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Biomedical subjects

Y Masui

Publications and source records attributed to Y Masui.

At least 109 records · Page 6Linked to original sources
PubMed1983

The germinal vesicle material required for sperm pronuclear formation is located in the soluble fraction of egg cytoplasm.

The chromatin of Xenopus laevis sperm nuclei was induced to decondense, swell and form mitotic chromosomes following its injection into mature Rana pipiens oocytes. In contrast, the sperm chromatin did not decondense or form mitotic chromosomes when injected into oocytes from which the germinal vesicle (GV) was removed prior to the initiation of maturation. Injection into enucleated oocytes of the material extracted from manually-isolated GVs restored their ability to decondense sperm nuclei. This soluble GV material was stable at 18 degrees C for 16 h but was inactivated by heating to 80 degrees C for 10 min. We examined the distribution of this GV material in a cytoplasmic preparation from activated eggs which can induce sperm pronuclear formation in vitro. The cytoplasmic preparation was separated into soluble and particulate fractions by centrifugation and then each fraction was injected into enucleated eggs to determine whether or not it restored the ability to decondense sperm nuclei. We found that the soluble, but not the particulate fraction could restore the ability to decondense sperm nuclei to enucleated oocytes. This result clearly indicates that the soluble fraction contains most of the GV material required for chromatin decondensation. However, since the soluble fraction fails to decondense sperm chromatin in vitro in the absence of material from the particulate fraction, sperm pronuclear formation appears to require both the soluble material derived from the GV and particulate material which can develop in the oocyte cytoplasm in the absence of the GV.

Animals↗
PubMed1983

Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.

A cell-free preparation of the cytoplasm from activated eggs of Rana pipiens induces, in demembranated sperm nuclei of Xenopus laevis, formation of a nuclear envelope, chromatin decondensation, initiation of DNA synthesis, and chromosome condensation. Both soluble and particulate cytoplasmic constituents are required to initiate these processes in vitro. The observed changes resemble processes occurring during fertilization and the mitotic cycle in early amphibian embryos. Therefore, this cell-free system may be useful in biochemical analysis of the interactions of nucleus and cytoplasm that control nuclear behavior.

Animals↗
PubMed1983

The induction of reversible and irreversible chromosome decondensation by protein synthesis inhibition during meiotic maturation of mouse oocytes.

We investigated the effects of puromycin on mouse oocyte chromosomes during meiotic maturation in vitro. Puromycin treatment for 6 hr at 100 micrograms/ml almost completely, but reversibly, suppressed [35S]methionine incorporation into oocyte protein at all stages of maturation tested. Nevertheless, oocytes treated at the germinal vesicle stage underwent germinal vesicle breakdown (GVBD) and chromosome condensation. These oocytes completed nuclear maturation to metaphase II (MII) if the inhibitor was withdrawn. Prolonged (24-hr) treatment, however, caused the chromosomes to degenerate. The chromosomes of oocytes treated shortly after GVBD for 6 hr remained condensed, but the oocytes failed to form a polar body. However, 24-hr treatment caused the chromosomes to decondense to form an interphase nucleus. Oocytes treated near MI for 6 hr gave off a polar body during the treatment, and their chromosomes decondensed to form a nucleus, which remained as long as the treatment was continued. However, if the puromycin was withdrawn, the chromosomes recondensed to a state morphologically similar to that at MII. Thus, the chromosome decondensation induced by protein synthesis inhibition at MI was reversible. Oocytes treated at MII, several hours after first polar body formation, also underwent chromosome decondensation to form a nucleus. In the continuous presence of puromycin, the chromosomes remained decondensed, but neither DNA synthesis nor mitosis occurred. However, following puromycin withdrawal, these oocytes synthesised DNA and underwent mitosis. Thus, protein synthesis inhibition at MII, by parthenogenetically activating the oocytes, caused irreversible chromosome decondensation. Based on these observations, we discussed the roles of protein synthesis in the regulation of oocyte chromosome behaviour during meiotic maturation.

Animals↗
PubMed1983

Effects of inserting eight amino acid residues into the major lipoprotein on its assembly in the outer membrane of Escherichia coli.

A DNA sequence consisting of 24 base pairs was inserted into the structural gene (lpp) coding for the major lipoprotein of the Escherichia coli outer membrane which was carried on a high-copy-number plasmid in which expression was regulated through a lac promoter-operator region. This modification resulted in the insertion of eight amino acid residues, Glu-Glu-Phe-Leu-Glu-Glu-Phe-Leu, between the glutamine residue at position 9 and the leucine residue at position 10 of the wild-type lipoprotein sequence. When production of the mutant lipoprotein was induced by a lac inducer, the cells became swollen, showed unusual morphology, and eventually lysed. When the membrane fraction was analyzed after the induction, the mutant lipoprotein was found to have been normally secreted across the cytoplasmic membrane and assembled in the outer membrane. This lipoprotein was modified with glycerol and palmitic acid and even formed the bound form, which was linked covalently to peptidoglycan. The major difference between the membrane-associated mutant lipoprotein and the wild-type lipoprotein was that the mutant lipoprotein became sensitive to trypsin treatment. These results indicate that the substantial alteration in mutant lipoprotein structure near the amino-terminal end does not interfere with modification of the amino-terminal cysteine residue or cleavage of the signal peptide by the prolipoprotein-specific signal peptidase. However, this mutant lipoprotein assembled in the outer membrane appears to have deleterious effects with respect to envelope structure and cellular morphology and viability.

Amino Acid Sequence↗
PubMed1982

Oscillatory activity of maturation promoting factor (MPF) in extracts of Rana pipiens eggs.

Dejellied Rana pipiens eggs were crushed by centrifugation and clear extracts were prepared by further centrifugation at 150,000 X G for 2 h. The extracts were kept at 0 degrees C for 2 to 3 weeks. At intervals during the storage period, 80-nl aliquots were injected into ovarian oocytes to assay maturation promoting factor (MPF). In most cases, MPF activity disappeared from the extracts on day 3 or 4, but reappeared on the following days and persisted 2 or 3 days at high levels before disappearing again. The cycle of MPF activity in extracts was repeated fairly regularly a few times during storage of the extracts. The average period of the oscillation was 5.05 + 1.25 days. If a single extract was assayed by oocytes from different frogs, all the assays exhibited exactly the same patterns of changes in MPF activity during storage of the extract. This indicates that the oscillations in MPF activity observed during storage of the extracts reflect changes in MPF activity intrinsic to the extracts, rather than fluctuations in the sensitivity of recipient oocytes to MPF. The reappearance of MPF activity in stored extracts was sensitive to Ca and dependent on Mg ions. The MPF activity that recurred periodically in cold-stored extracts was indistinguishable from that contained in fresh extracts, since oocytes induced to mature by the recurring MPF activity had chromosomes condensed to meiotic metaphase and were capable of cleavage when injected with a sperm suspension. The oscillatory behavior of MPF activity observed in this study may be comprehended on a conceptual basis by aid of a kinetic model.

Animals↗
PubMed1982

Sperm-induced cell cycle activities in blastomeres arrested by the cytostatic factor of unfertilized eggs in Rana pipiens.

Suspensions of lysolecithin-treated sperm were injected into blastomeres of two-cell embryos which had been arrested with the extract of unfertilized eggs containing cytostatic factor (CSF). Whereas the CSF-arrested blastomeres remained unchanged if no sperm were injected, those injected with sperm formed abortive cleavage furrows. The injected sperm exhibited characteristics similar to those associated with sperm injected into activated eggs. These were nuclear decondensation to the pronucleus, DNA synthesis, and late chromosome condensation to the metaphase stage. The activity of CSF-arrested blastomere cytoplasm to promote meiotic maturation when injected into ovarian oocytes rapidly decreased following sperm injection, but later fluctuated at low levels. It was suggested that the CSF-arrested blastomeres retained the potential to reinitiate cell cycle activities which could be evoked by sperm injection.

Animals↗
PubMed1982

Use of a lac promoter-operator fragment as a transcriptional control switch for expression of the constitutive lpp gene in Escherichia coli.

We constructed hybrid plasmids to allow controlled expression of the lpp gene coding for the outer membrane lipoprotein of Escherichia coli, which is otherwise expressed constitutively. This was achieved by the insertion of a DNA fragment carrying the lacUV5 promoter-operator region as a transcriptional control switch into the 5'-untranslated region of the lpp gene. When fully induced, the production of the lipoprotein, controlled under the tandem promoters of lppp-lacpo-lpp, increased approximately 3-fold compared to that under lacpo-lpp control. However, it was still only one-third of the lipoprotein production under the constitutive lpp expression. One such plasmid, pKEN125, carrying lppp-lacpo-lpp in pBR322 produced only a trace amount of the lipoprotein without induction in an E. coli lpp- cell. Upon the addition of isopropyl-beta-d-thiogalactoside, however, the amount of the lipoprotein reached almost 40% of the total membrane proteins. Cells carrying pKEN125 grew normally in the presence of the inducer, whereas cells carrying plasmid pKEN126 with tandem duplication of lppp-lacpo-lpp sequences in pBR322 lysed upon induction at high temperature. In cells with pKEN126 induced at high temperature, at least three new bands which were cross-reactive with antilipoprotein serum in addition to the mature lipoprotein were detected by pulse-labeling cells with [35S]methionine.

Base Sequence↗
PubMed1978

Induction of cleavage in nucleated and enucleated frog eggs by injection of isolated sea-urchin mitotic apparatus.

Mitotic apparatus (MA) were isolated in glycerol-dimethylsulphoxide solution (MTME) from zygotes of sea urchins (Stronglyocentrotus purpuratus). Freshly isolated MA were stored in 1/10 strength MTME for varying periods of time and were then injected into unfertilized frog (Rana pipiens) eggs. These injections induced 40-60% of the recipient frog eggs to initiate cleavage, resulting in the formation of blastula cell clusters. The cleavage-inducing activity of MA stored in 1/10 MTME at room temperature decreased with time of storage in 1/10 strength MTME, and disappeared by about 6 h. There was no change in the ultrastructure of MA during storage. MA isolated and stored in MTME at room temperature had a constant level of cleavage-inducing activity during the first 48 h of storage, but this activity slowly declined upon further storage; almost no activity was left after 3 weeks. MA isolated in hexylene glycol (HG) and immediately transferred into MTME were compared with MA isolated in MTME; both MA had the same cleavage-inducing activity on the day of isolation, after which the MA isolated in HG quickly lost activity. On the other hand, MA isolated and stored in HG had little cleavage-inducing activity when tested 3 h following isolation. Cleavage-inducing agent (CIA) isolated from frog brains induced cleavage and blastula formation when injected into nucleated frog eggs, but had no such activity when injected into enucleated frog eggs. MA isolated in MTME induced cleavage and blastula formation in enucleated frog eggs as well as in nucleated frog eggs. Cytological examination revealed that blastula cells which developed from MA-injected enucleated eggs contained Feulgennegative nuclei, whereas cells which developed from CIA-injected nucleated eggs contained Feulgen-positive nuclei. These results suggest that sea-urchin nuclear materials participate in mitosis in frog eggs. Isolated MA which had been stored in MTME for 3 weeks and which exhibited little cleavage-inducing activity were injected together with frog brain CIA into either normal or enucleated eggs; normal recipient eggs cleaved with significantly higher frequencies (70%) than those injected with CIA alone (40%). Furthermore, enucleated eggs injected with CIA alone failed to cleave, while those injected with MA and CIA together cleaved with significant frequencies (overall 29%). This result suggests a cooperative interaction between CIA and the inactivated MA to restore the cleavage-inducing activity of MA.

Animals↗