Biomedical subjects

M Hirabayashi

Publications and source records attributed to M Hirabayashi.

At least 19 recordsLinked to original sources

PubMed2004

Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure.

Rabbit zygotes at the pronuclear-stage were cryopreserved by vitrification using a gel-loading tip (GL-tip), Cryoloop or Cryotop. In GL-tip and Cryoloop methods, zygotes were first exposed to 10% ethylene glycol (EG)+10% DMSO in TCM199+20% fetal bovine serum (FBS) for 2 min, and then equilibrated for 30 s in a vitrification solution composed of 20% EG+20% DMSO+0.6 M sucrose in TCM199+20% FBS. In Cryotop method, zygotes were first exposed to 7.5% EG+7.5% DMSO+20% FBS in TCM199 for 3 min, and then equilibrated for 1 min in a vitrification solution composed of 15% EG+15% DMSO+0.5 M sucrose+20% FBS in TCM199. In vitro culture of vitrified-warmed zygotes using GL-tip and Cryoloop resulted in low cleavage rates (2 and 5%, respectively) and no development into blastocysts. In contrast, zygotes vitrified-warmed using Cryotop exhibited higher proportions of cleavage (58%) and development into blastocysts (24%). When compacted morulae or early blastocysts were vitrified by these three procedures, 80-93% of them exhibited blastocoele expansion or zona hatching during the subsequent 48 h of culture. Use of Cryotop instead of GL-tip or Cryoloop for zygote vitrification, without changing conditions of solutions and periods for exposure, equilibration and post-warm dilution, resulted in cleavage and blastocyst development rates of 88 and 45%, respectively. A longer exposure time (10 min) of zygotes to 7.5% EG+7.5% DMSO+20% FBS in TCM199 resulted in higher proportions of zygotes cleaving (94%) and developing into blastocysts (51%) after Cryotop vitrification. Proportions of post-warm zygotes (10-min exposure group) and fresh control zygotes developing into newborn offspring were 36 and 53%, respectively. Pronuclear-stage rabbit zygotes were successfully cryopreserved by vitrification using the Cryotop method.

Animals↗

PubMed2002

Ultrasound biomicroscopic study of ciliary body changes in the post-treatment phase of Vogt-Koyanagi-Harada disease.

AIMS: To investigate the usefulness of ultrasound biomicroscopy for evaluating changes in the ciliary body in patients with Vogt-Koyanagi-Harada disease. METHODS: Ultrasound biomicroscopy was used to evaluate 14 eyes of seven patients diagnosed with Vogt-Koyanagi-Harada disease. Cross sectional images of the ciliary body and thickness of the pars plana 3.0 mm posterior to the scleral spur were examined. Predicted thickness of the pars plana was obtained by multiple linear regression analysis of thickness in the acute phase and in the remission phase. RESULTS: In the active phase, the cross sectional images showed a shallow anterior chamber in eight of the 14 eyes, ciliochoroidal detachment in five eyes, and a thickened ciliary body in all 14 eyes. Internal reflectivity of the ciliary stroma was low, with ciliary processes being unclear in 13 eyes. One month after steroid treatment, slit lamp examination findings were normal in 14 eyes. 10 eyes of five patients were examined by ultrasound biomicroscopy at this stage. Ciliochoroidal detachment was no longer seen in any eye. Internal reflection of the ciliary stroma became relatively homogeneous, and the ciliary processes were seen, though not clearly. However, the pars plana remained thickened. The actual thickness was greater at 1 month after steroid treatment than the predicted thickness for the remission phase. In the remission phase, the internal reflection was homogeneous and the ciliary processes were delineated clearly in all 14 eyes. CONCLUSION: Objective, quantitative evaluation of the ciliary body is possible with ultrasound biomicroscopy during the course of Vogt-Koyanagi-Harada disease. Ultrasound biomicroscopy is useful in determining disease activity in the anterior segment and in monitoring the clinical course, and it may improve evaluation of the efficacy of treatment.

Adult↗

PubMed2002

Developmental competence and oxidative state of mouse zygotes heat-stressed maternally or in vitro.

Mammalian preimplantation embryos are sensitive to maternal and direct heat stress. However, the mechanisms by which heat stress affects early embryonic development in vivo or in vitro are unknown. This study examined whether heat-stress-induced loss of developmental competence in mouse embryos was mediated by physiological changes in the maternal environment or by high temperatures alone. After fertilization, zygotes at the same stage were heat-stressed at 39.5 degrees C for 12 h either maternally (measured by maternal rectal temperature) or directly in culture. Zygotes in each group were cultured at 37.5 degrees C for a further 84 h to assess their developmental ability. Neither type of heat stress affected the first cleavage rate. However, the proportion of embryos that developed to morulae or blastocysts was significantly lower in the maternally heat-stressed group, but not in the directly heat-stressed group. Moreover, maternal heat stress significantly reduced intracellular glutathione concentrations and enhanced hydrogen peroxide concentrations in both zygotes and two-cell embryos that were recovered immediately after heat stress or 12 h later, respectively. In contrast, direct heat stress had little effect on concentrations of glutathione or hydrogen peroxide in cultured early embryos. These results demonstrate that maternal heat stress at the zygote stage reduces the developmental ability of mouse embryos via physiological changes in the maternal environment that lead to an increase in intracellular oxidative stress on the embryo.

Animals↗

PubMed2002

Adaptation to sustained high plasma vasopressin in water and electrolyte homeostasis in the rat transgenic for the metallothionein-vasopressin fusion gene.

Prolonged exposure of tissues to a receptor agonist often leads to adaptive changes that limit the subsequent responsiveness of the tissue to the same agonist. Recently, we have generated rats transgenic for the metallothionein I-human arginine vasopressin (AVP) fusion gene (Tg), which produced high plasma AVP with relatively preserved renal water excretion, suggesting that there might be adaptive mechanism(s) for maintaining water and electrolyte homeostasis against chronic AVP oversecretion from the earliest stage of life. In this study, to investigate whether down-regulation of AVP V2 receptor (V2R), which could possibly be caused by long-standing high plasma AVP, participates in this adaptive mechanism(s), non-peptidic V2R antagonist OPC31260 was administered to reverse the down-regulation, and water loading was performed after V2R antagonist treatment had been withdrawn. Additionally, to confirm the down-regulation, Northern blotting analysis for V2R mRNA was carried out. Tg rats showed slightly decreased urine volume and water intake with an equivalent plasma [Na(+)] level (Tg 140.4 +/- 0.6 mEq/l; control 139.3 +/- 0.6 mEq/l) under basal conditions. After water loading using a liquid diet containing zinc, which stimulates the promoter region in the transgene, the urine increase showed only limited suppression with a dramatically increased plasma AVP level and mild hyponatremia (135.8 +/- 1.8 mEq/l) in Tg rats. When diet containing OPC31260 had been provided for 4 days until the day before the start of water loading, antidiuresis and hyponatremia (125.4 +/- 1.mEq/l) were significantly potentiated. V2R mRNA expression in kidney was significantly less in Tg rats than in control rats under basal conditions, and this suppression was restored by OPC31260 treatment to levels comparable with those of control rats. These results suggest that long-standing high plasma AVP causes V2R down-regulation, and it may play an important role in the adaptive mechanism(s) for maintaining water and electrolyte homeostasis in chronically AVP-overexpressing rats.

Adaptation, Physiological↗

PubMed2002

Overexpression of vasopressin in the rat transgenic for the metallothionein-vasopressin fusion gene.

Arginine vasopressin (AVP) is a major antidiuretic hormone, the overproduction of which causes diluting hyponatremia in humans and is called the syndrome of inappropriate antidiuresis (SIAD). To study physiological changes resulting from AVP overproduction and to develop an animal model of hyponatremia, the human AVP gene was expressed under the control of the metallothionein promoter in transgenic (Tg) rats. Analyses of AVP immunoreactivity (irAVP) in the tissues revealed that the transgene is expressed mainly in the central nervous system. Gel filtration showed that irAVP in the brain and plasma was properly processed AVP. AVP purified from the brains of both Tg and control rats also exerted equal bioactivity to generate cAMP in LLC-PK1 cells. The founder rats did not show any physical or anatomical abnormalities. Under basal conditions, Tg rats had high plasma AVP levels (Tg 13.8 +/- 2.5 pg/ml; control 2.7 +/- 1.2 pg/ml; n=6 in both groups; means +/- S.E.M.), decreased urine volume, and normal plasma [Na(+)]. Hypertonic saline injected i.p. did not affect AVP secretion in Tg rats. In response to a zinc-supplemented liquid diet, plasma AVP decreased in control rats, but increased in Tg rats (Tg 32.7 +/- 2.7 pg/ml; control 1.0+/-0.1 pg/ml; n=6), resulting in hyponatremia (Tg 135.2 +/- 2.5 mEq/l; control 140.8 +/- 0.4 mEq/l; n=6). To our knowledge, this is the first transgenic animal to show diluting hyponatremia. This transgenic rat may therefore provide a useful model in which to investigate various physiological alterations resulting from the oversecretion of AVP which involve SIAD, stress response, behavior, and blood pressure.

Animals↗

PubMed2001

New lattice-Boltzmann model for magnetic fluids.

A new type of two-dimensional lattice-Boltzmann model is developed in order to study the rheological properties of magnetic fluids. The appropriate extension of the lattice-Boltzmann scheme for magnetohydrodynamics makes it possible to deal with the rotation of the magnetic moment without losing the advantage of its simple algorithm. This model is suitable for simulating various behaviors of magnetic fluids influenced by the internal angular momentum.

Journal Article↗

PubMed2001

Green fluorescent protein-transgenic rat: a tool for organ transplantation research.

The purpose of this study is to evaluate green fluorescent protein (GFP) transgenic rats for use as a tool for organ transplantation research. The GFP gene construct was designed to express ubiquitously. By flow cytometry, the cells obtained from the bone marrow, spleen, and peripheral blood of the GFP transgenic rats consisted of 77, 91, and 75% GFP-positive cells, respectively. To examine cell migration of GFP-positive cells after organ transplantation, pancreas graft with or without spleen transplantation, heart graft with or without lung transplantation, auxiliary liver and small bowel transplantation were also performed from GFP transgenic rat to LEW (RT1(1)) rats under a 2-week course of 0.64 mg/kg tacrolimus administration. GFP-positive donor cells were detected in the fully allogenic LEW rats after organ transplantation. These results showed that GFP transgenic rat is a useful tool for organ transplantation research such as cell migration study after organ transplantation without donor cell staining.

Animals↗

PubMed2001

Effects of cryopreservation of pronuclear-stage rabbit zygotes on the morphological survival, blastocyst formation, and full-term development after DNA microinjection.

The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.

Animals↗

PubMed2001

VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration.

Neuronal cell death, abnormal protein aggregates, and cytoplasmic vacuolization are major pathologies observed in many neurodegenerative disorders such as the polyglutamine (polyQ) diseases, prion disease, Alzheimer disease, and the Lewy body diseases, suggesting common mechanisms underlying neurodegeneration. Here, we have identified VCP/p97, a member of the AAA+ family of ATPase proteins, as a polyQ-interacting protein in vitro and in vivo, and report on its characterization. Endogenous VCP co-localized with expanded polyQ (ex-polyQ) aggregates in cultured cells expressing ex-polyQ, with nuclear inclusions in Huntington disease patient brains, and with Lewy bodies in patient samples. Moreover, the expression of VCP mutants with mutations in the 2nd ATP binding domain created cytoplasmic vacuoles, followed by cell death. Very similar vacuoles were also induced by ex-polyQ expression or proteasome inhibitor treatment. These results suggest that VCP functions not only as a recognition factor for abnormally folded proteins but also as a pathological effector for several neurodegenerative phenotypes. VCP may thus be an ideal molecular target for the treatment of neurodegenerative disorders.

Adenosine Triphosphatases↗

PubMed2001

The phytotoxicity to tobacco plants of short-chain carboxylic acids at atmospheric concentration levels in urban areas.

In this paper, we describe the influence of monocarboxylic acids (formic acid and acetic acid) and dicarboxylic acids (succinic acid and adipic acid), which are usually contained in aerosol particles and fog water, on the growth of tobacco plant. Their influence was examined by spraying the acid solutions on intact plants and by administering them in a culture medium for suspension-cultured cells. Their growth rates suggest that the influence of short-chain monocarboxylic acids was not significant in both the intact plant experiment and the cell culture experiment. In contrast, dicarboxylic acids exhibited significant influence on the growth of intact plants and no influence on culture cells, indicating that their toxicity is exerted mainly on the tissue of leaf surface. Phytotoxicity of dicarboxylic acids is higher than that of monocarboxylic acids.

Acetic Acid↗

PubMed2001

A comparative study on the integration of exogenous DNA into mouse, rat, rabbit, and pig genomes.

Transgenic mammals, from small laboratory rodents to domestic animals, have been successfully produced to date, but their production efficiency within or across species has been variable. This is probably due to the differences in the type of injected DNA and/or technical procedures employed in each laboratory, as well as the reproductive characteristics of the species. Here we report the direct comparison of the efficiencies of producing transgenic mice, rats, rabbits and pigs by one technician using a fusion gene composed of the bovine alpha S1-casein promoter and human growth hormone (hGH) gene. Before the fusion gene was injected into the zygotes, high magnitude centrifugation to visualize the pronuclei was necessary for all of the pig zygotes and one-third of the rabbit zygotes, but not for mouse and rat zygotes. Post-injection survival of the mouse zygotes (67.1%) was lower than those of the rat, rabbit and pig zygotes (89.6 to 100%). The volume change of the pronucleus following DNA injection was the lowest in mice (50% increase), moderate in rabbits (148% increase), and the most prominent in rats (238% increase). The data from only 1 pig zygote indicated a 22% increase in the pronucleus volume by DNA injection. The PCR analyses of the tail DNA of new born offspring indicated that 0.8% (4/493), 4.8% (22/463), 0.8% (3/367) and 0.9% (2/221) of the injected eggs in mice, rats, rabbits and pigs, respectively, developed into transgenic offspring. Some of the founder animals in all four species expressed the transgene in the mammary gland which was confirmed in hGH mRNA by RT-PCR and/or hGH peptide in Witch's milk with ELISA. These results suggest that the maximum volume of DNA solution injectable into the pronucleus is a possible factor explaining the species differences in the production of transgenic animals.

Animals↗

PubMed2001

Production of transgenic rats using young Sprague-Dawley females treated with PMSG and hCG.

The aim of this study was to examine the effects of gonadotrophin treatments on estrus synchronization and superovulation in young Sprague-Dawley (SD) rats that had not yet exhibited defined estrus cycles (5 to 7 weeks old), and to produce transgenic rats using these females as embryo donors and recipients. In Experiment 1, female rats were injected with PMSG and hCG (12.5, 25, 50 and 100 IU/kg each) and were mated with stud males. The reproductive performance of young rats were highest when PMSG and hCG at doses of 25 IU/kg each were injected (delivery rate 87.5%, nursing rate 92.9%). In Experiment 2, female rats were injected with PMSG and hCG (100, 150 and 300 IU/kg each) to induce superovulation. More eggs were recovered from the rats injected with PMSG and hCG at 150 and 300 IU/kg than from those treated with 100 IU/kg (33.4 and 41.3 vs. 13.3 eggs per female, respectively; p < 0.05). In Experiment 3, pronuclear-stage zygotes from 150 IU/kg PMSG/hCG-treated rats were used for microinjection of the fusion gene of bovine alpha S1-casein gene promoter and human growth hormone gene (2.8 kb), and the microinjected zygotes were transferred into the oviduct ampullae of the 25 IU/kg PMSG/hCG-treated rats. Seventeen transgenic rats were obtained from the 334 DNA-injected zygotes (5.1%). These results indicate that recipients and embryo donors for the production of transgenic rats can be prepared by the appropriate PMSG and hCG treatments of young SD rats, regardless of their estrus stages.

Animal Husbandry↗

PubMed2000

Impaired estrogen sensitivity in bone by inhibiting both estrogen receptor alpha and beta pathways.

Although it is well established that estrogen deficiency causes osteoporosis among the postmenopausal women, the involvement of estrogen receptor (ER) in its pathogenesis still remains uncertain. In the present study, we have generated rats harboring a dominant negative ERalpha, which inhibits the actions of not only ERalpha but also recently identified ERbeta. Contrary to our expectation, the bone mineral density (BMD) of the resulting transgenic female rats was maintained at the same level with that of the wild-type littermates when sham-operated. In addition, ovariectomy-induced bone loss was observed almost equally in both groups. Strikingly, however, the BMD of the transgenic female rats, after ovariectomized, remained decreased even if 17beta-estradiol (E(2)) was administrated, whereas, in contrast, the decrease of littermate BMD was completely prevented by E(2). Moreover, bone histomorphometrical analysis of ovariectomized transgenic rats revealed that the higher rates of bone turnover still remained after treatment with E(2). These results demonstrate that the prevention from the ovariectomy-induced bone loss by estrogen is mediated by ER pathways and that the maintenance of BMD before ovariectomy might be compensated by other mechanisms distinct from ERalpha and ERbeta pathways.

Animals↗

PubMed2000

The Dakin-West reaction of N-alkoxycarbonyl-N-alkyl-alpha-amino acids employing trifluoroacetic anhydride.

The Dakin-West reaction of N-alkoxycarbonyl-N-alkyl-alpha-amino acids (1a-j) with trifluoroacetic anhydride in the presence of pyridine gave alpha-amido trifluoromethyl ketones (2a-j), in which probable intermediates were mesoionic 1,3-oxazolium-5-olates (munchnones). The diastereoselective reduction of 2a-f with NaBH4 gave the threo-aminoalcohols (5a-f), which may be explained by the Felkin-Anh model. This was confirmed by converting 5a-f into trans-5-trifluoromethyl-2-oxazolidinones (6a-f) in good yields.

Acetic Anhydrides↗

PubMed2000

Production of transgenic rabbits using centrifuged pronuclear zygotes.

Superovulation of female rabbits was induced by subcutaneous injection(s) of porcine FSH. Zygotes were recovered 17 to 19 hr after hCG injection and were classified into two categories under a microscope equipped with Nomarski interference-contrast optics at x 200 magnification: (A) zygotes with clearly visible pronuclei, or (B) zygotes with visualized pronuclei after 10 min centrifugation at 12,000 x g. No significant difference between strains was found in the proportion of category-A zygotes (JW 72.6% vs NZW 79.3%). Pronuclei of category-A zygotes were located in the center of the cytoplasm, and the pronuclei of category-B zygotes were slightly moved by centrifugation toward the mass of cytoplasmic lipid droplets. Exogenous DNA solution (5 microg/ml of fusion gene composed of bovine alphaS1-casein promoter and human growth hormone structural gene) was microinjected into the pronucleus of the JW zygotes. The pronucleus of category-A zygotes with a mean volume of 7.4 pl swelled up to 16.6 pl (132% increase), while that of category-B zygotes with a mean volume of 6.1 pl swelled up to 15.9 pl (148% increase). Nevertheless, similar proportions of category-A and category-B zygotes developed into offspring after transfer to recipient females (11.1 and 11.2%, respectively). The efficiency to produce hGH-carrying transgenic rabbits was 0.9% (2/235) from category-A zygotes and 0.5% (1/215) from category-B zygotes (P>0.05). To date, transgenic rabbits have been produced without centrifugation of pronuclear zygotes. However approximately 25% of fertilized rabbit zygotes can be used for DNA microinjection after they have been centrifuged to visualize their pronuclei.

Animals↗

PubMed2000

Nuclear transfer in cattle : effect of linoleic acid-albumin on freezing sensitivity of enucleated oocytes.

The effect of linoleic acid-albumin (LAA) supplementation to the media for IVM, enucleation, and activation on the developmental potential of bovine embryos produced by nuclear transfer (NT) into frozen-thawed cytoplasts was investigated. Blastomeres derived from morulae was placed in the perivitelline space of frozen-thawed cytoplasts, which were then fused by a DC pulse. The proportion of fused embryos was similar between groups with and without LAA (87 vs. 90%). The proportion of development to blastocysts of NT embryos derived from the media with LAA (14%) was higher than that without LAA (4%), indicating that LAA treatment of bovine oocytes during IVM, enucleation and activation can improve the ability of such cytoplasts after freezing and thawing to develop into blastocysts after NT.

Albumins↗

PubMed2000

Generation of transgenic rats with YACs and BACs: preparation procedures and integrity of microinjected DNA.

The aim of the present study was to investigate differences in the methods for preparing a large DNA fragment to be used for making transgenic rats from the standpoint of transgenic production efficiency and integrity of the introduced gene. In yeast artificial chromosome (YAC) transgenesis, three methods for preparing DNA for microinjection were compared: amplification of YAC in yeast (AMP), amplification of YAC in yeast and removal of the amplification element (AMP/RE), and no amplification of the YAC in yeast (AMP-). Production efficiency per microinjected ovum with DNA by the AMP method was four times higher than that by the AMP/RE and AMP-. Based on these results, we favor the AMP method in spite of the thymidine kinase gene-induced male sterility. In bacterial artificial chromosome (BAC) transgenesis, linear DNA fragments for microinjection prepared by three kinds of purification procedures were compared: Not I digestion and CsCl gradient ultra-centrifugation (Prep. 1), CsCl gradient ultra-centrifugation, Not I digestion, gel electrophoresis, and beta-agarase digestion (Prep. 2), and CsCl gradient ultra-centrifugation, Not I digestion, pulse field gel electrophoresis, and beta-agarase digestion (Prep. 3). Although the efficiency of producing transgenic rats was similar with all these three DNA preparations, integration of the intact DNA fragment only occurred with the Prep. 3 procedure. We therefore favor the Prep. 3 method for preparing BAC DNA fragments. These results indicate that the method used to prepare a large DNA fragment such as YAC and BAC DNAs is important in order to produce transgenic rats with an intact transgene.

Animals↗

PubMed1999

High-level expressing YAC vector for transgenic animal bioreactors.

The position effect is one major problem in the production of transgenic animals as mammary gland bioreactors. In the present study, we introduced the human growth hormone (hGH) gene into 210-kb human alpha-lactalbumin position-independent YAC vectors using homologous recombination and produced transgenic rats via microinjection of YAC DNA into rat embryos. The efficiency of producing transgenic rats with the YAC vector DNA was the same as that using plasmid constructs. All analyzed transgenic rats had one copy of the transgene and produced milk containing a high level of hGH (0.25-8.9 mg/ml). In transgenic rats with the YAC vector in which the human alpha-lactalbumin gene was replaced with the hGH gene, tissue specificity of hGH mRNA was the same as that of the endogenous rat alpha-lactalbumin gene. Thus, the 210-kb human alpha-lactalbumin YAC is a useful vector for high-level expression of foreign genes in the milk of transgenic animals.

Animals↗