delayed in cytokinesis. However, chromosome segregation was normal. CONCLUSION: Fission yeast PP2A regulatory subunit plays a critical role in cell morphogenesis, probably through regulation of the cytoskeletal network and cell wall synthesis.

Amino Acid Sequence↗

PubMed1996

Interferon-gamma promotes the survival and Fc epsilon RI-mediated histamine release in cultured human mast cells.

We examined the effects of interferon-gamma (IFN-gamma) on 100% pure human mast cells generated in suspension cultures of umbilical cord blood mononuclear cells in the presence of stem cell factor (SCF) and interleukin-6 (IL-6). When mast cells were suspended in serum-free medium without any cytokine after the withdrawal of SCF and IL-6, they died over a period of 5 days because of apoptosis. IFN-gamma in the cultures suppressed apoptosis and prolonged their survival in a dose-dependent manner. This survival-promoting effect of IFN-gamma was blocked by neutralizing antibodies to IFN-gamma or to IFN-gamma receptor (IFN-gamma R). When mast cells were incubated with IFN-gamma in serum-free medium for more than 4 hr during sensitization, immunoglobulin E (IgE)/anti-IgE antibody-induced histamine release was effectively enhanced. Polymerase chain reaction (PCR) amplification of the alpha-chain of IFN-gamma R (IFN-gamma R alpha) yielded products of the correct size predicted from the sequence of the receptor. In addition, flow cytometry using anti-IFN-gamma R monoclonal antibodies (mAbs) indicated that these mast cells bear IFN-gamma R on their surface. These findings suggested that IFN-gamma activates human mast cells via specific receptors in certain aspects of inflammatory reactions.

Antibodies↗

PubMed1996

Aberrant mitosis in fission yeast mutants defective in fatty acid synthetase and acetyl CoA carboxylase.

Two fission yeast temperature-sensitive mutants, cut6 and lsd1, show a defect in nuclear division. The daughter nuclei differ dramatically in size (the phenotype designated lsd, large and small daughter). Fluorescence in situ hybridization (FISH) revealed that sister chromatids were separated in the lsd cells, but appeared highly compact in one of the two daughter nuclei. EM showed asymmetric nuclear elongation followed by unequal separation of nonchromosomal nuclear structures in these mutant nuclei. The small nuclei lacked electron-dense nuclear materials and contained highly compacted chromatin. The cut6+ and lsd1+ genes are essential for viability and encode, respectively, acetyl CoA carboxylase and fatty acid synthetase, the key enzymes for fatty acid synthesis. Gene disruption of lsd1+ led to the lsd phenotype. Palmitate in medium fully suppressed the phenotypes of lsd1. Cerulenin, an inhibitor for fatty acid synthesis, produced the lsd phenotype in wild type. The drug caused cell inviability during mitosis but not during the G2-arrest induced by the cdc25 mutation. A reduced level of fatty acid thus led to impaired separation of non-chromosomal nuclear components. We propose that fatty acid is directly or indirectly required for separating the mother nucleus into two equal daughters.

Acetyl-CoA Carboxylase↗

PubMed1996

Fission yeast Nda1 and Nda4, MCM homologs required for DNA replication, are constitutive nuclear proteins.

The nda1+ and nda4+ genes of the fission yeast Schizosaccharomyces pombe encode proteins similar to budding yeast MCM2 and MCM5/CDC46, respectively, which are required for the early stages of DNA replication. The budding yeast Mcm proteins display cell-cycle dependent localization. They are present in the nucleus specifically from late M phase until the beginning of S phase, so that they were suggested to be components of a replication licensing factor, a positive factor for the onset of replication, which is thought to be inactivated after use, thus restricting replication to only once in a cell cycle. In the present study, we raised antibodies against Nda1 or Nda4 and identified 115 kDa and 80 kDa proteins, respectively. Their immunolocalization was examined in wild-type cells and in various cell-cycle mutants. Both Nda1 and Nda4 proteins remained primarily in the nucleus throughout the cell cycle. In mutants arrested in G1, S, and G2 phases, these proteins were also enriched in the nucleus. These results indicate that the dramatic change in subcellular localization as seen in budding yeast is not essential in fission yeast for the functions of Nda1 and Nda4 proteins to be executed. The histidine-tagged nda1+ gene was constructed and integrated into the chromosome to replace the wild-type nda1+ gene. The resulting His-tagged Nda1 protein was adsorbed to the Ni-affinity column, and co-eluted with the untagged Nda4 protein, suggesting that they formed a complex.

Animals↗

PubMed1996

A nitrogen starvation-induced dormant G0 state in fission yeast: the establishment from uncommitted G1 state and its delay for return to proliferation.

Fission yeast cells either remain in the mitotic cell cycle or exit to meiotic sporulation from an uncommitted G1 state dependent on the presence or absence of nitrogen source in the medium (Nurse and Bissett, 1981). We examined how heterothallic haploid cells, which cannot sporulate, behave under nitrogen-starvation for longer than 25 days at 26 degrees C. These cells were shown to enter a stable state (designated the dormant G0) with nearly full viability. Maintaining the dormant cells required glucose, suggesting that the cells remained metabolically active although cell division had ceased. They differed dramatically from mitotic and uncommitted G1 cells in heat resistance, and also in cytoplasmic and nuclear morphologies. After nitrogen replenishment, the initial responses of dormant G0 cells were investigated. The kinetics for reentry into the proliferative state were delayed considerably, and the changes in cell shape were enhanced particularly for those recovering from extended nitrogen starvation. A part of the delay could be accounted for by the duration of nuclear decondensation and cell elongation for the first cell division.

Cell Division↗