Resident macrophages activated by lipopolysaccharide (LPS) suppress muscle tension and initiate inflammatory response in the gastrointestinal muscle layer

A great number of macrophages is found to be evenly distributed in the muscle layer of the gastrointestinal tract. We investigated their effects on smooth muscle contraction and the initiation of immune reactions such as inflammatory responses. Macrophages were demonstrated by uptake of FITC-dextran and their ultrastructural features were elucidated by electronmicroscopy. Muscle layers of rats’ ileums were incubated with lipopolysaccharide (LPS) for 4 to 8 h and the force of smooth muscle contraction was measured. The induction effect of inducible nitric oxide synthase on macrophages was then checked by immunohistochemistry. The expression of Main Histocompatible Complex (MHC) class II was also examined. Macrophages in the muscle layer were confirmed as resident macrophages and were different from a population of dendritic cells. After incubation with LPS, macrophages begun to express iNOS and produced NO, and it reduced smooth muscle contraction. iNOS immunopositive cells increased in a time dependent manner. Macrophages also began to express MHC class II. The total number of macrophages did not alter after incubation. Results indicate that resident macrophages in the muscle layer induced iNOS as an inflammatory reaction,affected smooth muscle contraction, and initiated immune response in the smooth muscle layer of the gastrointestinal tract, when activated by LPS.journal articl

大学教職科目「特別活動論」の教育実践記録 ―人間関係形成と自治的活動を中心として―

text紀要論文 / Departmental Bulletin Paperdepartmental bulletin pape

Improved measurements of the branching fractions for B→Kπ, ππ, and KK̅ decays

journal articl

保谷民博関係人名録

departmental bulletin pape

siRNAs to the Internally Truncated LRP5 Receptor Inhibit Tumor Growth in Xenografted SCID Mice

<div><p>(A) Injection of sHPT-1 cells pretransfected for 24 h with siLRP5Δ666–809 or control siRNA. Arrows indicate representative parathyroid tumor growth at site of transplantation.</p> <p>(B) Parathyroid tumors from SCID mice (<i>n</i> = 35) injected with sHPT-1 cells pretransfected for 24 h with the indicated siRNAs were excised and weighed. *<i>p</i> < 0.05. The animals were monitored every day and killed after 8–9 wk.</p></div

Maintained Expression of the Internally Truncated LRP5 Receptor Is Required for Accumulation of Nonphosphorylated β-Catenin and Continued Cell Growth

<div><p>(A) Western blot analysis of active β-catenin [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040328#pmed-0040328-b025" target="_blank">25</a>], 60 h after transfection. The β-catenin–actin signal ratio is shown.</p> <p>(B) Effects on sHPT-1 cell growth. HeLa cells were used as control for toxic effects. *<i>p</i> < 0.05.</p> <p>(C) Flow cytometry analysis of sHPT-1 cells at 84 h after transfection after staining with annexin V–FITC and propidium iodide. Accumulation of dead cells in the upper left quadrant; population of late apoptotic cells (upper right quadrant) and early apoptotic cells (lower right quadrant).</p></div

The Internally Truncated LRP5 Receptor Regulates β-Catenin–Driven Transcription

<div><p>(A) Transient cotransfections of FOPFLASH or TOPFLASH TCF/β-catenin reporters, the CMV-LacZ reference plasmid, and indicated siRNAs to sHPT-1 cells (left panel). FOPFLASH contains mutated binding sites for TCF factors, while TOPFLASH does not [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040328#pmed-0040328-b022" target="_blank">22</a>]. Luciferase activities were normalized to β-galactosidase activities. Effects on endogenous <i>MYC</i> expression in sHPT-1 cells (right panel). *<i>p</i> < 0.05.</p> <p>(B) Western blot analysis of V5-tagged LRP5 and LRP5Δ666–809 transiently transfected to HEK293T cells. LRP5 and LRP5Δ666–809 were expressed at similar levels.</p> <p>(C) Western blot analysis of cytosolic nonphosphorylated active β-catenin in HEK293T cells, 24 h after transfection.</p> <p>(D) Endogenous <i>MYC</i> expression in transfected HEK293T cells. *<i>p</i> < 0.05.</p> <p>(E) ChIP of the <i>MYC</i> promoter in transfected HEK293T cells. An anti-active–β-catenin monoclonal antibody was used [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040328#pmed-0040328-b025" target="_blank">25</a>].</p></div

WNT3 Ligand and the Internally Truncated LRP5 Receptor Strongly Activates Transcription of the TOPFLASH TCF/β-Catenin Luciferase Reporter with Impaired DKK1 Inhibition

<div><p>(A) sHPT-1 cells cotransfected with TOPFLASH, LRP5wt, or LRP5Δ666–809 expression vectors and CMV-LacZ reference plasmid, followed by incubation in WNT1, WNT3, or WNT3A conditioned medium (CM). CM was from HEK293T cells transfected transiently with expression vectors for the various WNT ligands. The 11-fold (<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040328#pmed-0040328-g004" target="_blank">Figure 4</a>A) endogenous β-catenin activity is set to 1 (unstimulated, empty vector).</p> <p>(B) Representative RT-PCR analysis of RNA from three pHPT and three sHPT tumors using primers for WNT3. No expression of WNT1 was detected by the conditions used [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040328#pmed-0040328-b021" target="_blank">21</a>].</p> <p>(C) Cotransfection of TOPFLASH and CMV-LacZ reference plasmid to sHPT-1 cells. Incubation in WNT3 and DKK1 CM.</p> <p>(D) Cotransfection of TOPFLASH, CMV-LacZ reference plasmid, and LRP5wt or LRP5Δ666–809 expression vectors to HEK293T cells followed by incubation in WNT1, WNT3, or DKK1 CM. HEK293T cells do not express the internally truncated LRP5 receptor.</p></div

An Internally Truncated LRP5 Receptor Is Expressed in Parathyroid Tumors

<div><p>(A) RT-PCR analysis of RNA from pHPT tumors (<i>n</i> = 20) and sHPT tumors (<i>n</i> = 20) using primers located in exons 9 and 13 of LRP5. The wild-type or truncated LRP5 mRNAs were detected by primary PCR for most of the tumors or with nested PCR using an additional overlapping forward primer. The truncation comprised the last 93 bp of exon 9, all 227 bp of exon 10, and the first 106 bp of exon 11.</p> <p>(B) Nested RT-PCR of normal parathyroid tissue (<i>n</i> = 6), and parathyroid tumor cell line sHPT-1 [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040328#pmed-0040328-b008" target="_blank">8</a>] as marker (top panel). Nested RT-PCR analysis of 17 normal tissues, and the parathyroid tumor pHPT21 as marker (lower panel).</p> <p>(C) Immunoprecipitation followed by Western blot analysis of LRP5. Transiently expressed (HEK293T cells) LRP5 and LRP5Δ666–809 shown as size markers. sHPT-1, human parathyroid tumor cell line [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040328#pmed-0040328-b008" target="_blank">8</a>]; pHPT, pHPT tumor. The lower panel shows additional pHPT tumor samples analyzed on a 5% SDS–polyacrylamide gel, where the proteins separate more clearly.</p> <p>(D) A schematic structure of LRP5 is shown. The truncated mRNA contains an in-frame deletion of LRP5 between amino acids 666 and 809 (Δ666–809), encompassing the third YWTD β-propeller domain. The truncation (Δ666–809) is flanked by imperfect direct repeat sequences (horizontal arrows) with putative cryptic donor (Ac-GTG) and acceptor (AC-cT) RNA splice sites in exons 9 and 11, respectively (arrows). The Δ666–809 is between nucleotide positions 2039 and 2466 of the LRP5 mRNA (GenBank accession no. AF064548).</p></div

Specificity and Efficiency of siRNAs Transiently Transfected to the sHPT-1 Parathyroid Tumor Cell Line

<div><p>(A) Locations of siRNAs and probes for quantitative real-time PCR. tot mRNA PCR probe determines both LRP5wt and LRP5Δ666–809 transcripts.</p> <p>(B) Expression of LRP5wt and LRP5tot mRNA assessed by quantitative real-time PCR. (C) Immunoprecipitation and Western blot analysis of LRP5.</p></div