Modulation of Gene Expression by Human Cytosolic tRNase ZL through 5′-Half-tRNA

A long form (tRNase Z^L) of tRNA 3′ processing endoribonuclease (tRNase Z, or 3′ tRNase) can cleave any target RNA at any desired site under the direction of artificial small guide RNA (sgRNA) that mimics a 5′-half portion of tRNA. Based on this enzymatic property, a gene silencing technology has been developed, in which a specific mRNA level can be downregulated by introducing into cells a synthetic 5′-half-tRNA that is designed to form a pre-tRNA-like complex with a part of the mRNA. Recently 5′-half-tRNA fragments have been reported to exist stably in various types of cells, although little is know about their physiological roles. We were curious to know if endogenous 5′-half-tRNA works as sgRNA for tRNase Z^L in the cells. Here we show that human cytosolic tRNase Z^L modulates gene expression through 5′-half-tRNA. We found that 5′-half-tRNA^Glu, which co-immunoprecipitates with tRNase Z^L, exists predominantly in the cytoplasm, functions as sgRNA in vitro, and downregulates the level of a luciferase mRNA containing its target sequence in human kidney 293 cells. We also demonstrated that the PPM1F mRNA is one of the genuine targets of tRNase Z^L guided by 5′-half-tRNA^Glu. Furthermore, the DNA microarray data suggested that tRNase Z^L is likely to be involved in the p53 signaling pathway and apoptosis.     

Repression of Nascent Strand Elongation by Deregulated Cdt1 during DNA Replication in Xenopus Egg Extracts

Excess Cdt1 reportedly induces rereplication of chromatin in cultured cells and Xenopus egg extracts, suggesting that the regulation of Cdt1 activity by cell cycle-dependent proteolysis and expression of the Cdt1 inhibitor geminin is crucial for the inhibition of chromosomal overreplication between S phase and metaphase. We analyzed the consequences of excess Cdt1 for DNA replication and found that increased Cdt1 activity inhibited the elongation of nascent strands in Xenopus egg extracts. In Cdt1-supplemented extracts, overreplication was remarkably induced by the further addition of the Cdt1-binding domain of geminin (Gem79-130), which lacks licensing inhibitor activity. Further analyses indicated that fully active geminin, as well as Gem79-130, restored nascent strand elongation in Cdt1-supplemented extracts even after the Cdt1-induced stalling of replication fork elongation had been established. Our results demonstrate an unforeseen, negative role for Cdt1 in elongation and suggest that its function in the control of replication should be redefined. We propose a novel surveillance mechanism in which Cdt1 blocks nascent chain elongation after detecting illegitimate activation of the licensing system.     

Allelopathic effects of the dinophyte Prorocentrum minimum on the growth of the bacillariophyte Skeletonema costatum

We investigated growth interactions between the dinophyte Prorocentrum minimum and the bacillariophyte Skeletonema costatum using bi-algal cultures under axenic conditions. When low cell densities of P. minimum and high cell densities of S. costatum were inoculated into the same medium, growth of P. minimum was suppressed. Other inoculum combinations resulted in reduced S. costatum maximum cell densities. A mathematical model was used to simulate growth and interactions of P. minimum and S. costatum in bi-algal cultures. The model indicated that P. minimum always outcompeted S. costatum over time. Enriched filtrate from low-density P. minimum cultures significantly stimulated S. costatum growth, but enriched filtrate from high-density P. minimum cultures notably inhibited the growth of S. costatum. Growth of P. minimum was not affected by enriched filtrate from cultures of P. minimum at any density. Filtrates of P. minimum cultures were fractionated by ultrafiltration (molecular weight cutoff >3000 Da), and retentate that included polysaccharide(s) significantly inhibited the growth of S. costatum.     

Phosphoproteomic analysis of distinct tumor cell lines in response to nocodazole treatment

Here, we report for the first time a comparative phosphoproteomic analysis of distinct tumor cell lines in the presence or absence of the microtubule‐interfering agent nocodazole. In total, 1525 phosphorylation sites assigned to 726 phosphoproteins were identified using LC‐MS‐based technology following phosphopeptide enrichment. Analysis of the amino acid composition surrounding the identified in vivo phosphorylation sites revealed that they could be classified into two motif groups: pSer‐Pro and pSer‐Asp/Glu. Phosphoproteomic change resulting from nocodazole treatment varied among cell lines in terms of the numbers of total phosphopeptides identified, motif groups, and functional annotation groups; however, the cell lines were equally sensitive to nocodazole. The identified phosphoproteome subset contained major signaling proteins and proteins known to be involved in mitosis, but did not always exhibit the same changes in the tumor cells from nocodazole treatment. In spite of the complex changes observed in the phosphorylation of many of the proteins, possible common features induced by nocodazole were found, including phosphorylation of nucleophosmin (NPM) S254 and coatomer protein complex, subunit α (COPA) S173, suggesting that the events are not cell‐type specific but events generally occurring in mitosis or induced by a microtubule‐interfering agent. Further, temporal analysis of phosphoproteome change revealed that phosphorylation of NPM S254 and COPA S173 was observed from the early (6 h) and late (24 h) time point after nocodazole treatment, respectively, suggesting that NPM S254 may be involved in the induction of M‐phase arrest by nocodazole, whereas COPA S173 may be caused as a result of M‐phase arrest.     

Phenylethanoid oligoglycosides and acylated oligosugars with vasorelaxant activity from Cistanche tubulosa

The methanolic extract from the dried stems of Cistanche tubulosa (Schrenk) R. Wight was found to show an inhibitory effect on contractions induced by noradrenaline in isolated rat aortic strips. From the extract, new phenylethanoid oligoglycoside constituents, kankanosides F and G, and an acylated oligosugar, kankanose, were isolated together with 14 known compounds. The structures of these new compounds were determined on the basis of their chemical and physicochemical evidence. In addition, principal constituents, kankanoside F, kankanose, echinacoside, acteoside, and cistanoside F, showed vasorelaxant activity, and several structural requirements for the activity were clarified.     

Inhibitory effects of coumarin and acetylene constituents from the roots of Angelica furcijuga on D-galactosamine/lipopolysaccharide-induced liver injury in mice and on nitric oxide production in lipopolysaccharide-activated mouse peritoneal macrophages

The methanolic extract (200 mg/kg, p.o. and i.p.), principal coumarin constituents (isoepoxypteryxin, anomalin, and praeroside IV), and a polyacetylene constituent (falcarindiol) (25 mg/kg, i.p.) from the roots of Angelica furcijuga protected the liver injury induced by D-galactosamine (D-GalN)/lipopolysaccharide (LPS) in mice. In in vitro experiments, coumarin constituents (hyuganins A-D, anomalin, pteryxin, isopteryxin, and suksdorfin) and polyacetylene constituents [(-)-falcarinol and falcarindiol] substantially inhibited LPS-induced NO and/or TNF-alpha production in mouse peritoneal macrophages, and isoepoxypteryxin inhibited D-GalN-induced cytotoxicity in primary cultured rat hepatocytes. Furthermore, hyuganin A, anomalin, and isopteryxin inhibited the decrease in cell viability by TNF-alpha in L929 cells.     

Long‐term infection of adult mice with murine polyomavirus following stereotaxic inoculation into the brain

Murine polyomavirus is used in various models of persistent virus infection. This study was undertaken to assess the spatial and temporal patterns of MPyV infection in the brains of immunocompetent (BALB/c) and immunocompromised (KSN nude) mice. MPyV was stereotaxically microinfused into the brain parenchyma, and the kinetics of infection were examined by quantitative PCR. In BALB/c mice, the amount of viral DNA in the brain peaked at 4 days p.i. and then rapidly diminished. In contrast, MPyV DNA levels increased up to 4 days and then gradually decreased over the 30‐day observation period in the brain of KSN mice. In both mouse strains, viral DNA was readily detected around the sites of inoculation from 2 to 6 days p.i., and continued to be detected for up to 30 days p.i. In addition, MPyV infection did not lead to a drastic induction of innate immune response in the brains, nor did MPyV‐inoculated mice show any signs of disease. These results indicate that MPyV establishes an asymptomatic long‐term infection in the mouse brain.