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1.
Ayumi HASEGAWA Keiji MOCHIDA Toshiko TOMISHIMA Kimiko INOUE Atsuo OGURA 《The Journal of reproduction and development》2014,60(3):187-193
Successful in vitro fertilization (IVF) in mice has been achieved using spermatozoa at concentrations
specifically optimized for the experimental conditions, such as species and source of spermatozoa. Although IVF in mice is
mostly performed using about 80–500 µl drops, it is expected that the number of spermatozoa used for insemination can be
reduced by decreasing the size of the IVF drops. The present study was undertaken to examine the extent to which the number
of spermatozoa used for IVF could be reduced by using small droplets (1 µl). We devised the experimental parameters using
frozen–thawed spermatozoa from C57BL/6 mice in anticipation of broader applications to other mouse facilities. We found that
as few as 5 spermatozoa per droplet could fertilize oocytes (1 or 3 oocytes per droplet), although the fertilization rates
were low (13–15%). Practical fertilization rates (> 40%) could be achieved with frozen-thawed C57BL/6J spermatozoa, which
are sensitive to cryopreservation, when 20 sperm per droplet were used to inseminate 3 oocytes. Even with spermatozoa from a
very poor quality suspension (10% motility), about 25% of oocytes were fertilized. Our calculations indicate that the number
of inseminated spermatozoa per oocyte can be reduced to 1/96–1/240 by this method. In two separate embryo transfer
experiments, 60% and 47%, respectively, of embryos developed to term. Our microdroplet IVF method may be particularly
advantageous when only a limited number of motile spermatozoa are available because of inadequate freezing-thawing or genetic
reasons. 相似文献
2.
Allelic variation in high-molecular-weight glutenin subunit Loci of Glu-1 in Japanese common wheats 总被引:2,自引:0,他引:2
Seed storage proteins of 131 Japanese Norin wheat (Triticum aestivum) varieties were fractionated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis to determine allelic make-up in varieties at each of three loci that control high-molecular-weight
(HMW) glutenin subunits. Three alleles were identified at the Glu-A1 locus, six at the Glu-B1 locus and five at the Glu-D1
locus. Twenty-four different, major glutenin HMW subunits were identified and each contained three to five subunits and seventeen
different glutenin subunit patterns were observed for 19 subunits in the 131 Japanese Norin varieties. Fourteen alleles were
identified by comparison of subunit mobility with that previously found in hexaploid wheat. Japanese Norin varieties showed
a specific pattern of allelic variation in glutenin HMW subunits, different from that of Chinese and other country common
wheats in allelic frequency at Glu-1 loci.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
3.
Kimiko HONSHO Michiko HIROSE Masanori HATORI Lubna YASMIN Haruna IZU Shogo MATOBA Sumie TOGAYACHI Hiroyuki MIYOSHI Tadashi SANKAI Atsuo OGURA Arata HONDA 《The Journal of reproduction and development》2015,61(1):13-19
Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in
vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use. 相似文献
4.
Taguchi H Watanabe S Hirao T Akiyama H Sakai S Watanabe T Matsuda R Urisu A Maitani T 《Journal of agricultural and food chemistry》2007,55(5):1649-1655
Kiwifruit (Actinidia deliciosa and Actinidia chinensis) is allergenic to sensitive patients, and, under Japanese regulations, it is one of the food items that are recommended to be declared on food labeling as much as possible. To develop PCR-based methods for the detection of trace amounts of kiwifruit in foods, two primer pairs targeting the ITS-1 region of the Actinidia spp. were designed using PCR simulation software. On the basis of the known distribution of a major kiwifruit allergen (actinidin) within the Actinidia spp., as well as of reports on clinical and immunological cross-reactivities, one of the primer pairs was designed to detect all Actinidia spp. and the other to detect commercially grown Actinidia spp. (i.e., kiwifruit, Actinidia arguta, and their interspecific hybrids) except for Actinidia polygama. The specificity of the developed methods using the designed primer pairs was verified by performing PCR experiments on 8 Actinidia spp. and 26 other plants including fruits. The methods were considered to be specific enough to yield target-size products only from the target Actinidia spp. and to detect no target-size products from nontarget species. The methods were sensitive enough to detect 5-50 fg of Actinidia spp. DNA spiked in 50 ng of salmon testis DNA used as a carrier (1-10 ppm of kiwifruit DNA) and 1700 ppm (w/w) of fresh kiwifruit puree spiked in a commercial plain yogurt (corresponding to ca. 10 ppm of kiwifruit protein). These methods would be expected to be useful in the detection of hidden kiwifruit and its related species in processed foods. 相似文献
5.
NISHIMURA ET HAMILTON HB KOBARA TY TAKAHARA S OGURA Y DOI K 《Science (New York, N.Y.)》1959,130(3371):333-334
The heterozygous carrier state of a rare hereditary disease, acatalasemia, has been defined biochemically. Affected homozygotes have no blood catalase activity, whereas heterozygotes show activities intermediate between this inactivity and the activity of normal controls, without overlap. Pedigrees show a high frequency of consanguineous marriages. 相似文献
6.
Taguchi H Watanabe S Temmei Y Hirao T Akiyama H Sakai S Adachi R Sakata K Urisu A Teshima R 《Journal of agricultural and food chemistry》2011,59(8):3510-3519
Shrimp and crab are well-known as allergenic ingredients. According to Japanese food allergy labeling regulations, shrimp species (including prawns, crayfishes, and lobsters) and crab species must be differentially declared when ≥10 ppm (total protein) of an allergenic ingredient is present. However, the commercial ELISA tests for the detection of crustacean proteins cannot differentiate between shrimp and crab. Therefore, two methods were developed to discriminate shrimp and crab: a shrimp-PCR method with postamplification digestion and a crab-PCR method that specifically amplifies a fragment of the 16S rRNA gene. The sensitivity and specificity of both PCR methods were verified by experiments using DNA extracted from 15 shrimp species, 13 crab species, krill, mysid, mantis shrimp, other food samples (cephalopod, shellfish, and fish), incurred foods, and commercial food products. Both PCR methods could detect 5 pg of DNA extracted from target species and 50 ng of genomic DNA extracted from incurred foods containing 10 ppm (μg/g) total protein of shrimp or crab. The two PCR methods were considered to be specific enough to separately detect species belonging to shrimp and crab. Although false-positive and false-negative results were obtained from some nontarget crustacean species, the proposed PCR methods, when used in conjunction with ELISA tests, would be a useful tool for confirmation of the validity of food allergy labeling and management of processed food safety for allergic patients. 相似文献
7.
The variations in glucosidic linkage specificity observed in products of different glucansucrases appear to be based on relatively small differences in amino acid sequences in their sugar-binding acceptor subsites. Various amino acid mutations near active sites of DSRBCB4 dextransucrase from Leuconostoc mesenteroides B-1299CB4 were constructed. A triple amino acid mutation (S642N/E643N/V644S) immediately next to the catalytic D641 (putative transition state stabilizing residue) converted DSRBCB4 enzyme from the synthesis of mainly α-(1→6) dextran to the synthesis of α-(1→6) glucan containing branches of α-(1→3) and α-(1→4) glucosidic linkages. The subsequent introduction of mutation V532P/V535I, located next to the catalytic D530 (nucleophile), resulted in the synthesis of an α-glucan containing increased branched α-(1→4) glucosidic linkages (approximately 11%). The results indicate that mutagenesis can guide glucansucrase toward the synthesis of various oligosaccharides or novel polysaccharides with completely altered linkages without compromising high transglycosylation activity and efficiency. 相似文献
8.
9.
Mochizuki M Yachi A Ohshima T Ohuchi A Ishida T 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2008,70(6):563-569
Infectious tracheobronchitis (ITB), also known as the kennel cough, is a respiratory syndrome of dogs and usually appears to be contagious among dogs housed in groups. Etiologic agent of ITB is multiple and sometimes complex. In the present study, 68 household dogs showing clinical signs of respiratory infection were examined, and 20 dogs (29.4%) were found to be positive for either of following agents. Bordetella bronchiseptica (B.b.) was most frequently detected from nasal and oropharynx sites of 7 dogs (10.3%). Among the viruses examined, canine parainfluenza virus (CPIV) was detected with the highest frequency (7.4%). Other pathogens included in the order of frequency group 1 canine coronavirus (4.4%), canine adenovirus type 2 (2.9%), group 2 canine respiratory coronavirus (1.5%), and canine distemper virus (1.5%). Only 2 cases showed mixed infections. Neither influenza A virus nor canine bocavirus (minute virus of canines) was found in any dogs examined. These results indicate that both B.b. and CPIV are likely to be the principal etiologic agents of canine ITB in Japan, and they may be considered as the target for prophylaxis by vaccination. 相似文献
10.
Mami OIKAWA Shogo MATOBA Kimiko INOUE Satoshi KAMIMURA Michiko HIROSE Narumi OGONUKI Hirosuke SHIURA Michihiko SUGIMOTO Kuniya ABE Fumitoshi ISHINO Atsuo OGURA 《The Journal of reproduction and development》2013,59(3):231-237
In mice, one of the major epigenetic errors associated with somatic cell nuclear
transfer (SCNT) is ectopic expression of Xist during the preimplantation
period in both sexes. We found that this aberrant Xist expression could
be impeded by deletion of Xist from the putative active X chromosome in
donor cells. In male clones, it was also found that prior injection of
Xist-specific siRNA could significantly improve the postimplantation
development of cloned embryos as a result of a significant repression of
Xist at the morula stage. In this study, we examined whether the same
knockdown strategy could work as well in female SCNT-derived embryos. Embryos were
reconstructed with cumulus cell nuclei and injected with Xist-specific
siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment
successfully repressed Xist RNA at the morula stage, as shown by the
significant decrease in the number of cloud-type Xist signals in the
blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”)
and numbers of Xist RNA signals remained within single embryos. After
implantation, the dysregulated Xist expression was normalized
autonomously, as in male clones, to a state of monoallelic expression in both embryonic
and extraembryonic tissues. However, at term there was no significant improvement in the
survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective
in repressing the Xist overexpression in female cloned embryos but failed
to rescue them, probably because of an inability to mimic consistent monoallelic
Xist expression in these embryos. This could only be achieved in female
embryos by applying a gene knockout strategy rather than an siRNA approach. 相似文献