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Biomedical subjects

Y Hamaguchi

Publications and source records attributed to Y Hamaguchi.

At least 19 recordsLinked to original sources
PubMed2000

Molecular characterization of a novel nucleolar protein in starfish oocytes which is phosphorylated before and during oocyte maturation.

In response to 1-methyladenine, a maturation-inducing substance, starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown through activation of p34cdc2-cyclin B, which results in the dispersal of the nucleolus. Little information has been elucidated thus far on nucleolar proteins that are phosphorylated by p34cdc2-cyclin B during meiotic maturation. Here, we describe a novel nucleolar protein of the starfish Asterina pectinifera oocyte, which is designated ANO39 and which is phosphorylated during meiotic maturation. A full-length ANO39 cDNA of 2106 base pairs encodes a polypeptide of 346 amino acids having a calculated Mr of 39 005. The amount of ANO39 is kept nearly constant during oocyte maturation and embryogenesis up to the midgastrula stage. The transcript encoding ANO39 was present in growing oocytes but not in full-grown ones, as evidenced by Northern blot hybridization. Ser145 is specifically phosphorylated when ANO39 is incubated in vitro with purified starfish p34cdc2-cyclin B. This phosphorylation site corresponds to that is phosphorylated during meiotic maturation in vivo. Immunoblot analysis using phosphoserine145-specific antibody as a probe revealed that some populations of ANO39 of the immature oocytes at the G2 stage have been already phosphorylated on Ser145 and Ser145 is maximally phosphorylated during meiotic maturation.

Adenine↗
PubMed1999

New rapid flow cytometric method for the enumeration of nucleated red blood cells.

BACKGROUND: Nucleated red blood cells (NRBC) in blood specimens compromise the automated white blood cell (WBC) count on most hematology analyzers. This makes it necessary to correct the WBC count by subtracting separately counted NRBC by manual microscopy. In addition, it is clinically important to establish the non-physiological presence of NRBC in blood specimens because of their association with significant hematological and non-hematological disease. Unfortunately, manual microscopic methods lack sensitivity, specificity and reproducibility required for both. METHODS: We have developed a new, rapid flow cytometric method for the detection and enumeration of NRBC, based on two-color staining with anti-CD45-fluorescein-isothiocyanate (CD45-FITC) and propidium iodide (PI). EDTA anticoagulated blood samples are incubated for 30 min with CD45-FITC, followed by 30 sec acid-hypotonic lysis, containing PI and subsequent addition of an alkaline-hypertonic solution. The samples are thus ready for flow cytometric analysis. RESULTS: The method typically yields up to four populations, (1) red blood cell (RBC) ghosts, debris, lyse-resistant RBC, reticulocytes and platelets, (2) CD45(+) WBC unstained by PI, (3) CD45(+) WBC stained by PI, and (4) CD45(-)/PI(bright) NRBC. Manual microscopic reference NRBC counts of 25 patient specimens showed excellent correlation with flow cytometric NRBC determinations (y = 0.943x+0. 66; r(2) = 0.982). Performance for precision showed a mean coefficient of variation (CV) for the flow cytometric method of =10%, with a mean CV for manual NRBC counts of 40%. CONCLUSIONS: We conclude that this method is suitable for NRBC counting in peripheral blood specimens with improved performance in terms of accuracy, reproducibility when compared to manual microscopic methods.

Adult↗
PubMed1999

Telomerase enzyme activity and RNA expression in adriamycin-resistant human breast carcinoma MCF-7 cells.

Telomerase activity has been reported in cancer cells after treatment with antineoplastic agents. Assessment of telomerase activity could be a valuable tool to measure the reduction of aggression caused by chemotherapy. This study was designed to investigate the significance of telomerase for chemotherapy with respect to Adriamycin (ADM)-resistance. MCF-7 and its ADM-resistant line (AdrR) were treated with ADM, 5-fluorouracil (5FU) or taxotere (TAXO). Telomerase activity and human telomerase RNA component (hTR) were quantitatively measured by the telomeric repeat amplification protocol assay and RT-PCR, respectively. Cell counting and MTT assay were also performed. In MCF-7, enzyme activity was significantly reduced by ADM and 5FU treatments. In AdrR, 5FU and TAXO reduced enzyme activity, while ADM significantly increased the activity. No significant changes in hTR were seen in these two cell lines after treatment with any of these drugs. When Bcl-2 expression was examined after drug treatments, ADM increased Bcl-2 expression in AdrR cells, while not changing it in MCF-7 cells. We conclude that an unusual reaction of telomerase activity in AdrR may explain, at least in part, one of the mechanisms of the malignant biological behavior related with the drug-resistance to ADM.

Antineoplastic Agents, Phytogenic↗
PubMed1999

Complete regression of recurrent esophageal carcinoma with reduced expression of glutathione S-transferase-pi by treatment with continuous infusion of 5-fluorouracil and low-dose cisplatin infusion.

The mortality rate of recurrent esophageal carcinoma remains high because of its resistance to chemotherapy and radiation therapy. We present a patient with recurrent esophageal carcinoma, which dramatically disappeared after treatment with the combination of continuous infusion of 5-fluorouracil and low-dose cis-Diamminedichloroplatinum-II (cisplatin) infusion (FP therapy). Furthermore, we immunohistologically found that glutathione S-transferases (GST)-pi, a marker of resistance to cisplatin, was faintly expressed both in the endoscopical biopsy specimens of recurrent tumor and in the resected specimens of esophageal carcinoma and metastatic lymph nodes. FP therapy was suggested to be effective for recurrent esophageal carcinoma. Immunostaining for GST-pi might be a prospective marker for the sensitivity of esophageal carcinoma to FP therapy, particularly cisplatin.

Antimetabolites, Antineoplastic↗
PubMed1999

Function of a minus-end-directed kinesin-like motor protein in mammalian cells.

CHO2 is a mammalian minus-end-directed kinesin-like motor protein present in interphase centrosomes/nuclei and mitotic spindle fibers/poles. Expression of HA- or GFP-tagged subfragments in transfected CHO cells revealed the presence of the nuclear localization site at the N-terminal tail. This domain becomes associated with spindle fibers during mitosis, indicating that the tail is capable of interaction with microtubules in vivo. While the central stalk diffusely distributes in the entire cytoplasm of cells, the motor domain co-localizes with microtubules throughout the cell cycle, which is eliminated by mutation of the ATP-binding consensus motif from GKT to AAA. Overexpression of the full-length CHO2 causes mitotic arrest and spindle abnormality. The effect of protein expression was first seen around the polar region where microtubule tended to be bundled together. A higher level of protein expression induces more elongated spindles which eventually become disorganized by loosing the structural integrity between microtubule bundles. Live cell observation demonstrated that GFP-labeled microtubule bundles underwent continuous changes in their relative position to one another through repeated attachment and detachment at one end; this results in the formation of irregular number of microtubule focal points in mitotic arrested cells. Thus the primary action of CHO2 appears to cross-link microtubules and move toward the minus-end direction to maintain association of the microtubule end at the pole. In contrast to the full-length of CHO2, overexpression of neither truncated nor mutant polypeptides resulted in significant effects on mitosis and mitotic spindles, suggesting that the function of CHO2 in mammalian cells may be redundant with other motor molecules during cell division.

Animals↗
PubMed1998

Aster-forming abilities of the egg, polar body, and sperm centrosomes in early starfish development.

Using ionophore-activated starfish eggs, the maternal centrosome accompanied by the female pronucleus (the egg centrosome) was confirmed to possess the ability for aster formation but generally not the capacity for reproduction. Inhibition of pronuclear fusion and removal of the male pronucleus and/or the sperm centrosome revealed that the egg centrosome lost its ability when it was only in the vicinity of the sperm centrosome. Hence, the entity that inactivated the egg centrosome was determined to be the adjacent sperm centrosome. When the second polar body was transplanted into a fertilized egg, its maternal centrosome (the polar body centrosome) organized an aster during cleavage; moreover, it possessed reproductive capacity. Furthermore, three different patterns of aster formation characterizing the sperm, egg, and polar body centrosomes occurred at each corresponding nucleus in the absence of nuclear fusion. When various kinds of centrosomes approached each other through nuclear fusion, there was observed only a pattern of aster formation at the fusion site that characterized a certain centrosome. Consequently, the sperm, polar body, and egg centrosomes were superior, intermediate, and inferior, respectively, in their ability for aster formation.

Animals↗
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↗