Kazuharu Shimizu

MicroRNA-34a inhibits cell proliferation by repressing mitogen-activated protein kinase kinase 1 during megakaryocytic differentiation of K562 cells.

Phorbol 12-myristate 13-acetate (PMA) induces megakaryocytic differentiation of the human chronic myelocytic leukemia cell line K562. We examined the potential regulatory role of microRNAs (miRNAs) in this process. Genome-wide expression profiling identified 21 miRNAs (miRs) that were induced by the treatment of K562 cells with PMA. Among them, the expression of miR-34a, miR-221, and miR-222 was induced in the early stages and maintained throughout the late stages of differentiation. Cell signaling analysis showed that the activation of extracellular signal-regulated protein kinase (ERK) in response to PMA strongly induced miR-34a expression by transactivation via the activator protein-1 binding site in the upstream region of the miR-34a gene. Reporter gene assays identified mitogen-activated protein kinase kinase 1 (MEK1) as a direct target of miR-34a and c-fos as a direct target of miR-221/222. Although overexpression of the three miRNAs had little effect on cell differentiation, overexpression of miR-34a significantly repressed the proliferation of K562 cells with a concomitant reduction in MEK1 protein expression. Conversely, a locked nucleic acid probe against miR-34a significantly enhanced the proliferation of PMA-treated K562 cells. Taken together, the results show that PMA activates the MEK-ERK pathway and strongly induces miRNA-34a expression, which in turn inhibits cell proliferation by repressing the expression of MEK1. Thus, the results highlight an important regulatory role for miR-34a in the process of megakaryocytic differentiation, especially in the arrest of cell growth, which is a prerequisite for cells to enter differentiation.

High Tc Phase of Bi-Sr-Ca-Cu-O Superconductor

High Tc superconducting phase above 100 K in a Bi-Sr-Ca-Cu-O superconductor was found to appear in proportion to the time of the sintering just below the melting temperature. The changes of the magnetic susceptibility for this high Tc phase corresponded well to the behavior of 4.8° peak in X-ray diffraction pattern taken with Cu-Kα, which indicated that the high Tc phase contains triple Cu-O layers sandwiched by two Bi2O2 layers. The mechanism of the high Tc phase formation is proposed such that a disproportionation into triple and single Cu-O layers occurs from 80 K phase with double Cu-O layers to form the high Tc and a semiconducting phase, respectively.

Observation of the High-Tc Phase and Determination of the Pb Position in a Bi-Pb-Sr-Ca-Cu Oxide Superconductor

The high-Tc phase of a Bi-Pb-Sr-Ca-Cu oxide superconductor was observed directly by high-resolution transmission electron microscopy (HREM), and the atomic positions of Pb in the crystal were determined by high-resolution analytical electron microscopy (HRAEM) with a high spatial resolution 17 A using a probe 5 to 10 A in diameter. The HREM observation revealed that the crystal consisted solely of a triple Cu-O layered structure with c/2=18 A and without any intergrowth, and that the crystal structure was modulated along the b-axis. The HRAEM indicated that the Pb atoms were located in the Bi-O layers with an atomic ratio of Pb/Bi~0.1.

Formation of a 100 K Superconducting Bi(Pb)-Sr-Ca-Cu-O Film by a Spray Pyrolysis

A 100 K superconducting Bi(Pb)-Sr-Ca-Cu-O film was formed on a MgO(100) single crystal by a spray pyrolysis method. Fifteen hours heating of the as-sprayed film at 845°C in air was enough to give the superconducting film with a Tc zero higher than 100 K. An X-ray diffraction pattern showed that this film mainly consisted of the high-Tc phase with the orientation of the c-axis perpendicular to the surface.

Rapid quantitative profiling of N-glycan by the glycan-labeling method using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid.

Protein glycosylation is a crucial phenomenon for understanding protein functions, since its patterns and degree are associated with many biological processes, such as intercellular signaling and immune response. We previously reported a novel glycan-labeling method using a 3-ainoquinoline/α-cyano-4-hydroxycinnamic acid (3-AQ/CHCA) liquid matrix for highly sensitive detection by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS). In the present study, we examined the practicality of this method for qualitative and quantitative glycan profile analysis. We first investigated the reproducibility of the data for 16 N-glycans prepared from human epidermal growth factor receptor type 2 (HER2). All of the data obtained in intra-assays and interassays were highly correlated with statistical significance (R(2) > 0.9, p < 0.05). In addition, the HER2 glycosylation pattern differed significantly between different breast cancer cell lines SK-BR-3 and BT474 in a comparative analysis of profile data. Finally, the quantitative capability of this method was examined by using PA-labeled monosialylated N-glycan as an internal standard (IS). Using IS for AQ-labeled neutral and sialylated standard glycans, the ion peak intensity was highly linear (R(2) > 0.9) from 0.5 to 5000 fmol. Furthermore, using IS for HER2 N-glycans, all of the N-glycans were highly linear with their dilution factors (R(2) > 0.9). These results suggest that our developed AQ labeling method enabled rapid qualitative and quantitative analyses of glycans. This glycan analysis method should contribute to the field of biomarker discovery and biomedicine in applications such as quality control of biotechnology-based drugs.

Synthetic Pre-miRNA-Based shRNA as Potent RNAi Triggers.

RNA interference (RNAi) is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs) and DNA vector-based short hairpin RNAs (shRNAs) are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA) and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future.

Anodic oxidation effects on pyrolytic graphite surfaces in acid

Anodic oxidation effects on the basal and edge surfaces of pyrolytic graphite in acid were studied by laser Raman spectroscopy, and by a gas-phase chemical modification method coupled with X-ray photoelectron spectroscopy. Surface covering fractions were also studied by α-epichlorohydrin monolayer formation. It was found that the species of surface chemical groups added by anodic oxidation was closely related to the structure of the oxidized surface. In the case of the basal surface, carboxyl groups were added by anodic oxidation, being always accompanied by the destruction of its surface structure. On the other hand, in the case of the edge surface, hydroxyl groups were added without the destruction of its structure with a mild treatment. With more severe treatment, carboxyl groups were added and this addition was also accompanied by destruction of the surface structure. There was a range of treatment in which hydroxyl groups could be added without destroying the structure. Finally it was confirmed that both carboxyl and hydroxyl groups could make covalent bonds with epoxy groups of α-epichlorohydrin.

Effects of electrolyte on the structure of pyrolytic graphite surfaces in anodic oxidation

Anodic oxidation effects on the structure of the basal and edge surfaces of pyrolytic graphite in alkaline electrolytes have been studied. Laser Raman spectroscopy, a gas-phase chemical modification method, coupled with X-ray photoelectron spectroscopy and secondary ion-mass spectroscopy techniques, were used. Anodic oxidation of the surfaces of pyrolytic graphite in alkaline electrolytes does not cause destruction of their surface structure, even at a higher level of treatment, unlike oxidation of acid electrolytes. In alkaline electrolytes, the number of hydroxyl groups added on the edge surface gradually increases with the increase in treatment level, whereas the number of carboxyl groups does not increase. It was found that anodic oxidation in alkaline electrolytes has a wider permitted range of treatment, in which hydroxyl groups can be added without destroying the edge surface structure, than that found in acid electrolytes. On the other hand, the number of hydroxyl groups added by treating with alkaline electrolytes is smaller than that with acid electrolytes. At a higher treatment level with acid electrolytes, oxidation occurs, even to a depth of 40 nm from the edge surface, whereas with alkaline electrolytes, oxidation occurs only at the surface. On the basis of these results, the effects of electrolytes on the adhesion between carbon fibres and epoxy resin matrix are discussed.

Formation of as-deposited Y-Ba-Cu-O superconducting film by a high temperature spray pyrolysis method

An as-deposited Y-Ba-Cu-O superconducting film of which thickness was 7 µm was prepared by a spray pyrolysis method without post-annealing procedure. This film showed Tconset at 93 K and Tcend at 25 K. Its surface was smoother than that of a film heat treated at 950°C after the spraying process.

Interfacial debonding strength between the edge surfaces of pyrolytic graphite and epoxy resins

The edge surfaces of pyrolytic graphite have been treated by anodic oxidation and the interfacial debonding strength (IFDS) between the oxidized edge surfaces and epoxy resin measured. The unoxidized and oxidized edge surfaces were examined by X-ray photoelectron spectroscopy. The edge and epoxy resin surfaces after debonding test were examined by a field-emission scanning electron microscope. Carboxyl groups were expected to be added in great quantities by breaking the carbon-carbon bonds at the edge surface. On the other hand, hydroxyl groups could be added to edge carbon atoms at the edge surface without breaking the carbon-carbon bonds. The rise in IFDS with anodic oxidation does not correspond directly to the O/C ratios or the amount of carboxyl groups present on the edge surface. Hydroxyl groups added to the edge surface are considered to play an important role in improving the adhesion between the edge surface and epoxy resin through covalent bonds between hydroxyl and epoxy groups. In the case of epoxy resin used in this study, IFDS initially increased to 9 MPa with increasing -OH/C ratio up to about 0.02; thereafter it remains constant at this value. This strength is lower than those of PG and epoxy resin.