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 growthand maintenance of transplanted cells. To analyze the mechanisms of these interactions, asystemic fluorescent protein-expressing mouse is a useful recipient. In this study, wegenerated 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 clearlyidentified as EGFP-negative colonies in PgkEGFP-NOG mice. Immunohistochemical analysisrevealed that EGFP-expressing stromal tissues formed a complicated tumor microenvironmentwithin xenograft tissues. Moreover, a similar microenvironment was observed in human iPScell-derived teratomas. Collectively, these results indicated that a suitablemicroenvironment is essential for the growth and maintenance of xenotransplanted cells andthat PgkEGFP-NOG mice represent a useful animal model for analyzing the mechanisms ofmicroenvironment formation.     

Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes

Phosphoinositides (PI) play important regulatory roles in cell physiology. Localization and quantitation of PIs within the cell is necessary to understand their precise function. Currently, ectopic expression of green fluorescent protein (GFP)-fused PI-binding domains is used to visualize PIs localized to the cell membrane. However, ectopically expressed PI-binding domains may compete with endogenous binding proteins, thus altering the physiological functions of the PIs. Here, we establish a novel method for quantification and visualization of PIs in cells and tissue samples using PI-binding domains labeled with quantum dots (Qdot) as specific probes. This method allowed us to simultaneously quantify three distinct PIs, phosphatidylinositol 3,4,5-triphosphatase [PtdIns(3,4,5)P(3)), PtdIns(3,4)P(2), and PtdIns(4,5)P(2), in crude acidic lipids extracted from insulin-stimulated cells. In addition, the method allowed the PIs to be visualized within fixed cells and tissues. Sequential and spatial changes in PI production and distribution were detected in platelet-derived growth factor (PDGF)-stimulated NRK49F cells. We also observed accumulation of PtdIns(3,4)P(2) at the dorsal ruffle in PDGF-stimulated NIH3T3 cells. Finally, we found PtdIns(3,4,5)P(3) was enriched in lung cancer tissues, which also showed high levels of phosphorylated Akt. Our new method to quantify and visualize PIs is expected to provide further insight into the role of lipid signaling in a wide range of cellular events.     

Activation of prothrombin by two subtilisin-like serine proteases from Acremonium sp

Two novel subtilisin-like serine proteases (AS-E1 and -E2) that activate prothrombin have been identified in a culture of the fungus Acremonium sp. The enzymes were purified through repeated hydrophobic interaction chromatography. The N-terminal sequences of AS-E1 (34.4 kDa) and AS-E2 (32 kDa) showed high similarity to the internal sequences of two distinct subtilisin-like hypothetical proteins from Chaetomium globosum. Both enzymes proteolytically activated prothrombin to meizothrombin(desF1)-like molecules, while the activation cleavage seemed to occur at a site (Tyr(316)-Ile(317)) that is four residues proximal to the canonical Xa cleavage site (Arg(320)-Ile(321)). Both enzymes inhibited plasma clotting, possibly due to extensive degradation of fibrinogen and production of meizothrombin(desF1)-like molecule.     

Enzymatic activities of uridine and thymidine phosphorylase in normal and cancerous uterine cervical tissues

In this study, the preliminary analyses were conducted of enzymatic activities of uridine phosphorylase (UP) and thymidine phosphorylase (TP) in normal tissues and cancer tissues of the uterine cervix. The study was performed on 27 patients of cervical cancer, treated first in our hospital. Normal cervical tissues obtained from 15 patients undergoing hysterectomy for benign diseases were used as controls. The supernatant of the homogenated cervical tissues and the stroma (5-FU and ribose-1-P or deoxyribose-1-P) were analyzed by high performance liquid chromatography, and then the UP and TP activities calculated. TP activity was significantly greater than UP activity (P < 0.0001). Both UP and TP showed significantly greater activity in cancer tissues than in normal tissues (P < 0.0001). In the TP activity of the cancer tissues, there was no significant difference among the histological types, while the TP activity tended to be significantly higher in the cases with lymph node metastasis. These results showed that the TP-mediated route seemed important as the 5FU metabolic pathway in the uterine cervical tissues, and TP enzymatic activity might be associated with lymph node metastasis.     

Effects of selenium status and supplementary seleno-chemical sources on mouse T-cell mitogenesis

Although selenium is thought to be essential for various immune responses, the excess supplementation may have an adverse effect on certain immunological functions. The present study was designed to determine the effective chemical forms of selenium and their optimal levels on T-cell mitogenesis with splenic cells from mice given a selenium-deficient diet for 8 weeks to avoid effects of cellular selenium sources. Although selenium in tissues, except for spleen and thymus, was almost depleted by feeding selenium-deficient diet, the lymphoid organs still contained low levels of selenium. Both activities of cellular glutathione peroxidase (cGPx) and thioredoxin reductase (TR) in liver and splenic cells showed a tendency to decrease by selenium deficiency. However, splenic cells were tolerant against decrease of the selenoenzyme activities, and TR was also more tolerant than cGPx. T-cell proliferation of the selenium-insufficient splenic cells induced by concanavalin A was increased by addition of Na₂SeO₃, Na₂SeO₄, Na₂Se, seleno-dl-cystine, seleno-l-methionine and selenocystamine. Their promoting action was observed at levels lower than 0.1 μmol/L and was completely suppressed at the highest concentration (1 μmol/L), except for selenocystamine. Na₂SeO₃ was one of the efficient selenocompounds for the mitogenesis, which was concomitant with the significant induction of cGPx and TR. However, recovery of cGPx activity in the selenium-insufficient cells by supplementary Na₂SeO₃ was only partial, while TR activity was readily recovered from selenium deficiency. These results therefore indicate that only low levels of selenium is essential for T-cell mitogenesis even in selenium-insufficient splenic cells, and TR, which is readily recovered by Na₂SeO₃, may be the critical enzyme.     

Designer TGFβ superfamily ligands with diversified functionality

Transforming Growth Factor--beta (TGFβ) superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs), and Bone Morphogenetic Proteins (BMPs), are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer), to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values.     

Studies on the development of water maze-learning ability in rats (3). Effect of the learning schedule on learning acquisition

The effect of different learning schedules massed or distributed practice conditions (3 trials a day for 3 days), on water-filled multiple T-maze learning ability of 8-week-old SPF Wistar-Imamichi rats was investigated. Although the mean number of errors decreased day by day in both groups, the number of errors in a given day and the total number of errors in 3 days did not differ significantly between the two groups. A tendency toward a decrease in the number of errors was observed as the trials proceeded in the group with distributed practice but not in the group with massed practice. The result suggests that a certain time period for rest after each trial is necessary to acquire the memory.     

Coordinated ciliary beating requires Odf2-mediated polarization of basal bodies via basal feet

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Highlights? Mice expressing truncated Odf2 cough and sneeze due to primary ciliary dyskinesia ? Full-length Odf2 is needed for the formation of basal body-associated basal feet ? In the absence of basal feet, basal bodies fail to align with planar polarity cues ? Polarization of basal bodies by Odf2 is required for coordinated ciliary beating