Proliferation of donor mitochondrial DNA in nuclear transfer calves (Bos taurus) derived from cumulus cells

In embryos derived by nuclear-transfer (NT), fusion of donor cell and recipient oocyte caused mitochondrial heteroplasmy. Previous studies from other laboratories have reported either elimination or maintenance of donor-derived mitochondrial DNA (mtDNA) from somatic cells in cloned animals. Here we examined the distribution of donor mtDNA in NT embryos and calves derived from somatic cells. Donor mitochondria were clearly observed by fluorescence labeling in the cytoplasm of NT embryos immediately after fusion; however, fluorescence diminished to undetectable levels at 24 hr after nuclear transfer. By PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, donor mtDNAs were not detected in the NT embryos immediately after fusion (less than 3-4%). In contrast, three of nine NT calves exhibited heteroplasmy with donor cell mtDNA populations ranging from 6 to 40%. These results provide the first evidence of a significant replicative advantage of donor mtDNAs to recipient mtDNAs during the course of embryogenesis in NT calves from somatic cells.     

Determination of the absolute configurations of the anteiso acid moieties of glycoglycerolipid S365A isolated from Corynebacterium aquaticum

The absolute configurations of the two acid moieties, 12-methyltetradecanoate and 14-methylhexadecanoate, of glycoglycerolipid S365A isolated from Corynebacterium aquaticum were determined by an HPLC analysis after their conversion with the chiral fluorescent labeling reagents, (1S,2S)- and (1R,2R)-2-(2,3-anthracenedicarboximido)cyclohexanol. Both anteiso acids had the S configuration.     

Production of transgenic chimera rabbit fetuses using somatic cell nuclear transfer

We produced aggregate chimeric embryos between blastomeres from the somatic cell nuclear transfer (SCNT) embryos and blastomeres from normal embryos. The SCNT embryos were produced by fusing enucleated oocytes with GFP gene introduced fibroblast cells, which were derived from a day 16 fetus. GFP gene-introduced fibroblast cells were cultured and passaged four to 12 times over a period of 45-79 days before SCNT. After transferring them into pseudopregnant recipient rabbits, the 15-day postcoitus fetuses were collected. We examined the existence of the cells derived from SCNT embryos in the fetus stage of pregnancy to detect the GFP gene. Fetuses that were not collected continued to develop into newborn rabbits. Two hundred and thirty-six chimeric embryos were produced using 39 SCNT morula stage embryos, and these embryos were transferred to 11 recipient rabbits. As a result, 27 normally developed and 16 degenerated concepti were obtained. The GFP gene-positive signals were detected in one of the fetuses, two of the placentae, and two of the degenerated concepti. In this study, we found that the rabbit SCNT embryos have the ability to develop and differentiate in vivo. We also demonstrated a novel method of producing a transgenic rabbit using SCNT.     

Comparative biochemical studies of carotenoids in catfishes

The carotenoids of 12 species of Siluriformes fishes (eight families) were investigated from a comparative biochemical point of view. The patterns of carotenoids in catfishes belonging to the family Siluridae were quite different from those of the other seven families of catfishes (Bagridae, Amblycipitidae, Clariidae, Plotosidae, Ictaluridae, Callichthyidae and Malapteruridae). 7, 8-Dihydro-beta-carotene; 7, 8, 7', 8'- and 7, 8, 9, 10-tetrahydro-beta-carotene; (3R)-7', 8'-dihydro-beta-cryptoxanthin; 7, 8-dihydrolutein A; 7, 8-dihydrolutein B; parasiloxanthin; 7', 8'-dihydroparasiloxanthin; and 4 or 4'-hydroxyparasiloxanthin were characteristic carotenoids found in only one family, Siluridae, and these carotenoids accounted for 24-60% of total carotenoids. In catfishes belonging to the other seven families except Siluridae, the carotenoid patterns were very similar and the most predominant carotenoid was zeaxanthins (23-56%).     

Effects of UV dose on formation of spontaneously developing pocks in Streptomyces azureus ATCC14921

Spontaneously developing pocks (S pocks) of Streptomyces azureus ATCC14921 were formed by the both functions of conjugative plasmid pSA1 and lysogenic phage SAt2. The formation was affected by the dose of UV irradiation. The mean pock diameter in cultures treated with UV light at 0, 7.1, 14.2 and 21.3 x 10(2) microW. erg/cm, respectively, were 1.3, 0.4, 2.2, and 0.5 mm. The dose affected conjugative plasmid pSA1 related to pock formation. There was UV damage of autonomous pSA1 replicon and UV induction of the chromosomal integrated sequence. Increases and decreases in the amount of autonomous pSA1 replicon corresponded to increases and decreases, respectively, in the diameter of the pocks. Both pSA1 and SAt2 syntheses were developed in the large pocks (1.3 and 2.2 mm), but only SAt2 synthesis was developed in the pinhole pocks (0.4 and 0.5 mm).     

Constitutive tyrosine phosphorylation of ErbB-2 via Jak2 by autocrine secretion of prolactin in human breast cancer

Overexpression of the oncogene for ErbB-2 is an unfavorable prognostic marker in human breast cancer. Its oncogenic potential appears to depend on the state of tyrosine phosphorylation. However, the mechanisms by which ErbB-2 is constitutively tyrosine-phosphorylated in human breast cancer are poorly understood. We now show that human breast carcinoma samples with ErbB-2 overexpression have higher proliferative and metastatic activity in the presence of autocrine secretion of prolactin (PRL). By using a neutralizing antibody or dominant negative (DN) strategies or specific inhibitors, we also show that activation of Janus kinase Jak2 by autocrine secretion of PRL is one of the significant components of constitutive tyrosine phosphorylation of ErbB-2, its association with Grb2 and activation of mitogen-activated protein (MAP) kinase in human breast cancer cell lines that overexpress ErbB-2. Furthermore, the neutralizing anti-PRL antibody or erbB-2 antisense oligonucleotide or DN Jak2 or Jak2 inhibitor or DNRas or MAP kinase kinase inhibitor inhibits the proliferation of both untreated and PRL-treated cells. Our results indicate that autocrine secretion of PRL stimulates tyrosine phosphorylation of ErbB-2 by Jak2, provides docking sites for Grb2 and stimulates Ras-MAP kinase cascade, thereby causing unrestricted cellular proliferation. The identification of this novel cross-talk between ErbB-2 and the autocrine growth stimulatory loop for PRL may provide new targets for therapeutic and preventive intervention of human breast cancer.     

Activation of Rho signaling contributes to lysophosphatidic acid-induced contraction of intact ileal smooth muscle of guinea-pig

To elucidate the possible role of Rho A/Rho-kinase on lysophosphatidic acid (LPA)-induced contraction in intact guinea-pig ileal smooth muscle, we examined effects of pretreatment with a specific inhibitor of Rho-kinase (Y-27632) on the LPA-induced contraction and MLC20 phosphorylation. In addition, we investigated whether LPA actually elicits an activation of Rho A by studying subcellular distribution of Rho A in unstimulated and stimulated smooth muscles by LPA. LPA induced a less intense, but sustained, contraction compared with ACh, and was accompanied by significant increases in MLC20 phosphorylation. The effects of LPA on tension and MLC20 phosphorylation were inhibited by Y-27632. The ACh-induced contraction, but not increases in MLC20 phosphorylation, was partially inhibited by Y-27632. High K+-induced contraction was unaffected by the inhibitor. LPA stimulated translocation of Rho A from the cytosol to the membrane fraction of the muscle. Translocation of Rho A was also induced by ACh and high K+. These results suggest that LPA-induced contraction of intact ileal smooth muscle is dominated through activation of Rho A and Rho-kinase and subsequent increases in MLC20 phosphorylation.     

Pharmacological assessment of methamphetamine-induced behavioral hyperactivity mediated by dopaminergic transmission in planarian Dugesia japonica

The freshwater planarian Dugesia japonica has a simple central nervous system (CNS) and can regenerate complete organs, even a functional brain. Recent studies demonstrated that there is a great variety of neuronal-related genes, specifically expressed in several domains of the planarian brain. We identified a planarian dat gene, named it D. japonica dopamine transporter (Djdat), and analyzed its expression and function. Both in situ hybridization and immunofluorescence revealed that localization of Djdat mRNA and protein was the same as that of D. japonica tyrosine hydroxylase (DjTH). Although, dopamine (DA) content in Djdat(RNAi) planarians was not altered, Djdat(RNAi) planarians showed increased spontaneous locomotion. The hyperactivity in the Djdat(RNAi) planarians was significantly suppressed by SCH23390 or sulpiride pretreatment, which are D₁ or D₂ receptor antagonists, respectively. These results suggest that planarians have a Djdat ortholog and the ability to regulate dopaminergic neurotransmission and association with spontaneous locomotion.     

Fourteen-week feeding test of meat and milk derived from cloned cattle in the rat

Agricultural application of cloned livestock produced by nuclear transfer requires public and governmental understanding of food-safety issues. To determine whether physiological effects occurred in animals fed products derived from cloned cattle, we conducted long-term (14 week) trials feeding Crj:CD(SD)IGS rats meat and milk from cloned cattle. Diets containing meat and milk were equal in nutritional value to the basal diet (AIN93G). Urinalysis was performed at Weeks 4, 8 and 12; at the end of the feeding period, blood sampling and autopsies were conducted. During the feeding periods, there were no significant differences in general condition, death loss, growth, battery of functional observational tests and estrous cycles among groups given diets containing meat and milk powder from non-clone, embryonic clone and somatic clone cattle. Furthermore, no significant changes attributed to consumption of clone meat or milk were detected in urinalysis, hematological and blood chemical, gross pathological or histological examinations. Therefore, we concluded that the physiologic conditions of the rats were not affected by consumption of meat and milk from bovine clones.     

Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein engineered NADP+-dependent xylitol dehydrogenase

Effects of reversal coenzyme specificity toward NADP+ and thermostabilization of xylitol dehydrogenase (XDH) from Pichia stipitis on fermentation of xylose to ethanol were estimated using a recombinant Saccharomyces cerevisiae expressing together with a native xylose reductase from P. stipitis. The mutated XDHs performed the similar enzyme properties in S. cerevisiae cells, compared with those in vitro. The significant enhancement(s) was found in Y-ARSdR strain, in which NADP+-dependent XDH was expressed; 86% decrease of unfavorable xylitol excretion with 41% increased ethanol production, when compared with the reference strain expressing the wild-type XDH.