Involvement of signaling of VEGF and TGF-β in differentiation of sinusoidal endothelial cells during culture of fetal rat liver cells

Embryonic development of the liver is closely associated with vascular organization. However, little is known about the mechanisms of vascular differentiation during liver development. Our previous study showed that the maturation of sinusoidal endothelial cells (SECs) occurred during embryonic day 13.0 (E13.0) to E15.0. To improve our understanding of SEC differentiation, we examined here the expression of maturation markers, SE-1 and stabilin-2, in fetal livers and also attempted to establish an in vitro SEC differentiation system by culturing E13.5 fetal liver cells. Immunohistochemical examination of SE-1 and stabilin-2 expression during fetal rat liver development revealed that these differentiation markers were co-expressed in SECs in the late stage of liver development, although stabilin-2 was expressed in almost all vascular endothelial cells in the early stage. Liver cells from the E13.5 rat fetus were cultured in EBM-2 medium containing vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1) and VEGF plus SB-431542 (an inhibitor of the TGF-β1 receptor, activin receptor-like kinase 5 [ALK-5]). In vitro SEC differentiation, as indicated by the appearance of cells co-expressing SE-1 and stabilin-2 and of cells with cytoplasmic fenestrae in endothelial sheets, was induced by the addition of both VEGF and SB-431542, an inhibitor of the phosphorylation of Smad2/3 but not that of Smad1/5/8 in the cultured cells. These results indicate for the first time that both VEGF signaling and the blocking of the ALK-5-Smad2/3 signal pathway are important for the fetal differentiation of SECs.     

Transforming activity of purinergic receptor P2Y, G-protein coupled, 2 revealed by retroviral expression screening

Colorectal cancer (CRC) is one of the leading causes of cancer death in humans. In order to identify novel cancer-promoting genes in CRC, we here constructed a retroviral cDNA expression library from a CRC cell line RKO, and used it for a focus formation assay with mouse 3T3 fibroblasts, leading to the identification of 42 independent cDNAs. One of such cDNAs turned out to encode purinergic receptor P2Y, G-protein coupled, 2 (P2RY2). The oncogenic potential of P2RY2 was confirmed in vitro with the focus formation assay as well as soft agar-growth assay, and also in vivo with a tumorigenicity assay in nude mice. While our P2RY2 cDNA encodes a protein with two amino-acid substitutions compared to the reported one, we have confirmed that the wild-type P2RY2 has a strong transforming potential as well. These results indicate an unexpected role of P2RY2 in the carcinogenesis of human cancers.     

Identification of critical structural determinants responsible for octopamine binding to the alpha-adrenergic-like Bombyx mori octopamine receptor

Octopamine (OA) is a biogenic amine with a widespread distribution in the insect nervous system. OA modulates and/or regulates various behavioral patterns of insects as a neurotransmitter, neuromodulator, and neurohormone. OA receptors (OARs) belong to one of the families of G protein-coupled receptors (GPCRs). The binding of OA to OARs is coupled to the activation of the specific G proteins, which induces the release of intracellular second messengers such as cAMP and/or calcium. We previously reported the isolation of an OAR (BmOAR1) from Bombyx mori. In the study presented here, five mutated BmOAR1s were constructed with a point mutation in the putative binding crevice and expressed in HEK-293 cells. The S202A mutant receptor was found to retain the cAMP response to OA as does the wild-type receptor, but such function was impaired in the other four mutants (D103A, S198A, Y412F, and S198A/S202A). Furthermore, competition binding assays using [3H]OA and calcium mobilization assays gave results that were approximately consistent with those of the cAMP assays. Taken together, the results indicate that D103 and S198 are involved in the binding and activation of BmOAR1 with OA through electrostatic or hydrogen bond interactions, but S202 does not appear to participate in this process. Y412 seems to be involved in one of the active forms of BmOAR1. These findings should prove helpful in designing new pest control chemicals.     

Antitumor studies. Part 1: design, synthesis, antitumor activity, and AutoDock study of 2-deoxo-2-phenyl-5-deazaflavins and 2-deoxo-2-phenylflavin-5-oxides as a new class of antitumor agents

Novel 2-deoxo-2-phenyl-5-deazaflavins and 2-deoxo-2-phenylflavin-5-oxides were prepared as a new class of antitumor agents and showed significant antitumor activities against NCI-H 460, HCT 116, A 431, CCRF-HSB-2, andKB cell lines. In vivo investigation, 2-deoxo-10-methyl-2-phenyl-5-deazaflavin exhibited the effective antitumor activity against A 431 human adenocarcinoma cells transplanted subcutaneously into nude mouse. Furthermore, AutoDock study has been done by binding of the flavin analogs into PTK pp60(c-src), where a good correlation between their IC(50) and AutoDock binding free energy was exhibited. In particular, 2-deoxo-2-phenylflavin-5-oxides exhibited the highest potential binding affinity within the binding pocket of PTK.     

Sugar-inducible expression of the nucleolin-1 gene of Arabidopsis thaliana and its role in ribosome synthesis, growth and development

Animal and yeast nucleolin function as global regulators of ribosome synthesis, and their expression is tightly linked to cell proliferation. Although Arabidopsis contains two genes for nucleolin, AtNuc-L1 is the predominant if not only form of the protein found in most tissues, and GFP-AtNuc-L1 fusion proteins were targeted to the nucleolus. Expression of AtNuc-L1 was strongly induced by sucrose or glucose but not by non-metabolizable mannitol or 2-deoxyglucose. Sucrose also caused enhanced expression of genes for subunits of C/D and H/ACA small nucleolar ribonucleoproteins, as well as a large number of genes for ribosomal proteins (RPs), suggesting that carbohydrate availability regulates de novo ribosome synthesis. In sugar-starved cells, induction of AtNuc-L1 occurred with 10 mM glucose, which seemed to be a prerequisite for resumption of growth. Disruption of AtNuc-L1 caused an increased steady-state level of pre-rRNA relative to mature 25S rRNA, and resulted in various phenotypes that overlap those reported for several RP gene mutants, including a reduced growth rate, prolonged lifetime, bushy growth, pointed leaf, and defective vascular patterns and pod development. These results suggest that the rate of ribosome synthesis in the meristem has a strong impact not only on the growth but also the structure of plants. The AtNuc-L1 disruptant exhibited significantly reduced sugar-induced expression of RP genes, suggesting that AtNuc-L1 is involved in the sugar-inducible expression of RP genes.     

Rad50 is involved in MMS-induced recombination between homologous chromosomes in mitotic cells

The structural maintenance of chromosomes (SMC) family proteins (Smc1-Smc6) typically consist of two coiled-coil domains, an amino-terminal head domain, and a carboxyl-terminal tail domain. Rad50, a component of the Mre11/Rad50/Xrs2 (MRX) complex, has a similar domain structure to the SMC proteins. In Saccharomyces cerevisiae, the MRX complex appears to be essential for recombination between homologous chromosomes in meiotic cells, but not in cells undergoing vegetative growth. Here we provide for the first time evidence that Rad50, like Smc6, is required for the induction of recombination between homologous chromosomes in cells in the vegetative growth state upon exposure to methyl methanesulfonate. However, UV-induced recombination between homologous chromosomes is intact in both rad50 and smc6-56 mutant cells.     

High concentrations of D-amino acids in human gastric juice

Summary. The concentrations of free D- and L-amino acids were determined in the gastric juice from four groups: patients suffering from early gastric carcinoma with or without Helicobacter pylori infection, and patients without carcinoma but with peptic ulcers, duodenal ulcers or chronic gastritis with or without H. pylori infection. H. pylori is a bacterium associated with gastric inflammation and peptic ulcers and is a risk factor for stomach cancer. The highest D-amino acid ratios (free D-amino acid concentration to the total corresponding free D- and L-amino acid concentration) were 29%, 26%, 18%, 4% and 1% for proline, alanine, serine, aspartate and glutamate, respectively. The gastric juice levels of L-alanine, L-serine, L-proline, L-glutamate and D-alanine in the samples obtained from subjects bearing early gastric carcinoma and H. pylori were significantly higher than in the samples from the other three groups. Except for D-alanine, there was no correlation between the D-amino acid concentrations and presence of carcinoma or H. pylori.     

Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

Background

The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen.

Results

Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion.

Conclusions

The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0039-2) contains supplementary material, which is available to authorized users.