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Biomedical subjects

Y Masui

Publications and source records attributed to Y Masui.

At least 91 records · Page 5Linked to original sources
PubMed1987

cDNA cloning of IL-1 alpha and IL-1 beta from mRNA of U937 cell line.

Clones of cDNAs encoding growth inhibitory factors for human melanoma cell line A375 were isolated from cDNA library prepared by using mRNA derived from human histiocytic lymphoma cell line U937 induced with PMA and further stimulated with LPS. Cloning was achieved using Okayama-Berg cDNA expression vector system that permits expression of the inserted cDNA segments in mammalian cells. By assaying the transfected COS-1 cells supernatants and cell extracts, we isolated two distinct cDNA clones encoding growth inhibitory factors. It was determined by the nucleotide sequences of the inserts, the cDNAs corresponded to IL-1 alpha and -1 beta. Our results indicate U937 cells can be induced to produce both interleukin-1s.

Amino Acid Sequence↗
PubMed1987

Dose-dependent relationship between oocyte cytoplasmic volume and transformation of sperm nuclei to metaphase chromosomes.

We have studied the chromosome condensation activity of mouse oocytes that have been inseminated during meiotic maturation. These oocytes remain unactivated, and in those penetrated by up to three or four sperm, each sperm nucleus is transformed, without prior development of a pronucleus, into metaphase chromosomes. However, those penetrated by more than four sperm never transform any of the nuclei into metaphase chromosomes (Clarke, H. J., and Y. Masui, 1986, J. Cell Biol. 102:1039-1046). We report here that, when the cytoplasmic volume of oocytes was doubled or tripled by cell fusion, up to five or eight sperm nuclei, respectively, could be transformed into metaphase chromosomes. Conversely, when the cytoplasmic volume was reduced by bisection of oocytes after the germinal vesicle (GV) had broken down, no more than two sperm could be transformed into metaphase chromosomes. Thus, the capacity of the oocyte cytoplasm to transform sperm nuclei to metaphase chromosomes was proportional to its volume. The contribution of the nucleoplasm of the GV and the cytoplasm outside the GV to the chromosome condensation activity was investigated by bisecting oocytes that contained a GV and then inseminating the nucleate and anucleate fragments. The anucleate fragments never induced sperm chromosome formation, indicating that GV nucleoplasm is required for this activity. In the nucleate fragments, the capacity to induce sperm chromosome formation was reduced as compared with whole oocytes, in spite of the fact that the fragments contained the entire GV nucleoplasm. This implies that non-GV cytoplasmic material also was required for chromosome condensation activity. When inseminated oocytes were incubated in the presence of puromycin, the sperm nuclei were transformed into interphase-like nuclei, but no metaphase chromosomes developed. However, when protein synthesis resumed, the interphase nuclei were transformed to metaphase chromosomes. These results suggest that the transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of mouse oocytes requires both the nucleoplasm of the GV and non-GV cytoplasmic substances, including proteins synthesized during maturation.

Animals↗
PubMed1986

Nuclear-magnetic-resonance studies on the conformation of membrane-bound alpha-mating factor. Transferred nuclear Overhauser effect analysis.

The C-H proton resonances of alpha-mating factor, yeast pheromone, in 2H2O solution were assigned. The phase transition temperature of perdeuterated dipalmitoylglycerophosphocholine (suspension) was found to be 35.5 degrees C. In the presence of vesicles of this phospholipid, the exchange broadening and transferred nuclear Overhauser effect (TRNOE) of peptide proton resonances (at 50 degrees C) were analyzed. The mode of binding of this peptide with the phospholipid bilayer was elucidated. The N-terminal nine residues (Trp1-Gly9) are tightly bound to the bilayer, while the C-terminal four residues (Gln10-Tyr13) are left free in aqueous phase. This is consistent with the previous observation that the C-terminal three residues (Pro11-Tyr13) are not essential for the activity of this pheromone [Masui, Y. et al. (1977) Biochem. Biophys. Res. Commun. 78, 534-538]. Furthermore, from the TRNOE analyses, the conformation of the membrane-bound N-terminal part of alpha-mating factor was elucidated; the residues Trp1-Gln5 form a compact helical structure while the residues Lys7-Gly9 form an extended structure. A similar TRNOE was also observed for an active decapeptide analog Trp1-Gln10. This confirms the previous conclusion that the physiological activities of this pheromone and analog peptides are correlated with the conformations of membrane-bound peptide molecules [Higashijima, T. et al. (1983) FEBS Lett. 159, 229-232].

Fungal Proteins↗
PubMed1986

Chromosome condensation activity in the cytoplasm of anucleate and nucleate fragments of mouse oocytes.

The activity of maturation promoting factor (MPF) which causes chromosome condensation and subsequent oocyte maturation was investigated in mouse oocytes using polyethylene-glycol-mediated cell fusion technique. Fully grown oocytes were bisected at germinal vesicle (GV) stage or shortly after germinal vesicle breakdown (GVBD) into anucleate and nucleate fragments. After 2-3 or 15-17 hr of culture these fragments were fused with interphase blastomeres from two-cell embryos. It was found that almost all the anucleate oocyte fragments cultured for a short term (2-3 hr), regardless of whether they were produced at GV stage or after GVBD, induced premature chromosome condensation in the blastomere nuclei, whereas only about 20% of those cultured for a long term (15-17 hr) could do so. On the other hand, the nucleate fragments always retain the cytoplasmic activity to induce chromosome condensation. Thus we suggested that the MPF initially could appear in mouse oocytes independently of the GV, that the mixing of GV material with the oocyte cytoplasm following GVBD had no effect on the activity of MPF in anucleate fragments, and that oocyte chromosomes or some components associated with them could play a significant role in maintaining the MPF activity.

Animals↗
PubMed1986

Interactions between metaphase and interphase factors in heterokaryons produced by fusion of mouse oocytes and zygotes.

The cytoplasmic factor responsible for chromosome condensation was introduced into mouse zygotes at different times after fertilization by fusion of the zygotes with metaphase I oocytes. In 72% of heterokaryons obtained after fusion of early zygotes (14-18 hr post-human chorionic gonadotrophin (HCG) with oocytes, the male and female pronuclei of the zygote decondensed. At the same time, the oocyte chromosomes became enclosed in a nuclear envelope and decondensed to an interphase state. However, in the rest of the heterokaryons, the chromatin of the pronuclei condensed to metaphase chromosomes, thus resulting in three sets of chromosomes. Fusion of zygotes that had begun DNA synthesis (20-22 hr post-HCG) with oocytes induced chromosome condensation of the pronuclei in 76% of the cases. In some heterokaryons, however, the oocyte chromosome decondensed to an interphase state similar to the zygote pronuclei. Fusion between late zygotes (27-29 hr post-HCG) with oocytes resulted in chromosome condensation of the pronuclei in all heterokaryons. On the basis of these results, the formation of the pronuclei and their progression toward mitosis in the zygote may be explained by changing levels of a metaphase factor in the cell, or by a balance between interphase and metaphase factors.

Animals↗
PubMed1986

Microtubule cycles in oocytes of the surf clam, Spisula solidissima: an immunofluorescence study.

Oocytes of the surf clam, Spisula solidissima, underwent germinal vesicle breakdown and two meiotic divisions to give off polar bodies when they were fertilized or parthenogenetically activated with KCl. Fertilized eggs further proceeded to mitosis and cleaved, while parthenogenetically activated eggs remained uncleaved. We examined changes in microtubule-containing structures during meiotic divisions and subsequent mitotic processes by immunofluorescence. A monoclonal anti-tubulin antibody was applied to alcohol-fixed eggs from which the vitelline membrane had been removed by protease digestion. Up to the stage of second polar body formation, the pattern of microtubule organization in the first and second meiotic spindles was identical in both fertilized and parthenogenetically activated eggs. However, while fertilized eggs formed a sperm aster and mitotic spindles later, activated eggs formed only monaster- or ring-shaped microtubule-containing structures which underwent cycles of alternating formation and breakdown. Lactoorecin staining of parthenogenetically activated eggs revealed that the chromosome cycle could occur in these eggs, in phase with this microtubule cycle.

Animals↗
PubMed1986

Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse.

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.

Animals↗
PubMed1985

Functional and morphometric study of the liver in motor neuron disease.

In routine liver function tests, 23 of 44 patients with motor neuron disease (MND) had abnormal findings, and there was disturbance of unconjugated bilirubin metabolism in 10 of the 33 patients tested. Liver-biopsy specimens from 10 MND patients were compared by electron microscopic examination with specimens from age-matched controls who had chronic persistent hepatitis. The MND patients had a higher incidence of intramitochondrial inclusions, less abundant mitochondria in a given area of cytoplasm and enlarged mitochondria. Electron-probe X-ray microanalysis of hepatocytic lysosomes found copper in 8 of 13 MND patients, but not in the controls. These findings suggest that the pathogenetic processes in MND may involve not only motor neurons but also hepatic cells.

Adult↗
PubMed1985

Inhibition by dibutyryl cyclic AMP of the transition to metaphase of mouse oocyte nuclei and its reversal by cell fusion to metaphase oocytes.

Mouse oocytes at metaphase I of meiotic maturation were treated with puromycin, which caused the condensed chromosomes to become decondensed to form an interphase nucleus. The chromosomes returned to a metaphase state 6.3 hr after the oocytes were transferred to puromycin-free medium [H. J. Clarke and Y. Masui (1983) Dev. Biol. 97, 291-301]. In contrast, the chromosomes of the puromycin-treated oocytes remained decondensed within the nucleus if dibutyryl cyclic AMP (dbcAMP) was included in the puromycin-free medium. This implies that dbcAMP inhibited the development of conditions in the oocytes that were required for the transition to metaphase. The chromosomes of puromycin-treated oocytes that were incubated for 7.5 hr in dbcAMP-containing medium returned to metaphase just 1.9 hr after transfer to dbcAMP-free medium. Therefore, the protein synthesis-dependent process that is required for the transition to metaphase could occur in the presence of dbcAMP. Fusion to metaphase II oocytes, or to puromycin-treated oocytes that had returned to metaphase, rapidly induced transition of the nuclei of dbcAMP-inhibited oocytes to metaphase, despite the presence of the inhibitor. These results suggest that the transition of nuclei to metaphase can be induced by a cytoplasmic factor that is present in metaphase oocytes, and that dbcAMP inhibits the development of this factor.

Animals↗
PubMed1984

Nuclear-magnetic-resonance studies on the conformations of tridecapeptide alpha-mating factor from yeast Saccharomyces cerevisiae and analog peptides in aqueous solution. Conformation-activity relationship.

The conformation of tridecapeptide alpha-mating factor from yeast Saccharomyces cerevisiae in aqueous solution was analyzed, in comparison with those of active analog and inactive analog peptides. 270-MHz 1H-NMR spectra of these peptides were observed and the spectral patterns of main-chain N-H proton resonances were classified into three groups. alpha-mating factor and Trp1-bearing active peptides belong to the group A1, active des-Trp1-peptides belong to the group A2 while the peptides of group B are inactive. The main-chain N-H proton resonances of the groups A1 and A2 and side-chain N-H proton resonances were all assigned to individual residues. The 13C-NMR analysis of alpha-mating factor indicates that the Lys7-Pro8 and Gln10-Pro11 peptide bonds exclusively take the trans form. From the temperature and pH dependences of chemical shifts and Gd(III)-induced relaxation enhancements of amide proton resonances, alpha-mating factor is found to take partly a folded conformation in aqueous solution, with an alpha-helical form in the N-terminal domain and two beta-turn forms in the central and C-terminal domain. The pH dependence of fluorescence intensity indicates that, in this folded conformation, the C-terminal carboxylate group lies close to the N-terminal domain. The presence of the folded form in the N-terminal domain and the beta-turn form in the central domain correlates with the biological activity of alpha-mating factor and analog peptides. However, the folded conformation of alpha-mating factor is in equilibrium with predominantly unordered form, as found from the circular dichroism and NMR analyses. The N-H proton and C-alpha proton resonances of free alpha-mating factor as assigned in the present study allow the transferred nuclear Overhauser enhancement (NOE) analysis of the membrane-bound conformation that is more directly related with the activity.

Amides↗
PubMed1984

Secretion cloning vectors in Escherichia coli.

The DNA fragment coding for the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, has been inserted into the high-level expression vectors, pIN-III. A foreign DNA fragment can be cloned in any one of the three reading frames at the unique EcoRI, HindIII or BamHI sites immediately after the ompA signal peptide coding sequence. The cloned foreign gene is under the control of both the lpp promoter and the lac promoter-operator. The expression of the gene is regulated by the lac repressor produced by the same vectors. Using the pIN-III-ompA vector, the DNA fragment coding for only the mature portion of beta-lactamase was inserted into the EcoRI site. Upon induction of gene expression, beta-lactamase was secreted into the periplasmic space. The ompA signal peptide was correctly removed resulting in the production of beta-lactamase with four extra amino acid residues (Gly-Ile-Pro-Gly) at its amino terminus due to the linker sequence in the vector. After a 3-h induction, beta-lactamase was accumulated to 20% of total cellular protein without any detectable accumulation of pro-beta-lactamase. Using oligonucleotide-directed site-specific mutagenesis, we have also removed the linker sequence and upon induction of gene expression, beta-lactamase with the authentic NH2-terminal sequence was produced, in even larger amounts than the beta-lactamase with the linker sequence.

Amino Acid Sequence↗
PubMed1984

Effects of Ca2+ ions on the formation of metaphase chromosomes and sperm pronuclei in cell-free preparations from unactivated Rana pipiens eggs.

Nuclei transplanted into unactivated amphibian eggs are known to condense into metaphase chromosomes whereas those transplanted into activated eggs decondense and enlarge. We have made cell-free cytoplasmic preparations from Rana pipiens eggs which can induce demembranated Xenopus laevis sperm to undergo changes similar to those seen in intact eggs. Sperm chromatin which is incubated for 3 hr in unactivated egg preparations made using a buffer containing 3 mM EGTA is induced to form metaphase chromosomes. However, decondensed interphase nuclei are formed when chromatin is incubated in unactivated egg preparations made without EGTA as well as in activated egg preparations. When Ca2+ ions are added to unactivated egg preparations made with EGTA, the preparations lose the ability to induce metaphase chromosome formation and become capable of decondensing sperm chromatin. Once the ability to decondense chromatin has developed, either in unactivated or activated egg preparations, it cannot be suppressed by the addition of EGTA. However, decondensation of sperm chromatin in activated egg preparations can be suppressed by the addition of unactivated egg preparations made with EGTA. In this case, the incubated sperm chromatin is induced to form metaphase chromosomes. These results may indicate that the chromosome condensation activity of unactivated egg cytoplasm can be sustained in cell-free preparations when Ca2+ ion levels are kept low, but when Ca2+ ion levels increase this activity is lost and replaced by a new activity which can decondense chromatin. Since this change in cytoplasmic activities is comparable to that occurring in the intact egg following fertilization, these results suggest that Ca2+ ions play a crucial role during activation in altering the cytoplasmic activities which control nuclear behavior.

Animals↗
PubMed1984

Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs.

A cell-free cytoplasmic preparation from activated Rana pipiens eggs could induce in demembranated Xenopus laevis sperm nuclei morphological changes similar to those seen during pronuclear formation in intact eggs. The condensed sperm chromatin underwent an initial rapid, but limited, dispersion. A nuclear envelope formed around the dispersed chromatin and the nuclei enlarged. The subcellular distribution of the components required for these changes was examined by separating the preparations into soluble (cytosol) and particulate fractions by centrifugation at 150,000 g for 2 h. Sperm chromatin was incubated with the cytosol or with the particulate material after it had been resuspended in either the cytosol, heat-treated (60 or 100 degrees C) cytosol or buffer. We found that the limited dispersion of chromatin occurred in each of these ooplasmic fractions, but not in the buffer alone. Nuclear envelope assembly required the presence of both untreated cytosol and particulate material. Ultrastructural examination of the sperm chromatin during incubation in the preparations showed that membrane vesicles of approximately 200 nm in diameter, found in the particulate fraction, flattened and fused together to contribute the membranous components of the nuclear envelope. The enlargement of the sperm nuclei occurred only after the nuclear envelope formed. The pronuclei formed in the cell-free preparations were able to incorporate [3H]dTTP into DNA. This incorporation was inhibited by aphidicolin, suggesting that the DNA synthesis by the pronuclei was dependent on DNA polymerase-alpha. When sperm chromatin was incubated greater than 3 h, the chromatin of the pronuclei often recondensed to form structures resembling mitotic chromosomes within the nuclear envelope. Therefore, it appeared that these ooplasmic preparations could induce, in vitro, nuclear changes resembling those seen during the first cell cycle in the zygote.

Animals↗
PubMed1984

Roles of Ca ions and ooplasmic factors in the resumption of metaphase-arrested meiosis in Rana pipiens oocytes.

Fully grown amphibian oocytes are arrested at the diplotene stage of meiosis. When they undergo meiotic maturation, meiosis resumes and the oocyte chromosomes condense to metaphase. During this period, the oocyte cytoplasm develops 'chromosome condensation activity' (CCA), the ability to induce the formation of metaphase chromosomes from nuclei transplanted into the oocytes. The cytoplasm also produces 'maturation promoting factor' (MPF), the substance that induces meiotic maturation when injected into oocytes. Also, before meiosis is arrested again at the 2nd metaphase, the cytoplasm develops 'cytostatic factor' (CSF), the substance that causes metaphase arrest when injected into zygotes. Since CSF-arrested zygotes have properties similar to those of metaphase-arrested oocytes, including the potential to resume cell cycle activities, CSF appears to be a genuine factor that causes meiotic arrest. Following oocyte activation, meiosis is completed and the chromosomes decondense to form a pronucleus. During this period, the oocyte cytoplasm loses its CCA and develops 'chromosome decondensation activity' (CDA), the ability to decondense the chromatin of injected nuclei. Concomitantly, MPF and CSF disappear. Both MPF and CSF are inactivated by Ca2+ ions, in vitro. The sensitivity of CCA to Ca2+ and the requirement for Ca2+ during the development of CDA have also been demonstrated in vitro by incubating demembranated sperm nuclei in cell-free preparations from unactivated or activated oocytes. Preparations made from unactivated oocytes in the presence of EGTA exhibit CCA, whereas those made in the absence of EGTA, as well as those made from activated eggs, exhibit CDA. Unactivated ooplasmic preparations made using EGTA lose CCA and develop CDA when Ca2+ ions are added to them. However, at low Ca2+ concentrations CCA is sustained and, when unactivated and activated preparations are mixed, is able to overcome CDA. Therefore, it is likely that at low intracellular Ca2+ levels in unactivated oocytes, CSF is stable and CCA predominates over CDA, thus preventing oocyte chromosomes from decondensing. However, when Ca2+ levels are elevated during oocyte activation, CSF disappears and CCA is replaced by CDA. This change in cytoplasmic activities may allow meiosis to resume.

Animals↗