Search PubMed⌕ Search

Biomedical subjects

M Yanagida

Publications and source records attributed to M Yanagida.

At least 73 records · Page 4Linked to original sources
PubMed1998

Fission yeast cut mutations revisited: control of anaphase.

Studies of anaphase are approaching a golden age. Several different disciplines have contributed immensely to advances in our understanding of cell-cycle control and chromosome and spindle dynamics during mitosis. This article describes control of anaphase based on results obtained from Schizosaccharomyces pombe cut (cell untimely torn) mutants. These temperature-sensitive mutants were isolated by selection for uncoordinated mitosis with aberrant sister-chromatid separation and post-anaphase events. Characterization of some of the cut gene products has led to identification of novel molecular events related to chromosome condensation, sister-chromatid separation, anaphase-promoting proteolysis, fatty-acid metabolism, and cell-cycle arrest induced by stress or a replication block.

Anaphase↗
PubMed1998

Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle.

In higher eukaryotic cells, the spindle forms along with chromosome condensation in mitotic prophase. In metaphase, chromosomes are aligned on the spindle with sister kinetochores facing toward the opposite poles. In anaphase A, sister chromatids separate from each other without spindle extension, whereas spindle elongation takes place during anaphase B. We have critically examined whether such mitotic stages also occur in a lower eukaryote, Schizosaccharomyces pombe. Using the green fluorescent protein tagging technique, early mitotic to late anaphase events were observed in living fission yeast cells. S. pombe has three phases in spindle dynamics, spindle formation (phase 1), constant spindle length (phase 2), and spindle extension (phase 3). Sister centromere separation (anaphase A) rapidly occurred at the end of phase 2. The centromere showed dynamic movements throughout phase 2 as it moved back and forth and was transiently split in two before its separation, suggesting that the centromere was positioned in a bioriented manner toward the poles at metaphase. Microtubule-associating Dis1 was required for the occurrence of constant spindle length and centromere movement in phase 2. Normal transition from phase 2 to 3 needed DNA topoisomerase II and Cut1 but not Cut14. The duration of each phase was highly dependent on temperature.

Anaphase↗
PubMed1997

Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1.

Fission yeast Cut5/Rad4 plays a unique role in the genome maintenance as it is required for replication, replication checkpoint, and normal UV sensitivity. It is unknown, however, how Cut5 protein is linked to other checkpoint proteins, and what part it plays in replication and UV sensitivity. Here we report that Cut5 interacts with a novel checkpoint protein Crb2 and that this interaction is needed for normal genome maintenance. The carboxyl terminus of Crb2 resembles yeast Rad9 and human 53BP1 and BRCA1. Crb2 is required for checkpoint arrests induced by irradiation and polymerase mutations, but not for those induced by inhibited nucleotide supply. Upon UV damage, Crb2 is transiently modified, probably phosphorylated, with a similar timing of phosphorylation in Chk1 kinase, which is reported to restrain Cdc2 activation. Crb2 modification requires other damage-sensing checkpoint proteins but not Chk1, suggesting that Crb2 acts at the upstream of Chk1. The modified Crb2 exists as a slowly sedimenting form, whereas Crb2 in undamaged cells is in a rapidly sedimenting structure. Cut5 and Crb2 interact with Chk1 in a two-hybrid system. Moreover, moderate overexpression of Chk1 suppresses the phenotypes of cut5 and crb2 mutants. Cut5, Crb2, and Chk1 thus may form a checkpoint sensor-transmitter pathway to arrest the cell cycle.

Amino Acid Sequence↗
PubMed1997

Early and late events in Fc epsilon RI signal transduction in human cultured mast cells.

Protein tyrosine phosphorylation and other biochemical events have been shown to occur after cross-linking of Fc epsilonRI in rodent mast cells. To investigate the mechanism of Fc epsilonRI signal transduction in human mast cells, we used human cultured mast cells (HCMC) generated from cord blood cells in the presence of recombinant human stem cell factor and IL-6. We found that on cross-linking of Fc epsilonRI: 1) HCMC released histamine; 2) rapid tyrosine phosphorylation of multiple cellular substrates, including Syk, HS1, c-Cbl, ERK-1, and ERK-2, was observed; 3) intracellular Ca2+ and inositol phosphate production were increased within the first minute after Fc epsilonRI cross-linking; and 4) genistein, a tyrosine kinase inhibitor, inhibited both protein tyrosine phosphorylation and histamine release in a dose-dependent manner. These results were consistent with previous studies in rodent mast cells. In contrast, no tyrosine phosphorylation of phospholipase C gamma1 and Btk (Bruton's tyrosine kinase) were observed in our experimental conditions. These results suggest that the greater part of the early and late signaling events in HCMC is similar to those obtained with rodent mast cells and indicated that the requirement of tyrosine phosphorylation in the activation process of each of the signaling molecules might be different in HCMC and rodent mast cells. Our finding indicates that HCMC may be useful for analysis of Fc epsilonRI-mediated signal transduction in human mast cells.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Molecular cloning and cell cycle-dependent expression of mammalian CRM1, a protein involved in nuclear export of proteins.

Crm1 of Schizosaccharomyces pombe, a nuclear protein essential for proliferation and chromosome region maintenance, is a possible target of leptomycin B, an antifungal and antitumor antibiotic with cell cycle-arresting activity. cDNA encoding a human homolog of Crm1 was cloned. Human CRM1 (hCRM1) consisted of 1071 amino acids, of which the sequence showed 52% homology with S. pombe Crm1. hCRM1 weakly complemented the cold-sensitive mutation of S. pombe crm1-809, as did S. pombe crm1+. Overproduction of hCRM1 under the control of a series of nmt1 promoters suppressed cell proliferation in wild-type S. pombe in an expression level-dependent manner. A similar inhibitory effect was also observed for crm1+. Cells overproducing either hCRM1 or S. pombe Crm1 were distinctly larger than uninduced cells and contained compacted and fragmented nuclei. Furthermore, calcofluor staining demonstrated that most of these cells formed two septa per cell and accumulated a large amount of chitin or its related polysaccharides around the septa. Closely similar phenotypes between hCRM1- and S. pombe Crm1-induced cells indicate that the cloned cDNA encodes a functional homolog of S. pombe crm1+. Northern blot analyses with RNAs isolated from synchronized mammalian cells showed that the expression of mammalian CRM1 was initiated in late G1 and reached a peak at G2/M, although its protein level unchanged during the cell cycle. Transient expression of hCRM1 fused to the green fluorescent protein (GFP) in NIH3T3 cells showed that hCRM1 was localized preferentially in the nuclear envelope and was also detectable in the nucleoplasm and the cytoplasm. A crm1 mutation of S. pombe caused nuclear import of a GFP fusion protein containing a nuclear export signal but no change in the distribution of a GFP fusion protein containing a nuclear localization signal. All of these data suggest that CRM1 is a novel cell-cycle regulated gene that is essential for the nuclear export signal-dependent nuclear export of proteins.

3T3 Cells↗
PubMed1997

CRM1 is responsible for intracellular transport mediated by the nuclear export signal.

The discovery of nuclear export signals (NESs) in a number of proteins revealed the occurrence of signal-dependent transport of proteins from the nucleus to the cytoplasm. Although the consensus motif of the NESs has been shown to be a leucine-rich, short amino-acid sequence, its receptor has not been identified. A cytotoxin leptomycin B (LMB) has recently been suggested to inhibit the NES-mediated transport of Rev protein. Here we show that LMB is a potent and specific inhibitor of the NES-dependent nuclear export of proteins. Moreover, we have found a protein of relative molecular mass 110K (p110) in Xenopus oocyte extracts that binds to the intact NES but not to the mutated, non-functional NES. The binding of p110 to NES is inhibited by LMB. We show that p110 is CRM1, which is an evolutionarily conserved protein originally found as an essential nuclear protein in fission yeast and known as a likely target of LMB. We also show that nuclear export of a fission yeast protein, Dsk1, which has a leucine-rich NES, is disrupted in wild-type yeast treated with LMB or in the crm1 mutant. These results indicate that CRM1 is an essential mediator of the NES-dependent nuclear export of proteins in eukaryotic cells.

Animals↗
PubMed1997

Fission yeast Cut2 required for anaphase has two destruction boxes.

The fission yeast Schizosaccharomyces pombe cut2(+) gene is essential for sister chromatid separation. Cut2 protein, which locates in the interphase nucleus and along the metaphase spindle, disappears in anaphase with the same timing as mitotic cyclin destruction. This proteolysis depends on the APC (Anaphase-Promoting Complex)-cyclosome which contains ubiquitin ligase activity. The N-terminus of Cut2 contains two stretches similar to the mitotic cyclin destruction box. We show that both sequences (33RAPLGSTKQ and 52RTVLGGKST) serve as destruction boxes and are required for in vitro polyubiquitination and proteolysis. Cut2 with doubly mutated destruction boxes inhibits anaphase, whereas Cut2 with singly mutated boxes can suppress cut2 mutations. Strong expression of the N-terminal 73 residues containing the destruction boxes leads to the accumulation of endogenous cyclin and Cut2, and arrests cells in metaphase, whereas the same fragment with the mutated boxes does not. Cut2 proteolysis occurs in vitro using Xenopus mitotic extracts in the presence of functional destruction boxes. Furthermore, Cut2 is polyubiquitinated in an in vitro system using HeLa extracts, and this polyubiquitination requires the destruction boxes.

Anaphase↗
PubMed1997

DNA renaturation activity of the SMC complex implicated in chromosome condensation.

Chromosome condensation occurs in mitosis before the separation of sister chromatids, and requires DNA topoisomerase II and a group of proteins called SMCs. The resulting condensed chromosomes in metaphase have a complex hierarchical structure. SMCs, the components of condensed chromosomes, are also required for the separation of sister chromatids and gene dosage compensation, and are found in a range of organisms from yeasts to mammals. However, the mechanisms by which the SMCs contribute to chromosome condensation are unknown. We have studied chromosomes in fission-yeast SMC mutants cut3-477 and cut14-208, which remain largely non-condensed during mitosis at the restrictive temperature (36 degrees C). To test their role in DNA condensation, we isolated the proteins Cut3 and Cut14 as an oligomeric complex, and tested their interactions with isolated DNA. The complex efficiently promoted the DNA renaturation reactions (the winding up of single-strand DNAs into double helical DNA) as much as approximately 70-fold more efficiently than RecA, which is a bacterial protein with similar activity. The activity of the mutant complex was heat sensitive. As DNA winding by renaturation is a potential cause of supercoiling, the SMC complex may be implicated in promoting the higher-order DNA coiling found in condensed chromosomes.

Chromosomes, Fungal↗
PubMed1997

Mis6, a fission yeast inner centromere protein, acts during G1/S and forms specialized chromatin required for equal segregation.

Disorder in sister chromatid separation can lead to genome instability and cancer. A temperature-sensitive S. pombe mis6-302 frequently loses a minichromosome at 26 degrees C and abolishes equal segregation of regular chromosomes at 36 degrees C. The mis6+ gene is essential for viability, and its deletion results in missegregation identical to mis6-302. Mis6 acts before or at the onset of S phase, and mitotic missegregation defects are produced only after the passage of G1/S at 36 degrees C. Mis6 locates at the centromeres throughout the cell cycle. In the mutant, positioning of the centromeres becomes abnormal, and specialized chromatin in the inner centromeres, which give the smear micrococcal nuclease pattern in wild type, is disrupted. The ability to establish correct biorientation of sister centromeres in metaphase cells requires the Mis6-containing chromatin and originates during the passage of G1/S.

Amino Acid Sequence↗
PubMed1997

Phosphorylation of glial fibrillary acidic protein at the same sites by cleavage furrow kinase and Rho-associated kinase.

Site- and phosphorylation state-specific antibodies are useful to analyze spatiotemporal distribution of site-specific phosphorylation of target proteins in vivo. Using several polyclonal and monoclonal antibodies that can specifically recognize four phosphorylated sites on glial fibrillary acidic protein (GFAP), we have previously reported that Thr-7, Ser-13, and Ser-34 on this intermediate filament protein are phosphorylated at the cleavage furrow during cytokinesis. This observation suggests that there exists a protein kinase named cleavage furrow kinase specifically activated at metaphase-anaphase transition (Matsuoka, Y., Nishizawa, K., Yano, T., Shibata, M., Ando, S., Takahashi, T., and Inagaki, M. (1992) EMBO J. 11, 2895-2902; Sekimata, M., Tsujimura, K., Tanaka, J., Takeuchi, Y., Inagaki, N., and Inagaki, M. (1996) J. Cell Biol. 132, 635-641). Here we report that GFAP is phosphorylated specifically at Thr-7, Ser-13, and Ser-34 by Rho-associated kinase (Rho-kinase), which binds to the small GTPase Rho in its GTP-bound active form. The kinase activity of Rho-kinase toward GFAP is dramatically stimulated by guanosine 5'-(3-O-thio)-triphosphate-bound RhoA. Furthermore, the phosphorylation of GFAP by Rho-kinase results in a nearly complete inhibition of its filament formation in vitro. The possibility that Rho-kinase is a candidate for cleavage furrow kinase is discussed.

Antibodies↗
PubMed1997

A telomerase mutant defective in sister chromatid separation at mitosis.

The telomere is a functional domain of the chromosome, located at the extreme ends, and is essential for normal chromosome stability. Chromosomes lacking telomeres are inherited improperly, and mutations in the telomeric repeat sequences are thought to lead to senescence and possibly to cancer. The molecular mechanisms maintaining chromosomes by telomeres, however, have been unclear. Results recently reported by Kirk et al, offer an insight into new telomerase function. They have identified a novel telomerase mutation that blocks sister chromatid separation in mitosis.

Anaphase↗
PubMed1997

An immunohistochemical study on the tissue-specific localization of metallothionein in dogs.

To study the tissue specificity of metallothionein (MT) expression, tissues of dogs ranging in age from 1 day to 18 years were examined immunohistochemically. Of the organs examined, liver and kidney showed the strongest immunoreactivity; a comparable intensity of MT immunolabelling was noted in the two organs in adult animals. In the central nervous system, astrocytes and ependymal cells showed MT immunoreactivity. MT labelling was shown in the sustentacular cells of the olfactory epithelium, but immunoreactivity was slight in the epithelium of the respiratory tract. Slight MT immunoreactivity was demonstrated in the epithelium of a variety of glands (sweat, uterine, mammary, olfactory, perianal and thyroid) and in parietal cells of the fundic glands of the stomach. Sporadic MT immunolabelling was demonstrated in the columnar and goblet cells of the small and large intestines, surface mucosal cells of the stomach and epithelial cells of the hair follicles. These findings may help in understanding important features of MT, such as its dynamic induction mechanism, its systemic degradation pathway and the possible biological functions.

Animals↗
PubMed1997

The role of transforming growth factor-beta in PEG-rHuMGDF-induced reversible myelofibrosis in rats.

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) injected at a suprapharmacologic dose (100 microg/kg) daily for 5 d in normal rats caused marked increases in marrow megakaryocytes and platelet counts at 6-8 d followed by gradual decreases to control levels at 10-20 d. Interestingly, in addition to the expected thrombopoiesis, PEG-rHuMGDF was associated with myelofibrosis with a predominance of reticulin fibres at day 10 followed by complete normalization by day 20. At 6-8 d, the levels of transforming growth factor-beta1 (TGF-beta1) in the extracellular fluid of the marrow, the platelet poor plasma, and the platelet extract were increased 23-, 7- and 2-fold, respectively. The elevated levels of TGF-beta1 were gradually reduced to baseline levels at 13-20 d in accordance with the normalization of myelofibrosis and thrombopoiesis. An ultrastructural analysis showed that large fragments of megakaryocytes were deposited in the marrow parenchyma of PEG-rHuMGDF-treated rats at day 6. PEG-rHuMGDF administration at pharmacologic doses (1 and 10 microg/kg) did not induce the deposition of reticulin fibres in the marrow. These findings suggest that TGF-beta1 leaked from megakaryocytes is involved in the development of the PEG-rHuMGDF-induced myelofibrosis and that this is a reversible process related to the regulation of the excess production of platelets.

Animals↗
PubMed1997

A globular complex formation by Nda1 and the other five members of the MCM protein family in fission yeast.

BACKGROUND: In the fission yeast Schizosaccharomyces pombe, Nda1, Nda4, Mis5 and Cdc21 proteins belong to the MCM (minichromosome maintenance) protein family which is thought to have six members. Each MCM member is required for the early stages of DNA replication, and has a well-conserved central 200-amino acid domain containing a putative ATP binding motif. However, the precise molecular functions of MCM proteins are not yet clear. RESULTS: We investigated the physical interaction of Nda1 protein with the other fission yeast MCM proteins using specific antibodies. Immunoprecipitation of Nda1 protein leads to the co-precipitation of all the other members of the fission yeast MCM protein family. We purified the MCM protein complex by a combination of column chromatography. The native molecular weight of the MCM complex was estimated by gel filtration to be 560 kDa. The purified fraction contained nearly equal quantities of the six MCM proteins. Electron microscope observation showed that the MCM complex has a globular shape with a central cavity. CONCLUSIONS: We have developed a procedure to purify fission yeast MCM proteins in a native hetero-oligomeric complex form for the first time, which opens an avenue to further biochemical analysis.

Amino Acid Sequence↗
PubMed1997

Effect of a chymotrypsin-like inhibitor, TPCK, on histamine release from cultured human mast cells.

The involvement of endogenous proteases in the secretory process from human mast cells remains to be clarified. A chymotrypsin-like protease inhibitor, N-tosyl-L-phenylalanylchloromethyl ketone (TPCK), blocked both FceRI- and A23187-mediated histamine release from cultured human mast cells at concentrations above 1 microM. At 10 microM, the concentration that completely inhibited FceRI-mediated histamine release, TPCK did not inhibit the chymase activity of the lysate or that in intact cells. The addition of TPCK to cells 30 min before challenge did not affect FceRI- or A23187-mediated Ca2+ mobilization. These findings suggest that a TPCK-sensitive molecule distinct from chymase is involved in a late stage of the process of histamine release from mast cells in man.

Calcimycin↗