Caspase cleavage releases a nuclear protein fragment that stimulates phospholipid scrambling at the plasma membrane

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A Novel Enhanced Green FluorescentProtein-Expressing NOG Mouse for Analyzingthe Microenvironment of Xenograft Tissues

The interaction between transplanted cells and host tissues is important for the growth and maintenance of transplanted cells. To analyze the mechanisms of these interactions, a systemic fluorescent protein-expressing mouse is a useful recipient. In this study, we generated a novel NOG strain, which strongly expresses enhanced green fluorescent protein (EGFP; PgkEGFP-NOG), especially in the liver, kidney, gastrointestinal tract, and testis. Because the host tissues expressed EGFP, xenotransplanted human cancer cells were clearly identified as EGFP-negative colonies in PgkEGFP-NOG mice. Immunohistochemical analysis revealed that EGFP-expressing stromal tissues formed a complicated tumor microenvironment within xenograft tissues. Moreover, a similar microenvironment was observed in human iPS cell-derived teratomas. Collectively, these results indicated that a suitable microenvironment is essential for the growth and maintenance of xenotransplanted cells and that PgkEGFP-NOG mice represent a useful animal model for analyzing the mechanisms of microenvironment formation.     

MiR-376c Down-Regulation Accelerates EGF-Dependent Migration by Targeting GRB2 in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.     

Racemization in the Coupling Reaction with the Several Methods beside the Activated Ester Methods

The degree of racemization in the coupling reaction, Pro-Val + Pro, with the several other methods than the activated ester methods was measured and the results were compared with that in the coupling reaction, Leu-Phe + Val, as well as in the previous paper.¹⁾ In the azide and the mixed anhydride methods, no or almost no racemization was detected, whereas in the other tested methods of peptide synthesis (Pachornik-, DCCD- and phosphazo-methods) the significantly large racemization was observed. It can be attributed to the strong nucleo- philic N-atom in the penultimate amino acids (Pro) and the steric hindrance of C-terminal amino acid (Val), which are favourable to the formation of the oxazolone ring.

This assumption was further systematically confirmed in the synthesis of the other several tripeptides with the DCCD method. The separation of the diastereoisomers (LLL and LDL) of the resulting tripeptides by gas chromatography with a packed column was also here reported.     

Isolation of Crystalline Quinolinate Phosphoribosyltransferase from Alcaligenes eutrophus nov. subsp. quinoiinicus and Its Behavior on Polyacrylamide Gel Disc Electrophoresis

Quinolinate phosphoribosyltransferase (EC 2.4.2.19) was purified and crystallized from cell-free extracts of Alcaligenes eutrophus nov. subsp. quinoiinicus, IAM 12305 which was isolated in our laboratory from soil. The enzyme was labile in the cold, and all purification steps were performed at room temperature (10~15°C). The crystalline enzyme was certified to be homogeneous by ultracentrifugal analysis and starch-gel electrophoresis. On polyacrylamide gel disc electrophoresis, the crystalline enzyme showed a multiple profile, but it showed a single band by addition of a certain amount of glycerol and 2-mercaptoethanol. The adding effect of these compounds was discussed.     

MRI-Based Analysis of Intracerebral Hemorrhage in Mice Reveals Relationship between Hematoma Expansion and the Severity of Symptoms

Intracerebral hemorrhage (ICH) is featured by poor prognosis such as high mortality rate and severe neurological dysfunction. In humans, several valuables including hematoma volume and ventricular expansion of hemorrhage are known to correlate with the extent of mortality and neurological dysfunction. However, relationship between hematoma conditions and the severity of symptoms in animal ICH models has not been clarified. Here we addressed this issue by using 7-tesla magnetic resonance imaging (MRI) on collagenase-induced ICH model in mice. We found that the mortality rate and the performance in behavioral tests did not correlate well with the volume of hematoma. In contrast, when hemorrhage invaded the internal capsule, mice exhibited high mortality and showed poor sensorimotor performance. High mortality rate and poor performance in behavioral tests were also observed when hemorrhage invaded the lateral ventricle, although worsened symptoms associated with ventricular hemorrhage were apparent only during early phase of the disease. These results clearly indicate that invasion of the internal capsule or the lateral ventricle by hematoma is a critical determinant of poor prognosis in experimental ICH model in mice as well as in human ICH patients. MRI assessment may be a powerful tool to refine investigations of pathogenic mechanisms and evaluations of drug effects in animal models of ICH.     

Studies on Osmophilic Yeasts

Assimilation of galactose and maltose by Saccharomyces rouxii, which is a typical salt-tolerant yeast playing an important role in soy-brewing, was negligible or extremely poor in the medium containing 18% NaCl, although the assimilation in the ordinary medium was vigorous. The yeasts which were able to assimilate and ferment galactose, maltose and/or saccharose in the high-saline medium were limited to a few strains. From the studies on balances of fermentation products, it was revealed that ethanol yield based on sugar consumed in the 18% NaCl-medium was lower than that in the ordinary medium, suggesting that other fermentation products than ethanol should be accumulated. Analytical results of the fermented broths showed much polyalcohol production in the high-saline medium.     

Polyalcohol Production by Pichia miso in a Jar Fermentor

Cultural conditions for polyalcohol production by Pichia miso were examined in Waldhof type 20 liter-fermentor scale. The best result was obtained under conditions where the aeration rate was 1 volume per volume of the medium per minute with the stirring rate of 500r.p.m., (Kd=5×l0^-6 [g-mol of O₂/atm. min. ml.]); in 5 days incubation, Pichia miso completely dissimilated the glucose of a high concentration, 30%, and produced glycerol, D-arabitol and erythritol in a very high yield, 50% of sugar consumed. The greatest advantage compared with the shake flask culture is that the required fermentation time is shortened to half.