Self-Replication Reactions Dependent on Tertiary Interaction Motifs in an RNA Ligase Ribozyme

RNA can function both as an informational molecule and as a catalyst in living organisms. This duality is the premise of the RNA world hypothesis. However, one flaw in the hypothesis that RNA was the most essential molecule in primitive life is that no RNA self-replicating system has been found in nature. To verify whether RNA has the potential for self-replication, we constructed a new RNA self-assembling ribozyme that could have conducted an evolvable RNA self-replication reaction. The artificially designed, in vitro selected ligase ribozyme was employed as a prototype for a self-assembling ribozyme. The ribozyme is composed of two RNA fragments (form R1·Z1) that recognize another R1·Z1 molecule as their substrate and perform the high turnover ligation reaction via two RNA tertiary interaction motifs. Furthermore, the substrate recognition of R1·Z1 is tolerant of mutations, generating diversity in the corresponding RNA self-replicating network. Thus, we propose that our system implies the significance of RNA tertiary motifs in the early RNA molecular evolution of the RNA world.     

Vildagliptin Stimulates Endothelial Cell Network Formation and Ischemia-induced Revascularization via an Endothelial Nitric-oxide Synthase-dependent Mechanism

Dipeptidyl peptidase-4 inhibitors are known to lower glucose levels and are also beneficial in the management of cardiovascular disease. Here, we investigated whether a dipeptidyl peptidase-4 inhibitor, vildagliptin, modulates endothelial cell network formation and revascularization processes in vitro and in vivo. Treatment with vildagliptin enhanced blood flow recovery and capillary density in the ischemic limbs of wild-type mice, with accompanying increases in phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS). In contrast to wild-type mice, treatment with vildagliptin did not improve blood flow in ischemic muscles of eNOS-deficient mice. Treatment with vildagliptin increased the levels of glucagon-like peptide-1 (GLP-1) and adiponectin, which have protective effects on the vasculature. Both vildagliptin and GLP-1 increased the differentiation of cultured human umbilical vein endothelial cells (HUVECs) into vascular-like structures, although vildagliptin was less effective than GLP-1. GLP-1 and vildagliptin also stimulated the phosphorylation of Akt and eNOS in HUVECs. Pretreatment with a PI3 kinase or NOS inhibitor blocked the stimulatory effects of both vildagliptin and GLP-1 on HUVEC differentiation. Furthermore, treatment with vildagliptin only partially increased the limb flow of ischemic muscle in adiponectin-deficient mice in vivo. GLP-1, but not vildagliptin, significantly increased adiponectin expression in differentiated 3T3-L1 adipocytes in vitro. These data indicate that vildagliptin promotes endothelial cell function via eNOS signaling, an effect that may be mediated by both GLP-1-dependent and GLP-1-independent mechanisms. The beneficial activity of GLP-1 for revascularization may also be partially mediated by its ability to increase adiponectin production.     

Effect of leukotriene C4 exposure on ciliated cells of the nasal mucosa

To clarify the effects of leukotriene C4 (LTC4) on human ciliated epithelium, ciliary activity of the ethmoid sinus mucosa was measured photoelectrically in tissue culture. At concentrations ranging from 10⁻⁶M to 10⁻⁹M, LTC4 showed minimal effects on the ciliated epithelium during the initial 30 minutes of exposure; thereafter, ciliary inhibition was observed in a concentration- and time-dependent manner. Irrigation of the mucosa with culture medium 15 minutes after exposure prevented the LTC4-induced ciliary inhibition. However, irrigation 60 minutes after exposure failed to inhibit 10⁻⁸M LTC4-induced ciliary dysfunction and mucosal damage. The LTC4-induced ciliary inhibition was blocked in the presence of FPL-55712 and/or Ly-171883, both leukotriene receptor antagonists. L-serine and sodium tetraborate complex (SBC), a γ-glutamyl transpeptidase (γ-GTP) inhibitor, also inhibited the LTC4-induced ciliary inhibition. These findings indicate that LTC4 is converted to LTD4 by γ-GTP during 60 minutes of exposure, and LTC4 itself has minimal direct effects on the ciliated cells.     

Formation of non-spheroidal aggregates by adult rat hepatocytes on polyacrylamide coated surfaces

Summary Adult rat hepatocytes in primary culture formed floating, non-spheroidal aggregates on the surfaces coated with 1 % polyacrylamide. In the cells of aggregates, DNA synthetic capacity was limited, but albumin secreting capacity was maintained at least for 2 weeks. These properties were influenced by addition of serum.     

Next-generation sequencing analysis of lager brewing yeast strains reveals the evolutionary history of interspecies hybridization

The lager beer yeast Saccharomyces pastorianus is considered an allopolyploid hybrid species between S. cerevisiae and S. eubayanus. Many S. pastorianus strains have been isolated and classified into two groups according to geographical origin, but this classification remains controversial. Hybridization analyses and partial PCR-based sequence data have indicated a separate origin of these two groups, whereas a recent intertranslocation analysis suggested a single origin. To clarify the evolutionary history of this species, we analysed 10 S. pastorianus strains and the S. eubayanus type strain as a likely parent by Illumina next-generation sequencing. In addition to assembling the genomes of five of the strains, we obtained information on interchromosomal translocation, ploidy, and single-nucleotide variants (SNVs). Collectively, these results indicated that the two groups of strains share S. cerevisiae haploid chromosomes. We therefore conclude that both groups of S. pastorianus strains share at least one interspecific hybridization event and originated from a common parental species and that differences in ploidy and SNVs between the groups can be explained by chromosomal deletion or loss of heterozygosity.     

Rational design of crystal contact‐free space in protein crystals for analyzing spatial distribution of motions within protein molecules

Contacts with neighboring molecules in protein crystals inevitably restrict the internal motions of intrinsically flexible proteins. The resultant clear electron densities permit model building, as crystallographic snapshot structures. Although these still images are informative, they could provide biased pictures of the protein motions. If the mobile parts are located at a site lacking direct contacts in rationally designed crystals, then the amplitude of the movements can be experimentally analyzed. We propose a fusion protein method, to create crystal contact‐free space (CCFS) in protein crystals and to place the mobile parts in the CCFS. Conventional model building fails when large amplitude motions exist. In this study, the mobile parts appear as smeared electron densities in the CCFS, by suitable processing of the X‐ray diffraction data. We applied the CCFS method to a highly mobile presequence peptide bound to the mitochondrial import receptor, Tom20, and a catalytically relevant flexible segment in the oligosaccharyltransferase, AglB. These two examples demonstrated the general applicability of the CCFS method to the analysis of the spatial distribution of motions within protein molecules.     

Effect of Gonadectomy on the Androgen-Dependent Behavior of Ganglion Cell-Like Cells in Djungarian Hamsters (Phodopus sungorus)

Ganglion cell-like (GL) cells reside in the dermis of the ventral skin of mature male Djungarian hamsters (Phodopus sugorus) and express androgen receptor (AR). To assess whether GL cells have androgen-dependent behavior, we evaluated the histologic changes of GL cells after gonadectomy. Five male and 5 female hamsters were gonadectomized at the age of 4 wk and necropsied 14 wk later. The number, distribution, and proliferative activity of GL cells in the thoracoabdominal and dorsal skins were evaluated histologically and compared with those of corresponding intact animals. GL cells were more numerous, were distributed throughout the skin more widely, and had higher proliferative activity in the intact male hamsters than in their gonadectomized counterparts. Similar trends regarding these 3 parameters were seen in ovariectomized compared with intact female hamsters and between intact male and intact female hamsters. These results suggest that the GL cells of Djungarian hamsters demonstrate sex-associated differences in their distribution and proliferative activity and that androgen may be involved in the development of these cells.Abbreviations: AR, androgen receptor; GL cell, ganglion cell-like cellGanglion cell-like (GL) cells reside in the dermis of the abdominal and thoracic skin of mature male Djungarian hamsters (Phodopus sungorus).^1,7 GL cells have a small round nucleus with an apparent nucleolus and abundant basophilic foamy cytoplasm. These cells usually aggregate to form nests accompanied by various volumes of stromal collagen fibers. The nests increase in size and number with maturation. The nuclei of GL cells have a positive reaction for androgen receptor (AR), and the cytoplasm is positive for vimentin.^1,7 Although the morphologic characteristics and various immunophenotypes of GL cells have been documented, their behavior and role remain almost unclear. Some authors speculate that increased levels of testosterone may influence GL cell proliferation and the oncogenesis of atypical skin fibromas preferentially arising in this species.¹The current study aims to elucidate the androgen-dependent behavior of GL cells and compare the histologic changes of GL cells in gonadectomized Djungarian hamsters with those in intact control animals.