Improvement of a miRNA inhibitor by intracellular selection

ABSTRACT

Sequences surrounding the miRNA binding domain of the miRNA inhibitor LidNA were selected intracellularly. The library was transfected into cells, and then, inhibitors that were associated with argonaute 2 were selected. The potent inhibitors were slowly degraded intracellularly, while the lower-activity inhibitors were rapidly degraded. A combination of the selected sequences surrounding the miRNA binding domain enhanced miRNA inhibitory activity.     

Cyclic AMP and Ca2+ as regulators of zygote formation in the cellular slime mold Dictyostelium mucoroides

To analyze the mechanism of the sexual process (macrocyst formation) in the cellular slime mold Dictyostelium mucoroides-7 (Dm7), the effects of 3',5'-cyclic adenosine monophosphate (cAMP), conditioned medium (CM) factors, and various ions including Ca2+ on zygote formation were examined. The application of cAMP was found to inhibit the sexual cell fusion. In addition, the activity of fusion inhibitor(s) contained in CM was heat stable and lost by phosphodiesterase (PDE)-treatment, thus indicating that cAMP is the inhibitor, being in contrast to ethylene as a fusion activator. Pulse experiments using two cAMP analogues, 2'-deoxy-cAMP and 8-bromo-cAMP suggested that the signal transduction system through the cell surface cAMP receptor is of particular importance for regulation of the sexual fusion process. Among several ions having effects on zygote formation, Ca2+ seemed to be necessary both for the acquisition of fusion competence and for cell fusion itself. In the presence of Ca2+, K+ and Na+ had the opposite effects on zygote formation; K+ was stimulative, while Na+ inhibitory. The significance of these findings is discussed in relation to the regulatory mechanism of zygote formation.     

Structural improvement of LidNA: delta-type LidNA is a potent miRNA inhibitor constructed with unmodified DNA

ABSTRACT

Many miRNA inhibitors have been developed, including chemically modified oligonucleotides, such as 2′-O-methylated RNA and locked nucleic acid (LNA). Unmodified DNA has not yet been reported as a miRNA inhibitor due to relatively low DNA/miRNA binding affinity. We designed a structured DNA, LidNA, which was constructed with unmodified DNA, consisting of a complementary sequence to the target miRNA flanked by two structured DNA regions, such as double-stranded DNA. LidNA inhibited miRNA activity more potently than 2′-O-methylated RNA or LNA. To optimize LidNA, two double-stranded regions were joined, causing the molecule to assume a delta-like shape, which we termed delta-type LidNA. Delta-type LidNAs were developed to target endogenous and exogenous miRNAs, and exhibited potent miRNA inhibitory effects with a duration of at least 10 days. Delta-type LidNA-21, which targeted miR-21, inhibited the growth of cancer cell lines. This newly developed LidNA could contribute to miRNA studies across multiple fields.

Abbreviations: LidNA: DNA that puts a lid on miRNA function; LNA: locked nucleic acid; 3′-UTR: 3′-untranslated regions; RISC: RNA-induced silencing complex; MBL: Molecular beacon-like LidNA; YMBL: Y-type molecular beacon-like LidNA; TDMD: target-directed microRNA degradation.     

A tetracycline repressor-based mammalian two-hybrid system to detect protein-protein interactions in vivo

A mammalian two-hybrid system (termed as trM2H) was developed to detect protein-protein interactions in vivo, based on the reconstitution of the functions the of tetracycline repressor (TetR). The system is sensitive enough to detect protein-protein interactions with K_d up to 55 μM in mammalian cells, and the system can be regulated by small molecules. This system can be used as an efficient genetic selection system to map protein-protein interactions in mammalian cells.     

A role for RAD54B in homologous recombination in human cells.

In human somatic cells, homologous recombination is a rare event. To facilitate the targeted modification of the genome for research and gene therapy applications, efforts should be directed toward understanding the molecular mechanisms of homologous recombination in human cells. Although human genes homologous to members of the RAD52 epistasis group in yeast have been identified, no genes have been demonstrated to play a role in homologous recombination in human cells. Here, we report that RAD54B plays a critical role in targeted integration in human cells. Inactivation of RAD54B in a colon cancer cell line resulted in severe reduction of targeted integration frequency. Sensitivity to DNA-damaging agents and sister-chromatid exchange were not affected in RAD54B-deficient cells. Parts of these phenotypes were similar to those of Saccharomyces cerevisiae tid1/rdh54 mutants, suggesting that RAD54B may be a human homolog of TID1/RDH54. In yeast, TID1/RDH54 acts in the recombinational repair pathway via roles partially overlapping those of RAD54. Our findings provide the first genetic evidence that the mitotic recombination pathway is functionally conserved from yeast to humans.     

Phagocytosis and Exocytosis by Differentiating Dictyostelium discoideum Cells

While vegetative cells of the cellular slime mold Dictyosteliumdiscoideum engage in active phagocytosis, cells isolated frommigrating slugs of this organism were found to be almost lackingin this activity. However, when incubated under sparsely populatedconditions, these cells regained the phagocytic ability as theylost prespore specific antigen and dedifferentiated. Changesin phagocytic activity during the development were quantitativelyexamined. After cessation of feeding, phagocytic activity ofcells first increased but shortly afterward decreased rapidly,together with the initiation of aggregation. The loss of phagocyticability of a cell was accompanied by a change in cell shapefrom an isodiametric to an elongate form, as the cell becameaggregation competent. During such a transition, polystyrenebeads which had been ingested and retained by the cell wereexocytized. ¹ This paper is dedicated to the memory of Prof. Joji Ashida. (Received December 17, 1982; Accepted February 18, 1983)     

Angiogenesis after administration of basic fibroblast growth factor induces proliferation and differentiation of mesenchymal stem cells in elastic perichondrium in an in vivo model: mini review of three sequential republication-abridged reports

To date, studies on mesenchymal tissue stem cells (MSCs) in the perichondrium have focused on in vitro analysis, and the dynamics of cartilage regeneration from the perichondrium in vivo remain largely unknown. We have attempted to apply cell and tissue engineering methodology for ear reconstruction using cultured chondrocytes. We hypothesized that by inducing angiogenesis with basic fibroblast growth factor (bFGF), MSCs or cartilage precursor cells would proliferate and differentiate into cartilage in vivo and that the regenerated cartilage would maintain its morphology over an extended period. As a result of a single administration of bFGF to the perichondrium, cartilage tissue formed and proliferated while maintaining its morphology for at least 3 months. By day 3 post bFGF treatment, inflammatory cells, primarily comprising mononuclear cells, migrated to the perichondrial region, and the proliferation of matrix metalloproteinase 1 positive cells peaked. During week 1, the perichondrium thickened and proliferation of vascular endothelial cells was noted, along with an increase in the number of CD44-positive and CD90-positive cartilage MSCs/progenitor cells. Neocartilage was formed after 2 weeks, and hypertrophied mature cartilage was formed and maintained after 3 months. Proliferation of the perichondrium and cartilage was bFGF concentration-dependent and was inhibited by neutralizing antibodies. Angiogenesis induction by bFGF was blocked by the administration of an angiogenesis inhibitor, preventing perichondrium proliferation and neocartilage formation. These results suggested that angiogenesis may be important for the induction and differentiation of MSCs/cartilage precursor cells in vivo, and that morphological changes, once occurring, are maintained.     

Evaluation of acute corneal damage induced by 222-nm and 254-nm ultraviolet light in Sprague–Dawley rats

Two hundred twenty-two nanometres ultraviolet (UV) light produced by a krypton–chlorine excimer lamp is harmful to bacterial cells but not skin. However, the effects of 222-nm UV light exposure to the eye are not fully known. We evaluated acute corneal damage induced by 222- and 254-nm UV light in albino rats. Under deep anaesthesia, 6-week-old Sprague–Dawley albino rats were exposed to UV light. The exposure levels of corneal radiation were 30, 150, and 600?mJ/cm². Epithelial defects were detected by staining with fluorescein. Superficial punctate keratitis developed in corneas exposed to more than 150?mJ/cm² of UV light, and erosion was observed in corneas exposed to 600?mJ/cm² of UV light. Haematoxylin and eosin staining also showed corneal epithelial defects in eyes exposed to 254-nm UV light. However, no damage developed in corneas exposed to 222-nm UV light. Cyclobutane pyrimidine dimer-positive cells were observed only in normal corneas and those exposed to 254-nm UV light. Although some epithelial cells were stained weakly in normal corneas, squamous epithelial cells were stained moderately, and the epithelial layer that was detached from the cornea exposed to 600?mJ/cm² of light was stained intensely in corneas exposed to 254-nm UV light. In the current study, no corneal damage was induced by 222-nm UV light, which suggested that 222-nm UV light may not harm rat eyes within the energy range and may be useful for sterilising or preventing infection in the future.     

Dietary restriction reduces the incidence of 3-methylcholanthrene-induced tumors in mice: Close correlation with its potentiating effect on host T cell functions

Summary All mice treated with 3-methylcholanthrene (MC) suffered with tumor 114 days after treatment. However, 40% dietary restriction caused a great inhibition of tumor incidence. In order to understand the mechanisms by which dietary restriction decreased the occurrence of tumor in mice, we investigated the correlation between tumor incidence and host T cell immune responses. At 114 days after MC administration, the mice were sacrificed and their T cell immune responses were assessed. Flow cytometry studies demonstrated that dietary restriction caused a marked increase of the proportion of Thy1.2⁺, L3T4⁺ T cells in MC-treated diet-restricted mice. Consistent with this result, T cell responses against concanavalin A and interleukin-2 were also potentiated in spleen cells obtained from MC-treated diet-restricted mice, while spleen cells obtained from MC-treated unrestricted mice showed decreased T cell responses because of their tumor burden. Such potentiation of T cell functions by dietary restriction was also observed at earlier stages of MC-induced tumorigenesis. During the course of carcinogenesis, spleen cells obtained from diet-restricted mice showed decreased natural killer activity in vivo. However, in vitro induction of cytotoxic T cells was markedly augmented in MC-treated diet-restricted mice compared with unrestricted mice. These results strongly suggest that the increase of host T cell immune responses might be one of the major causes for the reduction of tumor occurrence by dietary restriction.