Search

Periocular povidone-iodine (PI) and polyvinyl alcohol-iodine (PAI) have had a major role in the prevention of endophthalmitis. The purpose of this study was to investigate the corneal epithelial toxicity of PAI in a rabbit eye model using corneal resistance (CR) measurement, which is a good indicator of cell barrier function. Rabbit eyes were administered PAI solution at 4-, 6-, 8-, or 16-fold dilution with physiological saline solution (saline) or saline alone (control), to the conjunctival sac with/without wash-out with saline. Corneal epithelial injury assessed by fluorescein staining and the CR ratio was measured at 10 minutes (min) to 96 hours (h) after the initial administration. Histological observation was performed in the eyes following the PAI or control administrations. At 120 min after administration of PAI solution, the CR ratio was decreased and superficial punctate keratopathy (SPK) was significantly increased in each of the PAI-administered groups compared to the control. Recovery of CR and SPK after administration of 6- or 8-fold dilution of PAI was significantly delayed in eyes that were not subsequently washed with saline compared with eyes that were. Pre- or post-instillation of 2% rebamipide ophthalmic suspension significantly reduced PAI induced-SPK and -decrease of CR ratio. The CR method was able to accurately and quantitatively evaluate fine corneal epithelial injury. It is suggested that eyes should be washed with saline solution after administration of PAI solution or the instillation of rebamipide to prevent or reduce corneal epithelial injury., Competing Interests: The authors have declared that no competing interests exist.

By functioning as an enzyme cofactor, hemoglobin component, and gene regulator, heme is vital for life. One mode of heme-regulated transcription involves amplifying the activity of GATA-1, a key determinant of erythrocyte differentiation. To discover biological consequences of the metal cofactor-transcription factor mechanism, we merged GATA-1/heme-regulated sectors of the proteome and transcriptome. This multi-omic analysis revealed a GATA-1/heme circuit involving hemoglobin subunits, ubiquitination components, and proteins not implicated in erythrocyte biology, including the zinc exporter Slc30a1. Though GATA-1 induced expression of Slc30a1 and the zinc importer Slc39a8, Slc39a8 dominantly increased intracellular zinc, which conferred erythroblast survival. Subsequently, a zinc transporter switch, involving decreased importer and sustained exporter expression, reduced intracellular zinc during terminal differentiation. Downregulating Slc30a1 increased intracellular zinc and, strikingly, accelerated differentiation. This analysis established a conserved paradigm in which a GATA-1/heme circuit controls trace metal transport machinery and trace metal levels as a mechanism governing cellular differentiation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mouse toll-like receptor 9 (TLR9) is an endosomal sensor for single-stranded DNA. TLR9 is transported from the endoplasmic reticulum to endolysosomes by a multiple transmembrane protein Unc93 homolog B1, and proteolytically cleaved at its ectodomain. The structure of TLR9 and its biochemical analyses have shown that the proteolytic cleavage of TLR9 ectodomain enables TLR9-dimerization and TLR9 activation. However, the requirement of TLR9 cleavage in vivo has not been studied. We here show that the 13 amino acids deletion at the cleavage site made TLR9 resistant to proteolytic cleavage. The deletion mutation in the Tlr9 gene impaired TLR9-dependent cytokine production in conventional dendritic cells from the mutant mice. Not only in vitro, in vivo production of inflammatory cytokines (TNF-α and IL-12p40), chemokine (CCR5/RANTES), and type I interferon (IFN-α) induced by administration of TLR9 ligand was also impaired. These results demonstrate that the TLR9 cleavage is required for TLR9 responses in vivo .

Objective: The aim of this study was to elucidate the long-term results of crossover bypass (CB) for iliac atherosclerotic lesions in the era of endovascular treatment (EVT). Methods: A retrospective multicenter cohort study was performed. CB was performed in 242 patients between 2003 and 2014 by vascular surgeons at multiple medical centers in Japan. Results: Perioperative mortality was 1.7%. Primary patency rates were 86% at 5 years and 82% at 8 years. Univariate analysis showed that critical limb ischemia (Rutherford class 4-6), vein graft, and superficial femoral artery occlusion were significantly associated with low primary patency. In multivariate analysis, only critical limb ischemia influenced primary patency. The secondary patency rate was 87% at both 5 and 8 years. The limb salvage rate was 98% at both 5 and 8 years. The overall survival rates were 71% at 5 years and 49% at 8 years. Conclusion: The long-term results of CB were good in our study, compared with previous reports. Our results suggest that CB remains an option for the arterial reconstruction in unilateral iliac occlusive disease after EVT failed.

Plasmacytoid dendritic cells (pDC) sense viral RNA through toll-like receptor 7 (TLR7), form self-adhesive pDC-pDC clusters, and produce type I interferons. This cell adhesion enhances type I interferon production, but little is known about the underlying mechanisms. Here we show that MyD88-dependent TLR7 signaling activates CD11a/CD18 integrin to induce microtubule elongation. TLR7 + lysosomes then become linked with these microtubules through the GTPase Arl8b and its effector SKIP/Plekhm2, resulting in perinuclear to peripheral relocalization of TLR7. The type I interferon signaling molecules TRAF3, IKKα, and mTORC1 are constitutively associated in pDCs. TLR7 localizes to mTORC1 and induces association of TRAF3 with the upstream molecule TRAF6. Finally, type I interferons are secreted in the vicinity of cell-cell contacts between clustered pDCs. These results suggest that TLR7 needs to move to the cell periphery to induce robust type I interferon responses in pDCs.

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) complex is essential for LPS recognition and induces innate immune responses against Gram-negative bacteria. As activation of TLR4/MD-2 is also critical for the induction of adaptive immune responses, TLR4/MD-2 agonists have been developed as vaccine adjuvants, but their efficacy has not yet been ascertained. Here, we demonstrate that a funiculosin (FNC) variant, FNC-RED, and FNC-RED and FNC derivatives are agonists for both murine and human TLR4/MD-2. FNC-RED induced nuclear factor-κB (NF-κB) activation via murine TLR4/MD-2, whereas FNC had no TLR4/MD-2 stimulatory activity. Biacore analysis revealed that FNC-RED binds to murine TLR4/MD-2 but not murine radioprotective 105 (RP105)/myeloid differentiation factor-1 (MD-1), another LPS sensor. FNC-RED induced CD14-independent expressions of pro-inflammatory cytokines and co-stimulatory molecules in murine macrophages and dendritic cells. In contrast, FNC-RED stimulation was reduced in CD14-dependent LPS responses, including dimerization and internalization of TLR4/MD-2 and IFN-β expression. FNC-RED-induced IL-12p40 production from murine dendritic cells was dependent on NF-κB but not MAPK pathway. In addition, fetal bovine serum augmented lipid A-induced NF-κB activation but blocked FNC-RED-mediated responses. Two synthetic phosphate group-containing FNC-RED and FNC derivatives, FNC-RED-P01 and FNC-P01, respectively, activated human TLR4/MD-2, unlike FNC-RED. Finally, computational analysis revealed that this species-specific activation by FNC-RED and FNC-RED-P01 resulted from differences in electrostatic surface potentials between murine and human TLR4/MD-2. We conclude that FNC-RED and its synthetic derivative represent a novel category of murine and human TLR4/MD-2 agonist., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

An enhancer with amalgamated E-box and GATA motifs (+9.5) controls expression of the regulator of hematopoiesis GATA-2. While similar GATA-2-occupied elements are common in the genome, occupancy does not predict function, and GATA-2-dependent genetic networks are incompletely defined. A "+9.5-like" element resides in an intron of Samd14 (Samd14-Enh) encoding a sterile alpha motif (SAM) domain protein. Deletion of Samd14-Enh in mice strongly decreased Samd14 expression in bone marrow and spleen. Although steady-state hematopoiesis was normal, Samd14-Enh -/- mice died in response to severe anemia. Samd14-Enh stimulated stem cell factor/c-Kit signaling, which promotes erythrocyte regeneration. Anemia activated Samd14-Enh by inducing enhancer components and enhancer chromatin accessibility. Thus, a GATA-2/anemia-regulated enhancer controls expression of an SAM domain protein that confers survival in anemia. We propose that Samd14-Enh and an ensemble of anemia-responsive enhancers are essential for erythrocyte regeneration in stress erythropoiesis, a vital process in pathologies, including β-thalassemia, myelodysplastic syndrome, and viral infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

To elucidate the dynamic features of a biologically relevant large-scale reaction network, we constructed a computational model of minimal protein synthesis consisting of 241 components and 968 reactions that synthesize the Met-Gly-Gly (MGG) peptide based on an Escherichia coli -based reconstituted in vitro protein synthesis system. We performed a simulation using parameters collected primarily from the literature and found that the rate of MGG peptide synthesis becomes nearly constant in minutes, thus achieving a steady state similar to experimental observations. In addition, concentration changes to 70% of the components, including intermediates, reached a plateau in a few minutes. However, the concentration change of each component exhibits several temporal plateaus, or a quasi-stationary state (QSS), before reaching the final plateau. To understand these complex dynamics, we focused on whether the components reached a QSS, mapped the arrangement of components in a QSS in the entire reaction network structure, and investigated time-dependent changes. We found that components in a QSS form clusters that grow over time but not in a linear fashion, and that this process involves the collapse and regrowth of clusters before the formation of a final large single cluster. These observations might commonly occur in other large-scale biological reaction networks. This developed analysis might be useful for understanding large-scale biological reactions by visualizing complex dynamics, thereby extracting the characteristics of the reaction network, including phase transitions.

A 77-year-old man with a history of stent implantation in the right common iliac artery(CIA) and the left external iliac artery(EIA) was admitted to our hospital for a rapid growth of an aneurysm( max 53 mm) at Th11 level of the descending aorta. Although thoracic endovascular aortic repair (TEVAR) was required, there were many problems about access rout. The infrarenal abdominal aorta and the left EIA were severely calcified, and the lumens of the right CIA stent(5.3 mm) and the left EIA stent( 4.3 mm) were small in size. Besides, the left CIA was short(13 mm). Therefore, TEVAR was performed by retrograde approach from the left internal iliac artery( IIA) with a tube graft conduit in the hybrid operation room. IIA is a useful option for an access rout in endovascular aortic repair.

Metal ion-containing macromolecules have fundamental roles in essentially all biological processes throughout the evolutionary tree. For example, iron-containing heme is a cofactor in enzyme catalysis and electron transfer and an essential hemoglobin constituent. To meet the intense demand for hemoglobin assembly in red blood cells, the cell type-specific factor GATA-1 activates transcription of Alas2, encoding the rate-limiting enzyme in heme biosynthesis, 5-aminolevulinic acid synthase-2 (ALAS-2). Using genetic editing to unravel mechanisms governing heme biosynthesis, we discovered a GATA factor- and heme-dependent circuit that establishes the erythroid cell transcriptome. CRISPR/Cas9-mediated ablation of two Alas2 intronic cis elements strongly reduces GATA-1-induced Alas2 transcription, heme biosynthesis, and surprisingly, GATA-1 regulation of other vital constituents of the erythroid cell transcriptome. Bypassing ALAS-2 function in Alas2 cis element-mutant cells by providing its catalytic product 5-aminolevulinic acid rescues heme biosynthesis and the GATA-1-dependent genetic network. Heme amplifies GATA-1 function by downregulating the heme-sensing transcriptional repressor Bach1 and via a Bach1-insensitive mechanism. Through this dual mechanism, heme and a master regulator collaborate to orchestrate a cell type-specific transcriptional program that promotes cellular differentiation., (© 2015 The Authors.)

Complex developmental programs require orchestration of intrinsic and extrinsic signals to control cell proliferation, differentiation, and survival. Master regulatory transcription factors are vital components of the machinery that transduce these stimuli into cellular responses. This is exemplified by the GATA family of transcription factors that establish cell type-specific genetic networks and control the development and homeostasis of systems including blood, vascular, adipose, and cardiac. Dysregulated GATA factor activity/expression underlies anemia, immunodeficiency, myelodysplastic syndrome, and leukemia. Parameters governing the capacity of a GATA factor expressed in multiple cell types to generate cell type-specific transcriptomes include selective coregulator usage and target gene-specific chromatin states. As knowledge of GATA-1 mechanisms in erythroid cells constitutes a solid foundation, we will focus predominantly on GATA-1, while highlighting principles that can be extrapolated to other master regulators. GATA-1 interacts with ubiquitous and lineage-restricted transcription factors, chromatin modifying/remodeling enzymes, and other coregulators to activate or repress transcription and to maintain preexisting transcriptional states. Major unresolved issues include: how does a GATA factor selectively utilize diverse coregulators; do distinct epigenetic landscapes and nuclear microenvironments of target genes dictate coregulator requirements; and do gene cohorts controlled by a common coregulator ensemble function in common pathways. This review will consider these issues in the context of GATA factor-regulated hematopoiesis and from a broader perspective., (© 2016 Elsevier Inc. All rights reserved.)

In this study, we evaluated the efficacy of a novel minipig strain, the Microminipig (MMPig), as an animal model for studying the pharmacokinetics of a mixture of 10 perfluoroalkyl acids (PFAAs). After a single oral dose was given, we found that the blood depuration of PFAAs (blood t1/2), which we calculated using first-order elimination curves, ranged from 1.6 to 86.6 days. Among the five body compartments analyzed, the liver was the greatest site of accumulation of perfluorooctanesulfonate and longer chain perfluorinated carboxylates such as perfluorodecanoic acid, perfluoroundecanoic acid and perfluorododecanoic acid. We observed an increasing accumulation trend of perfluorinated carboxylates in the organs associated with the fluorinated carbon chain length. The perfluorononanoic acid burden was the highest among the treated compounds 21 days after a single exposure, as 29% of the given perfluorononanoic acid dose was accumulated in the tissues. The persistence of PFAAs in edible pig tissues even after 21 days post-exposure raises concerns about the safety of swine products. This was the first study to use MMPigs to elucidate the pharmacokinetics of a group of environmental pollutants. We found that MMPigs could be excellent experimental animals for toxicological studies due to their easy handling, cost efficacy for target compounds and ease of waste treatment., (Copyright © 2015 John Wiley & Sons, Ltd.)

A 64-years-old man had cor triatriatum (Lucas-Schmidt type I A) with severe mitral regurgitation and atrial fibrillation. We perfomed resection of the anomalous septum between the accessory chamber and left atrium, and conducted mitral annuloplasty and maze procedure. Arrhythmia were not encountered after surgery. The maze procedure and resection of the anomalous septum with mitral surgery proved to be effective for atrial fibrillation with cor triatriatum.

A 74-year-old woman progressed to extensive aortic aneurysm after 2 years and 6 months from onset of type B dissection. A computed tomography scan revealed aortic aneurysm from ascending aorta to Th12 level of descending aorta. Her appearance was very frailty. Therefore, we performed 2-staged hybrid repair for this case. First, surgical total arch replacement with elephant trunk via median sternotomy was performed. On the 47th days after the 1st operation, thoracic endovascular aortic repair was performed. The spinal drainage was done for spinal cord protection. Postoperative course was uneventful without any complications. Considering a surgical stress, 2-staged hybrid repair using a stent graft was less-invasive than 1 staged graft replacement.

Toll-like receptor 7 (TLR7) senses microbial-derived RNA but can also potentially respond to self-derived RNA. To prevent autoimmune responses, TLR7 is thought to localize in endolysosomes. Contrary to this view, we show here that TLR7 is present on the cell surface of immune cells and that TLR7 responses can be inhibited by an anti-TLR7 antibody. The anti-TLR7 antibody is internalized with TLR7 and accumulates in endolysosomes as an immune complex. TLR7 responses in dendritic cells, macrophages and B cells are all inhibited by the anti-TLR7 antibody. Furthermore, the anti-TLR7 antibody inhibits in vivo cytokine production induced by a TLR7 ligand. Spontaneous TLR7 activation in Unc93b1(D34A/D34A) mice causes lethal inflammation. Progressive inflammation such as splenomegaly, thrombocytopenia and chronic active hepatitis are ameliorated by anti-TLR7 antibody treatment. These results demonstrate that cell surface TLR7 is a promising target for therapeutic intervention in autoimmune diseases.

Toll-like receptor 7 (TLR7) and TLR9 sense microbial single-stranded RNA (ssRNA) and ssDNA in endolysosomes. Nucleic acid (NA)-sensing in endolysosomes is thought to be important for avoiding TLR7/9 responses to self-derived NAs. Aberrant self-derived NA transportation to endolysosomes predisposes to autoimmune diseases. To restrict NA-sensing in endolysosomes, TLR7/9 trafficking is tightly controlled by a multiple transmembrane protein Unc93B1. In contrast to TLR7/9 trafficking, little is known about a mechanism underlying NA transportation. We here show that Mucolipin 1 (Mcoln1), a member of the transient receptor potential (TRP) cation channel gene family, has an important role in ssRNA trafficking into lysosomes. Mcoln1(-/-) dendritic cells (DCs) showed impaired TLR7 responses to ssRNA. A mucolipin agonist specifically enhanced TLR7 responses to ssRNAs. The channel activity of Mcoln1 is activated by a phospholipid phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), which is generated by a class III lipid kinase PIKfyve. A PIKfyve inhibitor completely inhibited TLR7 responses to ssRNA in DCs. Confocal analyses showed that ssRNA transportation to lysosomes in DCs was impaired by PIKfyve inhibitor as well as by the lack of Mcoln1. Transportation of TLR9 ligands was also impaired by the PIKfyve inhibitor. These results demonstrate that the PtdIns(3,5)P2-Mcoln1 axis has an important role in ssRNA transportation into lysosomes in DCs., (© The Japanese Society for Immunology. 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

TLR3 senses viral dsRNA in endolysosomes. The TLR3 ectodomain is cleaved by proteases such as cathepsins in endolysosomes. It remains controversial whether the N-terminal fragment of TLR3 ectodomain (TLR3N) is cleaved off or remains associated with the C-terminal TLR3 fragment (TLR3C). In addition to endosomes, TLR3 is reported to be expressed on the surface of human fibroblasts, but not of human monocyte-derived dendritic cells. Less is known about roles of TLR3N and cell surface TLR3 in dsRNA sensing. In this study, we show the cleavage site of the TLR3 ectodomain and cell surface expression of TLR3 on mouse primary immune cells. TLR3C, which started at 343S, was associated with TLR3N. Both TLR3N and TLR3C were required for activation of IFN-β and NF-κB promoters by dsRNA, demonstrating that dsRNA is sensed by the TLR3N+C complex. Newly established mAbs to mouse TLR3 revealed that cell surface TLR3 was highly expressed on splenic CD8(+) dendritic cells and marginal zone B cells. Cell surface expression of TLR3 on these cells was dependent on the TLR-specific transporter Unc93B1. Although cell surface TLR3 was only weakly expressed on macrophages, TLR3 mAb specifically enhanced TLR3 responses to dsRNA. These results demonstrate that dsRNA is sensed by the TLR3N+C complex and that cell surface TLR3 is a promising target for modulating TLR3 responses., (Copyright © 2014 by The American Association of Immunologists, Inc.)

TLR4/MD-2 senses lipid A, activating the MyD88-signaling pathway on the plasma membrane and the TRIF-signaling pathway after CD14-mediated TLR4/MD-2 internalization into endosomes. Monophosphoryl lipid A (MPL), a detoxified derivative of lipid A, is weaker than lipid A in activating the MyD88-dependent pathway. Little is known, however, about mechanisms underlying the attenuated activation of MyD88-dependent pathways. We here show that MPL was impaired in induction of CD14-dependent TLR4/MD-2 dimerization compared with lipid A. Impaired TLR4/MD-2 dimerization decreased CD14-mediated TNFα production. In contrast, MPL was comparable to lipid A in CD14-independent MyD88-dependent TNFα production and TRIF-dependent responses including cell surface CD86 up-regulation and IFNβ induction. Although CD86 up-regulation is dependent on TRIF signaling, it was induced by TLR4/MD-2 at the plasma membrane. These results revealed that the attenuated MPL responses were due to CD14-initiated responses at the plasma membrane, but not just to responses initiated by MyD88, that is, MPL was specifically unable to induce CD14-dependent TLR4/MD-2 dimerization that selectively enhances MyD88-mediated responses at the plasma membrane., (© The Japanese Society for Immunology. 2013. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The operative procedure of extensive aortic aneurysm with ischemic coronary artery disease is controversial. We report a case of arch and descending thoracic aortic aneurysm replacement with coronary artery bypass grafting(CABG)via left thoracotomy. A 70-year-old man followed up by hepatic disease was diagnosed with expanding aortic thoracic aneurysm at the other hospital. He had admission to our hospital for surgical intervention. Computed tomography(CT)revealed arch and descending thoracic aortic aneurysm, and coronary arteriography (CAG) revealed #7 90% and #13 75% stenosis. We performed arch and descending thoracic aortic aneurysm replacement with CABG via left thoracotomy. Replaced synthetic graft and bypass grafts were patent on the postoperative CT. He was discharged at 15th postoperative day with no morbidity.

Serum amyloid A (SAA) 3 is a major component of the acute phase of inflammation. We previously reported that SAA3 served as an endogenous peptide ligand for TLR4 to facilitate lung metastasis. Because these experiments were performed with SAA3 recombinant proteins purified from Escherichia coli or mammalian cells, we could not rule out the possibility of LPS contamination. In this study, we used SAA3 synthetic peptides to eliminate the presence of LPS in SAA3. We found that the SAA3 synthetic peptide (aa 20-86) (20-86) stimulated cell migration and activated p38 in a manner dependent on TLR4, MD-2, and MyD88. SAA3 (20-86) also activated NF-κB and Rho small GTPase. Using surface plasmon resonance analysis, the binding constant KD values between SAA3 (20-86) or SAA3 (43-57) and TLR4/MD-2 protein highly purified by the baculovirus system were 2.2 and 30 μM, respectively. FLAG-tagged SAA3 tightly bound to protein A-tagged MD-2, but not to TLR4 in baculovirus coinfection experiments. Although SAA3 (20-86) caused a low, but appreciable level of endocytosis in TLR4, it induced the upregulation of both IL-6 and TNF-α, but not IFN-β1. An i.v. injection of SAA3 (43-57) induced the lung recruitment of CD11b(+)Gr-1(+) cells at an estimated serum concentration around its KD value toward TLR4/MD-2. Taken together, these results suggest that SAA3 directly binds MD-2 and activates the MyD88-dependent TLR4/MD-2 pathway.

Papillary muscle rupture associated with acute myocardial infarction (AMI) is well known, but it's incidence is rare. We report a case of mitral valve repair with artificial chordae for partial papillary muscle rupture after AMI. A 75-year-old man underwent percutaneous coronary intervention(PCI) for right coronary artery because of AMI about 2 months ago at another hospital, and suffered from dyspnea 1 week after PCI. He had emergency admission to our hospital for cardiac failure. Echo-cardiogram revealed severe mitral regurgitation due to posterior papillary muscle rupture. We performed mitral valve repair with neochorda implantation to left ventricular wall of papillary muscle rupture site and ring annuloplasty. Mitral regurgitation was well controlled on postoperative echo-cardiogram. He was discharged at 25th postoperative day with no morbidity.

A small number of transcription factors, including Oct-3/4 and Sox2, constitute the transcriptional network that maintains pluripotency in embryonic stem (ES) cells. Previous reports suggested that some of these factors form a complex that binds the Oct-Sox element, a composite sequence consisting of closely juxtaposed Oct-3/4 binding and Sox2 binding sites. However, little is known regarding the components of the complex. In this study we show that Sall4, a member of the Spalt-like family of proteins, directly interacts with Sox2 and Oct-3/4. Sall4 in combination with Sox2 or Oct-3/4 simultaneously occupies the Oct-Sox elements in mouse ES cells. Overexpression of Sall4 in ES cells increased reporter activities in a luciferase assay when the Pou5f1- or Nanog-derived Oct-Sox element was included in the reporter. Microarray analyses revealed that Sall4 and Sox2 bound to the same genes in ES cells significantly more frequently than expected from random coincidence. These factors appeared to bind the promoter regions of a subset of the Sall4 and Sox2 double-positive genes in precisely similar distribution patterns along the promoter regions, suggesting that Sall4 and Sox2 associate with such Sall4/Sox2-overlapping genes as a complex. Importantly, gene ontology analyses indicated that the Sall4/Sox2-overlapping gene set is enriched for genes involved in maintaining pluripotency. Sall4/Sox2/Oct-3/4 triple-positive genes identified by referring to a previous study identifying Oct-3/4-bound genes in ES cells were further enriched for pluripotency genes than Sall4/Sox2 double-positive genes. These results demonstrate that Sall4 contributes to the transcriptional network operating in pluripotent cells together with Oct-3/4 and Sox2.

We previously reported that 14-day exposure to 7-chlorinated benz[a]anthracene (7-Cl-BaA), a new environmental pollutant, selectively induced hepatic cytochrome P450 (CYP)1A2 in rats, although treatment with its parent, benz[a]anthracene (BaA), induced CYP1A1, CYP1A2, and CYP1B1. In this study, to better understand the relative contribution of chlorination to the toxicity of polycyclic aromatic hydrocarbons (PAHs), we investigated the organ-specific distributions of 7-Cl-BaA and BaA in F334 rats. After 14 days of oral administration of 7-Cl-BaA or BaA at a concentration of 1 or 10 mg/kg body weight/day, both chemicals were detected in their plasma, which was collected 24 hr after the last administration, even at the lower dosage. Dose-dependent accumulation patterns were observed in the liver, muscle, kidney, spleen, heart, and lung. The 7-Cl-BaA concentrations in the organs were higher than those of the BaA. Furthermore, at the end of the exposure, 7-Cl-BaA specifically regulated several CYP genes in the heart more so than in other organs, although these inductions were not significant in the BaA treatment. 7-Cl-BaA might also stimulate the metabolic pathways of chemicals other than AhR-mediated metabolism, which is specific to normal PAHs, because of the alterations of CYP2J4, CYP4B1, and CYP17A1 expression in rats. In conclusion, our results imply that the chlorination of PAHs may change their organ-specific distribution and consequently alter their toxicological impacts compared to their parent PAHs.

Lolitrem B, a causative toxin for ryegrass staggers, is produced by Neotyphodium lolii infecting perennial ryegrass (Lolium perenne). Japanese black cattle have been suspected to be more sensitive to lolitrem B than to other strains, and there has been a concern about the public health hazard of eating beef contaminated with lolitrem B. We carried out a feeding experiment to examine the sensitivity of Japanese black cattle to lolitrem B and the residual level of lolitrem B in several animal tissues. Japanese black steers were fed a 0, 500, 750, 1000, 1500 or 2000 µg kg(-1) diet of lolitrem B provided by endophyte-infected perennial ryegrass straw for 12 weeks. All six animals in the 2000 µg kg(-1) diet group exhibited ryegrass staggers symptoms. Furthermore, two out of three animals in the 1500 µg kg(-1) diet group, three out of six animals in the 1000 µg kg(-1) diet group and one out of three animals in the 750 µg kg(-1) diet group presented clinical signs of ryegrass staggers. These results suggest that a daily intake of 18 µg kg(-1) body weight of lolitrem B can produce ryegrass staggers in Japanese black steers. Perirenal fat tissues of the steers from those groups having one or more animals exhibiting ryegrass staggers symptoms contained approximately 150 ng g(-1) of lolitrem B, while only small amounts of lolitrem B were detected in muscle, liver and kidney. Because the residual amount of lolitrem B in tissues of Japanese black cattle is small, the exposure to lolitrem B in consumers of the beef is likely to be low.

A cell surface heterodimer Toll-like receptor 4 (TLR4)/MD-2 senses lipopolysaccharide (LPS), a principal membrane component of Gram-negative bacteria. LPS binds to MD-2 and induces dimerization of TLR4/MD-2. Dimerized TLR4 activates downstream signaling. TLR4 polymorphism replacing Asp299 with Gly and Thr399 with Ile (D299G/T399I) causes LPS hyporesponsiveness, and is associated with a variety of infectious and noninfectious diseases. However, a molecular mechanism underlying the LPS hyporesponsiveness remains controversial. We here asked whether the TLR4 polymorphism influenced cell surface expression of TLR4/MD-2, ligand-dependent TLR4/MD-2 dimerization or TLR4/MD-2 responses to a weak agonist monophosphoryl lipid A (MPL). A newly established anti-TLR4 mAb detected D299G/T399I TLR4/MD-2 on Ba/F3 cells, whereas a previous anti-TLR4 mAb did will this fit on the line above?, suggesting that the D299G/T399I polymorphism caused a conformational change in TLR4. Hyporesponsiveness of D299G/T399I TLR4/MD-2 was much more apparent when cells were stimulated with MPL than with lipid A. MPL-dependent TLR4/MD-2 dimerization was impaired by the D299G/T399I polymorphism. The D299G/T399I polymorphism did not alter LPS-binding to soluble TLR4/MD-2, but impaired its dimerization. These results suggest that the D299G/T399I TLR4 polymorphism impairs TLR4/MD-2 responses by altering ligand-dependent dimerization.

West Nile virus (WNV) is endemic throughout Africa, Eurasia, America, and Australia and has important implications for avian, horse, and human health. In these regions, dead birds are monitored for the presence of WNV through immunohistochemistry (IHC) and PCR. However, a number of the tools for IHC are inadequate owing to their cross-reactivity to other Japanese encephalitis serogroup viruses. Here we have established eight monoclonal antibodies (MAbs) to WNV. Four of them bound to the envelope protein, three of them bound to nonstructural protein 1 (NS1), and one bound to precursor membrane protein (prM), as shown by Western blot analysis. The anti-NS1 MAbs and the anti-prM MAb did not cross-react with Japanese encephalitis virus (JEV), Murray valley encephalitis virus, or St. Louis encephalitis virus in an indirect enzyme-linked immunosorbent assay. One NS1-specific MAb, SHW-32B1, and the previously reported NS1-specific MAb, SHW-7A11, were shown by IHC to specifically detect the cytoplasm of degenerated cells in the heart and brain of a WNV-infected goose. Neither of these MAbs were shown by IHC to cross-react with degenerated cells in the brain of a JEV-infected pig. These MAbs are the first reported anti-NS1 MAbs that can be used for WNV-specific IHC using formalin-fixed, paraffin-embedded sections. They may be useful for WNV research and surveillance.

In this study, we investigated the suppressive effect of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (DL-PCBs) transfer from the feed to the eggs of laying hens by using activated carbon as a feed additive. Four groups of six hens (White Leghorn egg-layers; age, 11weeks) were housed as two control groups and two exposure groups for a period of 20weeks. Two control groups were fed with either the basal feed "Control" or basal feed additing activated carbon "Control+C". Another two exposure groups were fed with feed contaminated (about 6ng TEQ kg(-1) feed) by standard solutions of PCDDs/PCDFs and DL-PCBs "Exposure" alone and contaminated feed adding activated carbon "Exposure+C". There was no significant effect on each groups for the growth rate, biochemical blood components, and egg production: these were around the standard levels for poultry in general. Moreover the results in this study showed the availability of activated carbon as a feed additive owing to the reduction in the risk of food pollution by PCDDs/PCDFs and DL-PCBs. The concentration in the eggs of the Exposure group gradually increased following the start of egg-laying but reached a steady state after about 1month. In contrast, the concentration for the Exposure+C group was stationary and below the maximum EU level (6pgTEQg(-1)fat). In comparison to the Exposure group, the Exposure+C group showed a significant decline in the percentage of bioaccumulation into the egg. This reduction due to activated carbon was also observed in the muscle and abdominal fat. The reductions were compound- and congener-dependent for DL-PCBs as follows: PCDDs/PCDFs, non-ortho-PCBs, and mono-ortho-PCBs were more than 90%, 80%, and 50%, respectively, irrespective of the type of tissues. Fat soluble vitamin concentrations in the eggs of the Exposure+C group showed lower trends than the Exposure group. The γ-tocopherol and α-tocopherol concentrations in eggs of Exposure+C group showed a significant reduction of about 40%. However, the addition of activated carbon into animal feed could obviate the remote potential for accidents causing unintentional food pollution with PCDDs/PCDFs and DL-PCBs., (Copyright © 2012. Published by Elsevier Ltd.)

The expression of genes involved in xenobiotic detoxification is under the control of the circadian clock. The aryl hydrocarbon receptor (AhR) is one of the transcription factors responsible for the induction of detoxification enzymes in response to xenobiotic toxins, and the expression of AhR has been suggested to be regulated by a circadian oscillator. In this study, we investigated whether toxin-mediated activation of the AhR signaling pathway was modulated by CLOCK protein, a key component of the mammalian circadian clock. The expression of AhR and its DNA binding ability in the lungs of wild-type mice showed significant 24-h oscillation. Clock mutant (Clk/Clk) mice, producing CLOCK protein deficient in transcriptional activity, failed to show significant oscillation in the expression of AhR. The mRNA levels of AhR in the lungs of Clk/Clk mice were significantly lower than in wild-type mice. A single intraperitoneal injection of benzo[α]pyrene, a ligand of AhR, induced the expression of Cyp1a1 in the lungs of wild-type mice, but the induction varied depending on the benzo[α]pyrene injection time. The dosing time-dependency of benzo[α]pyrene-induced Cyp1a1 expression was also modulated by Clock gene mutation. These findings suggest that CLOCK protein affects the toxin-induced expression of detoxification enzymes through modulating the activity of AhR. Our present findings provide a molecular link between the circadian clock and xenobiotic detoxification., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Helicobacter pylori is a common cause of gastroduodenal inflammatory diseases such as chronic gastritis and peptic ulcers and also an important factor in gastric carcinogenesis. Recent reports have demonstrated that bacterial inflammatory processes, such as stimulation with H. pylori lipopolysaccharide (LPS), initiate atherosclerosis. To establish the structures responsible for the inflammatory response of H. pylori LPS, we synthesized various kinds of lipid A structures (i.e., triacylated lipid A and Kdo-lipid A compounds), with or without the ethanolamine group at the 1-phosphate moiety, by a new divergent synthetic route. Stereoselective α-glycosylation of Kdo N-phenyltrifluoroacetimidate was achieved by use of microfluidic methods. None of the lipid A and Kdo-lipid A compounds were a strong inducer of IL-1β, IL-6, or IL-8, suggesting that H. pylori LPS is unable to induce acute inflammation. In fact, the lipid A and Kdo-lipid A compounds showed antagonistic activity against cytokine induction by E. coli LPS, except for the lipid A compound with the ethanolamine group, which showed very weak agonistic activity. On the other hand, these H. pylori LPS partial structures showed potent IL-18- and IL-12-inducing activities. IL-18 has been shown to correlate with chronic inflammation, so H. pylori LPS might be implicated in the chronic inflammatory responses induced by H. pylori. These results also indicated that H. pylori LPS can modulate the immune response: NF-κB activation through hTLR4/MD-2 was suppressed, whereas production of IL-18 and IL-12 was promoted., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Toll-like receptor (TLR)4/MD-2, a sensor for LPS, delivers the MyD88-dependent signal from the cell surface, then traffics to endolysosomes and delivers the TRIF/TICAM-1-dependent signal. Both signals are thought to be dependent on cell surface TLR4/MD-2. Although TLR4/MD-2 is located also in recycling endosomes, the Golgi apparatus or the endoplasmic reticulum, little is known about a role for intracellular TLR4/MD-2 in LPS responses. We here studied intracellular LPS sensing in macrophages. PRAT4A (protein associated with TLR4 A) is a cochaperone for a general chaperone gp96 and required for cell surface expression of TLR4/MD-2. Cell surface TLR4/MD-2 was undetectable on PRAT4A(-/-) thioglycollate-elicited peritoneal macrophages (P-Macs) and bone marrow-derived macrophages (BM-Macs). LPS responses were all abolished in PRAT4A(-/-) P-Macs, whereas a part of LPS responses remained detectable in PRAT4A(-/-) BM-Macs. Of note, LPS responses in PRAT4A(-/-) BM-Macs were not necessarily dependent on TRIF/TICAM-1 signaling. PRAT4A(-/-) BM-Macs showed unimpaired production of both TRIF/TICAM-1-dependent chemokine RANTES (CCL5) and MyD88-dependent chemokine MCP-1 (CCL2). Moreover, up-regulation of co-stimulatory molecules, CD40 and CD86 was not altered. In contrast, TRIF/TICAM-1-dependent production of type I IFN was profoundly impaired. In response to heat-killed bacteria Escherichia coli, BM-Macs also required PRAT4A-independent TLR4/MD-2 for production of MCP-1 (CCL2) and RANTES (CCL5) and for up-regulation of CD40 and CD86, indicating that intracellular TLR4/MD-2 is able to sense phagocytosed bacteria and activate immune responses. These results demonstrate that intracellular TLR4/MD-2 is responsible for unique set of LPS responses.

Seven miniature pigs were injected intravenously with deoxynivalenol (DON) at 1 mg/kg body weight; afterward, the number of leukocytes in peripheral blood, the luminol-dependent chemiluminescence of neutrophils, the serum or plasma concentration of cytokines and acute-phase proteins were evaluated to determine the effects of acute exposure to DON on inflammatory responses. White blood cell counts were transiently increased at 3, 6, and 12 hr post-injection (PI) due to the increased number of neutrophils. The luminol-dependent chemiluminescence value of neutrophils was significantly elevated at 24 hr PI, indicating the activation of the bactericidal function of neutrophils. Significant increases of interleukin (IL)-8 and tumor necrosis factor-α at 3 hr PI and IL-6 at 6 hr PI were detected in the serum. The concentration of haptoglobin and serum amyloid A was significantly increased at 24 hr PI. These results suggest that acute exposure to DON induced a temporary recruitment of neutrophils in the peripheral blood by IL-8 and subsequent activation of the bactericidal function, and a transient increase of proinflammatory cytokines and acute-phase proteins, indicating the immunomodulatory effects of DON in pigs.

Linker for activation of T cells (LAT) is a dually palmitoylated transmembrane adaptor protein essential for T cell development and activation. However, whether LAT palmitoylation and/or lipid raft localization are required for its function is controversial. To address this question, we used a combination of biochemical, imaging, and genetic approaches, including LAT retrovirus-transduced mouse T cells and bone marrow chimeric mice. A nonpalmitoylated, non-lipid raft-residing mutant of transmembrane LAT could not reconstitute T cell development in bone marrow chimeric mice. This mutant was absent from the plasma membrane (PM) and was restricted mainly to the Golgi apparatus. A chimeric, nonpalmitoylated LAT protein consisting of the PM-targeting N-terminal sequence of Src kinase and the LAT cytoplasmic domain (Src-LAT) localized as a peripheral membrane protein in the PM, but outside lipid rafts. Nevertheless, Src-LAT restored T cell development and activation. Lastly, monopalmitoylation of LAT on Cys(26) (but not Cys(29)) was required and sufficient for its PM transport and function. Thus, the function of LAT in T cells requires its PM, but not raft, localization, even when expressed as a peripheral membrane protein. Furthermore, LAT palmitoylation functions primarily as a sorting signal required for its PM transport.

One-day-old male chickens were exposed via oral gavage to mixtures of perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), and perfluorodecanoate (PFDA) at either a low dose (0.1 mg/kg body weight [b.w.]) or a high dose (1.0 mg/kg b.w.), or a saline/ethanol vehicle control, three times a week for 3 weeks. After 3 weeks of exposure, half of the chicks were sacrificed and the other half were allowed to depurate for a further 3 weeks. No dose-dependent statistically significant differences in body/organ weights were observed among treatment and control groups after 3 weeks of exposure or after three 3 of depuration. Neither 15 histological nor 14 measured plasma biochemical parameters were significantly different in chicks from the exposed groups and vehicle controls. PFOS, PFDA, and PFOA concentrations in blood/liver/kidney samples were measured throughout the exposure and depuration periods at different time intervals. PFOS and PFDA accumulated at much higher concentrations than PFOA during the experimental periods. Interestingly, PFOS and PFDA accumulation patterns in the blood were similar during the exposure and depuration periods. The half-lives for each PFC at the 0.1 and 1.0 mg/kg doses were, respectively, approximately 15 and 17 days for PFOS, 11 and 16 days for PFDA, and 3.9 and 3.9 days for PFOA. PFDA accumulation in organs was greater than or similar to that of PFOS: the liver was the main target during exposure and the blood was the main reservoir during depuration. These results indicate that exposure to a 1.0-mg mixture of PFOS/PFDA/PFOA/kg b.w. has no adverse effect on juvenile chickens.

A 78-year-old male with aortitis syndrome was referred to our hospital for the treatment of unstable angina pectoris with ischemic mitral regurgitation, which was diagnosed by transthoracic echocardiography and coronary artery angiography. Computed tomography showed segmental wall thickness of thoracic and abdominal aorta He underwent an emergent coronary artery bypass grafting. The postoperative course was uneventful without any neurological complications. Postoperative echocardiogram and coronary artery angiography showed good mitral valve function and all patent bypass grafts. He was discharged 33 days after surgery. At 26 months after surgery, he is well without limitation of daily activities and any evidence of myocardial ischemia.

Toll-like receptor 4 (TLR4) activates two distinct signaling pathways inducing production of proinflammatory cytokines or type I interferons (IFNs), respectively. MyD88 and TIRAP/Mal are essential adaptor molecules for the former but not for the latter pathway. In contrast, TRIF/TICAM-1 and TRAM/TICAM-2 are essential for both. TIRAP is a sorting adaptor molecule recruiting MyD88 to activated TLR4 in the plasma membrane. TRAM is thought to bridge between TLR4 and TRIF by physical association. Little is known, however, how TRAM interacts with TLR4 or with TRIF during LPS response. Here, we show that TRAM recruits TRIF to the plasma membrane. Moreover, LPS induces upregulation of TLR4-association with TRAM and their subsequent translocation into endosome/lysosome. The internalized signaling complex consisting of TLR4 and TRAM colocalizes with TRAF3, a signaling molecule downstream of TRIF, in endosome/lysosome. These results suggest that TLR4 activates TRIF-signaling in endosome/lysosome after relocation from the cell surface.

Toll-like receptors (TLR) recognize a variety of microbial products and activate defense responses. Pathogen sensing by TLR2/4 requires accessory molecules, whereas little is known about a molecule required for DNA recognition by TLR9. After endocytosis of microbes, microbial DNA is exposed and recognized by TLR9 in lysosomes. We here show that cathepsins, lysosomal cysteine proteases, are required for TLR9 responses. A cell line Ba/F3 was found to be defective in TLR9 responses despite enforced TLR9 expression. Functional cloning with Ba/F3 identified cathepsin B/L as a molecule required for TLR9 responses. The protease activity was essential for the complementing effect. TLR9 responses were also conferred by cathepsin S or F, but not by cathepsin H. TLR9-dependent B cell proliferation and CD86 upregulation were apparently downregulated by cathepsin B/L inhibitors. Cathepsin B inhibitor downregulated interaction of CpG-B with TLR9 in 293T cells. These results suggest roles for cathepsins in DNA recognition by TLR9.

Specific-pathogen-free chickens inoculated with H5N1 highly pathogenic avian influenza (HPAI) viruses isolated in Japan in 2004 were investigated pathologically. The chickens inoculated intravenously with the viruses died within 26 hr after inoculation. Macroscopically, minimal necrosis of the tip of the comb, and hemorrhages of the palpebral conjunctiva, liver, cerebellum, and muscles were rarely observed. Histologically, dead chickens had minimal focal necrosis of hepatocytes with fibrinous thrombi in sinusoids, mild necrosis of splenic ellipsoids with fibrinous exudation, minimal necrosis of the brain, mild necrosis of epidermal cells of the comb with congestion of the lamina propria, and hemorrhages and edema of the lamina propria of the conjunctiva. Virus antigens were seen in the sinusoidal endothelial cells and hepatocytes in the liver, the capillary endothelial cells of the spleen, the capillary endothelial cells and cardiac myocytes in the heart, the capillary endothelial cells and necrotic nerve cells in the brain, the capillary endothelial cells in the lamina propria of the comb, the renal tubular epithelial cells, and the pancreatic acinar cells. The chickens inoculated by natural infectious routes died within 1-4 days after inoculation. Macroscopically, some chickens had hemorrhages in the conjunctiva, edematous swelling of the face and wattles, hydropericardium, hemorrhages of the proventriculus and bursa of Fabricius, increased secretion of tracheal mucus, and congestion and edema of lungs. Histologic lesions by natural infectious routes were similar to those by intravenous inoculation, except for the pancreatic necrosis. This study suggests H5N1 HPAI viruses isolated in Japan in 2004 cause pathologic conditions similar to natural cases.

A 65-year-old man was referred to our hospital to treat recent anterior myocardial infarction. Coronary artery angiography showed acute occlusion of left anterior descending coronary artery (LAD) and chronic occlusion of right coronary artery. After emergent percutaneous coronary intervention for LAD, drug-refractory electrical storm necessitating frequent electrical defibrillating cardioversion occurred. This patient successfully underwent surgical cryoablation, left ventriculoplasty and coronary revascularization. At 2 years and 10th month after the operation, he is well without limitation of daily activities and any evidence of myocardial ischemia and ventricular tachycardia.

Second harmonic generation (SHG), electrochemical quartz microbalance (EQCM), and cyclic voltammetry are applied to clarify the structure and properties of Cu adlayers formed in the presence of Keggin polytungstate anions. For 0.02-10 mM CuSO4 solutions, no pronounced suppression of underpotential copper deposition (Cu UPD) by 0.1-10 mM H3PW12O40 (PW12) or H4SiW12O40 (SiW12) is observed in electrochemical experiments. Moreover, coadsorption with polyanions results in an increase of charge in the Cu UPD region. EQCM data demonstrate high surface coverage with polytungstate in the overall potential range and their pronounced co-adsorption with Cu2+ cations under open circuit. The unusual potential dependence of EQCM response of polytungstates is discovered and discussed in terms of anion interactions with adsorbed hydrogen. The SHG response of Cu UPD demonstrates a non-linear dependence on Cu surface coverage, which is interpreted in terms of discontinuous submonolayers consisting of 2D Cu islands. The additives of PW12 or SiW12 decrease copper SHG response at low and high CuSO4 concentrations, with minor effect for a mid range of concentrations. In all mixed solutions, the potential dependence of the SHG response remains typical for Cu UPD, not for polytungstates. SHG transients measured under potential step mode demonstrate that the initial non-steady-state SHG behavior of the adlayer is more close to the behavior of polytungstates, but typical copper features appear at longer wavelength. These facts favor the hypothesis of Cu adatom penetration through anionic adlayers and formation of a metal submonolayer at the vacant areas between large quasi-spherical polyanions, with subsequent transformation into a Pt/Cu/polytungstate layered structure.

A 77-year-old female with unstable angina pectoris was referred to our hospital for further evaluation of multiple aortic aneurysms. Computed tomography showed descending thoracic (65 mm), thoracoabdominal (40 mm) and infra-renal abdominal aneurysm (50 mm). Initially, this patient underwent off pump coronary revascularization. On 11 days after initial surgery, descending thoracic aneurysm ruptured, followed by emergent descending thoracic and thoraco-abdominal aneurysm repair. Two months later from this aortic repair, this patient successfully underwent abdominal aortic aneurysm repair. At 3 years and 7th month after the last operation, she is well without limitation of daily activities and any evidence of myocardial ischemia.

Immune cells express multiple Toll-like receptors (TLRs) that are concomitantly activated by a variety of pathogen products. Although there is presumably a need to coordinate the expression and function of TLRs in individual cells, little is known about the mechanisms governing this process. We show that a protein associated with TLR4 (PRAT4A) is required for multiple TLR responses. PRAT4A resides in the endoplasmic reticulum, and PRAT4A knockdown inhibited trafficking of TLR1 and TLR4 to the cell surface and ligand-induced trafficking of TLR9 to lysosomes. Other cell-surface molecules were expressed normally on immunocytes from PRAT4A-/- mice. There was impaired cytokine production to TLR ligands, except to the TLR3 ligand poly(I:C), and to whole bacteria. Activation of antigen-specific T helper type 1 responses were also defective. Moreover, PRAT4A-/- bone marrow chimeric mice were resistant to lipopolysaccharide-induced sepsis. These results suggest that PRAT4A regulates the subcellular distribution and response of multiple TLRs and is required for both innate and adaptive immune responses.

A 59-year-old male with congestive heart failure caused by impaired left ventricular function after coronary artery bypass grafting (CABG) was referred to our hospital, and massive ischemic mitral regurgitation was detected by echocardiography. This patient underwent on-pump beating-heart mitral valve repair without aortic cross-clamp successfully through right thoracotomy. Postoperative echocardiography revealed no mitral regurgitation. The patient recovered uneventfully and was discharged on the 17th postoperative day. At 6th month after the operation, he is well without mitral regurgitation.

Skeletal muscle satellite cells play key roles in postnatal muscle growth and regeneration. To study molecular regulation of satellite cells, we directly prepared satellite cells from 8- to 12-week-old C57BL/6 mice and performed genome-wide gene expression analysis. Compared with activated/cycling satellite cells, 507 genes were highly upregulated in quiescent satellite cells. These included negative regulators of cell cycle and myogenic inhibitors. Gene set enrichment analysis revealed that quiescent satellite cells preferentially express the genes involved in cell-cell adhesion, regulation of cell growth, formation of extracellular matrix, copper and iron homeostasis, and lipid transportation. Furthermore, reverse transcription-polymerase chain reaction on differentially expressed genes confirmed that calcitonin receptor (CTR) was exclusively expressed in dormant satellite cells but not in activated satellite cells. In addition, CTR mRNA is hardly detected in nonmyogenic cells. Therefore, we next examined the expression of CTR in vivo. CTR was specifically expressed on quiescent satellite cells, but the expression was not found on activated/proliferating satellite cells during muscle regeneration. CTR-positive cells reappeared at the rim of regenerating myofibers in later stages of muscle regeneration. Calcitonin stimulation delayed the activation of quiescent satellite cells. Our data provide roles of CTR in quiescent satellite cells and a solid scaffold to further dissect molecular regulation of satellite cells. Disclosure of potential conflicts of interest is found at the end of this article.

Background: Pulmonary sarcoma arising from the pulmonary artery is a rare disease and its prognosis is disastrous., Methods: Five patients who underwent surgery for pulmonary artery sarcoma were reviewed., Results: All patients except one were initially diagnosed with pulmonary embolism. One patient with preoperative profound shock could not wean from cardiopulmonary bypass. Two patients are still surviving for 36 and 30 months, respectively., Conclusions: Early diagnosis and complete surgical resection is perhaps the best way to improve patients with pulmonary artery sarcoma.

To evaluate the potential pathogenicity to mammals of the recent H5N1 avian influenza A virus, viruses recovered from dead mice infected with A/chicken/Yamaguchi/7/2004 isolated in Japan were examined. All recovered viruses from the brains of dead mice infected with this strain (without any prior adaptation to mice) had substituted the amino acid at position 627 of the PB2 protein from glutamic acid to lysine. Their mouse lethality had increased by approximately 5 x 10(4) times over that of the original virus. Histopathological analysis reinforced the finding that these variants caused more rapid and severe damage to mice than the original virus. This revealed that it might be useful to characterize the recovered virus to assess its potential pathogenicity to mammals.

TLRs recognize microbial products. Their subcellular distribution is optimized for microbial recognition. Little is known, however, about mechanisms regulating the subcellular distribution of TLRs. LPS is recognized by the receptor complex consisting of TLR4 and MD-2. Although MD-2, a coreceptor for TLR4, enhances cell surface expression of TLR4, an additional mechanism regulating TLR4 distribution has been suggested. We show here that PRAT4A, a novel protein associated with TLR4, regulates cell surface expression of TLR4. PRAT4A is associated with the immature form of TLR4 but not with MD-2 or TLR2. PRAT4A knockdown abolished LPS responsiveness in a cell line expressing TLR4/MD-2, probably due to the lack of cell surface TLR4. PRAT4A knockdown down-regulated cell surface TLR4/MD-2 on dendritic cells. These results demonstrate a novel mechanism regulating TLR4/MD-2 expression on the cell surface.

LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe(119) to Lys(132) (Arg(132) in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly(59) was found to be a novel critical amino acid for LPS binding outside the region 119-132. LPS signaling is thought to be triggered by ligand-induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe(126) or Gly(129) impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition.

LPS is recognized by a heterodimer consisting of TLR4 and its coreceptor MD-2. LPS signal causes excessive inflammation and tissue damage. In this study, we show that a mAb to TLR4/MD-2 protected mice from acute lethal hepatitis caused by LPS/d-galactosamine. The protective effect of the mAb was not due to inhibition of LPS response, because serum TNF-alpha, which was induced by LPS and caused lethal hepatitis, was 10 times up-regulated by the mAb pretreatment. Moreover, this mAb induced antiapoptotic genes in liver in a TLR4/MD-2-dependent manner. These results demonstrated that an agonistic mAb to TLR4/MD-2 protected mice from LPS/d-galactosamine-induced acute lethal hepatitis by delivering a protective signal activating NF-kappaB through TLR4/MD-2.

Palmitoylation is a protein modification for trafficking to lipid raft. Without palmitoylation, linker for activation of T cells (LAT), an adaptor molecule mediating T cell receptor signaling, is unable to localize in lipid rafts and to mediate T cell activation. We here show a novel role for palmitoylation in LAT trafficking to the plasma membrane and in the stability of the LAT protein. The human LAT mutant lacking palmitoylation was unable to traffic to the plasma membrane despite the presence of transmembrane portion. The mouse LAT mutant lacking palmitoylation was unstable and susceptible to degradation via the proteasome pathway. The human LAT mutant became unstable when the extracellular portion was swapped for that from mouse, indicating that both palmitoylation and the extracellular portion regulate the stability of LAT. These results suggest that palmitoylation has an important role in trafficking to the plasma membrane and the stability of LAT.

We examined the pathogenicity for chickens of two H5N1 avian influenza viruses isolated in Japan, A/chicken/ Yamaguchi/7/2004 (Ck/Yamaguchi/7/04) isolated from outbreaks in commercial layer chickens, and A/duck/Yokohama/aq10/ 2003 (Dk/Yokohama/aq10/03) isolated from duck meat imported from China. All chickens inoculated intranasally with either strain died, and the viruses were reisolated from all organs examined. However, both the mean time of onset of clinical signs and the mean death time of Ck/Yamaguchi/7/04 were shorter than those of Dk/Yokohama/aq10/03.