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Y Hamaguchi

Publications and source records attributed to Y Hamaguchi.

At least 19 recordsLinked to original sources
PubMed1992

Genomic organization of mouse J kappa recombination signal binding protein (RBP-J kappa) gene.

We have isolated a cDNA clone (RBP-2) for the protein (RBP-J kappa) which binds to immunoglobulin recombination signals with 23-base pair spacers (Matsunami, N., Hamaguchi, Y., Yamamoto, Y., Kuze, K., Kangawa, K., Matsuo, H., Kawaichi, M., and Honjo, T. (1989) Nature 342, 934-937). During further screening of a cDNA library from the same mouse pre-B cell line (38B9), we have isolated a second cDNA clone (RBP-2N) which differs from RBP-2 in its 5' sequence. RNase protection assays indicated that the RBP-2N type mRNA was produced in 10-20 times the quantity as RBP-2 mRNA. To elucidate the relationship between these two mRNAs, we analyzed the genomic organization of the RBP-J kappa gene. Southern hybridization of mouse genomic DNA detected at least 7 EcoRI fragments hybridizing to an RBP-2 cDNA probe, suggesting a complex structure for the RBP-J kappa gene. Cloning of each EcoRI fragment revealed one functional RBP-J kappa gene and three related genes. The functional gene was composed of 11 exons and spanned at least 50 kilobase pairs. The sequence of exon 1 and its 5'-flanking region contained a GC-rich promoter-like region but no apparent TATA box. The initiation site of transcription was heterogeneous, and the two types of mRNA are produced from the same exon by transcription initiation at different sites and by different usage of splice signals. Two of the three related genes were processed pseudogenes with scattered stop codons. The other was also a processed gene with a sequence exactly the same as that of RBP-2, except that this gene lacked the sequence corresponding to the first exon of the functional gene.

Animals↗
PubMed1992

Mitotic apparatus formation and cleavage induction by micromanipulation of the nucleus and centrosome: the centrosome forms a spindle together with only the chromosomes at a short distance.

We micromanipulated the nucleus and centrosomes in the zygote of the starfish, Asterina pectinifera, in order to investigate their roles in mitotic apparatus formation and cleavage induction. The zygote cleaved without spindle formation when its nucleus was removed. When one or two centrosomes were transplanted, they formed asters in the recipient cell, which cleaved into three or four blastomeres so that each blastomere might contain one centrosome or aster. When one centrosome was removed, a half-spindle formed in the manipulated cell, which did not cleave until the other centrosome was duplicated. When both centrosomes were removed, no microtubular structures such as the spindle and the aster appeared in the manipulated cell, which failed to cleave. These results indicate that two centrosomes or more in the cell induce cleavage with or without the nucleus and that one centrosome or less does not induce cleavage. It is also concluded that the centrosome(s) together with the nucleus forms a half-spindle or bipolar spindle. However, from the experiments of nucleus transplantation and displacement, spindle formation is found to depend on the distance between chromosomes and centrosomes. The half-spindle formed when the distance from the centrosome to the chromosomes was shorter than 22 microns; on the other hand, when the distance was longer than 22 microns, the nucleus remained apart from the aster, which means that the functional range of the astral microtubule's ability to engage chromosomes was 22 microns from the centrosome.

Animals↗
PubMed1992

Biochemical and immunological characterization of the DNA binding protein (RBP-J kappa) to mouse J kappa recombination signal sequence.

We have investigated whether J kappa recombination signal sequence (RS) binding protein (RBP-J kappa) has any partial catalytic activities involved in the VDJ recombination reaction, such as cleavage, ligation, and bending of DNA. Murine RBP-J kappa protein purified by J kappa-RS affinity chromatography did not show DNA cleavage activities but contained a strong DNA ligase activity. To obtain a large amount of purified RBP-J kappa protein, recombinant RBP-J kappa was synthesized in Escherichia coli as a fusion protein and also in silkworm cells. Although recombinant RBP-J kappa produced in silkworm cells could bind J kappa-RS, it failed to show either ligase or DNA bending activity. Since the DNA affinity-purified RBP-J kappa has the ligase activity, the RBP-J kappa protein may form a complex with a ligase in vivo. We have raised monoclonal antibodies against the RBP-J kappa fusion protein which was synthesized in E. coli and unable to bind J kappa-RS. Using the anti-RBP-J kappa monoclonal antibody we have shown that the RBP-J kappa protein is expressed ubiquitously in mammalian tissues. The ubiquitous expression of the RBP-J kappa protein is consistent with the hypothesis that the RBP-J kappa protein may have dual function [Furukawa et al. (1991) J. Biol. Chem. 266, 23334-23340].

Animals↗
PubMed1992

High survival of rabbit morulae after vitrification in an ethylene glycol-based solution by a simple method.

Rabbit morulae were exposed to a vitrification solution-modified PBS [PB1] medium containing 40% ethylene glycol + 18% Ficoll + 0.3 M sucrose (EFS) for 2, 5, or 10 min at 20 degrees C and were vitrified in liquid nitrogen. When morulae were rapidly warmed, 96% had an intact zona pellucida. When embryos were cultured after removal of the mucin coat, high proportions of them formed blastocoel (79-100%), but the percentage of embryos developed to fully expanded blastocysts decreased with increased exposure time 87%, 40%, and 17%). The survival rate of morulae vitrified after removal of the mucin coat was lower than that of mucin-intact embryos. To assess the development potential in vivo, 131 embryos were vitrified after 2 min of exposure to EFS solution; all the embryos were recovered and 120 were transferred to recipients without removal of the mucin coat, resulting in 78 (65%) full-term fetuses or young. This simple method, which yields high survival both in vitro and in vivo, will be of practical use for vitrifying rabbit embryos.

Animals↗
PubMed1992

Neutrophil elastase and its complex with alpha 1-antitrypsin in the pathogenesis of chronic suppurative otitis media.

Neutrophil elastase (NE) and its complex with alpha 1-antitrypsin were quantified in ear discharges from 15 patients with chronic suppurative otitis media (CSOM), and their levels were compared to those in middle ear effusions from 10 pediatric patients with chronic otitis media with effusion (OME). The localization of immunoreactive NE was also examined by immunocytologic study. The mean value of total NE was 161.8 +/- 29.5 micrograms/mL in CSOM, which was significantly higher than that in OME (16.5 +/- 6.7 micrograms/mL). The mean value of NE-alpha 1-antitrypsin complex was 13.7 +/- 8.8 micrograms/mL in CSOM and 7.8 +/- 4.0 micrograms/mL in OME. There was no significant difference between culture-positive and culture-negative samples in CSOM. Immunoreactive NE could be observed in the neutrophils of ear discharges, and extracellular release of NE was also observed. Ear discharges in CSOM contain a considerable amount of uncomplexed NE, of which the level does not depend on active bacterial infections. Uncomplexed NE seems to play a role in the chronicity of CSOM.

Adolescent↗
PubMed1992

Rotational movement of a spermatozoon around its long axis.

The rotational movement of a spermatozoon around its longitudinal axis was investigated by two methods: by observing a spermatozoon attached vertically to a coverslip by the tip of its head, and by observing a spermatozoon freely swimming in a medium by means of 'double-focal microscopy', which yielded simultaneous images at two different focal planes. Similar results were obtained by these two methods. Sea urchin, starfish, medaka, human, golden hamster and bull spermatozoa rolled in both clockwise and counterclockwise directions, although there was a large difference in the proportion of spermatozoa rolling in each direction in the different species. The majority of sea urchin and starfish spermatozoa rolled in a clockwise direction when an observer viewed the cell from its anterior end, whereas the majority of medaka, golden hamster, human and bull spermatozoa rolled in a counterclockwise direction relative to the same observer. Moreover, some spermatozoa occasionally changed their rotational direction. These results suggest that the mechanism regulating the direction of rotation of the spermatozoa is lax. As rotational movement of a spermatozoon around its longitudinal axis is due to the three-dimensional component of the beat of the flagellum, the direction of the three-dimensional movement presumably changes as the spermatozoa swim.

Animals↗
PubMed1992

Relationship between direction of rolling and yawing of golden hamster and sea urchin spermatozoa.

As a first step towards understanding the function and mechanism of spiral movement of spermatozoa swimming through a medium, the direction of rolling (rotational movement of the spermatozoa around their long axis) and that of yawing (circular motion of spermatozoa upon the surface of a glass microscope slide and coverslip) were examined for golden hamster and sea urchin spermatozoa. Most golden hamster spermatozoa yawed clockwise over the upper surface of a glass slide when viewed from above, whereas in most sea urchin spermatozoa yawing was counterclockwise. Under the lower surface of a coverslip, the direction of yaw of golden hamster or of sea urchin spermatozoa was reversed. Most golden hamster spermatozoa rolled counterclockwise as seen from the anterior end, whereas all examined sea urchin spermatozoa rolled clockwise relative to the observer. On the basis of quantitative analysis of the proportion of spermatozoa rolling (or yawing) clockwise to those rolling (or yawing) counterclockwise, a close relationship between the direction of rolling motion and that of yawing motion was shown for both golden hamster and sea urchin spermatozoa.

Animals↗
PubMed1992

[The quantitation of Pseudomonas aeruginosa elastase in suppurative chronic otitis media using a sensitive ELISA method].

A sensitive sandwich ELISA method has been developed in order to quantitate the Pseudomonas aeruginosa elastase (PE) of ear discharge from chronic suppurative otitis media (CSOM) patients. Samples were incubated with EDTA-2Na before ELISA in order to inhibit the PE activity which hydrolyzes anti-PE IgG antibody into smaller molecular fragments. Quantitation of PE in middle ear effusions (MEE) from 10 patients with chronic otitis media with effusion (OME) were also performed. In CSOM, 12 of 14 samples revealed a significant amount of PE from 0.6 microliter/ml to 62.1 microliters/ml, which was significantly higher than those in MEE (p less than 0.05). In MEE, 8 of 10 samples were under the detection limit. Two samples in CSOM with Pseudomonas aeruginosa infection had high levels of PE. The quantitation was linear, with a concentration from 5 ng PE/sample to 500 ng PE/sample. This ELISA system is a sensitive method for quantitation of PE requiring only very small samples.

Adolescent↗
PubMed1992

[A study on reaction mechanism of sodium lauryl sulfate-hemoglobin (SLS-Hb), Part 1].

The cyanmethemoglobin (HiCN) method has been adopted as the international standard procedure for hemoglobin (Hb) determinations due to the accuracy and stability of result. However, the presence of potassium cyanide (KCN) and potassium ferricyanide (K3-Fe(CN)6) in the reagents has raised problems of laboratory and environmental pollution. In 1981, Oshiro and colleagues developed a cyanide free method of Hb determination that is based on a low toxicity compound Sodium Lauryl Sulfate (SLS). The SLS-Hb method provides stable SLS-Hb formation through the following steps. 1) Reaction of SLS to erythrocytic membrane (disruption of the erythrocytic membrane). 2) Conformation change of Hb by SLS. 3) Iron oxidation by oxygen (Fe2+----Fe2+). 4) Formation of stable SLS-Hb (coordination of SLS). The paper presents several findings on the reaction mechanism of the SLS-Hb method.

Blood Cell Count↗
PubMed1992

[Rapid spontaneous postpartum remission in a case of chronic inflammatory demyelinating polyradiculoneuropathy associated with pregnancy].

A 28-year-old woman developed numbness and weakness of the hands and arms when she was 8 months pregnant, and weakness worsened gradually. However, weakness started to spontaneously subside immediately after delivery and she felt almost recovered several hours later. But weakness and numbness recurred one week after delivery and she was admitted to our hospital. Neurological examination revealed moderate weakness and disturbance of the deep and cutaneous sensations in the upper and lower extremities, and marked decrease of the deep tendon reflexes. The upper extremities were more severely affected. Nerve conduction study showed marked decrease in the motor conduction velocities. Cerebrospinal fluid showed increase of protein without pleocytosis. Teased preparation of the biopsied sural nerve showed occasional internodal segments with thin myelination, indicating demyelination and remyelination. A diagnosis of chronic inflammatory demyelinating polyradiculoneuropathy was made. Treatment with prednisolone markedly improved the weakness and hyporeflexia as well as the cerebrospinal fluid protein. Postpartum rapid remission may have been produced by rapid increase of endogenous steroid hormone in the blood by its massive excretion during delivery.

Adult↗
PubMed1991

Cloning and characterization of a protein binding to the J kappa recombination signal sequence of immunoglobulin genes.

A protein with molecular weight of 60,000 that binds to the recombination signal sequence (RS) of the immunoglobulin J kappa segment was purified from the nuclear extract of a murine pre B cell line 38B9. This binding protein was found in lymphoid cell lines but not in non-lymphoid cell lines. The Kd value of the J kappa RS binding protein to the J kappa RS was 1 nM. The cDNA clone (RBP-2) was isolated based on partial amino-acid sequence of this protein. This cDNA encodes 526 amino-acid residues, and its sequence does not show extensive overall homology with any known proteins, but displays an interesting homology to a 40-residue region that is conserved among a subset of site specific recombinase (integrase family).

Amino Acid Sequence↗
PubMed1991

Chromosomal behavior in starfish (Asterina pectinifera) zygotes under the effect of aphidicolin, an inhibitor of DNA polymerase.

When calf thymus histones were labeled fluorescently and microinjected into oocytes of the starfish, Asterina pectinifera, the labeled histones visualized chromosomes during maturation division and cleavage. In doing so, we confirmed the previously reported phenomenon that chromosomes became incompetent at the first cleavage in the aphidicolin-treated egg, although cleavage itself took place. Moreover, we found that chromosomes were aligned at the equator of the metaphase spindle of the first cleavage and that they did not separate into two groups at all, but made a lump in the middle of the spindle. Chromosomes finally entered one blastomere, although they did not participate in the following karyokinesis. DNA and microtubules were examined by cytochemistry and immunofluorescence in order to investigate the relation between chromosome movement and the microtubular cytoskeleton. The mitotic apparatus developed and grew in the aphidicolin-treated cells in the same manner as those in normal cells without normal chromatin condensation or chromosome movement during the first cleavage. However, the mitotic apparatus consisted of two asters without the spindle formed at subsequent cleavages. Electron microscopic study revealed that chromosomes did not condense normally and kinetochores were not detected during the first cleavage. These results indicate that the dynamic changes in microtubular structures during mitosis have poor relation with the chromosome behavior such as prophase chromosome condensation and anaphase chromosome movement.

Anaphase↗
PubMed1991

ELISA to determine immunoreactive Pseudomonas aeruginosa elastase in chronic suppurative otitis media.

A sensitive sandwich ELISA has been developed to measure the levels of Pseudomonas aeruginosa elastase (PE) in ear discharges from chronic suppurative otitis media (CSOM) patients. Preincubation of the sample with EDTA-2Na before ELISA was employed to inhibit PE activity which hydrolyzes the anti-PE IgG antibody into a smaller molecular form. The PE levels of 10 middle ear effusions (MEE) from chronic otitis media with effusion were also measured. In CSOM, 9 of 10 samples had significant PE levels, ranging from 6.8 to 62.1 micrograms/ml, which were significantly higher than those in MEE (p less than 0.01), the majority of which was below the detection limit. Two samples of CSOM with the P. aeruginosa infection showed high PE levels. This sandwich ELISA for the measurement of PE is a very sensitive method requiring only a small sample amount.

Enzyme-Linked Immunosorbent Assay↗
PubMed1991

Change in the heterogeneous distribution of tubulin isotypes in mitotic microtubules of the sea urchin egg by treatment with microtubule depolymerizing or stabilizing drugs.

In the mitotic sea urchin egg, the spindle microtubules were composed of different tubulin isotypes from those of astral microtubules using monoclonal antibodies [Oka et al. (1990) Cell Motil. Cytoskeleton, 16, 239-250]. Three of the antibodies, D2D6, DM1B, and YL1/2, were specific for spindle microtubules, astral microtubules and reactive with both microtubules, respectively. The mitotic sea urchin egg was treated with microtubule depolymerizing (colcemid and nocodazole) and stabilizing (hexylene glycol) drugs and change in the heterogeneous distribution of the tubulin isotypes was investigated by the immunofluorescence procedure using these three monoclonal anti-tubulin antibodies. We observed that: (1) the microtubule depolymerizing drugs caused quick depolymerization of most mitotic microtubules, and a small number of spindle microtubules remaining were stained with all three antibodies; (2) hexylene glycol induced many microtubules in the mitotic apparatus, which was stained with D2D6 but was not stained with DM1B; (3) hexylene glycol also induced a great number of miniasters in the cytoplasm, and they were stained with three antibodies. These results suggest that these drugs altered the distribution of tubulin isotypes in the mitotic microtubules during depolymerization or polymerization within a short time.

Animals↗
PubMed1991

Propagation of transient Ca2+ increase in sea urchin eggs upon fertilization and its regulation by microinjecting EGTA solution.

Upon fertilization, the concentration of intracellular Ca2+ (Cai) in sea urchin eggs increased up to 3 microM when measured with fura-2, a fluorescent Ca indicator and the increase in Cai traversed from the sperm entry point as a wave over the entire egg at the mean propagation velocities of 5.0 microns/sec in C. japonicus egg and 5.3 microns/sec in H. pulcherrimus egg. However, the velocity was not uniform; i.e., it was rapid in the vicinity of the sperm entry point and the opposite point, but slow in the central region of the egg. Microinjecting a Ca-EGTA buffer and an IP3 solution into the C. japonicus egg induced the transient Cai increase more rapidly than that upon fertilization, due perhaps to the diffusion of the injectates. In order to investigate Ca2+ release during Cai increase upon fertilization, EGTA solutions were microinjected into unfertilized or fertilizing eggs. Microinjecting 100 mM EGTA (final concentration of 1 mM) not only suppressed the transient Cai increase, but also reduced the increased Cai rapidly, and never induced egg activation after insemination, whereas 10 mM EGTA (final concentration of 0.1 mM) did not significantly affect the Cai increase or the activation. Ca2+ released upon fertilization was estimated to be 150-170 microM in the egg cytoplasm from the amount of microinjected EGTA and fura-2. It was concluded that although more than 150 microM of Ca2+ was released intracellularly upon fertilization, Cai increased to only a few microM because most of the released Ca2+ was sequestered by intracellular Ca2+ binding substances.

Animals↗
PubMed1991

A quantitative study of the ciliary area of experimental otitis media with effusion in cats.

A quantitative electron microscopic study was performed to determine the extent of the ciliary surface area of the middle ear epithelium in cats. In normal cats, 97.8% of the middle ear mucosa in the hypotympanum and 45.9% in the upper part of the bulla were covered by cilia. Eustachian tube obstruction (ETO) significantly reduced the ciliary area to 73.1% in the hypotympanum and to 16.2% in the upper part of bulla at 1 week after ETO. Instillation of Escherichia coli lipopolysaccharide (LPS) or cat polymorphonuclear leukocyte (PMN) lysate into the middle ear cavity followed by ETO did not reduce the ciliary area, compared with ETO induced alone. Our results indicate that ETO reduces the ciliary area of the middle ear mucosa and that LPS or neutrophils do not enhance reduction of the ciliary area compared to ETO induced alone in this experimental condition.

Animals↗