Biomedical subjects

M Yanagida

Publications and source records attributed to M Yanagida.

At least 19 recordsLinked to original sources

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

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

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↗

PubMed1996

Ascorbic acid 2-O-alpha-glucoside, a stable form of ascorbic acid, rescues human keratinocyte cell line, SCC, from cytotoxicity of ultraviolet light B.

Environmental exposure to ultraviolet light B (UVB, wave lengths 290-320 nm) of the solar spectrum causes major damage, including an inflammatory response, in skin. In the present study, we estimated the ability of a stable derivative of ascorbic acid, ascorbic acid 2-O-alpha-glucoside (AA-2G), to reduce UVB damage, using the human keratinocyte cell line, SCC, established from squamous cell carcinoma. By pre- (9 h) and post-cultivation with AA-2G, a significant preventive effect on the decrease in the absolute number of surviving cells by exposure to UVB (typical dose, 20 mJ/cm2) was measurable by a neutral red-uptake assay. The release of lactate dehydrogenase from the cell membrane damaged by UVB was inhibited by AA-2G. In agarose gel electrophoresis, relatively high molecular weight DNA fragments were detected in irradiated cells after 6 h post-irradiation, suggesting that the mechanism of cell death was necrosis. Quantitative analysis of DNA content by flow cytometry indicated that AA-2G suppressed both an increase in debris with degraded nuclei and a decrease in cells in G1 and S phases, but not in the G2/M phase, by UVB exposure. These data suggest that AA-2G shows a photoprotective effect against UVB-induced damage in human epithelial cells.

Ascorbic Acid↗

PubMed1996

Characteristics of histamine release from cultured human mast cells.

BACKGROUND: The mast cell is one of the important cells in the pathogenesis of allergic disorders. However, isolating human mast cells is a laborious procedure. Recently, cultured human mast cells raised from umbilical cord blood cells have become available. It is necessary to examine whether these cells are useful in investigating the role of mast cells in human diseases. OBJECTIVE: The phenotype of mast cells depends on their anatomical sites. To examine which phenotype of mast cells these cultured mast cells most closely resemble, their ability to release was investigated. METHODS: The mast cells were raised from human umbilical cord blood cells in the presence of stem cell factor and interleukin-6. To determine the mast cell subtypes, the mast cells were immunocytochemically stained for tryptase and chymase. The cultured mast cells were then stimulated with various secretagogues, and histamine release was measured by a fluorometric technique using high-performance liquid chromatography. RESULTS: The immunocytochemical staining for mast cell proteases revealed that virtually all cells contained tryptase, the definitive marker of mast cells, and that about a quarter of the cells contained chymase. Anti-IgE effectively stimulated these mast cells to release histamine in a dose-dependent, time-dependent manner. The release was completed in about 30 min. One of the non-specific stimuli, calcium ionophore A23187, also induced histamine release in a dose-dependent, time-dependent manner. In contrast, compound 48/80 and substance P failed to induce histamine release from these cells. CONCLUSION: Cultured human mast cells resemble lung mast cells in their ability to release histamine. They will help in studying the functional properties of human mast cells and may contribute to clarifying the pathophysiology of human allergic diseases.

Antibodies, Anti-Idiotypic↗

PubMed1995

Schizosaccharomyces pombe atf1+ encodes a transcription factor required for sexual development and entry into stationary phase.

We describe the identification and characterization of a transcription factor encoded by the atf1+ gene of the fission yeast Schizosaccharomyces pombe. The factor Atf1, contains a bZIP domain at its C-terminus with strong homology to members of the ATF/CREB family of mammalian factors and in vitro binds specifically to ATF/CRE recognition sites. Furthermore the ATF-like binding activity detected in extracts from fission yeast cells is entirely lost upon deletion of the atf1+ gene. Upon growth to saturation, fission yeast cells exit the mitotic cycle and enter a G0-like stationary phase. However, on rich medium, entry of atf1- cells into stationary phase is restricted and they rapidly lose viability; this does not occur on minimal medium unless cAMP levels are raised. Thus stationary phase entry appears to be regulated negatively by cAMP and positively by Atf1. atf1- cells are also sterile and this sterility appears to be due to a combination of two defects: first, upon nitrogen starvation the majority of atf1- cells fail to arrest in the G1 phase of the cell cycle and second, the induction of ste11+ expression is lost. Thus expression of ste11+ represents a second example of an event that is negatively regulated by the cAMP pathway and positively regulated by Atf1. Despite their close association however, these two regulatory pathways function independently and Atf1 activity is not directly modulated by cAMP levels or mutations that alter the activity of components of the cAMP signalling pathway. Thus Atf1 is a transcription factor that plays an important role in the response of cells to adverse environmental conditions, which is to exit the mitotic cell cycle and either sexually differentiate or enter a resting state.

Activating Transcription Factor 1↗

PubMed1995

Effects of T-helper 2-type cytokines, interleukin-3 (IL-3), IL-4, IL-5, and IL-6 on the survival of cultured human mast cells.

Although stem cell factor (SCF) has been identified as a critical cytokine for the development of human mast cells from their progenitors, the effects of other cytokines on human mast cells are less well understood. We examined the effects of several cytokines on the survival of human mast cells of 100% purity generated in suspension cultures of umbilical cord blood mononuclear cells in the presence of 100 ng/mL recombinant human (rh) SCF and interleukin-6 (IL-6). Mast cells suspended in conventional serum-containing medium died over a period of 2 to 6 days after the withdrawal of SCF and IL-6. The cells became pyknotic and underwent DNA fragmentation characteristic of apoptosis. The addition of SCF, IL-3, IL-4, IL-5, or IL-6 to the cultures in both serum-containing and serum-free medium prolonged their survival in a dose-dependent manner. Some other cytokines, such as IL-2, IL-9, IL-10, IL-11, tumor necrosis factor-alpha, transforming growth factor-beta 1, and nerve growth factor, had no survival-promoting effect at 100 ng/mL. Preincubation of mast cells with SCF, IL-4, IL-5, or IL-6 for 24 hours during sensitization with IgE enhanced IgE/anti-IgE antibody-induced histamine release from mast cells, whereas IL-3 showed a negligible effect. Polymerase chain reaction amplification of alpha-chains of IL-3 receptor (R), IL-4 R, IL-5 R, and IL-6 R yielded products of the correct size predicted from the sequence of each receptor. The binding assay using 125I-labeled IL-3 indicated that these mast cells bear receptors for IL-3. These findings suggest that IL-3, Il-4, IL-5, and IL-6, which are mainly produced by T-helper 2 lymphocytes, might regulate the functions of human mast cells in vivo via specific receptors in allergic reactions.

Antibodies, Anti-Idiotypic↗

PubMed1995

A novel protein kinase gene ssp1+ is required for alteration of growth polarity and actin localization in fission yeast.

Temperature-sensitive suppressor mutants were isolated from two fission yeast mutants defective in cell shape control: ppe1, encoding a type 2A-like protein phosphatase, and sts5, one of 11 staurosporine-supersensitive mutants. Complementation tests showed that suppression was due to two chromosomal loci, ssp1 and ssp2. Cells of the ssp1 mutant grown at the restrictive temperature arrested uniformly with an elongated cell body and a 2C content of DNA. Interestingly, these mutant cells grew only in a monopolar manner. At a specific point in the G2 phase of the cell cycle, wild-type cells exhibit a drastic alteration in growth polarity, from mono- to bipolar. This change coincides with the distribution of cortical actin from one end of the cell to both ends. In the ssp1 mutant cells, cortical actin was localized only at one end, suggesting that the mutant fails to change growth polarity. Nucleotide sequence determination showed that ssp1+ encodes a novel protein kinase. Ectopic overexpression of ssp1+ resulted in an altered cell morphology and cortical actin was randomly dispersed within the cells. Immunocytological analysis revealed that the protein was primarily localized in the cytoplasm and that half of the protein existed in an insoluble fraction. These results show that the dynamics of actin-based growth polarity during the cell cycle are regulated, at least in part, by a novel set of protein kinases and phosphatases.

Actins↗

PubMed1995

p93dis1, which is required for sister chromatid separation, is a novel microtubule and spindle pole body-associating protein phosphorylated at the Cdc2 target sites.

Fission yeast cold-sensitive (cs) dis1 mutants are defective in sister chromatid separation. The dis1+ gene was isolated by chromosome walking. The null mutant showed the same phenotype as that of cs mutants. The dis1+ gene product was identified as a novel 93-kD protein, and its localization was determined by use of anti-dis1 antibodies and green fluorescent protein (GFP) tagged to the carboxyl end of p93dis1. The tagged p93dis1 in living cells localizes along cytoplasmic microtubule arrays in interphase and the elongating anaphase spindle in mitosis, but association with the short metaphase spindle microtubules is strikingly reduced. In the spindle, the tagged p93dis1 is enriched at the spindle pole bodies (SPBs). Time-lapse video images of single cells support the localization shift of p93dis1 to the SPBs in metaphase and spindle microtubules in anaphase. The carboxy-terminal fragment, which is essential for Dis1 function, accumulates around the mitotic SPB. We propose that these localization shifts of p93dis1 in mitosis facilitates sister chromatid separation by affecting SPB and anaphase spindle function.

Amino Acid Sequence↗

PubMed1995

A large circular minichromosome of Schizosaccharomyces pombe requires a high dose of type II DNA topoisomerase for its stabilization.

We have constructed circular minichromosomes, ranging in size from 36 to 110 kb, containing the centromeric repeats of Schizosaccharomyces pombe cen3. Comparison of their mitotic stability showed that the circular minichromosomes became more unstable with increasing in size, however, a linear cen3 minichromosome, which is almost the same size as the largest circular one tested, does not show such instability. High levels of expression of the top2+ (type II DNA topoisomerase; topo II) but not top1+ gene (type I DNA topoisomerase) suppressed the instability of the largest circular minichromosome, whereas partial inactivation of topo II dramatically destabilized the minichromosome. A mutant topo II, defective in nuclear localization but still retaining its in vitro relaxation activity, did not stabilize the circular minichromosome. These results indicate that endogenous type II DNA topoisomerase is insufficient for accurate segregation of the circular minichromosome. In addition, the replication of the minichromosomal DNA appears to proceed normally, because the presence of the unstable minichromosome did not cause G2 delay. A likely cause of the instability is intertwining of the minichromosome DNA possibly occurring after DNA replication. An interaction between topo II and the centromeric repeats is implied by the finding that multiple copies of the centromeric repeat, dg-dh, affect stability of the minichromosome similarly to top2+ gene dosage.

Chromosomes, Fungal↗

PubMed1995

Frontier questions about sister chromatid separation in anaphase.

Sister chromatid separation in anaphase is an important event in the cell's transmission of genetic information to a descendent. It has been investigated from different aspects: cell cycle regulation, spindle and chromosome dynamics within the three-dimensional cell architecture, transmission fidelity control and cellular signaling. Integrated studies directed toward unified understanding are possible using multidisciplinary methods with model organisms. Ubiquitin-dependent proteolysis, protein dephosphorylation, an unknown function by the TPR repeat proteins, chromosome transport by microtubule-based motors and DNA topological change by DNA topoisomerase II are all necessary for progression from metaphase to anaphase. Chromosome condensation, mitotic kinetochore function and spindle formation require a larger number of proteins, which are prerequisites for successful sister chromatid separation. Factors that help to retain sister chromatid connection after replication and prevent premature separation remain to be determined. Although sister chromatid separation occurs in anaphase, gene functions in other cell cycle stages also ensure the progression of correct chromatid separation.

Anaphase↗

PubMed1995

The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability.

Spindle formation in fission yeast occurs by the interdigitation of two microtubule arrays extending from duplicated spindle pole bodies which span the nuclear membrane. By screening a bank of temperature-sensitive mutants by anti-tubulin immunofluorescence microscopy, we previously identified the sad1.1 mutation (Hagan, I., and M. Yanagida. 1990. Nature (Lond.). 347:563-566). Here we describe the isolation and characterization of the sad1+ gene. We show that the sad1.1 mutation affected both spindle formation and function. The sad1+ gene is a novel essential gene that encodes a protein with a predicted molecular mass of 58 kD. Deletion of the gene was lethal resulting in identical phenotypes to the sad1.1 mutation. Sequence analysis predicted a potential membrane-spanning domain and an acidic amino terminus. Sad1 protein migrated as two bands of 82 and 84 kD on SDS-PAGE, considerably slower than its predicted mobility, and was exclusively associated with the spindle pole body (SPB) throughout the mitotic and meiotic cycles. Microtubule integrity was not required for Sad1 association with the SPB. Upon the differentiation of the SPB in metaphase of meiosis II, Sad1-staining patterns similarly changed from a dot to a crescent supporting an integral role in SPB function. Moderate overexpression of Sad1 led to association with the nuclear periphery. As Sad1 was not detected in the cytoplasmic microtubule-organizing centers activated at the end of anaphase or kinetochores, we suggest that Sad1 is not a general component of microtubule-interacting structures per se, but is an essential mitotic component that associates with the SPB but is not required for microtubule nucleation. Sad1 may play a role in SPB structure, such as maintaining a functional interface with the nuclear membrane or in providing an anchor for the attachment of microtubule motor proteins.

Amino Acid Sequence↗

PubMed1995

A novel essential fission yeast gene pad1+ positively regulates pap1(+)-dependent transcription and is implicated in the maintenance of chromosome structure.

Fission yeast pap1+ gene encodes an AP-1-like transcription factor, whose overexpression can confer resistance to staurosporine, a protein kinase inhibitor. We have previously identified a target gene (p25) for pap1+, and shown that, crm1+, which is required for maintenance of higher order chromosome structure, negatively regulates pap1-dependent transcription. In this study, we have characterized a novel gene, pad1+, which was isolated as a multicopy plasmid capable of conferring staurosporine-resistance. We showed that high copy pad1+ induces transcriptional activation of the p25 gene and that the induction by pad1+ is dependent on the pap1+ gene. Furthermore, a cis-element analysis of the 5'-region of the p25 gene showed that two elements (an AP-1 site and a 14 bp palindrome sequence) where pap1 binds in vitro is essential for the induction by pad1+. These results indicate that pad1 can positively regulate pap1-dependent transcription. Through an electromobility shift assay we showed that overexpression of pad1+ is not capable of enhancing the DNA-binding activity of pap1 directly. The pad1+ gene encodes a 35 kDa protein that has significant identity (68%) to Caenorhabditis elegans F37A4.5, and is also similar to mouse Mov34 and human C6.1A. Gene disruption experiments have demonstrated that pad1+ is essential for viability. A disruption mutant of pad1+ obtained after spore germination exhibited an elongated cell body with abberantly folded chromosomes. A mitotic plasmid loss experiment also produced similar cells having an abnormal chromosome structure. These suggest that pad1+ may play an important role in higher order chromosome structure. Taken concurrently with our previous results, two essential genes pad1+ and crm1+ regulate pap1-dependent transcription; pad1+ and crm1+ are positive and negative regulators, respectively.

Amino Acid Sequence↗

PubMed1995

Fission yeast TPR-family protein nuc2 is required for G1-arrest upon nitrogen starvation and is an inhibitor of septum formation.

Fission yeast nuc2+ gene encodes a protein of a tetratricopeptide repeat (TPR) family which is conserved throughout evolution. We previously showed that nuc2 is required for exit from the mitotic metaphase. In this study, we present evidence which shows that nuc2 has two additional roles in the cell cycle. We showed that the nuc2 mutant is sterile even at the permissive temperature and septation occurs in the absence of chromosome separation at the restrictive temperature. The nuc2 mutant fails to arrest at the G1 phase upon nitrogen starvation at the permissive temperature which is a prerequisite for conjugation. Upon starvation, however, the nuc2 mutant ceased division normally and induced starvation-dependent gene expression. Therefore, the nuc2 mutant is deficient only for failure to block DNA replication upon starvation. At the lower restrictive temperature, the nuc2 mutant showed a 'cut' phenotype where septation and cytokinesis takes place without the completion of mitosis. Ectopic overexpression of the nuc2+ gene caused multiple rounds of S and M phases in the complete absence of septum formation. We propose that nuc2 is a novel cell cycle regulator essential for three events; firstly for exit from mitosis, secondly for DNA replication restraint under nutrient starvation and thirdly for inhibition of septation and cytokinesis until the completion of mitosis.

Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome↗

PubMed1995

A highly sensitive enzyme immunoassay for G-CSF in human plasma.

We have developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to measure granulocyte colony-stimulating factor (G-CSF) in human plasma. This ELISA employs a combination of a mouse monoclonal antibody (MAb) as the first antibody and an affinity-purified sheep polyclonal antibody conjugated with beta-D-galactosidase as the second antibody. The coefficients of intra- and interassay variations were 2.2 to 3.6% and 8.3 to 10.2%, respectively. The assay had no cross-reactivity with four other human cytokines. Plasma G-CSF levels in healthy volunteers could easily be measured because the detection limit was 0.5 pg/mL. The mean plasma G-CSF concentration in 57 healthy volunteers (aged 19 to 47; 27 males and 30 females) was 10.5 +/- 4.5 pg/mL standard deviation [SD]). There was no statistically significant relationship between plasma G-CSF concentration and absolute neutrophil counts in those healthy volunteers (r = 0.259; p = 0.052).

Adult↗