New clade of silicified bolidophytes that belong to Triparma (Bolidophyceae,Stramenopiles)

The small phytoplankton genus Triparma belongs to the class Bolidophyceae and contains two distinct forms: silicified species and naked flagellated species (formerly Bolidomonas). Recent studies showed that four silicified species/strains (Triparma laevis f. inornata, T. laevis f. longispina, T. strigata, and T. aff. verrucosa) belong to a single clade that is paraphyletic, because it also contains an unclassified flagellated strain, and is sister to a flagellated species, T. eleuthera. In this study, we isolated and characterized two new strains of silicified species to test the phylogenetic unity of silicified bolidophytes. The isolates were identified as T. retinervis strains because they possessed fine areolation on the cell wall. 18S rDNA and rbcL phylogenetic analyses demonstrated that T. retinervis formed a new silicified clade that is sister to the flagellated species T. pacifica. This reveals that there are at least two distinct clades including both silicified and flagellated Triparma species.     

Partial silencing of fucosyltransferase 8 gene expression inhibits proliferation of Ishikawa cells,a cell line of endometrial cancer

Endometrial cancer is the most common gynecologic malignancy and is associated with increased morbidity each year, including young people. However, its mechanisms of proliferation and progression are not fully elucidated. It is well known that abnormal glycosylation is involved in oncogenesis, and fucosylation is one of the most important types of glycosylation. In particular, fucosyltransferase 8 (FUT8) is the only FUT responsible for α1, 6-linked fucosylation (core fucosylation), and it is involved in various physiological as well as pathophysiological processes, including cancer biology. Therefore, we aimed to identify the expression of FUT8 in endometrial endometrioid carcinoma and investigate the effect of the partial silencing of the FUT8 gene on the cell proliferation of Ishikawa cells, an epithelial-like endometrial cancer cell line. Quantitative real-time PCR analysis showed that FUT8 gene expression was significantly elevated in the endometrial endometrioid carcinoma, compared to the normal endometrium. The immunostaining of FUT8 and Ulex europaeus Agglutinin 1 (UEA-1), a kind of lectin family specifically binding to fucose, was detected endometrial endometrioid carcinoma. The proliferation assay showed FUT8 partial knockdown by transfection of siRNA significantly suppressed the proliferation of Ishikawa cells, concomitant with the upregulation in the gene expressions associated with the interesting pathways associated with de-ubiquitination, aspirin trigger, mesenchymal-epithelial transition (MET) et al. It was suggested that the core fucosylation brought about by FUT8 might be involved in the proliferation of endometrial endometrioid carcinoma cells.     

Suppression of Coronary Atherosclerosis by Helix B Surface Peptide,a Nonerythropoietic,Tissue-Protective Compound Derived from Erythropoietin

Erythropoietin (EPO), a type I cytokine originally identified for its critical role in hematopoiesis, has been shown to have nonhematopoietic, tissue-protective effects, including suppression of atherosclerosis. However, prothrombotic effects of EPO hinder its potential clinical use in nonanemic patients. In the present study, we investigated the antiatherosclerotic effects of helix B surface peptide (HBSP), a nonerythropoietic, tissue-protective compound derived from EPO, by using human umbilical vein endothelial cells (HUVECs) and human monocytic THP-1 cells in vitro and Watanabe heritable hyperlipidemic spontaneous myocardial infarction (WHHLMI) rabbits in vivo. In HUVECs, HBSP inhibited apoptosis (≈70%) induced by C-reactive protein (CRP), a direct mediator of atherosclerosis. By using a small interfering RNA approach, Akt was shown to be a key molecule in HBSP-mediated prevention of apoptosis. HBSP also attenuated CRP-induced production of tumor necrosis factor (TNF)-α and matrix metalloproteinase-9 in THP-1 cells. In the WHHLMI rabbit, HBSP significantly suppressed progression of coronary atherosclerotic lesions as assessed by mean cross-sectional stenosis (HBSP 21.3 ± 2.2% versus control peptide 38.0 ± 2.7%) and inhibited coronary artery endothelial cell apoptosis with increased activation of Akt. Furthermore, TNF-α expression and the number of M1 macrophages and M1/M2 macrophage ratio in coronary atherosclerotic lesions were markedly reduced in HBSP-treated animals. In conclusion, these data demonstrate that HBSP suppresses coronary atherosclerosis, in part by inhibiting endothelial cell apoptosis through activation of Akt and in association with decreased TNF-α production and modified macrophage polarization in coronary atherosclerotic lesions. Because HBSP does not have the prothrombotic effects of EPO, our study may provide a novel therapeutic strategy that prevents progression of coronary artery disease.     

An In Vitro ES Cell-Based Clock Recapitulation Assay Model Identifies CK2α as an Endogenous Clock Regulator

We previously reported emergence and disappearance of circadian molecular oscillations during differentiation of mouse embryonic stem (ES) cells and reprogramming of differentiated cells, respectively. Here we present a robust and stringent in vitro circadian clock formation assay that recapitulates in vivo circadian phenotypes. This assay system first confirmed that a mutant ES cell line lacking Casein Kinase I delta (CKIδ) induced ∼3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKIδ null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2α) homozygous mutant ES cell line developed significantly longer (about 2.5 hours) periods of circadian clock oscillations after in vitro or in vivo differentiation. Moreover, revertant ES cell lines in which mutagenic vector sequences were deleted showed nearly wild type periods after differentiation, indicating that the abnormal circadian period of the mutant ES cell line originated from the mutation in the CK2α gene. Since CK2α deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2α in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygous mutant ES cells, demonstrating that an ES cell-based in vitro assay is available for circadian genetic screening.     

Molecular dynamics simulation of diffusion of hydrogen in binary hydrogen–tetrahydrofuran hydrate

The binary structure II hydrogen–tetrahydrofuran (THF) hydrate was studied with molecular dynamics simulation. The simulations were carried out at 300, 310 K and 10.1 MPa, and with various contents of hydrogen and THF. The migrations of hydrogen molecules from cage to cage were observed. The migration process of hydrogen was also analysed, and the diffusion coefficients of hydrogen in the hydrate were calculated. The calculated diffusion coefficients qualitatively agreed with the experimental data. Double and quintet occupancies of hydrogen molecules were observed in the small and large cages, respectively, without changing the hydrate structure.     

Structural Basis for the Different Stability and Activity between the Cdk5 Complexes with p35 and p39 Activators

Cyclin-dependent kinase 5 (Cdk5) is a brain-specific membrane-bound protein kinase that is activated by binding to the p35 or p39 activator. Previous studies have focused on p35-Cdk5, and little is known regarding p39-Cdk5. The lack of functional understanding of p39-Cdk5 is due, in part, to the labile property of p39-Cdk5, which dissociates and loses kinase activity in nonionic detergent conditions. Here we investigated the structural basis for the instability of p39-Cdk5. p39 and p35 contain N-terminal p10 regions and C-terminal Cdk5 activation domains (AD). Although p35 and p39 show higher homology in the C-terminal AD than the N-terminal region, the difference in stability is derived from the C-terminal AD. Based on the crystal structures of the p25 (p35 C-terminal region including AD)-Cdk5 complex, we simulated the three-dimensional structure of the p39 AD-Cdk5 complex and found differences in the hydrogen bond network between Cdk5 and its activators. Three amino acids of p35, Asp-259, Asn-266, and Ser-270, which are involved in hydrogen bond formation with Cdk5, are changed to Gln, Gln, and Pro in p39. Because these three amino acids in p39 do not participate in hydrogen bond formation, we predicted that the number of hydrogen bonds between p39 and Cdk5 was reduced compared with p35 and Cdk5. Using substitution mutants, we experimentally validated that the difference in the hydrogen bond network contributes to the different properties between Cdk5 and its activators.     

Rap1 potentiates endothelial cell junctions by spatially controlling myosin II activity and actin organization

Reorganization of the actin cytoskeleton is responsible for dynamic regulation of endothelial cell (EC) barrier function. Circumferential actin bundles (CAB) promote formation of linear adherens junctions (AJs) and tightening of EC junctions, whereas formation of radial stress fibers (RSF) connected to punctate AJs occurs during junction remodeling. The small GTPase Rap1 induces CAB formation to potentiate EC junctions; however, the mechanism underlying Rap1-induced CAB formation remains unknown. Here, we show that myotonic dystrophy kinase–related CDC42-binding kinase (MRCK)-mediated activation of non-muscle myosin II (NM-II) at cell–cell contacts is essential for Rap1-induced CAB formation. Our data suggest that Rap1 induces FGD5-dependent Cdc42 activation at cell–cell junctions to locally activate the NM-II through MRCK, thereby inducing CAB formation. We further reveal that Rap1 suppresses the NM-II activity stimulated by the Rho–ROCK pathway, leading to dissolution of RSF. These findings imply that Rap1 potentiates EC junctions by spatially controlling NM-II activity through activation of the Cdc42–MRCK pathway and suppression of the Rho–ROCK pathway.     

Sensitivity to RNA interference-mediated gene silencing in intact and ligated larvae of the armyworm, Mythimna separata (Lepidoptera: Noctuidae)

RNA interference (RNAi) is a common tool for analysis of gene function in both model and non-model insects, but it is becoming evident that RNAi efficiency varies considerably from species to species. We examined RNAi efficiency in larvae of the armyworm Mythimna separata (Walker) using multiple genes and tissues. First, we showed that five different target genes exhibited distinct tissue distribution patterns by quantitative determination of mRNA in total hemocytes, foregut, midgut, hindgut, Malpighian tubules and fat body: neuroglian mRNA was most abundant in fat body; inhibitor of apoptosis proteins mRNA was found to be ubiquitous; aquaporin 4 mRNA was most enriched in hindgut; cueball and prophenoloxidase 2 were mainly expressed in hemocytes. Second, we assessed sensitivity to gene silencing by double-strand RNA injection of these five genes in the six different tissues. We found that these genes generally showed refractoriness to double-strand RNA-mediated gene knockdown irrespective of the tissue tested. Finally, we demonstrated that appreciable gene knockdown was achieved at least in the adhering hemocyte fraction when larval isolated abdomen was prepared by ligation and subjected to dsRNA injection. Our study thus added detailed information on the refractoriness of larval tissues of a lepidopteran insect to gene silencing through RNAi and provided a new potential approach to improve RNAi efficiency.