Importin-beta mediates Cdc7 nuclear import by binding to the kinase insert II domain, which can be antagonized by importin-alpha

We investigated the nuclear import mechanism of Cdc7, which is essential for the initiation of DNA replication. Here we report that importin-beta binds directly to Cdc7 via the Kinase Insert II domain, promoting its nuclear import. Although both importin-alpha and -beta bind to Cdc7 via the Kinase Insert II domain in a mutually independent manner, the binding affinity of Cdc7 for importin-beta is approximately 10 times higher than for importin-alpha at low protein concentrations of an equimolar ratio. Immunodepletion of importin-beta, but not importin-alpha, abrogates Cdc7 nuclear import, and the addition of importin-beta to the importin-depleted cytosol restores Cdc7 nuclear import. Furthermore, transduction of anti-importin-beta, but not anti-importin-alpha antibodies, into live cells inhibits Cdc7 nuclear import. Unexpectedly, we found that Cdc7 nuclear import is inhibited by competitive binding of importin-alpha to Cdc7. Further studies by site-directed mutagenesis suggest that Lys306 and Lys309 within the Kinase Insert II domain are critical for Cdc7 nuclear localization.     

Beneficial effect of 30Kc6 gene expression on production of recombinant interferon-β in serum-free suspension culture of CHO cells

The Bombyx mori 30Kc gene is known to have anti-apoptotic activity and can enhance the cell growth and expression of recombinant proteins in anchorage-dependent CHO cell cultures. In this study, an interferon-β (IFN-β)-producing CHO cell line, which expresses the recombinant 30Kc6 gene, was constructed to investigate the effect of 30Kc6 expression on the production of IFN-β in serum-free suspension culture. The 30Kc6 expressing cell line showed lower apoptotic activity and prolonged cell viability under apoptotic conditions induced by the addition of sodium butyrate, staurosporine, or the removal of serum. The 30Kc6 expressing cell line also suppressed the loss of mitochondrial membrane potential induced under these conditions. It was observed that viability, and production of IFN-β were also enhanced by 30Kc6 expression in serum-free suspension cultures. These results indicate that the 30Kc6 gene can positively affect the viability and production of recombinant therapeutic proteins in serum-free suspension cultures of CHO cell lines.     

Micropropagation of Salix pseudolasiogyne from nodal explants

The effect of phytohormones on the breaking of dormancy of axillary buds in Salix pseudolasiogyne and their subsequent proliferation from nodal explants were examined. Nodal explants obtained from a 20–year-old S. pseudolasiogyne tree were cultured either on woody plant basal medium (WPM) or WPM supplemented with benzyladenine (BA, 2.2/4.4 μM), zeatin (1.1/2.2 μM), gibberillic acid (GA₃, 2.9 and 14.5 μM), and GA₃ + BA (2.9 + 4.4 μM). Although axillary shoots developed in all the media, a higher percentage bud break occurred on BA supplemented media. To corroborate the results, endogenous levels of cytokinins [Cks, N ⁶-isopentenyladenine (iP), zeatin riboside (t-ZR), dihydrozeatinriboside (DHZR)] and abscisic acid (ABA) were determined. On BA supplemented media, the levels of zeatin type (Z-type) of Cks were higher than those of isopentenyladenine type of Ck in the explants, while the ABA level was low. Axillary shoots did not grow well and became necrotic upon subculture to fresh basal WPM. In order to improve shoot growth, they were subcultured twice at a 4-week interval on to WPM supplemented with BA (2.2/4.4 μM), GA₃ (1.4 μM), or GA₃ + BA (1.4 + 4.4/2.9 + 4.4 μM). Maximal shoot growth (93%) was achieved on WPM supplemented with 2.2 μM BA. Comparative analyses of endogenous Cks revealed that higher Cks (Z-type Cks) were present in actively growing shoots. Rooting was readily achieved when the shoots were subcultured to WPM without phytohormones. The rooted plants were acclimatized well upon transplantation.     

Parallelized Nanopillar Perovskites for Semitransparent Solar Cells Using an Anodized Aluminum Oxide Scaffold

Semitransparent solar cells have attracted significant attention for practical applications, such as windows in buildings and automobiles. Here, semitransparent, highly efficient, 1D nanostructured perovskite solar cells are demonstrated employing anodized aluminum oxide (AAO) as a scaffold layer. The parallel nanopillars in the perovskite layer enable construction of haze‐free semitransparent devices without any hysteresis behavior. By controlling the pore size in the AAO, the volume occupied by the perovskite layer can be precisely varied, and the color neutrality of the resulting devices can be achieved. With the incorporation of a transparent cathodic electrode (indium tin oxide) with a buffer layer (MoO_x), a highly efficient semitransparent nanopillared perovskite solar cell is achievable with a power‐conversion efficiency of 9.6% (7.5%) and a whole device average visible light transmittance of 33.4% (41.7%). To determine the role of the scaffold layer in improving the photoelectrical properties of the cell, impedance spectroscopy analyses are performed, revealing that the AAO‐structured perovskite layer suppresses internal ion diffusion and enables critical improvements in long‐term stability under continuous illumination.     

The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virus-infected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.     

Effects of endocytosis inhibitory drugs on rubella virus entry into VeroE6 cells

It has been suggested that infectious entry of rubella virus (RV) is conducted by receptor mediated endocytosis. To explore the cellular entry mechanism of RV, inhibitory effects of drugs affecting various endocytic pathways on RV entry into VeroE6 cells were analyzed. Results showed that RV infectious entry into VeroE6 cells is mediated by clathrin-dependent endocytosis and not by caveolae-mediated endocytosis. Moreover, chemical inhibition of macropinocytosis such as treatments of amiloride, actin and microtubule-disrupting drug significantly reduced RV infection. Considering that macropinocytosis is inducible endocytosis by cellular stimulations, clathrin-mediated endocytosis is likely to be a major route of RV infectious entry.     

Enzymatic analysis of an amylolytic enzyme from the hyperthermophilic archaeon Pyrococcus furiosus reveals its novel catalytic properties as both an alpha-amylase and a cyclodextrin-hydrolyzing enzyme

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13. This amylolytic enzyme, designated PFTA (Pyrococcus furiosus thermostable amylase), was cloned and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimum temperature of 90 degrees C. The substrate specificity of PFTA suggests that it possesses characteristics of both alpha-amylase and cyclodextrin-hydrolyzing enzyme. Like typical alpha-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose. However, it could also attack and degrade pullulan and beta-cyclodextrin, which are resistant to alpha-amylase, to primarily produce panose and maltoheptaose, respectively. Furthermore, acarbose, a potent alpha-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes. These results confirm that PFTA possesses novel catalytic properties characteristic of both alpha-amylase and cyclodextrin-hydrolyzing enzyme.     

Biosynthesis and emission of insect-induced methyl salicylate and methyl benzoate from rice

Two benzenoid esters, methyl salicylate (MeSA) and methyl benzoate (MeBA), were detected from insect-damaged rice plants. By correlating metabolite production with gene expression analysis, five candidate genes encoding putative carboxyl methyltransferases were identified. Enzymatic assays with Escherichia coli-expressed recombinant proteins demonstrated that only one of the five candidates, OsBSMT1, has salicylic acid (SA) methyltransferase (SAMT) and benzoic acid (BA) methyltransferase (BAMT) activities for producing MeSA and MeBA, respectively. Whereas OsBSMT1 is phylogenetically relatively distant from dicot SAMTs, the three-dimensional structure of OsBSMT1, which was determined using homology-based structural modeling, is highly similar to those of characterized SAMTs. Analyses of OsBSMT1 expression in wild-type rice plants under various stress conditions indicate that the jasmonic acid (JA) signaling pathway plays a critical role in regulating the production and emission of MeSA in rice. Further analysis using transgenic rice plants overexpressing NH1, a key component of the SA signaling pathway in rice, suggests that the SA signaling pathway also plays an important role in governing OsBSMT1 expression and emission of its products, probably through a crosstalk with the JA signaling pathway. The role of the volatile products of OsBSMT1, MeSA and MeBA, in rice defense against insect herbivory is discussed.