Construction of a Modified Hairpin Ribozyme for Investigations of the Structure-Function Relationship

Abstract

We have constructed a new three domain hairpin ribozyme, in which domain II is connected with domain I' by a helix and a nucleotidic linker to enhance the active bent conformation. The insertion of a nucleotide linker into domain I' increased the cleavage activity more than into domain II. Furthermore, Rev responsive elements (RRE) were introduced in the helix connecting domains. The cleavage activities of the hairpin ribozyme containing the RRE were not affected by the presence of the Rev.     

Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.     

Evaluation of mental stress by physiological indices derived from finger plethysmography

Background

Quantitative evaluation of mental stress is important to prevent stress-related disorders. Finger plethysmography (FPG) is a simple noninvasive method to monitor peripheral circulation, and provides many physiological indices. Our purpose is to investigate how FPG-derived indices reflect on mental stress, and to clarify any association between these physiological indices and subjective indices of mental stress.

Methods

Thirty-one healthy women (mean age, 22 years ± 2) participated. The participants rested by sitting on a chair for 10 min. They then performed a computerized version of the Stroop color-word conflict test (CWT) for 10 min. Finally, they rested for 10 min. FPG was recorded throughout the experiment. The participants completed a brief form of the Profile of Mood States (POMS) questionnaire before and after the test. Using the FPG data, we conducted chaos analysis and fast Fourier transform analysis, and calculated chaotic attractors, the largest Lyapunov exponent, a high-frequency (HF) component, a low-to-high-frequency (LF/HF) ratio, finger pulse rate and finger pulse wave amplitude.

Results

The HF component decreased and the LF/HF ratio increased significantly during the test (P < 0.01), while the confusion subscale of POMS increased after the test (P < 0.05). During testing, finger pulse rate significantly increased (P < 0.001), and the finger pulse wave amplitude decreased (P < 0.001). The attractor size reduced during testing and returned to a baseline level afterwards. Although the largest Lyapunov exponent showed no significant change during testing, significant negative correlation with the tension-anxiety subscale of POMS was observed at the beginning (P < 0.01). A significant negative correlation between the LF/HF ratio and two subscales was also observed in the beginning and middle of the test (P < 0.05). There were no correlations during the rest periods.

Conclusions

The physiological indices derived from FPG were changed by mental stress. Our findings indicate that FPG is one of the easiest methods to evaluate mental stress quantitatively. In particular, the largest Lyapunov exponent and the LF/HF ratio might be associated with acute mental stress. Farther examination is needed to find any association between the physiological indices and various types of mental stress.     

Myosin phosphatase-targeting subunit 1 controls chromatid segregation

Myosin phosphatase is a heterotrimeric holoenzyme consisting of myosin phosphatase-targeting subunit 1 (MYPT1), a catalytic subunit of PP1Cβ, and a 20-kDa subunit of an unknown function. We have previously reported that myosin phosphatase also controls mitosis, apparently by antagonizing polo-like kinase 1 (PLK1). Here we found that depletion of MYPT1 by siRNA led to precocious chromatid segregation when HeLa cells were arrested at metaphase by a proteasome inhibitor, MG132, or by Cdc20 depletion. Consistently, cyclin B1 and securin were not degraded, indicating that the chromatid segregation is independent of the anaphase-promoting complex/cyclosome. Precocious segregation induced by MYPT1 depletion requires PLK1 activity because a PLK1 inhibitor, BI-2536, blocked precocious segregation. Furthermore, the expression of an unphosphorylatable mutant of SA2 (SCC3 homologue 2), a subunit of the cohesin complex, prevented precocious chromatid segregation induced by MYPT1 depletion. It has been shown that SA2 at centromeres is protected from phosphorylation by PP2A phosphatase recruited by Shugoshin (Sgo1), whereas SA2 along chromosome arms is phosphorylated by PLK1, leading to SA2 dissociation at chromosome arms. Taken together, our results suggest that hyperactivation of PLK1 caused by MYPT1 reduction could override the counteracting PP2A phosphatase, resulting in precocious chromatid segregation. We propose that SA2 at the centromeres is protected by two phosphatases. One is PP2A directly dephosphorylating SA2, and the other is myosin phosphatase counteracting PLK1.     

E6AP ubiquitin ligase mediates ubiquitylation and degradation of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.     

A genome-wide gain-of-function analysis of rice genes using the FOX-hunting system

The latest report has estimated the number of rice genes to be approximately 32,000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13,980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12,000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene,confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.     

Genetic diversity of Microcystis cyanophages in two different freshwater environments

Bacteriophages rapidly diversify their genes through co-evolution with their hosts. We hypothesize that gene diversification of phages leads to locality in phages genome. To test this hypothesis, we investigated the genetic diversity and composition of Microcystis cyanophages using 104 sequences of Ma-LMM01-type cyanophages from two geographically distant sampling sites. The intergenetic region between the ribonucleotide reductase genes nrdA and nrdB was used as the genetic marker. This region contains the host-derived auxiliary metabolic genes nblA, an unknown function gene g04, and RNA ligase gene g03. The sequences obtained were conserved in the Ma-LMM01 gene order and contents. Although the genetic diversity of the sequences was high, it varied by gene. The genetic diversity of nblA was the lowest, suggesting that nblA is a highly significant gene that does not allow mutation. In contrast, g03 sequences had many point mutations. RNA ligase is involved in the counter-host’s phage defense mechanism, suggesting that phage defense also plays an important role for rapid gene diversification. The maximum parsimony network and phylogenic analysis showed the sequences from the two sampling sites were distinct. These findings suggest Ma-LMM01-type phages rapidly diversify their genomes through co-evolution with hosts in each location and eventually provided locality of their genomes.     

Systemic Preconditioning by a Prolyl Hydroxylase Inhibitor Promotes Prevention of Skin Flap Necrosis via HIF-1-Induced Bone Marrow-Derived Cells

Background

Local skin flaps often present with flap necrosis caused by critical disruption of the blood supply. Although animal studies demonstrate enhanced angiogenesis in ischemic tissue, no strategy for clinical application of this phenomenon has yet been defined. Hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in ischemic vascular responses, and its expression is induced by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). We assessed whether preoperative stabilization of HIF-1 by systemic introduction of DMOG improves skin flap survival.

Methods and Results

Mice with ischemic skin flaps on the dorsum were treated intraperitoneally with DMOG 48 hr prior to surgery. The surviving area with neovascularization of the ischemic flaps was significantly greater in the DMOG-treated mice. Significantly fewer apoptotic cells were present in the ischemic flaps of DMOG-treated mice. Interestingly, marked increases in circulating endothelial progenitor cells (EPCs) and bone marrow proliferative progenitor cells were observed within 48 hr after DMOG treatment. Furthermore, heterozygous HIF-1α-deficient mice exhibited smaller surviving flap areas, fewer circulating EPCs, and larger numbers of apoptotic cells than did wild-type mice, while DMOG pretreatment of the mutant mice completely restored these parameters. Finally, reconstitution of wild-type mice with the heterozygous deficient bone marrow cells significantly decreased skin flap survival.

Conclusion

We demonstrated that transient activation of the HIF signaling pathway by a single systemic DMOG treatment upregulates not only anti-apoptotic pathways but also enhances neovascularization with concomitant increase in the numbers of bone marrow-derived progenitor cells.     

Jellyfish mucin may have potential disease-modifying effects on osteoarthritis

Background  

We aimed to study the effects of intra-articular injection of jellyfish mucin (qniumucin) on articular cartilage degeneration in a model of osteoarthritis (OA) created in rabbit knees by resection of the anterior cruciate ligament. Qniumucin was extracted from Aurelia aurita (moon jellyfish) and Stomolophus nomurai (Nomura's jellyfish) and purified by ion exchange chromatography. The OA model used 36 knees in 18 Japanese white rabbits. Purified qniumucin extracts from S. nomurai or A. aurita were used at 1 mg/ml. Rabbits were divided into four groups: a control (C) group injected with saline; a hyaluronic acid (HA)-only group (H group); two qniumucin-only groups (M groups); and two qniumucin + HA groups (MH groups). One milligram of each solution was injected intra-articularly once a week for 5 consecutive weeks, starting from 4 weeks after surgery. Ten weeks after surgery, the articular cartilage was evaluated macroscopically and histologically.     

Palynological record of climate change during the last deglaciation from the Song Hong (Red River) delta, Vietnam

The palynological sequence from core ND-1 on the Song Hong delta in the northern Vietnam reveals the climate change during the last deglaciation. The identified pollen in the core is dominated by Castanopsis (Lithocarpus), Elaeocarpus, Ficus, Piperaceae and Quercus. High percentages of temperate taxa including the conifers Dacrydium, Podocarpus, Pinus, Cupressaceae, Txodiaceae and Cryptomeria, and broad-leaved taxa of Carpinus, Alnus, Juglans, Carya, Ulmus, Fagus, Ilex, Castanea, Quercus, which have a mainly upland distribution, is possibly associated with a lowering of the montane vegetation boundary. On the other hand, intensified river influence can also result in an increase in the temperate allochthonous taxa. After analyzing the magnitude of river influence on pollen assemblages during the sedimentary environment evolution, we extracted the climate information and used this to reconstruct climate change during the last deglaciation. A cooler climate during 14.5-10.9 cal. kyr BP than at present is indicated by higher percentage of temperate types than at present under a similar river influence on the floodplain. The Younger Dryas cooling event is recognized by widely developed grasslands between 12.9 and 11.6 cal. kyr BP, adding to the evidences for this period from both terrestrial and marine records in regions influenced by the East Asian monsoon. Two other cooling periods, 9.4-9.0 cal. kyr BP and 6.5-5.2 cal. kyr BP, are indicated by increases of temperate pollen taxa such as Cryptomeria, Alnus, Quercus, and Castanea.