6,7-Dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidines and their derivatives as novel corticotropin-releasing factor 1 receptor antagonists

To identify an orally active corticotropin-releasing factor 1 receptor antagonist, a series of 6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidines and their derivatives were designed, synthesized and evaluated. An in vitro study followed by in vivo and pharmacokinetic studies of these heterotricyclic compounds led us to the discovery of an orally active CRF1 receptor antagonist. The results of a structure-activity relationship study are presented.     

Endogenous synthesis of corticosteroids in the hippocampus

Background

Brain synthesis of steroids including sex-steroids is attracting much attention. The endogenous synthesis of corticosteroids in the hippocampus, however, has been doubted because of the inability to detect deoxycorticosterone (DOC) synthase, cytochrome P450(c21).

Methodology/Principal Findings

The expression of P450(c21) was demonstrated using mRNA analysis and immmunogold electron microscopic analysis in the adult male rat hippocampus. DOC production from progesterone (PROG) was demonstrated by metabolism analysis of ³H-steroids. All the enzymes required for corticosteroid synthesis including P450(c21), P450(2D4), P450(11β1) and 3β-hydroxysteroid dehydrogenase (3β-HSD) were localized in the hippocampal principal neurons as shown via in situ hybridization and immunoelectron microscopic analysis. Accurate corticosteroid concentrations in rat hippocampus were determined by liquid chromatography-tandem mass spectrometry. In adrenalectomized rats, net hippocampus-synthesized corticosterone (CORT) and DOC were determined to 6.9 and 5.8 nM, respectively. Enhanced spinogenesis was observed in the hippocampus following application of low nanomolar (10 nM) doses of CORT for 1 h.

Conclusions/Significance

These results imply the complete pathway of corticosteroid synthesis of ‘pregnenolone →PROG→DOC→CORT’ in the hippocampal neurons. Both P450(c21) and P450(2D4) can catalyze conversion of PROG to DOC. The low nanomolar level of CORT synthesized in hippocampal neurons may play a role in modulation of synaptic plasticity, in contrast to the stress effects by micromolar CORT from adrenal glands.     

Evidence for mesenchymal-epithelial transition associated with mouse hepatic stem cell differentiation

Mesenchymal-epithelial transition events are related to embryonic development, tissue construction, and wound healing. Stem cells are involved in all of these processes, at least in part. However, the direct evidence of mesenchymal-epithelial transition associated with stem cells is unclear. To determine whether mesenchymal-epithelial transition occurs in liver development and/or the differentiation process of hepatic stem cells in vitro, we analyzed a variety of murine liver tissues from embryonic day 11.5 to adults and the colonies derived from hepatic stem/progenitor cells isolated with flow cytometry. The results of gene expression, immunohistochemistry and Western blot showed that as liver develops, the expression of epithelial markers such as Cytokeratin18 and E-cadherin increase, while expression of mesenchymal markers such as vimentin and N-cadherin decreased. On the other hand, in freshly isolated hepatic stem cells, the majority of cells (65.0%) co-express epithelial and mesenchymal markers; this proportion is significantly higher than observed in hematopoietic cells, non-hematopoietic cells and non-stem cell fractions. Likewise, in stem cell-derived colonies cultured over time, upregulation of epithelial genes (Cytokeratin-18 and E-cadherin) occurred simultaneously with downregulation of mesenchymal genes (vimentin and Snail1). Furthermore, in the fetal liver, vimentin-positive cells in the non-hematopoietic fraction had distinct proliferative activity and expressed early the hepatic lineage marker alpha-fetoprotein. CONCLUSION: Hepatic stem cells co-express mesenchymal and epithelial markers; the mesenchymal-epithelial transition occurred in both liver development and differentiation of hepatic stem/progenitor cells in vitro. Besides as a mesenchymal marker, vimentin is a novel indicator for cell proliferative activity and undifferentiated status in liver cells.     

Maturation of vomeronasal receptor neurons in vitro by coculture with accessory olfactory bulb neurons

To analyze the mechanisms of perception and processing of pheromonal signals in vitro, we previously developed a new culture system for vomeronasal receptor neurons (VRNs), referred to as the vomeronasal pocket (VN pocket). However, very few VRNs were found to express the olfactory marker protein (OMP) and to have protruding microvilli in VN pockets, indicating that these VRNs are immature and that VN pockets are not appropriate for pheromonal recognition. To induce VRN maturation in VN pockets, we here attempted to coculture VN pockets with a VRN target-accessory olfactory bulb (AOB) neurons. At 3 weeks of coculture with AOB neurons, the number of OMP-immunopositive VRNs increased. By electron microscopy, the development of microvilli in VRNs was found to occur coincidentally with OMP expression in vitro. These results indicate that VRN maturation is induced by coculture with AOB neurons. The OMP expression of VRNs was induced not only by AOB neurons but also by neurons of other parts of the central nervous system (CNS). Thus, VRN maturation requires only CNS neurons. Since the maturation of VRNs was not induced in one-well separate cultures, the nonspecific induction of OMP expression by CNS neurons suggests the involvement of a direct contact effect with CNS in VRN maturation.     

Peptides binding to a Gb3 mimic selected from a phage library

Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides.     

Cleavage and survival of Xenopus embryos exposed to 8 T static magnetic fields in a rotating clinostat

In this study, we examined cleavage and survival of fertilized Xenopus embryos exposed to 8 T static magnetic fields (SMFs). We investigated fertilized Xenopus embryos exposed to magnetic field either in static chamber or in a rotating culture system. Our results showed that the exposure to the strong magnetic field of 8 T changed the third cleavage furrow from the usual horizontal one to a perpendicular one; however, when the direction of gravity was randomized by exposing embryos to magnetic field in a rotating culture system, the third cleavage furrow were formed horizontally, a finding which suggests that the observed distortion of the third cleavage furrow in magnetism-exposed embryos was accomplished by altering gravity effects which were elicited by diamagnetic force due to high gradient magnetic field. Our results also showed that the exposure to the strong magnetic field did not damage survival. These results demonstrate that SMF and altering gravity cause distortion of the third cleavage furrow and show that effects of exposing cleavage embryos to magnetic field were transient and did not affect the post-cleavage development. We also showed that strong magnetic field is not hazardous to the cleavage and blastula-gastrula transition of developing embryonic cells.     

Stimulation effect of Dnmt3L on the DNA methylation activity of Dnmt3a2

Quantification of DNA methyltransferases Dnmt3a and Dnmt3a2, and Dnmt3L in isolated male gonocytes in day 16.5 embryos confirmed that not Dnmt3a but Dnmt3a2 and Dnmt3L were the major Dnmt3s. The expression level of Dnmt3L constituted 5- to 10-fold molar excess compared to that of Dnmt3a2. The stimulation property of the DNA methylation activity of Dnmt3a2 with Dnmt3L towards substrate DNA in naked or nucleosomes was similar to that of Dnmt3a. However, the DNA methylation activity of not Dnmt3a but Dnmt3a2 was severely inhibited at the physiological salt concentration. Interestingly, the activity of Dnmt3a2 was significantly detected in the presence of Dnmt3L even at the physiological salt concentration. This indicates that Dnmt3a2 functions only in the presence of Dnmt3L in male gonocytes, and may explain why Dnmt3L is required specifically in mouse gonocytes for DNA methylation.