A soluble form of human nectin-2 impairs exocrine secretion of pancreas and formation of zymogen granules in transgenic mice

Transgenic mouse lines expressing a soluble form of human nectin-2 (hNectin-2Ig Tg) exhibited distinctive elevation of amylase and lipase levels in the sera. In this study, we aimed to clarify the histopathology and to propose the transgenic mouse lines as new animal model for characteristic pancreatic exocrine defects. The significant increase of amylase and lipase levels in sera of the transgenic lines approximately peaked at 8 weeks old and thereafter, plateaued or gradually decreased. The histopathology in transgenic acinar cells was characterized by intracytoplasmic accumulation of abnormal proteins with decrease of normal zymogen granules. The hNectin-2Ig expression was observed in the cytoplasm of pancreatic acinar cells, which was consistent with zymogen granules. However, signals of hNectin-2Ig were very weak in the transgenic acinar cells with the abnormal cytoplasmic accumulaion. The PCNA-positive cells increased in the transgenic pancreas, which suggested the affected acinar cells were regenerated. Acinar cells of hNectin-2Ig Tg had markedly small number of zymogen granules with remarkable dilation of the endoplasmic reticulum (ER) lumen containing abundant abnormal proteins. In conclusion, hNectin-2Ig Tg is proposed as a new animal model for characteristic pancreatic exocrine defects, which are due to the ER stress induced by expression of mutated cell adhesion molecule that is a soluble form of human nectin-2.     

FOXO1 delays skeletal muscle regeneration and suppresses myoblast proliferation

Unloading stress, such as bed rest, inhibits the regenerative potential of skeletal muscles; however, the underlying mechanisms remain largely unknown. FOXO1 expression, which induces the upregulated expression of the cell cycle inhibitors p57 and Gadd45α, is known to be increased in the skeletal muscle under unloading conditions. However, there is no report addressing FOXO1-induced inhibition of myoblast proliferation. Therefore, we induced muscle injury by cardiotoxin in transgenic mice overexpressing FOXO1 in the skeletal muscle (FOXO1-Tg mice) and observed regeneration delay in skeletal muscle mass and cross-sectional area in FOXO1-Tg mice. Increased p57 and Gadd45α mRNA levels, and decreased proliferation capacity were observed in C2C12 myoblasts expressing a tamoxifen-inducible active form of FOXO1. These results suggest that decreased proliferation capacity of myoblasts by FOXO1 disrupts skeletal muscle regeneration under FOXO1-increased conditions, such as unloading.     

Temperature dependence on the pesticide sampling rate of polar organic chemical integrative samplers (POCIS)

Laboratory experiments were performed to determine the sampling rates of pesticides for the polar organic chemical integrative samplers (POCIS) used in Japan. The concentrations of pesticides in aquatic environments were estimated from the accumulated amounts of pesticide on POCIS, and the effect of water temperature on the pesticide sampling rates was evaluated. The sampling rates of 48 pesticides at 18, 24, and 30 °C were obtained, and this study confirmed that increasing trend of sampling rates was resulted with increasing water temperature for many pesticides.     

Intercellular signaling between ameloblastoma and osteoblasts

Ameloblastoma is an odontogenic tumor located in the bone jaw with clinical characteristics of extensive bone resorption. It is a locally invasive tumor with a high recurrence rate despite adequate surgical removal. In bone disease, tumors and other cells including osteoblasts, osteoclasts, and osteocytes in the bone microenvironment contribute to the pathogenesis of tumor growth. However, the effect of osteoblasts on ameloblastoma cells is not well-understood, and there has been limited research on interactions between them.This study investigated interactions between ameloblastoma cells and osteoblasts using a human ameloblastoma cell line (AM-3 ameloblastoma cells) and a murine pre-osteoblast cell line (MC3T3-E1 cells). We treated each cell type with the conditioned medium by the other cell type. We analyzed the effect on cytokine production by MC3T3-E1 cells and the production of MMPs by AM-3 cells. Treatment with AM-3-conditioned medium induced inflammatory cytokine production of IL-6, MCP-1, and RANTES from MC3T3-E1 cells. The use of an IL-1 receptor antagonist suppressed the production of these inflammatory cytokines by MC3T3-E1 cells stimulated with AM-3-conditioned medium. The MC3T3-E1-conditioned medium triggered the expression of MMP-2 from AM-3 cells. Furthermore, we have shown that the proliferation and migration activity of AM-3 cells were accelerated by MC3T3-E1 conditioned media.In conclusion, these intercellular signalings between ameloblastoma cells and osteoblasts may play multiple roles in the pathogenesis of ameloblastoma.     

Plasmodium yoelii yoelii 17XNL constitutively expressing GFP throughout the life cycle

Plasmodium yoelii is a rodent parasite commonly used as a model to study malaria infection. It is the preferred model parasite for liver-stage immunological studies and is also widely used to study hepatocyte, erythrocyte and mosquito infection. We have generated a P. yoelii yoelii 17XNL line that is stably transfected with the green fluorescent protein (gfp) gene. This parasite line constitutively expresses high levels of GFP during the complete parasite life cycle including liver, blood and mosquito stages. These fluorescent parasites can be used in combination with fluorescence activated cell sorting or live microscopy for a wide range of experimental applications.     

Effects of fibrillin-1 degradation on microfibril ultrastructure

Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.     

Constitutively active parathyroid hormone receptor signaling in cells in osteoblastic lineage suppresses mechanical unloading-induced bone resorption

Multiple signaling pathways participate in the regulation of bone remodeling, and pathological negative balance in the regulation results in osteoporosis. However, interactions of signaling pathways that act comprehensively in concert to maintain bone mass are not fully understood. We investigated roles of parathyroid hormone receptor (PTH/PTHrP receptor) signaling in osteoblasts in unloading-induced bone loss using transgenic mice. Hind limb unloading by tail suspension reduced bone mass in wild-type mice. In contrast, signaling by constitutively active PTH/PTHrP receptor (caPPR), whose expression was regulated by the osteoblast-specific Col1a1 promoter (Col1a1-caPPR), suppressed unloading-induced reduction in bone mass in these transgenic mice. In Col1a1-caPPR transgenic (Tg) mice, hind limb unloading suppressed bone formation parameters in vivo and mineralized nodule formation in vitro similarly to those observed in wild-type mice. In addition, serum osteocalcin levels and mRNA expression levels of type I collagen, Runx2 and Osterix in bone were suppressed by unloading in both wild-type mice and Tg mice. However, in contrast to unloading-induced enhancement of bone resorption parameters in wild-type mice, Col1a1-caPPR signaling suppressed, rather than enhanced, osteoclast number and osteoclast surface as well as urinary deoxypyridinoline excretion upon unloading. Col1a1-caPPR signaling also suppressed mRNA expression levels of RANK and c-fms in bone upon unloading. Although the M-CSF and monocyte chemoattractant protein 1 (MCP-1) mRNA levels were enhanced in control Tg mice, these levels were suppressed in unloaded Tg mice. These results indicated that constitutive activation of PTH/PTHrP receptor signaling in osteoblastic cells suppresses unloading-induced bone loss specifically through the regulation of osteoclastic activity.     

Integration of biological networks and gene expression data using Cytoscape

Cytoscape is a free software package for visualizing, modeling and analyzing molecular and genetic interaction networks. This protocol explains how to use Cytoscape to analyze the results of mRNA expression profiling, and other functional genomics and proteomics experiments, in the context of an interaction network obtained for genes of interest. Five major steps are described: (i) obtaining a gene or protein network, (ii) displaying the network using layout algorithms, (iii) integrating with gene expression and other functional attributes, (iv) identifying putative complexes and functional modules and (v) identifying enriched Gene Ontology annotations in the network. These steps provide a broad sample of the types of analyses performed by Cytoscape.     

Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting

Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.     

Prediction of GPCR-G protein coupling specificity using features of sequences and biological functions

Understanding the coupling specificity between G protein-coupled receptors （GPCRs） and specific classes of G proteins is important for further elucidation of receptor functions within a cell. Increasing information on GPCR sequences and the G protein family would facilitate prediction of the coupling properties of GPCRs. In this study, we describe a novel approach for predicting the coupling specificity between GPCRs and G proteins. This method uses not only GPCR sequences but also the functional knowledge generated by natural language processing, and can achieve 92.2% prediction accuracy by using the C4.5 algorithm. Furthermore, rules related to GPCR-G protein coupling are generated. The combination of sequence analysis and text mining improves the prediction accuracy for GPCR-G protein coupling specificity, and also provides clues for understanding GPCR signaling.