Induction of pluripotency by injection of mouse trophectoderm cell nuclei into blastocysts following transplantation into enucleated oocytes

Pluripotency of mouse trophectoderm (TE) cells was examined using a nuclear transfer technique. We transferred a TE cell to an enucleated oocyte and cultured the reconstituted oocyte to be blastocyst stage. Then a portion of the inner cell mass (ICM) isolated from the TE-origin blastocyst was injected into the cavity of a fertilized blastocyst to produce a chimeric embryo, which was transferred to a recipient female. Of 319 oocytes reconstituted with TE cells, 263 (82.4%) had a single nucleus (1PN), 3 (0.9%) had 2 nuclei (2PN) and 53 (16.6%) had a nucleus with a polar body (1PN1PB). Although the oocytes with 1PN and 2PN developed to blastocysts (81 of 263, 30.8% and 1 of 3, respectively), only those with 1PN were used to produce chimeric blastocysts. After the transfer of chimeric embryos to recipient females, 7 (28%) of 25 conceptuses analyzed at midgestation showed chimerism. Of those 5 (71%), 6 (86%) and 4 (57%) chimeric conceptuses showed distribution of donor nuclei in the fetus, membrane and placenta, and the distributions were 10 to 65, 10 to 50 and 10 to 15%, respectively. Of the 23 young obtained, 7 (30%; 2 males and 5 females) were coat color chimeras. The contributions of donor nuclei were detected in the brain, lung, heart, liver, kidney, testis, ovary and blood. Each coat-color chimeric mouse was mated with CD-1 male or female mice, but no germ line chimera was obtained. When ICM cells were used as the control nuclear donor, the contribution was equivalent to those of TE cells. In conclusion, pluripotency of mouse TE cells on a somatic line was induced, and chimeric young were obtained using a nuclear transplantation technique.     

Mitochondrial DNA heteroplasmy in calves cloned by using adult somatic cell

Adult somatic cell cloned calves were produced by somatic cell nuclear transfer prepared by fusion of cultured ear fibroblast from a Holstein cow into enucleated oocytes of Luxi Yellow cow. In order to determinate the source of mitochondrial DNA of cloned calves, we designed the breed-specific PCR primers by aligning the known D-loop sequences of Bos taurus and analyzed the displacement loop sequences of five live cloned calves by breed-specific primers PCR. The results demonstrated that mtDNA originated from Holstein breed and that from Luxi breed co-exist in all five live calves.     

In vitro development and mitochondrial fate of macaca-rabbit cloned embryos

Interspecies cloning may be used as an effective method to conserve highly endangered species and to support the development of non-human primate animal models for studying therapeutic cloning and nuclear-cytoplasm interaction. The use of the monkey model for biomedical research can avoid legal, ethical, and experimental limitations encountered in a clinical situation. We describe in this study the in vitro development of macaca-rabbit embryos produced by fusing macaca fibroblasts with enucleated rabbit oocytes and examine the fate of mitochondrial DNA in these embryos. We show that macaca-rabbit cloned embryos can develop to the blastocyst stage when cultured in vitro in HECM(10) +10% FBS and that mitochondrial DNA derived from donor somatic cells was detectable in cloned embryos throughout preimplantation development. These results suggest that (1) macaca fibroblast nuclei can dedifferentiate in enucleated metaphase II rabbit oocytes; (2) HECM(10) +10% FBS can break through the development block and support the development of macaca-rabbit cloned embryos to blastocysts; and (3) donor-cell-derived mitochondrial DNA is not eliminated until blastocyst stage.     

Cholinergic regulation of the corpora allata in adult male loreyi leafworm Mythimna loreyi

The direct effect of acetylcholine on the activation of the corpora allata (CA) was investigated in the adult male loreyi leafworm, Mythimna loreyi. Acetylcholine, in the presence of the choline esterase inhibitor physostigmine (50 microM), elicited a stimulatory effect on juvenile hormone acids (JHAs) release from the CA. Maximum effect was obtained at concentrations of 10 and 50 microM. Repeated administration of 10 microM acetylcholine on the same CA did not elicit similar stimulatory effect. Since JHA release can be significantly activated by carbachol and not by nicotine, this cholinergic effect is likely to belong to the muscarinic type. The effect of acetylcholine was significantly antagonized by gallamine triethiodide (M(2) antagonist) and 4-DAMP (M(3) antagonist), pirenzepine (M(1) antagonist), and tropicamide (M(4) antagonist) were ineffective. It is concluded that in the adult male M. loreyi, the cholinergic regulation of CA is most likely via M(2) and M(3) muscarinic receptors.     

Subcellular targeting and agonist-induced site-specific phosphorylation of endothelial nitric-oxide synthase

The endothelial isoform of nitric-oxide synthase (eNOS) undergoes a complex pattern of covalent modifications, including acylation with the fatty acids myristate and palmitate as well as phosphorylation on multiple sites. eNOS acylation is a key determinant for the reversible subcellular targeting of the enzyme to plasmalemmal caveolae. We transfected a series of hemagglutinin epitope-tagged eNOS mutant cDNAs deficient in palmitoylation (palm(-)) and/or myristoylation (myr(-)) into bovine aortic endothelial cells; after treatment with the eNOS agonists sphingosine 1-phosphate or vascular endothelial growth factor, the recombinant eNOS was immunoprecipitated using an antibody directed against the epitope tag, and patterns of eNOS phosphorylation were analyzed in immunoblots probed with phosphorylation state-specific eNOS antibodies. The wild-type eNOS underwent agonist-induced phosphorylation at serine 1179 (a putative site for phosphorylation by kinase Akt), but phosphorylation of the myr(-) eNOS at this residue was nearly abrogated; the palm(-) eNOS exhibited an intermediate phenotype. The addition of the CD8 transmembrane domain to the amino terminus of eNOS acylation-deficient mutants rescued the wild-type phenotype of robust agonist-induced serine 1179 phosphorylation. Thus, membrane targeting, but not necessarily acylation, is the critical determinant for agonist-promoted eNOS phosphorylation at serine 1179. In striking contrast to serine 1179, phosphorylation of eNOS at serine 116 was enhanced in the myr(-) eNOS mutant and was markedly attenuated in the CD8-eNOS membrane-targeted fusion protein. We conclude that eNOS targeting differentially affects eNOS phosphorylation at distinct sites in the protein and suggest that the inter-relationships of eNOS acylation and phosphorylation may modulate eNOS localization and activity and thereby influence NO signaling pathways in the vessel wall.     

Apolipoprotein E and H polymorphisms in Mongolian Buryat: allele frequencies and relationship with plasma lipid levels

A Buryat population consisting of seven tribal groups in eastern Mongolia has been screened to determine the frequency distribution of different apolipoprotein E and H alleles (APOE and APOH, genes) coding for common isoforms and their association with quantitative plasma lipid levels. Allele frequencies at the APOE locus in 125 healthy Buryat aged 17 to 73 years were highest for APOE*3 (0.804), followed by APOE*4 (0.164) and APOE*2 (0.032). The APOH locus had high frequencies of APOH*2 (0.912) and APOH*3 (0.088). APOH*1 was not detected. No significant differences were observed in the overall APOE allele frequencies between the Buryat and the Siberian Evenki, Inuits, and Indians in Asia, or with some European whites. The frequency distribution of the overall APOH alleles of the Buryat was similar to that of the Japanese in Asia. Overall plasma lipid levels of the Buryat (males aged 20 to 73 years, females aged 21 to 64 years) were considerably lower, comparable to those of the Evenki. The APOE*4/E*3 males had significantly high total- and LDL-cholesterol levels compared with the APOE*3/E*3 males (p < 0.025 and p < 0.01, respectively). No significant effects of the APOH genotypes on any of the plasma lipid levels were observed. In particular, our data regarding APOE suggest that the Buryat are genetically close in allele frequencies to the Evenki and Inuits, but differ from them in the association of genotype APOE*4/E*3 with cholesterol levels.     

Blazeispirane and protoblazeispirane derivatives from the cultured mycelia of the fungus Agaricus blazei

Two blazeispirane derivatives including blazeispirols G and I were isolated from the cultured mycelia of the fungus Agaricus blazei Murill and were established to be (20S, 22S, 23R, 24S)-14 beta,22: 22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9-triene-11 alpha,23-diol and (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9,11-tetraene-23,28-diol by comparison of extensive 1D and 2D NMR spectral data with that of blazeispirol A. Furthermore, four blazeispirol derivatives blazeispirols, U, V, V(1) and Z(1) were isolated form the same source described above. Their structures were determined to be (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-23-hydroxyergosta-4,6,8,11-tetraen-3-one, (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-6 alpha,7 alpha,23-trihydroxyergosta-4,8,11-trien-3-one, (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-6 beta,7 alpha,23-trihydroxyergosta-4,8,11-trien-3-one and (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-23-hydroxy-4,5-seco-ergosta-6,8-diene-3,5-dione by extensive 1 D and 2D NMR spectral data.     

Highly sensitive time-resolved fluorometric determination of estrogens by high-performance liquid chromatography using a beta-diketonate europium chelate

A new fluorescent europium chelate labeling reagent, 5-(4"-chlorosulfo-1',1"-diphenyl-4'-yl)-1,1,1,2,2-pentafluoro-3,5-pentanedione (CDPP), was synthesized for the time-resolved fluorometric detection of HPLC. The label can be directly bound to amino or phenolic hydroxyl groups of analytes with its chlorosulfonyl group, and the labeled analytes are separated on a HPLC column. After separation, EuCl(3), TOPO (tri-n-octylphosphine oxide), and Triton X-100 were added by post-column introduction to the eluent, and the fluorescence of the europium chelate was measured with the time-resolved fluorometric detector. Estrone (E1), 17beta-estradiol (E2), ethynylestradiol (EE2) and estriol (E3) were measured with the detection limits of 0.65, 0.65, 0.65 and 0.60 ng/ml, respectively. The recovery for river water samples was in the range of 86.0-105.1% with the RSD of 1.9-5.8%. The method was applied to the analysis of a river water sample and estrone (E1) was determined to be 2.1 ng/l. The results and processing have been compared with those of a GC-MS method and a high degree of correlation (r> or =0.98) was observed.