Activation of Notch1 promotes development of human CD8 single positive T cells in humanized mice

Notch1 mutations are found in more than 50% of human T cell acute lymphoblastic leukemia (T-ALL) cells. However, the functions of Notch1 for human T cell development and leukemogenesis are not well understood. To examine the role of Notch1, human hematopoietic stem cells (HSCs), which had been transduced with a constitutively active form of Notch1 (ICN1), were transplanted into severely immunodeficient NOD/Shi-scid-IL2rγ^null (NOG) mice. We found that the great majority of the ICN1-expressing hematopoietic cells in the bone marrow expressed surface markers for T cells, such as CD3, CD4, and CD8, and that this T cell development was independent of the thymus. Accordingly, phenotypically mature CD8⁺ single positive (SP) T cells were observed in the spleen. Furthermore, T-ALL developed in one NOG recipient mouse out of 26 that had been secondary transferred with the T cells developed in the first NOG mice. These results indicate that Notch1 signaling in HSCs promotes CD8⁺ SP T cell development, and that T cell leukemogenesis may require additional oncogenic factors other than Notch1 activation.     

Use of bicistronic vectors in combination with flow cytometry to screen for effective small interfering RNA target sequences

The efficacy and specificity of small interfering RNAs (siRNAs) are largely dependent on the siRNA sequence. Since only empirical strategies are currently available for predicting these parameters, simple and accurate methods for evaluating siRNAs are needed. To simplify such experiments, target genes are often tagged with reporters for easier readout. Here, we used a bicistronic vector expressing a target gene and green fluorescent protein (GFP) to create a system in which the effect of an siRNA sequence was reflected in the GFP expression level. Cells were transduced with the bicistronic vector, expression vectors for siRNA and red fluorescent protein (RFP). Flow cytometric analysis of the transduced cells revealed that siRNAs for the target gene silenced GFP from the bicistronic vector, but did not silence GFP transcribed without the target gene sequence. In addition, the mean fluorescence intensities of GFP on RFP-expressing cells correlated well with the target gene mRNA and protein levels. These results suggest that this flow cytometry-based method enables us to quantitatively evaluate the efficacy and specificity of siRNAs. Because of its simplicity and effectiveness, this method will facilitate the screening of effective siRNA target sequences, even in high-throughput applications.     

Exhaustive crystal structure search and crystal modeling of beta-chitin

An exhaustive search of the crystal structure of beta-chitin was carried out by simultaneously optimizing all the structural parameters based on published X-ray diffraction data and stereochemical criteria. The most probable structure was characterized by a parallel-up chain polarity, a gg orientation of hydroxymethyl groups and an intermolecular hydrogen bond along the a-axis, which essentially reproduced the original structure proposed by Gardner and Blackwell. The proposed crystal structure was subsequently subjected to crystal modeling using the AMBER force field. The probable orientation of hydroxyl groups and their motional behaviors is proposed based on calculations for the crystal models identified. Solvated crystal models exhibited a slightly deformed structure with the formation of appreciable numbers of hydrogen bonds along the b-axis.     

Starch phosphorus content in potato (Solanum tuberosum L.) cultivars and its effect on other starch properties

The research presented herein provides valuable data with respect to the phosphorus content of starches from many potato (Solanum tuberosum L.) cultivars using an energy-dispersive X-ray fluorescence technique. In all starches examined, the phosphorus content ranged from 308 to 1244 ppm. Furthermore, the estimation of the starch characteristics of representative samples differing manifestly in their phosphorus content indicated that enhancing the starch phosphate resulted in significant increases in the swelling power, peak viscosity, and breakdown and significant but small increases in the onset and peak temperatures of gelatinization. Other starch quality parameters, such as the amylose content, median granule size, and the gelatinization enthalpy, did not change significantly due to the degree of phosphate substitution of starch.     

Effects of compressive force on the differentiation of pluripotent mesenchymal cells

The purpose of this study was to determine the effect of mechanical stress on the differentiation of the pluripotent mesenchymal cell line C2C12. C2C12 cells were cultured continuously under compressive force (0.25-2.0 g/cm(2)). After mechanical stress loading, the levels of expression of mRNAs and proteins for phenotype-specific markers of osteoblasts (Runx2, Msx2, Dlx5, Osterix, AJ18), chondroblasts (Sox5, Sox9), myoblasts (MyoD), and adipocytes (PPAR gamma) were measured by real-time polymerase chain reaction analysis and Western blot analysis, respectively. The expression of activated p38 mitogen-activated protein kinase (p38 MAPK) was measured by Western blotting and/or ELISA. Loading 0.5 g/cm(2) of compressive force significantly increased the expression levels of Runx2, Msx2, Dlx5, Osterix, Sox5, and Sox9. In contrast, the expression levels of AJ18, MyoD, and PPAR gamma were decreased by exposure to 0.5 g/cm(2) of compressive force. Loading 0.5 g/cm(2) of compressive force also induced the phosphorylation of p38 MAPK. SB203580, which is a specific inhibitor of p38 MAPK, inhibited the compressive force-induced phosphorylation of p38 MAPK and partially blocked compressive force-induced Runx2 mRNA expression. These results demonstrate that compressive force stimulation directs the differentiation pathway of C2C12 cells into the osteoblast and chondroblast lineage via activated phosphorylation of p38 MAPK.     

Role of Src-family kinases in formation and trafficking of macropinosomes

Src-family kinases that localize to the cytoplasmic side of cellular membranes through lipid modification play a role in signaling events including membrane trafficking. Macropinocytosis is an endocytic process for solute uptake by large vesicles called macropinosomes. Although macropinosomes can be visualized following uptake of fluorescent macromolecules, little is known about the dynamics of macropinosomes in living cells. Here, we show that constitutive c-Src expression generates macropinosomes in a kinase-dependent manner. Live-cell imaging of GFP-tagged c-Src (Src-GFP) reveals that c-Src associates with macropinosomes via its N-terminus continuously from their generation at membrane ruffles, through their centripetal trafficking, to fusion with late endosomes and lysosomes. Fluorescence recovery after photobleaching (FRAP) of Src-GFP shows that Src-GFP is rapidly recruited to macropinosomal membranes from the plasma membrane and intracellular organelles through vesicle transport even in the presence of a protein synthesis inhibitor. Furthermore, using a HeLa cell line overexpressing inducible c-Src, we show that following stimulation with epidermal growth factor (EGF), high levels of c-Src kinase activity promote formation of macropinosomes associated with the lysosomal compartment. Unlike c-Src, Lyn and Fyn, which are palmitoylated Src kinases, only minimally induce macropinosomes, although a Lyn mutant in which the palmitoylation site is mutated efficiently induces macropinocytosis. We conclude that kinase activity of nonpalmitoylated Src kinases including c-Src may play an important role in the biogenesis and trafficking of macropinosomes.     

Prostaglandin E(2) protects human lung fibroblasts from cigarette smoke extract-induced apoptosis via EP(2) receptor activation

Prostaglandin E(2) (PGE(2)) has been shown to have a strong cytoprotective effect, inhibiting apoptosis. In the present study, we evaluated whether PGE(2) has a protective effect on cigarette smoke extract (CSE)-induced apoptosis in human lung fibroblasts. Apoptosis was assessed by various methods, including DNA content analysis. CSE (15%-20%) led to apoptosis and induced imbalance in favor of pro- over anti-apoptotic protein expression and activated caspases. PGE(2) blocked CSE-induced apoptosis and modulated the balance of pro- and anti-apoptotic proteins and decreased the activation of caspases. This anti-apoptotic effect was mediated via EP(2) receptor activation as the EP(2) agonist butaprost mimicked PGE(2) activity and siRNA for the EP(2) receptor blocked it. An adenylyl cyclase inhibitor was found to abolish the PGE(2)-mediated cytoprotective effect. Correspondingly, c-AMP analogs blocked CSE-induced apoptosis. Consistently, the protein kinase A (PKA) inhibitor KT-5720 abolished PGE(2)-mediated protection. PGE(2) and butaprost phosphorylated Bad and KT-5720 blocked phosphorylation. These results suggest that PGE(2) inhibits CSE-induced apoptosis via EP(2) receptor activation and activation of PKA, which leads to an alteration in the balance between pro- and anti-apoptotic factors. Through such a mechanism, PGE(2) may alter survival of cells in the smoke-exposed lungs, thus affecting the pathogenesis of cigarette smoke-induced disease.