EP2 and EP4 receptors on muscularis resident macrophages mediate LPS-induced intestinal dysmotility via iNOS upregulation through cAMP/ERK signals

Intestinal resident macrophages play an important role in gastrointestinal dysmotility by producing prostaglandins (PGs) and nitric oxide (NO) in inflammatory conditions. The causal correlation between PGs and NO in gastrointestinal inflammation has not been elucidated. In this study, we examined the possible role of PGE(2) in the LPS-inducible inducible NO synthase (iNOS) gene expression in murine distal ileal tissue and macrophages. Treatment of ileal tissue with LPS increased the iNOS and cyclooxygenase (COX)-2 gene expression, which lead to intestinal dysmotility. However, LPS did not induce the expression of iNOS and COX-2 in tissue from macrophage colony-stimulating factor-deficient op/op mice, indicating that these genes are expressed in intestinal resident macrophages. iNOS and COX-2 protein were also expressed in dextran-phagocytized macrophages in the muscle layer. CAY10404, a COX-2 inhibitor, diminished LPS-dependent iNOS gene upregulation in wild-type mouse ileal tissue and also in RAW264.7 macrophages, indicating that PGs upregulate iNOS gene expression. EP(2) and EP(4) agonists upregulated iNOS gene expression in ileal tissue and isolated resident macrophages. iNOS mRNA induction mediated by LPS was decreased in the ileum isolated from EP(2) or EP(4) knockout mice. In addition, LPS failed to decrease the motility of EP(2) and EP(4) knockout mice ileum. EP(2)- or EP(4)-mediated iNOS expression was attenuated by KT-5720, a PKA inhibitor and PD-98059, an ERK inhibitor. Forskolin or dibutyryl-cAMP mimics upregulation of iNOS gene expression in macrophages. In conclusion, COX-2-derived PGE(2) induces iNOS expression through cAMP/ERK pathways by activating EP(2) and EP(4) receptors in muscularis macrophages. NO produced in muscularis macrophages induces dysmotility during gastrointestinal inflammation.     

IA mutant functional antigen-presenting cell lines

We describe a protocol for the selection of mutant cells with an altered pattern of Ia antigenic determinants and antigen-presenting properties from a homogeneous population of functional antigen-presenting cells (APC). The APC line used in this work was obtained by fusing lipopolysaccharide-stimulated B cells from (BALB/c x A/J)F1 donors with cells from the M12.4.1 BALB/c B lymphoma cell line. The resulting hybridomas, including TA3, retained the potent antigen-presenting activity of the parental B lymphoma line and expressed Ia antigens and immune response gene-determined antigen-presenting properties of the A/J type. Mutants of TA3 were obtained by subjecting the cells to negative immunoselection with one monoclonal anti-(alpha) 1-Ak antibody and complement followed by positive immunoselection via electronic cell sorting with a second monoclonal alpha I-Ak or alpha I-Ek antibody. Two types of mutants were obtained. One, A8, appeared to have undergone a fairly limited alteration, since it lost only some of the I-Ak antigenic determinants; the second type appeared to have lost the entire I-Ak molecule but to have retained the I-E molecule. Functional studies with the A8 mutant demonstrated that the loss of a limited number of I-Ak determinants correlated with the loss of a specific I-Ak-encoded restriction element, since A8 failed to present a specific antigen, hen egg lysozyme (HEL), to a HEL-specific I-Ak-restricted T cell hybridoma but retained some capacity to present a second antigen, poly(Glu60Ala30Tyr10) (GAT), to a GAT-specific I-Ak-restricted T cell hybridoma. These results indicate that Ia antigens are the products of immune response gene loci. The availability of such mutants should allow an examination of the relationship between the structure of an Ia molecule and the antigens with which it is co-recognized by T cells.     

Identification and Characterization of a Novel Lysophosphatidic Acid Receptor, p2y5/LPA6

p2y5 is an orphan G protein-coupled receptor that is closely related to the fourth lysophosphatidic acid (LPA) receptor, LPA₄. Here we report that p2y5 is a novel LPA receptor coupling to the G₁₃-Rho signaling pathway. “LPA receptor-null” RH7777 and B103 cells exogenously expressing p2y5 showed [³H]LPA binding, LPA-induced [³⁵S]guanosine 5′-3-O-(thio)triphosphate binding, Rho-dependent alternation of cellular morphology, and G_s/13 chimeric protein-mediated cAMP accumulation. LPA-induced contraction of human umbilical vein endothelial cells was suppressed by small interfering RNA knockdown of endogenously expressed p2y5. We also found that 2-acyl-LPA had higher activity to p2y5 than 1-acyl-LPA. A recent study has suggested that p2y5 is an LPA receptor essential for human hair growth. We confirmed that p2y5 is a functional LPA receptor and propose to designate this receptor LPA₆.     

Exogenous seeding of cerebral β-amyloid deposition in βAPP-transgenic rats

Deposition of the amyloid-β (Aβ) peptide in senile plaques and cerebral Aβ angiopathy (CAA) can be stimulated in Aβ-precursor protein (APP)-transgenic mice by the intracerebral injection of dilute brain extracts containing aggregated Aβ seeds. Growing evidence implicates a prion-like mechanism of corruptive protein templating in this phenomenon, in which aggregated Aβ itself is the seed. Unlike prion disease, which can be induced de novo in animals that are unlikely to spontaneously develop the disease, previous experiments with Aβ seeding have employed animal models that, as they age, eventually will generate Aβ lesions in the absence of seeding. In the present study, we first established that a transgenic rat model expressing human APP (APP21 line) does not manifest endogenous deposits of Aβ within the course of its median lifespan (30?months). Next, we injected 3-month-old APP21 rats intrahippocampally with dilute Alzheimer brain extracts containing aggregated Aβ. After a 9-month incubation period, these rats had developed senile plaques and CAA in the injected hippocampus, whereas control rats remained free of such lesions. These findings underscore the co-dependence of agent and host in governing seeded protein aggregation, and show that cerebral Aβ-amyloidosis can be induced even in animals that are relatively refractory to the spontaneous origination of parenchymal and vascular deposits of Aβ.     

The erroneous courtship hypothesis: do insects really engage in aerial wars of attrition?

Males of various flying insects perform conspicuous aerial interactions around their mating stations. The broadly accepted interpretation of their aerial interaction is a war of attrition, where two contestants perform costly displays, and the one that reaches its cost threshold earlier gives up. The implicit but important requirement in this model is that some forces that match the intensity of display of the two contestants are necessary, and failure to enforce matching allows foul contestants that delay or stop their display to avoid paying contest costs. In addition, wars of attrition require flying insects to distinguish the sex of flying conspecifics because their aerial interactions begin when intruders fly into the territory. We investigated past research on the behaviour of odonates and butterflies aiming to clarify whether the two prerequisites of wars of attrition are fulfilled: (1) contestants can inflict substantial costs on nondisplaying opponents and (2) contestants can discriminate the sex of flying conspecifics. In odonates, we found an abundance of evidence suggesting that contests involve physical attack and that the ability of sexual discrimination is sufficient. Therefore, wars of attrition may occur in territorial odonates. In butterflies, however, we could not find any evidence that the two prerequisites are filled. The aerial interactions of butterflies are better interpreted as courtship between sexually active males (the erroneous courtship hypothesis). Based on these results, we discuss future directions of research on the aerial contests of flying insects.     

Membrane-tethered MUC1 mucin is phosphorylated by epidermal growth factor receptor in airway epithelial cells and associates with TLR5 to inhibit recruitment of MyD88

MUC1 is a membrane-tethered mucin glycoprotein expressed on the apical surface of mucosal epithelial cells. Previous in vivo and in vitro studies established that MUC1 counterregulates airway inflammation by suppressing TLR signaling. In this article, we elucidate the mechanism by which MUC1 inhibits TLR5 signaling. Overexpression of MUC1 in HEK293 cells dramatically reduced Pseudomonas aeruginosa-stimulated IL-8 expression and decreased the activation of NF-κB and MAPK compared with cells not expressing MUC1. However, overexpression of MUC1 in HEK293 cells did not affect NF-κB or MAPK activation in response to TNF-α. Overexpression of MyD88 abrogated the ability of MUC1 to inhibit NF-κB activation, and MUC1 overexpression inhibited flagellin-induced association of TLR5/MyD88 compared with controls. The MUC1 cytoplasmic tail associated with TLR5 in all cells tested, including HEK293T cells, human lung adenocarcinoma cell line A549 cells, and human and mouse primary airway epithelial cells. Activation of epidermal growth factor receptor tyrosine kinase with TGF-α induced phosphorylation of the MUC1 cytoplasmic tail at the Y46EKV sequence and increased association of MUC1/TLR5. Finally, in vivo experiments demonstrated increased immunofluorescence colocalization of Muc1/TLR5 and Muc1/phosphotyrosine staining patterns in mouse airway epithelium and increased Muc1 tyrosine phosphorylation in mouse lung homogenates following P. aeruginosa infection. In conclusion, epidermal growth factor receptor tyrosine phosphorylates MUC1, leading to an increase in its association with TLR5, thereby competitively and reversibly inhibiting recruitment of MyD88 to TLR5 and downstream signaling events. This unique ability of MUC1 to control TLR5 signaling suggests its potential role in the pathogenesis of chronic inflammatory lung diseases.     

Functional expression of an scFv on bacterial magnetic particles by in vitro docking

A Gram-negative, magnetotactic bacterium, Magnetospirillum magneticum AMB-1 produces nano-sized magnetic particles (BacMPs) in the cytoplasm. Although various applications of genetically engineered BacMPs have been demonstrated, such as immunoassay, ligand–receptor interaction or cell separation, by expressing a target protein on BacMPs, it has been difficult to express disulfide-bonded proteins on BacMPs due to lack of disulfide-bond formation in the cytoplasm. Here, we propose a novel dual expression system, called in vitro docking, of a disulfide-bonded protein on BacMPs by directing an immunoglobulin Fc-fused target protein to the periplasm and its docking protein ZZ on BacMPs. By in vitro docking, an scFv–Fc fusion protein was functionally expressed on BacMPs in the dimeric or trimeric form. Our novel disulfide-bonded protein expression system on BacMPs will be useful for efficient screening of potential ligands or drugs, analyzing ligand–receptor interactions or as a magnetic carrier for affinity purification.     

Microbiological Redox Potential Control to Improve the Efficiency of Chalcopyrite Bioleaching

The effect of controlling the redox potential (E_h) on chalcopyrite bioleaching kinetics was studied as a new aspect of redox control during chalcopyrite bioleaching, and its mechanism was investigated by employing the “normalized” solution redox potential (E_normal) and the reaction kinetics model. Different E_h ranges were established by use of different acidophiles (Sulfobacillus acidophilus YTF1; Sulfobacillus sibiricus N1; Acidimicrobium ferrooxidans ICP; Acidiplasma sp. Fv-AP). Cu dissolution was very susceptible to real-time change in E_h during the reaction. It was found that efficiency of bioleaching of chalcopyrite can be effectively evaluated on the basis of E_normal, since it is normalized for real-time fluctuations of concentrations of major metal solutes during bioleaching. For steady Cu solubilization during bioleaching at a maximum rate, it was important to maintain a redox potential range of 0 ≤ E_normal ≤ 1 (?0.35 mV optimal) at the mineral surface by employing a “weak” ion-oxidizer. This led to a copper recovery of > 75%. At higher E_normal levels (E_normal > 1 by “strong” microbial Fe²⁺ oxidation), Cu solubilization was slowed by diffusion through the product film at the mineral surface (< 50% Cu recovery) caused by low reactivity of the chalcopyrite and by secondary passivation of the chalcopyrite surface, mainly by jarosite.     

Proteomic analysis of acrylamide gel separated proteins immobilized on polyvinylidene difluoride membranes following proteolytic digestion in the presence of 80% acetonitrile

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) combined with mass spectrometry (MS) is a highly accurate and sensitive means of identifying proteins. We have developed a novel method for digesting proteins on polyvinylidene difluoride (PVDF) membranes for subsequent matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis. After Tricine sodium dodecyl sulfate (SDS)-PAGE, separated proteins were electroblotted onto PVDF membranes in a semidry discontinuous buffer system, visualized by staining with Coomassie Blue, excised, digested with trypsin or lysC in 80% acetonitrile, and then analyzed by MALDI-TOF MS. This method has several advantages over in-gel digestion in terms of sample handling, sensitivity, and time. We identified 105 fmol of Bacillus subtilis SecA and 100 approximately 500 fmol of standard proteins. We also analyzed the submembrane protein fraction solubilized by 1% n-dodecyl-beta-D-maltoside from B. subtilis membranes after separation by 2-D PAGE, and identified 116 protein spots. This method can detect proteins at the 10 approximately 50 fmol level by pooling more than ten identical electroblotted protein spots.