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M Hirabayashi

Publications and source records attributed to M Hirabayashi.

At least 37 records · Page 2Linked to original sources
PubMed1999

Analysis of control elements for position-independent expression of human alpha-lactalbumin YAC.

A major problem in the production of transgenic animal bioreactors using microinjections is the low production rate of high-expressing transgenic animals due to the position effect. We previously reported that transgenic rats carrying the 210 kb yeast artificial chromosome (YAC) including the human alpha-lactalbumin gene express the transgene in a position-independent manner. The 210 kb YAC was thought to have all the elements necessary for position-independent expression. In this paper, we constructed fragmented YAC clones and a cosmid clone, and produced transgenic rats to analyze these elements. Transgenic rats with both the 50 kb upstream and downstream regions of the alpha-lactalbumin gene had position-independent expression. Transgenic rats with the 20 kb upstream and downstream regions, however, had position-dependent expression. Therefore, all the elements necessary for position-independent expression are thought to be located in the 50 kb upstream to 50 kb downstream region of the alpha-lactalbumin gene. Furthermore, we replaced the human alpha-lactalbumin promoter with the bovine alphaS1-casein promoter in the 210 kb YAC and produced transgenic rats. Position-dependent expression was observed. The elements required for position-independent expression of the bovine alphaS1-casein gene are different from those required for the human alpha-lactalbumin gene, despite the fact that the two genes have the same tissue and developmental specificity.

Animals↗
PubMed1999

Effects of timing of oocyte cryopreservation on in vitro development of nuclear-transferred bovine zygotes.

The utility of cryopreserved bovine oocytes as recipient cytoplasts for nuclear transfer (NT) was examined. In vitro-matured (IVM), metaphase-II oocytes were enucleated by mechanical suction and activated parthenogenetically. The cytoplasts were fused with blastomeres of in vitro-produced day-5 morulae by a DC electropulse, and then cultured up to 8 days (non-frozen controls; group I). Oocytes were frozen-thawed in 1.5-M ethylene glycol and 0.1-M sucrose before enucleation (group II), after enucleation (group III), after enucleation and aging culture (group IV), or after activation (group V). In group I, 91% of IVM oocytes could be used for NT and 89% of them fused successfully. Finally, 36% of the fused zygotes developed into blastocysts. The proportions of morphologically normal oocytes after thawing in groups IV and V (70 and 69%, respectively) were higher than in group III (56%), and the proportion of IVM oocytes used for NT in group IV (56%) was higher than those in groups II, III, and V (33%, 35%, and 38%, respectively). Fusion rates of the NT zygotes in groups III, IV, and V (90%, 88%, and 88%, respectively) were higher than the rate in group II (75%). Rates of development into blastocysts of the fused zygotes in groups II, III, IV, and V were 0%, 3%, 2%, and 6%, respectively (P < 0.05, group II vs. groups III, IV, and V). Developmental kinetics and cell numbers of the blastocysts were similar among the groups. It was suggested that timing of oocyte cryopreservation is among the factors influencing efficiency of production of cloned embryos in cattle.

Animals↗
PubMed1999

Production of transgenic rats using cryopreserved pronuclear-stage zygotes.

We investigated the application of cryopreserved pronuclear-stage zygotes for the production of transgenic rats. Most of the pronuclear-stage zygotes cryopreserved by conventional two-step freezing or vitrification appeared morphologically normal, but the proportion of frozen zygotes that developed into fetuses following transfer (59.7-60.2%) was higher than that of vitrified zygotes (5.5-22.1%). When the frozen-thawed zygotes were used for DNA microinjection, 97.5% survived after DNA microinjection and 25.1% of the transferred zygotes developed into fetuses. These proportions were comparable to those of the fresh control zygotes (97.0 and 30.0%, respectively). The integration efficiency of the exogenous DNA into fetuses was similar between the frozen group (3.3% per injected zygote) and the control group (3.5%). These results indicate that pronuclear-stage rat zygotes can be successfully cryopreserved by conventional two-step freezing for production of transgenic rats.

Animals↗
PubMed1999

Triggering of neuronal cell death by accumulation of activated SEK1 on nuclear polyglutamine aggregations in PML bodies.

BACKGROUND: A novel class of inherited human neurodegenerations is now known to be caused by expanded CAG repeats encoding polyglutamines. Polyglutamine-containing protein fragments have been shown to accumulate as aggregates in the nucleus and in the cytoplasm, and to induce cell death when expressed in cultured cells, leading to the proposal that polyglutamine aggregation is an important step in the pathogenesis. Supporting this, nuclear inclusions containing expanded polyglutamines have been identified in neurones from the brains of patients and in neurones from transgenic mouse models of this class of neural disorders. RESULTS: We analysed the consequences of polyglutamine expression in PC12 neuronal cells. Activated SEK1 accumulated with nuclear but not cytoplasmic polyglutamine aggregations, which consequently triggers cell death. Cell death induced by polyglutamine expression was inhibited by a dominant-negative SEK1 (DN-SEK1), but not by DN-SEK1 tagged with a nuclear export signal. Steady state SEK1 expression itself was enhanced two to three-fold. Nuclearly aggregated polyglutamines, which were identified in PML bodies, co-localized with not only activated SEK1 but also activated c-Jun. We also observed that nuclear inclusion-positive neurones from brains with Huntington's disease expressed SEK1. CONCLUSIONS: This study provides molecular links between the neurodegeneration observed in polyglutamine diseases, cell death signalling kinase cascades and nuclear subdomains related to cell death. We propose that the nuclear PML bodies containing polyglutamine aggregates activate the SEK1-JNK kinase cascade, resulting in the transduction of a death signal.

Animals↗
PubMed1999

Developmental competence, after transfer to recipients, of porcine oocytes matured, fertilized, and cultured in vitro.

The objective of this study was to evaluate the developmental ability of early porcine embryos produced in vitro and transferred to recipient gilts. Porcine cumulus-oocyte complexes were matured in modified North Carolina State University-37 solution for 44-46 h (in vitro maturation, IVM). In vitro fertilization (IVF) was performed with frozen-thawed epididymal spermatozoa. Inseminated oocytes were cultured in vitro (IVC) for 0, 24, or 48 h in modified NCSU-37 solution. Embryos were surgically transferred to the oviducts of recipients in which estrus had been synchronized with eCG and hCG. On the 29th day post-IVF, the uteri of some recipients were surgically examined for pregnancy; then pregnant females were hysterectomized in order to examine number and weight of the fetuses. Developmental rates to fetuses for IVM/IVF oocytes cultured for 24 and 48 h were significantly lower (p < 0.05, 1.7% and 2.0%, respectively) than that of IVM/IVF oocytes without IVC (6.7%). However, the weights of fetuses (1.0-1.2 g) did not differ among the experimental groups. The other recipients were examined for pregnancy using an ultrasound pregnancy detector, and pregnant females were allowed to go to term. Healthy piglets were delivered by some recipients to which embryos cultured for 0 or 24 h had been transferred; however, no farrow was obtained from embryos cultured for 48 h before the transfer. The results indicate that the viability of in vitro-produced porcine embryos is decreased by IVC after IVF; however, these embryos have competence to develop to term. An improved IVC system of porcine IVM/IVF oocytes is needed to generate advances in this field.

Animals↗
PubMed1999

Cerebral blood flow during treatment for pulmonary hypertension.

AIM: To determine if the haemodynamics of systemic and cerebral circulation are changed during treatment for persistent pulmonary hypertension of the newborn (PPHN). METHODS: Fifteen term newborn piglets with hypoxia induced pulmonary hypertension were randomly assigned either tolazoline infusion (Tz), hyperventilation alkalosis(HAT), and inhaled nitric oxide (iNO). Mean pulmonary arterial pressure (PAP), mean systemic arterial pressure (SAP), and cerebral blood flow volume (CBF) were measured. RESULTS: During hypoxic breathing, PAP increased significantly in all groups. After treatment PAP decreased significantly in all groups, but no significant difference was observed between groups. SAP decreased significantly only in the Tz group, and CBF reduced significantly only in the HAT group. On the other hand, iNO did not change SAP or CBF. CONCLUSION: Inhaled NO might be ideal for the resolution of pulmonary hypertension.

Alkalosis, Respiratory↗
PubMed1999

Establishment of SV40-tsA58 transgenic rats as a source of conditionally immortalized cell lines.

To isolate a variety of rat cell lines with differentiated functions, we established transgenic rat lines expressing the temperature-sensitive large T-antigen of simian virus 40 (SV40) tsA58 mutant under the control of the SV40 large T-antigen itself. We microinjected the DNA into 564 eggs of Wistar rat and 23 independent transgenic candidates were obtained. Ten pups died before weaning and eight transgenic rats could not transmit the transgene to the progeny. Finally, five lines of the transgenic rat were established. Although one line (#1511-6) had low reproductivity, the other four lines reproduced normally. Three out of the four lines (#1507-2, #1509-7, #1519-8) appeared normal but the other line had tumors in the brain and subcutaneous tissue at 3 weeks of age (#1511-6), and in the kidneys and subcutaneous tissue at 18 to 19-weeks of age (#1507-5). Fibroblast cells prepared from transgenic fetuses of lines #1507-5 and #1519-8 expressed the transgene and exhibited temperature-dependent growth. Both of the lines (#1507-5 and #1519-8) were successfully generated to be homozygous by sibling mating of transgenic offspring. These transgenic rat lines have bred through many generations and have been established to be a ready source of novel conditionally immortalized cell lines.

Animals↗
PubMed1998

Fermentation of soybean meal with Aspergillus usamii improves zinc availability in rats.

Soybean meal was fermented with Aspergillus usamii to improve zinc availability through the degradation of phytic acid. Rats fed a diet containing fermented soybean meal showed greater femoral zinc than did animals fed a diet containing regular soybean meal. Zinc solubility in the small intestine was higher in the rats fed fermented soybean meal than in the rats fed regular soybean meal. These results suggested that fermentation with Aspergillus usamii improved zinc availability in dietary soybean meal, which was induced by the increase of zinc solubility in the small intestine. Adding the same amount of phytate that was contained in the regular soybean meal-based diet did not affect the amount of zinc present in rats fed a fermented soybean meal-based diet with sodium phytate. Phytase activity was found in fermented soybean meal, and this activity may degrade added phytate in fermented soybean meal-based diet.

Animal Feed↗
PubMed1998

Effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes.

The effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h in TCM199 supplemented with 5% fetal bovine serum (FBS) in 3% CO2 in air. The oocytes were first exposed to 20% ethylene glycol solution and were subjected to vitrification in a solution containing 40% ethylene glycol, 18% Ficoll-70 and 0.3 M sucrose. After warming in 20 degrees C water, oocytes which had been vitrified at less than 24-h of IVM were again cultured to complete the 24-h of IVM period. Oocytes were then incubated with frozen-thawed spermatozoa in Brackett and Oliphant (BO) medium containing 60 micrograms/ml heparin and 0.25% BSA for 20 h. In vitro fertilization rates of oocytes vitrified-warmed at 0, 6, 12 and 24-h IVM were 75.2, 68.0, 82.0 and 72.4%, respectively, comparable to the rates for unvitrified control oocytes (80.6%). A higher incidence of polyspermic fertilization was observed in oocytes vitrified at 24-h IVM (44.9 vs 22.6% in the control group, P < 0.05). Vitrification of oocytes at 12-h IVM seemed to be better than that of other IVM groups, since the normal fertilization rate of all treated oocytes was the highest (36.0%) among the vitrification groups. Developmental competence of the oocytes following vitrification and in vitro fertilization (12-h IVM group) was examined by cell-free culture of presumptive zygotes up to 9 d in modified synthetic oviduct fluid (mSOF) in 5% CO2, 5% O2 and 90% N2. The cleavage rate of zygotes from vitrified oocytes 48 h after insemination was 29.8%, which was lower than that of the control group (57.0%, P < 0.05). Development to blastocysts from the vitrified oocytes (4.8%) was much lower than that of the control group (27.0%, P < 0.05). These results indicate that cryopreservation of bovine oocytes by vitrification may be affected by their maturation stage in vitro, and that developmental competence to blastocysts of cleaved oocytes following vitrification may be impaired compared with unvitrified control oocytes.

Animals↗
PubMed1998

Cryopreservation of in vitro-derived bovine blastocysts microinjected with foreign DNA at the pronuclear stage.

Days 6 and 7 bovine blastocysts derived from in vitro-fertilized and DNA-injected zygotes (day of IVF = Day 0) were cryopreserved either by conventional two-step freezing or by vitrification. Foreign DNA used for microinjection was the green fluorescent protein gene under the control of the immediate early promoter of human cytomegalovirus. All blastocysts were produced by an in vitro system and were harvested on Days 6 and 7. The proportion of DNA-injected zygotes developing into blastocysts on Days 6 and 7 (total 8%) was lower than that of nontreated zygotes (total 19%; P < 0.01). After cryopreservation in 1.5 M ethylene glycol, the survival rates of DNA-injected blastocysts assessed by re-expansion at 24 h of culture (Day 6: 59%, Day 7: 71%) were comparable with those of nontreated blastocysts (Day 6: 76%, Day 7: 71%). The post-thaw hatching rate within 72 h of culture of DNA-injected Day 7 blastocysts (38%) was not different from that of nontreated Day 7 blastocysts (40%), but the hatching rate of DNA-injected Day 6 blastocysts (23%) was lower than that of nontreated Day 6 blastocysts (47%; P < 0.05). After vitrification in 7.2 M ethylene glycol, 0.0026 M Ficoll-70 and 0.3 M sucrose, the survival and hatching rates of DNA-injected Day 7 blastocysts (61 and 28%, respectively) were similar to those of nontreated Day 6 (71 and 33%, respectively) and Day 7 (75 and 36%, respectively) blastocysts. However, the post-warming survival rate of DNA-injected Day 6 blastocysts was only 30%, and none of the blastocysts hatched (P < 0.01). The mean cell number of DNA-injected Day 6 blastocysts (100.3 +/- 36.4 cells) was lower than that of nontreated Day 6 blastocysts (130.5 +/- 37.1 cells; P < 0.01), while those of DNA-injected and nontreated Day 7 blastocysts were not different (111.2 +/- 42.8 and 119.6 +/- 31.4 cells, respectively). These results indicate that Day 7 IVMFC bovine blastocysts derived from DNA-injected zygotes can be successfully cryopreserved by conventional two-step freezing or vitrification.

Animals↗
PubMed1998

Fermentation of soybean meal with Aspergillus usamii reduces phosphorus excretion in chicks.

Fermentation with Aspergillus usamii almost completely degrades phytate phosphorus in soybean meal. Phosphorus excretion was investigated in chicks fed a fermented soybean meal-based diet. Thirty chicks were fed one of three experimental diets; a control soybean meal-based diet (total phosphorus, 5.2 g/kg; nonphytate phosphorus, 2.3 g/kg), a control soybean meal-based diet with added inorganic phosphorus (total phosphorus, 7.1 g/kg; nonphytate phosphorus, 4.0 g/kg), or a fermented soybean meal-based diet without supplemental inorganic phosphorus (total phosphorus, 5.8 g/kg; nonphytate phosphorus, 3.9 g/kg) for 4 wk. Body weight gain, the amount of retained phosphorus, and femoral phosphorus content were lower in the control group than in the phosphorus-supplied group and the fermented soybean meal group. The latter two groups showed similar body weight gain and femoral phosphorus content. On the other hand, phosphorus excretion was markedly more in the phosphorus-supplied group than in the other groups. As a result, phosphorus retention (percentage of intake) was lower in the phosphorus-supplied group than in the fermented soybean meal group. In conclusion, fermentation improved phosphorus bioavailability in soybean meal and supplemental inorganic phosphorus was not necessary for the fermented soybean meal-based diet, which remarkably reduced phosphorus excretion.

Animals↗
PubMed1998

Fermentation of soybean flour with Aspergillus usamii improves availabilities of zinc and iron in rats.

Soybean flour was fermented with Aspergillus usamii to improve the availabilities of dietary zinc and iron through the degradation of phytate. Three kinds of experimental diets that differed in protein sources were prepared: one consisting of 40% fermented soybean flour (RS diet), one consisting of 40% fermented soybean flour (FS diet), and one consisting of 20% regular soybean flour and 20% fermented soybean flour (RF diet). Zinc solubilities in the upper and the lower segments of the small intestine were higher in rats fed the FS diet than in rats fed the RS diet. The FS group showed higher solubility of iron in the lower small intestine than the RS group did. Zinc concentrations in the femur and plasma and iron concentrations in the liver and plasma were higher in the FS group than in the RS group. These results suggested that the fermentation of soybean flour improved the availabilities of dietary zinc and iron, which may be induced by increasing the solubilities of these minerals in the small intestine through the reduction of phytate content. Femoral and plasma zinc concentrations in the RF group were higher than in the RS group, but lower than in the FS group. No difference was noted in liver and plasma iron concentrations between the RF group and the FS group. Although phytase activity in FS degrades phytate in the RF diet, higher activity may be needed to degrade phytate completely.

Animals↗
PubMed1997

Transgenic rescue from embryonic lethality and renal carcinogenesis in the Eker rat model by introduction of a wild-type Tsc2 gene.

We recently reported that a germ-line insertion in the rat homologue of the human tuberous sclerosis gene (TSC2) gives rise to dominantly inherited cancer in the Eker rat model. In this study, we constructed transgenic Eker rats with introduction of a wild-type Tsc2 gene to ascertain whether suppression of the Eker phenotype is possible. Rescue from embryonic lethality of mutant homozygotes (Eker/Eker) and suppression of N-ethyl-N-nitrosourea-induced renal carcinogenesis in heterozygotes (Eker/+) were both observed, defining the germ-line Tsc2 mutation in the Eker rat as embryonal lethal and tumor predisposing mutation. To the best of our knowledge, this is the first report of rescue from a naturally occurring dominantly inherited cancer. This transgenic rescue system will be useful to analyze Tsc2 gene function, its relation to tumorigenesis in vivo, and genetic-environmental interactions in carcinogenesis.

Animals↗
PubMed1997

Position-independent and high-level expression of human alpha-lactalbumin in the milk of transgenic rats carrying a 210-kb YAC DNA.

The level of expression of transgenes in transgenic animals varies among lines, and is often much lower than that of endogenous genes (position effects). In order to surmount position effects and establish a more efficient production system of transgenic animals producing pharmaceutical proteins in their milk, transgenic rats carrying 210-kb YAC DNA containing the human alpha-lactalbumin gene were produced. Three transgenic lines transmitted the transgene to the next generation. They had one copy of the alpha-lactalbumin gene and secreted human alpha-lactalbumin in their milk at concentrations of 2.0-4.3 mg/ml. No position effect was seen. The transgene was expressed specifically in the mammary gland of the transgenic rats. The 210-kb region is thought to contain all the DNA elements required for proper expression of the human alpha-lactalbumin gene. The YAC carrying the human alpha-lactalbumin gene is a potential vector for the expression of foreign genes in the mammary gland.

Animals↗
PubMed1997

Viability of transgenic rat embryos after freezing and thawing.

In-vivo viability of frozen-thawed embryos derived from transgenic rats, as well as the transmission and the expression of transgenes in the resultant newborn rats, was investigated. Three strains of transgenic rats, carrying human growth hormone gene connected downstream to the promoter region of the bovine alpha-lactalbumin gene (alpha LA/hGH), bovine beta-casein gene (beta CN/hGH) or bovine alpha-S1 casein gene (alpha S1CN/hGH), were used. Two-cell stage embryos (non-transgenic Wistar female x heterozygous transgenic male) were placed in 10% (v/v) dimethylsulfoxide (DMSO) solution and cooled from -7 to -30 degrees C at -0.5 degree C/min before being plunged into liquid nitrogen. After 2 to 4 years storage, the embryos were thawed by rapid warming. The intact embryos were transferred into the oviducts of Day 1 pseudopregnant recipients. The postthaw survival rate of frozen embryos was high in all 3 transgenic strains (88 to 92%), which was similar to that of control (non-transgenic) frozen embryos (95%). Development to newborn rats following transfer of embryos derived from the 3 strains (64 to 68%) was also similar to that of control embryos (60%). These transgenes (alpha LA/hGH, beta CN/hGH and alpha S1CN/hGH) were detected in the DNA extracts from tail tissue of the newborn rats, but the transmission rates (41, 23 and 32%, respectively) were lower than 50% which is expected in the Mendelian fashion. In a transgenic line carrying alpha S1CN/hGH, hGH levels of secretion into the milk of transgenic newborn rats derived from frozen-thawed embryos and her transgenic offspring were the same mg/ml-level as that of their founder rat. Two-step freezing of embryos derived from transgenic rats was therefore an effective method for the long-term cryopreservation of transgene.

Animals↗
PubMed1996

Activation of glutathione transferase P gene by lead requires glutathione transferase P enhancer I.

Glutathione transferase P (GST-P) is specifically induced in rat liver and kidney by lead cation. The increase of GST-P mRNA after lead administration is blocked by actinomycin D, suggesting that GST-P production by lead is regulated at the transcriptional level. To further determine which part of the flanking region of the GST-P gene has the lead-responsive cis-element in vivo, we utilized transgenic rats with five different constructs having GST-P and/or chloramphenicol acetyl-transferase coding sequence. We studied the effect of lead on these transgenic rats and on transfected NRK (normal rat kidney) cells and found that GST-P induction by lead is indeed regulated at the transcriptional level and that the GST-P enhancer I (GPEI) enhancer is an essential cis-element required for the activation of the GST-P gene by lead. GPEI consists of two AP-1 (c-Jun/c-Fos heterodimer) site-like sequences that are palindromically arranged and can bind AP-1, c-jun mRNA in the liver increased after lead administration and GST-P, and c-Jun had patchy expression in the same hepatocytes 24 h after lead exposure. These results suggest that activation of the GST-P gene by lead is mediated in major part by enhancer GPEI and that AP-1 may be involved at least partially. GPEI has been shown to have essential sequence information for the trans-activation of the GST-P gene during chemical hepatocarcinogenesis of the rat (Morimura, S., Suzuki, T., Hochi, S., Yuki, A., Nomura, K., Kitagawa, T., Nagatsu, I., Imagawa, M., and Muramatsu, M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2065-2068; Suzuki, T., Imagawa, M., Hirabayashi, M., Yuki, A., Hisatake, K., Nomura, K., Kitagawa, T., and Muramatsu, M. (1995) Cancer Res. 55, 2651-2655). The present study establishes that the same enhancer element does operate in the activation of the GST-P gene by lead regardless of the trans-activators involved.

Animals↗
PubMed1996

Transgene expression in mammary glands of newborn rats.

Transgene expression in the mammary glands of newborn rats was studied to establish an early selection system for transgenic animals producing exogenous proteins in their milk during lactation. A fusion gene composed of the bovine alpha S1 casein gene promoter and the human growth hormone gene was microinjected into rat embryos. Transgenic lines that produced human growth hormone in their milk were established and used in this study. Immediately after birth, and without any hormone treatment, human growth hormone was found in the extracts of mammary glands from both male and female rats derived from the line secreting human growth hormone in their milk. The expression of the transgene in mammary glands of newborn rats was also detected by the presence of human growth hormone mRNA. Nontransgenic newborn rats did not express the human growth hormone gene in their mammary glands, while the mRNA for rat alpha casein, an endogenous milk protein, was found in all mammary glands from both transgenic and nontransgenic neonates. These results show that analyzing the expression of transgenes in the mammary glands of neonates is a valuable tool to select the desired transgenic animals and to shorten the selection schedules establishing the transgenic animals.

Animals↗