Temporal regulation of Cre recombinase activity in neural stem cells

Neural stem cells are known to give rise to distinct subtypes of neurons and glial cells over time by changing their competency. However, precise characterization of neural stem cells at various developmental stages remains to be performed. For such analysis, a tool to manipulate neural stem cells at different time points is necessary. Here, we generated transgenic mice that express Cre-ER(T2) in the ventricular zone of the developing nervous system under the control of the nestin promoter and enhancer (Nes-CreER(T2)). In mice expressing Cre-ER(T2) at appropriate levels, Cre recombinase activity was mostly inactive but efficiently activated by tamoxifen within 1 day. When such mice were crossed with the ROSA-26 or Z/EG reporter mice, neural stem cells were permanently labeled after administration of tamoxifen. Thus, Nes-CreER(T2) mice offer a powerful tool to manipulate neural stem cells genetically at desired time points.     

The effect of serotonin agonist 1-(trifluoromethylphenyl)-piperazine on corticotropin releasing factor and arginine vasopressin in rat hypothalamic nuclei

Effects of 1-(m-trifluoromethylphenyl)-piperazine, a serotonin agonist, were examined on rat plasma levels of adrenocorticotropin (ACTH) and arginine vasopressin (AVP), and on hypothalamic contents of corticotropin releasing factor (CRF) and AVP, to investigate the role of brain serotonin in ACTH regulation. Both plasma ACTH and AVP levels increased markedly 30 min after injection of the compound and were still elevated at 80 min. CRF and AVP contents in the median eminence decreased 30 min after injection but returned to the basal levels by 80 min. The AVP content in the supraoptic nucleus was elevated 80 min after injection. The CRF and aVP content did not significantly change in the paraventricular, suprachiasmatic and arcuate nuclei. Serotonin or 1-(m-trifluoromethylphenyl)-piperazine did not stimulate the release of ACTH in pituitary cell cultures. These results suggest that both CRF and AVP were secreted into the portal vessels by 1-(m-trifluoromethylphenyl)-piperazine to release ACTH from the anterior pituitary and that both the ACTH and AVP release were stimulated via the brain serotonergic mechanism.     

Chromosomal diagnosis in each individual blastomere of 5- to 10-cell bovine embryos derived from in vitro fertilization

Chromosomal normality and sex were diagnosed in each blastomere of bovine embryos derived from in vitro fertilization (IVF). Bovine embryos developing to the 5- to 10-cell stage were separated into individual blastomeres with 0.5% protease. After treatment with 100 ng/mL vinblastine sulfate for 8 to 10 h, they were prepared for chromosome samples. In total, 33 bovine embryos and 185 blastomeres were examined. Chromosomal normality was analyzed in 43.8% (81/185) of the blastomeres and 60.6% (20/33) of the embryos; while chromosomal anomalies were found in 16 (80%, 16/20) of the embryos, 5 haploid embryos and 11 mosaic (n/2n) embryos. Mosaicism characteristic of the opposite sex in X-and Y-chromosomes was found in 2 haploid embryos, and that of a Y-chromosome and of XX chromosomes in 1 n/2n embryo. Various sex-chromosome compositions were also observed in the other 10 chromosomal mosaic n/2n embryos.     

Growth hormone-releasing peptide-2 stimulates secretion and synthesis of adrenocorticotropic hormone in mouse pituitary

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides which induce strong GH release in both animals and humans. Among them, GHRP-2 is known to stimulate GH release by acting at both hypothalamic and pituitary sites, but also induces adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRP-2 may stimulate ACTH release directly via GHRP receptor type 1a in ACTH-producing tumors. GHRP-2 increases ACTH secretion in rat in vivo, but not ACTH release from rat primary pituitary cells. In the present study, in order to elucidate the mechanism underlying ACTH secretion by GHRPs, mouse pituitary cells were stimulated by GHRP-2. GHRP receptor mRNA was expressed in the mouse pituitary, and GHRP-2 directly stimulated secretion and synthesis of ACTH in the mouse anterior pituitary cells. GHRP-2 increased intracellular cyclic AMP production. H89, a potent protein kinase A (PKA) inhibitor, and bisindolylmaleimide I, a selective protein kinase C (PKC) inhibitor, inhibited the GHRP-2-induced ACTH release, and that H89, but not bisindolylmaleimide I, inhibited the GHRP-2-induced proopiomelanocortin mRNA levels. Together, the GHRP-2-induced ACTH release was regulated via both PKA and PKC pathways in the mouse pituitary cells, while ACTH was synthesized by GHRP-2 only via the PKA pathway.     

Development-dependent expression of isozymogens of monkey pepsinogens and structural differences between them

The developmental changes in the expression of monkey pepsinogens and structural differences between the polypeptides were investigated. Monkey pepsinogens included five different components, namely, pepsinogens A-(1-4) and progastricsin. Their respective relative levels and specific activities changed significantly during development. The sequential expression of genes for type-A pepsinogens was particularly noteworthy. Pepsinogen A-3 was the major zymogen at the newborn stage, accounting for nearly half of the total pepsinogens at this stage. Pepsinogen A-2 became predominant at the 4-month stage, and pepsinogen A-1 predominated at the juvenile and adult stages. Enzymatic properties of pepsinogens A-1, A-2 and A-3 were similar but not identical to those of pepsinogen A-4 and progastricsin, in particular with respect to the activation processes. Each pepsin digested various protein substrates but some differences in specificity were evident. cDNA clones for five pepsinogens were isolated, and the nucleotide sequences were determined. Each cDNA contained leader, pro, and pepsin regions that encoded 15, 47, and 326 amino acid residues, respectively, with the exception of the cDNA for progastricsin in which the pro and pepsin regions encoded 43 and 329 amino acid residues, respectively. Type-A pepsinogens exhibited a high degree of similarity, with over 96% of bases in the nucleotide sequences of the protein-coding regions being identical. Northern analysis revealed that the level of expression of genes for type-A pepsinogens and for progastricsin was significant at the fetal stage and increased with development.     

PPARgamma ligands suppress proliferation of human urothelial basal cells in vitro

Expression of peroxisome proliferator-activated receptor (PPAR) gamma in the human urinary tract through embryonic development suggests its possible roles in the development, proliferation, and differentiation of uroepithelium. Little is known, however, about physiological roles of PPARgamma in the urinary tract. We investigated effects of PPARgamma ligands on the proliferation of normal human urothelial cells and stromal cells cultivated from surgical specimens. Active proliferation in vitro as well as high molecular weight cytokeratin expression indicated that cultured urothelial cells possess basal cell phenotype. PPARgamma protein, expressed predominantly in the epithelial layer of the normal human urinary tract in vivo, was abundantly expressed in urothelial cells but barely detectable in stromal cells in vitro. Natural ligand for PPARgamma, 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), as well as synthetic ones, troglitazone and pioglitazone, suppressed proliferation of the urothelial cells dose-dependently. These effects were PPARgamma specific because clofibrate or PGF(2alpha) did not affect proliferation of urothelial cells. Neither 9-cis retinoic acid or all-trans retinoic acid (ATRA) at 1 microM showed any synergism on the antiproliferative effects of PPARgamma ligands. Urothelial cells treated with PPARgamma ligands showed drastic morphologic changes and cell cycle arrest at G0/G1 phase accompanied with increased mRNA level of a cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Since 15d-PGJ(2) is present in vivo during the resolution phase of inflammation, these results indicated that PPARgamma might be involved in the terminal phase of urothelial re-epithelialization processes.