Biomedical subjects

Y Masui

Publications and source records attributed to Y Masui.

At least 37 records · Page 2Linked to original sources

PubMed1996

Calcium-dependent development of secondary cytostatic factor (2 degrees CSF) from Xenopus laevis oocytes and zygotes.

Fresh cytosols extracted from unfertilised eggs of Xenopus laevis contain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca2+ and is designated primary CSF (1 degrees CSF). During storage of Ca2(+)-containing cytosols at 2 degrees C, a stable CSF activity appears which is designated secondary CSF (2 degrees CSF). In the present study, we report that 2 degrees CSF activity can be induced in cytosols extracted from stage VI oocytes, unfertilised eggs, electrically activated eggs or blastulae, in the presence of Ca2+. Both the intensity and the rate of 2 degrees CSF development are dependent on the concentration of Ca2+ ions added to the cytosol. At Ca2+ concentrations of 5-10 mM, 2 degrees CSF activity reaches a maximum in about 7 days. Secondary CSF is relatively resistant to heat but loses all activity after 5 min at 70 degrees C. When stored at-80 degrees C, 2 degrees CSF activity remains detectable for about 6 weeks. Cytological observations show that blastomeres arrested by microinjection of 2 degrees CSF developed in cytosols of unfertilised eggs, activated eggs or blastulae contain metaphase chromosomes embedded in a bipolar spindle that has no asters developed at its poles. In contrast, blastomeres arrested by 2 degrees CSF in cytosols of stage VI oocytes contain condensed chromosomes but no spindle is formed. The mechanisms of the development of 2 degrees CSF in Ca2(+)-containing cytosols and its mode of action are discussed.

Animals↗

PubMed1996

The role of microtubules and inositol triphosphate induced Ca2+ release in the tyrosine phosphorylation of mitogen-activated protein kinase in extracts of Xenopus laevis oocytes.

Microsomal fractions of Xenopus oocytes release preloaded 45 Ca2+ when treated with inositol triphosphate (InsP3). The effective concentration of InsP3 required for half-maximal release (EC50) is 59 nM and maximal release occurs at approximately 2 microM InsP3. Uptake and release of 45 Ca2+ are not altered by the catalytic subunit of protein kinase A, dibutyrl cyclic adenosine monophosphate, protein kinase A peptide inhibitor or nocodazole. In contrast, taxol decreases the sensitivity of the microsomal fraction to InsP3, shifting the EC50 for InsP3-induced Ca2+ release from 59 to 259 nM. In lysates of oocytes, InsP3-induced Ca2+ release causes the tyrosine phosphorylation of a 42,000 (M(r) 42k) protein identified as 42k mitogen-activated protein (MAP) kinase. InsP3-induced tyrosine phosphorylation of MAP kinase is prevented by BAPTA and taxol, but not by nocodazole. Thus, microtubule polymerisation modifies InsP3-induced Ca2+ release, thereby inhibiting phosphorylation of MAP kinase.

Animals↗

PubMed1995

Maintenance of metaphase in colcemid-treated mouse eggs by distinct calcium- and 6-dimethylaminopurine (6-DMAP)-sensitive mechanisms.

In mouse eggs, the arrest at meiotic metaphase II is released by the fertilization-induced increase in intracellular calcium. When eggs treated with the microtubule inhibitor colcemid are fertilized or exposed to the calcium ionophore A23187, normal calcium increases occur, but the eggs remain at metaphase. However, when colcemid-treated eggs are fertilized or A23187-treated and then exposed to the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), they enter interphase. Although colcemid-treated eggs require a calcium signal and exposure to 6-DMAP, colcemid-treated embryonic cells are released from metaphase by treatment with 6-DMAP, but not by exposure to A23187. These results suggest that two distinct mechanisms maintain metaphase: one is the calcium-sensitive mechanism involving cytostatic factor, which normally maintains metaphase II arrest in eggs; the other mechanism, which may require the activity of 6-DMAP-sensitive kinases, maintains metaphase in the absence of spindle assembly in both mitotic cells and eggs.

Adenine↗

PubMed1995

Regulation of the appearance of division asynchrony and microtubule-dependent chromosome cycles in Xenopus laevis embryos.

Divisions of animal-cap blastomeres dissociated from Xenopus laevis embryos are synchronous mostly up to 12th cleavage or the 13th cell cycle, but become asynchronous afterward, during the midblastula transition (MBT), and at the same time, chromosome cycles become microtubule-dependent and are arrested in mitosis if treated with nocodazole. To investigate causes for these changes in cell-cycle control, we observed division synchrony in animal-cap blastomeres dissociated from embryos whose nucleocytoplasmic ratio (N/C) had been altered by constriction of zygotes or by delaying nucleation into zygote halves and compared their mitotic indices in the presence and absence of nocodazole. Thus, we found that asynchronous divisions always commenced when N/C reached the value of 128 to 256 times that of an animal blastomere of the 32-cell embryo, corresponding to the 12th and 13th cycles of a normal embryo, while the number of synchronous cycles became variable, ranging from 9 to 14, depending on the initial N/C. Treatment with alpha-amanitin or cycloheximide did not alter the number of synchronous cycles. However, the time at which the mitotic index of nocodazole-treated blastomeres first exceeded that of control remained constant, at 3 to 5 hr after 5th cleavage, regardless of the initial N/C. Thus, chromosome cycles of blastomeres first became sensitive to nocodazole at a variable N/C, ranging from 8 to 1024 times that of an animal blastomere of the 32-cell embryo. The timing of the appearance of nocodazole sensitivity was unaffected by alpha-amanitin treatment, whereas it was markedly delayed following cycloheximide treatment. These results suggest that the commencement of division asynchrony is N/C-dependent, whereas the development of microtubule-dependent cell cycles is age-dependent, most likely being programmed by the translation of stored mRNAs.

Amanitins↗

PubMed1995

Metaphase arrest in newly matured or microtubule-depleted mouse eggs after calcium stimulation.

In mouse eggs arrested at meiotic metaphase II, the increase in intracellular calcium that results from fertilisation induces nuclear formation in both newly ovulated and older eggs. In contrast, the calcium increase that results from exposure to the calcium ionophore A23187 induces nuclear formation in older, but not young, newly ovulated eggs. When treated with the microtubule inhibitor colcemid, and fertilised, young eggs remained at metaphase, but many older eggs formed nuclei, although older eggs treated with colcemid and A23187 remained at metaphase. However, young A23187-treated eggs, young colcemid-treated fertilised eggs, and older colcemid- and A23187-treated eggs, formed nuclei when treated, in addition, with the protein synthesis inhibitor cycloheximide, or the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP). The possibility is discussed that metaphase in newly matured eggs and microtubule-depleted eggs may be maintained by similar mechanisms involving short-lived phosphorylated proteins.

Animals↗

PubMed1995

Purification and properties of two chitinases from Streptomyces sp. J-13-3.

Two chitinases (Chi-A and Chi-B) purified from Streptomyces sp. J-13-3 had the same molecular weights (31,000) and enzymatic properties (optimum pH and temperature of pH 6.0 and 45 degrees C) but had significantly different isoelectric points (3.9 for Chi-A, 3.5 for Chi-B). Chi-A and -B had identical N-terminal amino acid sequences (ADXAAAWNASSVYTGGGSASYNGHN), similar amino acid compositions, and immunological cross-reactivities. A concomitant decrease of Chi-A and increase of Chi-B was observed in their productions during cultivation.

Amino Acid Sequence↗

PubMed1995

Structure and action of MIP (Mytilus inhibitory peptide)-related tetrapeptides synthesized with a multipeptide synthesizer.

Using a multipeptide synthesizer we synthesized 19 peptide libraries, each of which consisted of 19 MIP-related tetrapeptides, and isolated a number of peptides, which have an inhibitory effect on phasic contraction of the ABRM of Mytilus, from the libraries. To the present, the structures of about 30 species of the peptides were determined, and the peptides with the determined structures were synthesized. The structure and action of each synthetic peptide was compared with those of others to explain structure-activity relationship of MIPs.

Amino Acid Sequence↗

PubMed1994

Enhancement of mouse egg activation by the kinase inhibitor, 6-dimethylaminopurine (6-DMAP).

Metaphase arrest in vertebrate eggs is maintained by the action of cytostatic factor (CSF) on maturation promoting factor (MPF). In amphibian eggs, the kinase inhibitor, 6-dimethylaminopurine (6-DMAP) inactivates both CSF and MPF, resulting in the release from metaphase and entry into interphase. In the mouse, 6-DMAP induces nuclear formation in maturing oocytes, but not in eggs at metaphase II. We found that 6-DMAP accelerated the transition to interphase in mouse eggs treated with the parthenogenetic agents A23187, cycloheximide, or phorbol 12-myristate 13-acetate (PMA). Newly ovulated eggs, refractory to full activation by A23187, entered interphase when treated with A23187 and 6-DMAP in combination. Occasional batches of eggs formed nuclei in response to 6-DMAP alone. These results are discussed with regard to the possible effects of 6-DMAP on MPF and CSF.

Adenine↗

PubMed1994

Incorporation of norleucine at methionine positions in recombinant human macrophage colony stimulating factor (M-CSF, 4-153) expressed in Escherichia coli: structural analysis.

Expression of the 17.5-kDa truncated form of human recombinant macrophage colony stimulating factor (rM-CSF, 4-153) in Escherichia coli is complicated by the replacement of methionine residues by norleucine. In order to detect and quantitate this mistranslational event, the intact and the S-carboxyamidomethylated proteins were analyzed by amino acid analysis, automated Edman amino acid sequencing, and electrospray mass spectrometry. In addition, the endoproteinase Glu-C generated peptides were subjected to amino acid sequencing, high-performance liquid chromatography, and electrospray ionization mass spectrometry. The extent of norleucine substitution in different batches of rM-CSF varied between 0% and 20%. The relative instability of methionine residues needs to be considered when calculating the extent of norleucine substitution at methionine positions. The mass spectrometry of the intact rM-CSF allowed for examination of the distribution of multiply substituted methionine to norleucine species, and it enabled detection and quantitation of the norleucine incorporation down to the approximately 3% level. Selective ion chromatograms of molecular ions of interest obtained in reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry of proteolytic fragments offered a reliable and fast method of detection and quantitation of norleucine-containing peptides. Norleucine residues were uniformly distributed among all four methionine positions (10, 27, 61, and 65). A substitution of methionine by its structural norleucine analog does not have any effect on the activity of the refolded rM-CSF dimers.

Amino Acid Sequence↗

PubMed1994

Single instillation of hydroxypropylcellulose-doxorubicin as treatment for superficial bladder carcinoma.

A single instillation of hydroxypropylcellulose (HPC)-doxorubicin (20 mg/20 ml) was performed in 20 patients with superficial bladder carcinoma. The therapeutic effect was assessed by cystoscopy at 14-30 days after the instillation, and the residual tumor tissue was resected by transurethral resection (TUR) when possible. The results obtained for the therapeutic effect were as follows: a complete response (CR), in 7 cases (35%); a reduction in size of more than 50% (partial response, PR), in 6 cases (30%); and a reduction of less than 25% in size (no change, NC), in 7 cases (35%). Combined intravesical instillation of HPC-doxorubicin and local hyperthermia using a Thermotron RF-8 was performed in 11 patients with recurrent superficial bladder carcinoma. The total number of treatment courses ranged from three to five per patient. The results obtained for the effect of this combined treatment were as follows: a CR, in 6 cases (54.5%); a PR, in 3 cases (27.3%); and NC, in 2 cases (18.2%). Therefore, the combination of intravesical instillation of HPC-doxorubicin and local hyperthermia was more effective against superficial bladder carcinoma than the single instillation of the chemotherapeutic agent alone.

Administration, Intravesical↗

PubMed1994

Effect of IL-1 receptor antagonist and antiserum to TNF-alpha on LPS-induced plasma ACTH and corticosterone rise in rats.

Using an antiserum against tumor necrosis factor (TNF)-alpha and an interleukin (IL-1) receptor antagonist, we studied putative roles of these cytokines in mediating the endotoxin-induced elevation of plasma adrenocorticotropic hormone (ACTH) and corticosterone levels in freely moving rats. Intravenous administration of Escherichia coli lipopolysaccharide (LPS) increased plasma ACTH and corticosterone levels in a dose-dependent manner. The plasma corticosterone reached to its highest level among a series of experiments after the administration of even the smallest dose (0.03 microgram/kg) tested. Plasma ACTH and corticosterone levels in these rats were completely inhibited by the intravenous administration of anti-murine TNF-alpha-rabbit antiserum (anti-TNFAS) after the administration of LPS but not by the intravenous administration of IL-1 receptor antagonist (IL-1RA). On the other hand, both recombinant human IL-1RA and anti-TNFAS significantly inhibited plasma ACTH increase stimulated with 10 micrograms/kg LPS. These findings indicate that 1) when the plasma corticosterone increase induced by intravenous LPS remains below its maximum, the effect is exclusively mediated by TNF-alpha, and 2) when a larger amount of LPS is administered, both IL-1 beta and TNF-alpha participate at least in part in the hypothalamic-pituitary-adrenal axis activation.

Adrenocorticotropic Hormone↗

PubMed1994

A rapid method for whole mount preparations of mammalian oocytes and early embryos.

Whole mounts of mouse oocytes and embryos are useful for observing intracellular structures while preserving morphological integrity. This method is inconvenient for rapid processing of a large number of specimens because washing each specimen in a protein-free solution is required prior to transfer into the fixative. We have developed a new fixative which does not cause protein precipitation which can be added directly to the culture medium. Specimens can be preserved in culture dishes for at least one month, and processed for cytological observation at a convenient time. When stained with hematoxylin, details of cellular structures such as nuclei, nucleoli, chromosomes and spindle microtubules can be observed while maintaining the organization of the organelles.

Animals↗

PubMed1994

[Detection of neural crest tumors by 123I-MIBG scintigraphy].

From January 1993 to January 1994, scintigraphy with 123I-MIBG and/or 131I-MIBG were performed in 22 patients and their scintigraphic usefulness was evaluated. Iodine-123 MIBG and 131I-MIBG scintigrams were obtained 24 hours after injection of 222 MBq of 123I-MIBG and 48 hours after injection of 20 MBq of 131I-MIBG, respectively. In two patients with pheochromocytoma, the 123I-MIBG and 131I-MIBG scans were performed and both images were compared. In a patient with single intraadrenal pheochromocytoma, the lesion not detected with 131I-MIBG was clearly visualized with 123I-MIBG. In the other patient with multiple metastatic pheochromocytoma, much more lesions were distinctly demonstrated on the 123I-MIBG images than on the 131I-MIBG images. All of the lesions were detected with 123I-MIBG in a patient with pheochromocytoma, 3 patients with neuroblastoma and a patient with medullary thyroid cancer. Most of the normal adrenal glands (86%) were visualized on the 123I-MIBG scintigrams, in 7 patients without neural crest tumor and adrenal diseases, while 131I-MIBG scintigraphy failed to visualize normal adrenal glands in 2 hypertensive patients. The main reason for the superiority of 123I-MIBG to 131I-MIBG is considered to be as follows: 1) higher specific activity of 123I-MIBG. 2) the larger amount of 123I-MIBG used. 3) gamma ray energy of 123I is ideal for gamma camera. In conclusion, 123I-MIBG appears to be a more suitable imaging agent than 131I-MIBG in depicting neural crest tumors.

3-Iodobenzylguanidine↗

PubMed1993

Mechanisms of experimental cancer cachexia. Local involvement of IL-1 in colon-26 tumor.

In the colon-26 (C-26) tumor model, the cytokine IL-6 is an important factor involved in experimental cancer cachexia. Recent in vitro data indicated that IL-1 plays a role in the interaction between host macrophages and C-26 cells that express IL-1R, resulting in the amplification of tumor IL-6 production. To investigate the role of IL-1 on the development of C-26 cachexia in vivo, the effect of specific blockade of the action of IL-1 with reagents against IL-1R was evaluated. Both IL-1R antagonist (IL-1RA) and the mAb 35F5 directed against IL-1R type I, prevented binding of radioactive IL-1, and inhibited IL-1-induced IL-6 synthesis by the C-26 cell line. Whereas a systemic administration of these reagents did not reverse weight loss in C-26-bearing mice, intratumoral injections of IL-1RA significantly reduced cachexia. Furthermore, body composition analysis confirmed that this treatment improved lean tissue and fat, as well as hypoglycemia and serum IL-6 level. The fact that the treatment did not change the tumor burden suggests that it affected the host directly. These results support the hypothesis that, at the microenvironment of the C-26 tumor, IL-1 is involved in the cachexia endured by the host.

Animals↗

PubMed1993

Changes in protein association with intracellular membranes of Xenopus laevis oocytes during maturation and activation.

Intracellular membranes isolated from fully grown immature oocytes, mature oocytes (eggs) and activated eggs of Xenopus laevis were fractionated through a discontinuous sucrose density gradient into light, intermediate and heavy fractions. Electron microscopy showed that the light and intermediate fractions consisted mainly of smooth membranes, while the heavy fraction consisted mainly of rough membranes and mitochondria. Variations in the proteins associate with samples taken at different stages were observed by SDS-PAGE. The following differences were consistently observed: a 200 kDa protein was present only in the intermediate fraction of activated eggs, 29 and 44 kDa proteins were present only in the intermediate fractions of immature oocytes and activated eggs, and 120 and 145 kDa proteins were present only in the heavy fractions of mature oocytes and activated eggs. Examination of Western blots showed that cyclins A and B2 did not associate with membrane fractions at any stage of meiosis. Instead, cyclin A was present in the cytosols of mature oocytes and cyclin B2 was present in the cytosols of immature and mature oocytes. c-mos protein was detected in the cytosols and occasionally in the light fractions of mature oocytes and activated eggs. While alpha- and beta-tubulins were detected in the light and intermediate fractions at all the stages of meiosis examined, only beta-tubulin was present in the heavy fraction. beta-tubulin present in the heavy fraction was detected only at interphase, i.e. in immature oocytes and activated eggs, and not in mature oocytes. Using immunogold labelling we confirmed these results and found evidence to suggest that beta-tubulin associates with the rough endoplasmic reticulum of interphase cells by a linking protein.

Animals↗

PubMed1993

The structure of recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor derived from mammalian cells.

The structure of recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor expressed in CHO cells was investigated. The bioactive protein ([-32-153]M-CSF), expressed from a nucleotide sequence that encoded a signal peptide of 32 amino acids and N-terminal amino acids numbers 1-153, was heterogeneous in terms of molecular mass, as analyzed by SDS-PAGE, because of the presence of N-linked sugar moieties. The primary structure of the polypeptide was determined by sequence analysis and amino acid analysis of the fragments obtained from lysylendopeptidase digests of reduced and alkylated M-CSF, and from pepsin digests of the intact molecule. A sugar chain was located only at Asn-122 of the two putative sites of N-glycosylation that were present per subunit. The homodimeric structure appeared to have seven disulfide bonds, formed by inter- or intra-molecular linkages, since there were no free thiol groups in the molecule. The assignment of disulfide bonds by sequence analysis using peptide fragments indicated the combinations of Cys7-Cys90, Cys48-Cys139, and Cys102-Cys146. Gel-filtration analysis of Ser31[-32-153]M-CSF, in which the remaining Cys31 was replaced by Ser and which was expressed in COS cells, suggested that the mutein existed as a monomer. Our study shows that the disulfide-bond pairings of [-32-153]M-CSF that is expressed and post-translationally modified in mammalian cells are identical to those of Escherichia coli-derived [3-153]M-CSF with only one intermolecular disulfide bond, namely, Cys31-Cys31.

Alkylation↗

PubMed1993

One-shot intravesical instillation of the mucous adhesive anticancer agent hydroxypropylcellulose-doxorubicin for the treatment of superficial bladder carcinoma is sufficient to determine antitumor effects.

A 20 mg/20 ml dose of the membrane adhesive anticancer preparation, hydroxypropylcellulose-doxorubicin (HPC-doxorubicin), was instilled into urinary bladders through a catheter for the treatment of superficial bladder carcinomas (Ta-T1). After 14-30 days, the effects of the drug on tumors was examined by cystoscopy, and the residual tumor tissue where reduction in size was observed was resected by transurethral resection (TUR). Therapeutic effects were as follows: a complete response (CR) was found in 6 cases (37.5%), a reduction in size of more than 50% (partial response = PR) in 4 cases (25%), and a less than 25% reduction (no change = NC) in 6 cases (37.5%). In no case was progression of the disease noted. In CR cases, cold cup punch biopsy revealed no tumor cells remaining at the site where the tumor was present before the instillation. Side effects such as bladder irritation were found in 4 cases, but they were only temporary in nature. The one-dose HPC-doxorubicin approach developed by ourselves allowed detection of antitumor effects between 14 and 30 days after the instillation and therefore determination of sensitivities at an earlier stage than with instillation of the conventional water-soluble type of doxorubicin hydrochloride.

Administration, Intravesical↗

PubMed1993

[A study of testicular tumor on magnetic resonance imaging].

Magnetic resonance imaging (MRI) was performed on 13 testes of 12 patients with testicular abnormalities. In all cases, scrotal palpation and initial clinical course had suggested malignant testicular tumors. The normal testes showed homogeneous intermediate signal intensity on T1-weighted images and high signal intensity on T2-weighted images such as the appearance of the corpus cavernosum penis. Ten patients proved to have testicular tumors. In eight of them, the tumors showed homogeneous intermediate signal intensity on T1-weighted images, while in the other two, these images showed heterogeneous signal intensity. In four patients, tumors were of homogeneous low signal intensity on T2-weighted images, while in the other six, these images showed heterogeneous signal intensity. The rate of correspondence about local staging of testicular tumor between MRI and pathological diagnosis was 70 percent. In three patients whose testes were undoubtedly malignant according to clinical findings and examination data, MRI enabled us to diagnose one case of testicular atrophy. However, MRI suggested benign disease in the other two patients; nevertheless, surgery was performed as indicated by the other findings.

Adolescent↗