Search PubMed⌕ Search

Biomedical subjects

Y Hamaguchi

Publications and source records attributed to Y Hamaguchi.

At least 37 records · Page 2Linked to original sources
PubMed1998

Displacement of cleavage plane in the sea urchin egg by locally applied taxol.

Taxol enhanced assembly and stability of microtubules in the mitotic apparatus and subsequently inhibited chromosome movement and cleavage when injected in the sea urchin egg as reported previously [Y. Hamaguchi et al., 1987: Cell Struct. Funct. 12:43-52]. In this study, to examine the local effect of taxol on cleavage, taxol was injected in small doses. When taxol was injected into the cortical region of the equatorial plane, the birefringence (BR) of the mitotic apparatus near the injection site increased, chromosome movement became slow near this site, and then cleavage furrow formation was inhibited in the cortex near the site, although chromosome movement and cleavage furrow formation were apparently normal in the other side of the cell. When taxol was injected in the polar cortex, BR of the mitotic apparatus near the injection site was enhanced and then the cleavage furrow was displaced toward this site from the equator. Accordingly, resultant blastomeres were unequal in size. The enhancement of microtubule assembly and stabilization of microtubule dynamics, which were detected as the increase in BR were confirmed by immunofluorescence microscopy with anti-tubulin antibody. Consequently, it was found that taxol injection caused local suppression of dynamics of microtubules in the cell, thereby modifying cleavage furrow formation.

Animals↗
PubMed1998

RNA chip: quality assessment of RNA by microchannel linear gel electrophoresis in injection-molded plastic chips.

Two major components of rRNA (18S and 28S rRNA) were separated by electrophoresis in injection-molded acrylic chips with a microchannel 100 microm in width, 40 microm in depth, and with 1 cm of separation distance. Microchannels were filled with 4 g/L hydroxypropylmethylcellulose as sieving polymer and 5 mg/L ethidium bromide for RNA staining. The fluorescent signals were detected by a fluorescent microscope equipped with a photometer and 590 nm emission filter. The assay is rapid (<3 min), reproducible, RNase-free, and requires only 1-2 microL of sample. The detection limit was approximately 10 mg/L (10 ng/microL), 100-fold lower than that for conventional agarose gel electrophoresis. Because only 0.1 nL of the loaded sample was used for electrophoresis, the detectable peaks of rRNA in the separation were derived from less RNA than in a single cell. Because the quality of RNA is critical for RNA-related diagnostic tests, disposable plastic chips will be useful for quality assessment of RNA.

Animals↗
PubMed1998

Direct reverse transcription-PCR on oligo(dT)-immobilized polypropylene microplates after capturing total mRNA from crude cell lysates.

To simplify gene expression analysis, oligo(dT)-immobilized polypropylene microplates were used serially to capture mRNA, synthesize cDNA, and amplify specific genes. The amounts of immobilized oligonucleotide, hybridized mRNA, and synthesized cDNA were quantified fluorometrically using either Yoyo-1 or AttoPhos. The immobilized oligonucleotides captured approximately 40-55% of mRNA directly from crude cell lysates. Hybridized mRNA was then amplified by one-step reverse transcription (RT)-PCR with rTth polymerase or two-step PCR with initial cDNA synthesis followed by PCR, where the latter exhibited more sensitivity. In two-step RT-PCR, microplates can be reused for multiple PCRs with the same or different primer sets because synthesized cDNA was covalently attached to the plates at its 5' end. We believe this microplate may be acceptable as a platform for various mRNA expression analyses, including basic research, drug screening, and molecular toxicology, as well as for molecular pathological diagnostics.

Animals↗
PubMed1997

Division of polar bodies induced by their enlargement in the starfish Asterina pectinifera.

The first polar body (FPB), which is formed at the first meiotic division during oogenesis, does not generally divide. We made a hypothesis that the amount of cytoplasm was not sufficient for the FPB to perform cell division, in spite of the same amount of genomes and centrosome as those of the secondary oocyte. To examine this hypothesis, hexylene glycol (HG) at a low concentration was applied to oocytes of the starfish Asterina pectinifera during the first meiotic division. Hence, FPBs were enlarged in their diameters, some of them divided once, and the division rate increased in proportion as their diameter extended. We further hypothesized that the difference between the second polar body (SPB) and the egg would be only the amount of cytoplasm and that if SPBs were enlarged, they would become eggs. When the secondary oocytes were treated with HG, large SPBs were obtained. Some of them, however, divided, and resultant daughter cells divided moreover, whereas eggs would not cleave unless they were fertilized. We discuss here the reason why the centrosome distributed during maturation division began to function in enlarged PBs.

Animals↗
PubMed1997

Effects of intracellular pH on the mitotic apparatus and mitotic stage in the sand dollar egg.

The effect of change in intracellular pH (pHi) on mitosis was investigated in the sand dollar egg. The pHi in the fertilized egg of Scaphechinus mirabilis and Clypeaster japonicus, which was 7.34 and 7.31, respectively, changed by means of treating the egg at nuclear envelope breakdown with sea water containing acetate and/or ammonia at various values of pH. The mitotic apparatus at pHi 6.70 became larger than that of normal fertilized eggs; that is, the mitotic spindle had the maximal size, especially in length at pHi 6.70. The spindle length linearly decreased when pHi increased from 6.70 to 7.84. By polarization microscopy, the increase in birefringence retardation was detected at slightly acidic pHi, suggesting that the increase in size of the spindle is caused by the increase in the amount of microtubules in the spindle. At pHi 6.30, the organization of the mitotic apparatus was inhibited. Furthermore, slightly acidic pHi caused cleavage retardation or inhibition. By counting the number of the eggs at various mitotic stages with time after treating them with the media, it is found that metaphase was persistent and most of the S. mirabilis eggs were arrested at metaphase under the condition of pHi 6.70. It is concluded that at slightly acidic pH, the microtubules in the spindle are stabilized and more microtubules assembled than those in the normal eggs.

Acids↗
PubMed1997

Regulation of intracellular pH in sea urchin eggs by medium containing both weak acid and base.

To establish a method of pHi regulation and to understand the pH regulation mechanism in the cell, we investigated the pHi response of unfertilized or fertilized eggs of sea urchin, applying sea water containing both weak permeant acid, acetic acid and/or base, ammonia, to eggs. Pyranine was employed as a pH indicator to measure intracellular pH (pHi) by microfluorometry. The unfertilized/fertilized eggs had a pHi of 6.80/7.34 and 6.81/7.32 for Schaphechinus mirabilis and Hemicentrotus pulcherrimus, respectively. With the addition of both acetic acid and ammonia to the media, pHi changed linearly against extracellular pH (pHo) between 6-8 and was almost equal to pHo at the concentration of 20 mM acetate and ammonia. This mixed application was proved to be available for regulating pHi at the desired value within a wide range involving the original pHi by a single solution system. pHi after the treatment was dependent on various factors, such as the concentration of the weak acid and base, the pHi before the treatment, and pH buffering power in the cytoplasm. The latter was estimated to be 43 mM and 58 mM in unfertilized and fertilized eggs, respectively, from the measurement of pHi change induced by microinjecting a HEPES solution, assuming that the pH buffering power is caused by phosphate.

Acetic Acid↗
PubMed1997

The inhibition of motility of demembranated spermatozoa by anti-tubulin antibodies.

The effects of monoclonal anti-tubulin antibodies on the motility of demembranated and reactivated sea urchin spermatozoa were investigated. Two out of ten antibodies examined significantly reduced the motility of spermatozoa, both in motile rate and swimming speed. The binding patterns of the two antibodies YL1/2 and TUB2.1 to the axoneme were studied by immunoblot, immunofluorescence, and immuno-electron microscopy. YL1/2 bound to the axoneme in a specific pattern; signals were very intense in the tail, rich in the proximal portion, and scarce in the middle part of the axoneme. Because the inhibitory effects of the antibody on the motility of spermatozoa with fully long flagella and short flagella were similar, the inhibition was probably due to the binding of the antibody to the proximal portion of the flagellum. TUB2.1 evenly bound to the axoneme by immunofluorescence and immunoelectron microscopy. On the other hand, the eight antibodies which did not affect sperm motility, did not bind to unfixed axonemes, although epitopes for these antibodies were detected abundantly in the axoneme.

Animals↗
PubMed1997

[Current status and problems of home parenteral nutrition for terminal cancer patients from the viewpoint of indication].

Since June 1993, we have been performing home parenteral nutrition (HPN) for end-stage cancer patients. We studied how many patients could be treated with HPN, why they were not able to be treated with HPN in the terminal stage cancer patients, 158 cases, who admitted and died in our ward from June 1993 to April 1997. Eighty-six patients (54.4%) were considered to not be indicated for HPN, due to general weakness (28 cases), dyspnea (17 cases), need for medical care other than HPN (34 cases), and inability to understand this treatment (1 case). Fifty-six patients (35.4%) were considered to have been able to be treated with HPN and could have had returned home. Fifteen patients (26.8%) were given HPN. Four cases wanted to stay in the hospital after they knew the truth and the prognosis of their disease. Another four patients became weak while preparing for HPN and could not go home. Eighty percent of patients could not to be treated with HPN because of the factors of their family. Eight patients had no family to cared for them. Twelve patients were refused to the truth of their disease. The families of 13 cases refused to accept and care for them in their home. We may conclude that co-operation among patients, their family and the medical staff is the most important factor in providing HPN for end-stage cancer patients.

Adult↗
PubMed1996

Calcium-dependent bidirectional power stroke of the dynein arms in sea urchin sperm axonemes.

Active sliding between doublet microtubules of sea urchin sperm axonemes that were demembranated with Triton X-100 in the presence or absence of calcium was induced with ATP and elastase at various concentrations of Ca2+ to examine the effects of Ca2+ on the direction of the power stroke of the dynein arms. Dark-field light microscopy of microtubule sliding revealed that the sliding from the axonemes demembranated with Triton and millimolar calcium and disintegrated with ATP and elastase showed various patterns of sliding disintegration, including loops of doublet microtubules formed near the head or the basal body. These loops were often thicker than the remaining axonemal bundle. In contrast, only thinner loops were found from the axonemes demembranated with Triton in the absence of calcium and disintegrated with ATP and elastase at high Ca2+ concentrations. Electron microscopic examination of the direction of microtubule sliding showed that the doublet microtubules in the axonemes demembranated in the presence of millimolar calcium moved toward the base of the axonemes by the dynein arms on the adjacent doublet microtubule as well as by their own dynein arms. Doublet microtubules in the axonemes demembranated in the absence of calcium moved toward the base of the axonemes only by their own dynein arms. Similar observations have been obtained from the axonemes from which the outer dynein arms were selectively extracted. From these observations, we can conclude that the dynein arms generate force in both directions and this feature of the dynein arms arises from at least the inner dynein arms.

Animals↗
PubMed1995

[Polymyositis with rimmed vacuoles in a case of chronic thyroiditis].

We report a 37-year-old woman who developed chronic thyroiditis, and polymyositis with rimmed vacuoles (RVs). She developed paresthesia of glove and stocking type, muscle weakness of the lower extremities, and slowness in motion. On examination, her lower legs were atrophic and weak, more prominent on the right, while proximal muscles in the upper and lower extremities were slightly affected. Deep tendon reflexes of the upper and lower extremities were normal except for the absent Achilles tendon reflexes. Serum CK was high (1,809 IU/l), and laboratory data indicated chronic thyroiditis and hypothyroidism. Muscle CT showed severe fatty changes in the gastrocnemius and soleus muscles, more marked on the right, and slight atrophy of the thigh muscles. Electromyography and nerve conduction velocity study revealed peripheral neuropathy. Muscle biopsy revealed numerous myofibers with RVs, together with myofiber necrosis, phagocytosis and lymphocyte infiltration, consistent with myositis. Nerve biopsy was indicative of slight axonal degeneration. A diagnosis of polymyositis with RVs in combination with chronic thyroiditis was made. Prednisolone combined with levothyroxine markedly improved muscle weakness though atrophy and weakness of the lower legs were barely improved. Distal-dominant asymmetric affection of the leg muscles and many myofibers with RV on the biopsied muscle are atypical and rare findings of polymyositis. Associated chronic thyroiditis and neuropathy may be related to the formation of marked rimmed-vacuolar degeneration of myofibers in the present case.

Adult↗
PubMed1994

Site-directed mutagenesis study on DNA binding regions of the mouse homologue of Suppressor of Hairless, RBP-J kappa.

To map regions important for DNA binding of the mouse homologue of Suppressor of Hairless or RBP-J kappa protein, mutated mouse RBP-J kappa cDNAs were made by insertion of oligonucleotide linkers or base replacement. DNA binding assays using the mutated proteins expressed in COS cells showed that various mutations between 218 Arg and 227 Arg decreased the DNA binding activity drastically. The DNA binding activity was not affected by amino acid replacements within the integrase motif of the RBP-J kappa protein (230His-269His). Replacements between 291Arg and 323Tyr affected the DNA binding activity slightly but reproducibly. These results indicate that the region encompassing 218Arg-227Arg is critical for the DNA binding activity of RBP-J kappa. This region did not show any significant homology to motifs or domains of the previously described DNA binding proteins. Using a truncation mutant protein RBP-J kappa was shown to associate with DNA as a monomer.

Alternative Splicing↗
PubMed1994

Recognition sequence of a highly conserved DNA binding protein RBP-J kappa.

DNA binding specificity of the RBP-J kappa protein was extensively examined. The mouse RBP-J kappa protein was originally isolated as a nuclear protein binding to the J kappa type V(D)J recombination signal sequence which consisted of the conserved heptamer (CACTGTG) and nonamer (GGTTTTTGT) sequences separated by a 23-base pair spacer. Electrophoretic mobility shift assay using DNA probes with mutations in various parts of the J kappa recombination signal sequence showed that the RBP-J kappa protein recognized the sequence outside the recombination signal in addition to the heptamer but did not recognize the nonamer sequence and the spacer length at all. Database search identified the best naturally occurring binding motif (CACTGTGGGAACGG) for the RBP-J kappa protein in the promoter region of the m8 gene in the Enhancer of split gene cluster of Drosophila. The binding assay with a series of m8 motif mutants indicated that the protein recognized mostly the GTGGGAA sequence and also interacted weakly with ACT and CG sequences flanking this hepta-nucleotide. Oligonucleotides binding to the RBP-J kappa protein were enriched from a pool of synthetic oligonucleotides containing 20-base random sequences by the repeated electrophoretic mobility shift assay. The enriched oligomer shared a common sequence of CGTGGGAA. All these data indicate that the RBP-J kappa protein recognizes a unique core sequence of CGTGGGAA and does not bind to the V(D)J recombination signal without the flanking sequence.

Animals↗
PubMed1994

Different reactivity with monoclonal anti-tubulin antibodies between native and fixed mitotic microtubules in sea urchin eggs.

The effect on fixation on the reactivities of mitotic microtubules with monoclonal anti-tubulin antibodies was investigated by the indirect immunofluorescence procedure. All of the seven antibodies used intensely stained mitotic microtubules in sea urchin eggs lysed and fixed with methanol at -20 degrees C, whereas only two of them stained the stabilized microtubules in the lysed eggs before the fixation. The other five did not stain the mitotic microtubules even after microtubule components other than tubulin were removed by treating the lysed eggs with 0.4 M KCl solution containing taxol. These results exclude the possibility that the fixation affects proteins, which interact with microtubules including microtubule-associated proteins (MAPs) and interfere with the binding of monoclonal antibodies with tubulin, and strongly suggest that the fixation directly affects the three-dimensional conformation of tubulin. Furthermore, microinjection of these antibodies indicated the results as follows [combining the results reported previously; Oka et al., 1990: Cell Struct. Funct. 15: 373-378]: The antibodies which stained mitotic microtubules stabilized in the lysed eggs induced disassembly of native mitotic microtubules in the living eggs, but those which did not stain the stabilized microtubules did not disassemble the native microtubules. From these results, it is suggested that the monoclonal antibodies which stain microtubules in the eggs lysed but not fixed are useful for microinjection experiments.

Animals↗
PubMed1994

Sperm-egg fusion in the sea urchin is blocked in Mg(2+)-free seawater.

Magnesium ions as well as calcium ions are required for successful fertilisation in sea urchins. In the absence of Mg2+ spermatozoa attached to the egg plasma membrane, their acrosomal processes passing through the vitelline envelope, but could not enter the egg cytoplasm (Sano et al., Dev. Growth Differ. 22, 531-41, 1980). Such an individual spermatozoon was observed microscopically to resume entry into the egg immediately after the addition of a sufficient amount of Mg2+ to the surrounding medium. Neither any change in membrane potential nor an increase in intracellular Ca2+ concentration of the egg was observed after insemination in the absence of Mg2+, although both could be observed after the addition of Mg2+. The sperm heads did not show fluorescence when attached to the surface of an egg previously microinjected with mithramycin A in Mg-free seawater, indicating that there was no connection between the sperm and the egg. Therefore, occurrence of fertilisation potential must be a post-fusional event. These results suggest that Mg2+ are indispensable for fusion between the sperm acrosomal membrane and the egg plasma membrane.

Animals↗