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Cystic fibrosis (CF), the most common lethal inherited disorder caused by mutation in the gene encoding the CF transmembrane regulator (CFTR), is characterized by chronic inflammation that ultimately leads to death from respiratory failure. In CF patients, up-regulation of toll-like receptor-2 (TLR2), a pattern recognition receptor that senses CF-pathogenic bacteria Staphylococcus aureus peptidoglycan (PGN), in airway epithelial cells is observed, and enhanced proinflammatory responses towards PGN may result in detrimental effects in CF patients. Here, we showed that curcumin, a well known anti-inflammatory agent derived from the curry spice turmeric, inhibits TLR2 expression in CF bronchial epithelial cell line, CFBE41o- cells. Strong suppression of TLR2 gene and protein expression was observed at more than 40 µM of curcumin treatment in CFBE41o- cells. Consistent with decreased expression of TLR2, PGN-dependent interleukin-8 (IL-8) gene up-regulation was markedly reduced by 40 µM of curcumin treatment. Strong reductions of TLR2 gene expression and function were also observed in primary human CF bronchial epithelial cells, but not in human non-CF primary cells. Interestingly, curcumin treatment decreased nuclear expression of transcription factor specificity protein 1 (SP1), a factor that is critical for increased basal TLR2 expression in CF cell line and primary cells. Finally, curcumin-dependent SP1 reduction was diminished by anti-oxidant N-acetylcystein (NAC) and proteasomal inhibitor MG-132, suggesting the crucial roles of oxidative and proteasomal degradation pathways. Taken together, our study shows that curcumin down-regulates TLR2 gene expression and function in CF bronchial epithelial cells possibly by accelerating SP1 degradation via an oxidative process.

We have been developing a simulation program for use with soil–wheel interaction problems by coupling Finite Element Method (FEM) and Discrete Element Method (DEM) for which a wheel is modeled by FEM and soil is expressed by DEM. Previous two-dimensional FE–DEM was updated to analyze the tractive performance of a flexible elastic wheel by introducing a new algorithm learned from the PID-controller model. In an elastic wheel model, four structural parts were defined using FEM: the wheel rim, intermediate part, surface layer, and wheel lugs. The wheel rigidity was controlled by varying the Young’s Modulus of the intermediate part. The tractive performance of two elastic wheels with lugs for planetary rovers of the European Space Agency was analyzed. Numerical results were compared with experimentally obtained results collected at DLR Bremen, Germany. The FE–DEM result was confirmed to depict similar behaviors of tractive performance such as gross tractive effort, net traction, running resistance, and wheel sinkage, as in the results of experiments. Moreover, the tractive performance of elastic wheels on Mars was predicted using FE–DEM. Results clarified that no significant difference of net traction exists between the two wheels.

Airway mucus hyperproduction and fluid imbalance are important hallmarks of cystic fibrosis (CF), the most common life-shortening genetic disorder in Caucasians. Dysregulated expression and/or function of airway ion transporters, including cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC), have been implicated as causes of CF-associated mucus hypersecretory phenotype. However, the contributory roles of other substances and transporters in the regulation of CF airway pathogenesis remain unelucidated. Here, we identified a novel connection between CFTR/ENaC expression and the intracellular Zn2 + concentration in the regulation of MUC5AC, a major secreted mucin that is highly expressed in CF airway. CFTR-defective and ENaC-hyperactive airway epithelial cells specifically and highly expressed a unique, alternative splice isoform of the zinc importer ZIP2/SLC39A2 (ΔC-ZIP2), which lacks the C-terminal domain. Importantly, ΔC-ZIP2 levels correlated inversely with wild-type ZIP2 and intracellular Zn2 + levels. Moreover, the splice switch to ΔC-ZIP2 as well as decreased expression of other ZIPs caused zinc deficiency, which is sufficient for induction of MUC5AC; while ΔC-ZIP2 expression per se induced ENaC expression and function. Thus, our findings demonstrate that the novel splicing switch contributes to CF lung pathology via the novel interplay of CFTR, ENaC, and ZIP2 transporters., Highlights • Zinc deficiency is a common feature in both CFTR-defective (CF) and ENaC-hyperactive (CF-like) airway epithelial cells. • A splice switch from WT-ZIP2 to ΔC-ZIP2 as well as other ZIPs down-regulation caused zinc deficiency in CF and CF-like cells. • Lower intracellular Zn2 + levels contributed to CF-associated MUC5AC hypersecretion in airway epithelial cells. The role of zinc in the pathogenesis of CF lung disease is not well understood. We utilized human CF patient-derived cell lines and primary cells as well as murine CF model, and identified zinc deficiency as a common characteristic in CF models. Down-regulation of several zinc importers (ZIPs) in CF cells caused zinc deficiency, which is sufficient for induction of MUC5AC, a major secreted mucin that exacerbates CF pathogenesis. Especially, strong contribution of ΔC-ZIP2, a novel ZIP2 splice isoform, in the regulation of CF-associated MUC5AC hypersecretion was clearly demonstrated. The study refined the importance of zinc in airway homeostasis.

Single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) is one of the immunoglobulin-like membrane proteins that is crucial for negative regulation of toll-like receptor 4 (TLR4) and interleukin-1 receptor. Despite the importance of understanding its expression and function, knowledge is limited on the regulatory mechanism in the epithelial tissues, such as the liver, lung, and gut, where its predominant expression is originally described. Here, we found expression of SIGIRR in non-epithelial innate immune cells, including primary peripheral blood monocytes, polymorphonuclear neutrophils, monocytic RAW264 cells, and neutrophilic-differentiated HL-60 cells. Consistent with previous findings in epithelial tissues, SIGIRR gene and protein expression were also down-regulated by LPS treatment in a time-dependent manner in primary blood monocytes and polymorphonuclear neutrophils. A reduction was also observed in RAW264 and differentiated HL-60 cells. Notably, exogenous introduction of the dominant negative form of TLR4 and siRNA of p38 resulted in inhibition of LPS-induced SIGIRR down-regulation, whereas treatment with p38 activator anisomycin showed a dose-dependent decrease in SIGIRR expression, suggesting TLR4-p38 signal as a critical pathway for LPS-induced SIGIRR down-regulation. Finally, reporter gene and chromatin immunoprecipitation assays demonstrated that Sp1 is a key factor that directly binds to the proximal promoter of SIGIRR gene and consequently regulates basal SIGIRR expression, which is negatively regulated by the LPS-dependent TLR4-p38 pathway. In summary, the data precisely demonstrate how LPS down-regulates SIGIRR expression and provide a role of LPS signal that counteracts Sp1-dependent basal promoter activation of SIGIRR gene via TLR4-p38 pathway in non-epithelial innate immune cells.

Protease-antiprotease imbalance and oxidative stress are considered to be major pathophysiological hallmarks of severe obstructive lung diseases including chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), but limited information is available on their direct roles in the regulation of pulmonary phenotypes. Here, we utilized βENaC-transgenic (Tg) mice, the previously established mouse model of severe obstructive lung diseases, to produce lower-mortality but pathophysiologically highly useful mouse model by backcrossing the original line with C57/BL6J mice. C57/BL6J-βENaC-Tg mice showed higher survival rates and key pulmonary abnormalities of COPD/CF, including mucous hypersecretion, inflammatory and emphysematous phenotypes and pulmonary dysfunction. DNA microarray analysis confirmed that protease- and oxidative stress-dependent pathways are activated in the lung tissue of C57/BL6J-βENaC-Tg mice. Treatments of C57/BL6J-βENaC-Tg mice with a serine protease inhibitor ONO-3403, a derivative of camostat methylate (CM), but not CM, and with an anti-oxidant N-acetylcystein significantly improved pulmonary emphysema and dysfunction. Moreover, depletion of a murine endogenous antioxidant vitamin C (VC), by genetic disruption of VC-synthesizing enzyme SMP30 in C57/BL6J-βENaC-Tg mice, exaggerated pulmonary phenotypes. Thus, these assessments clarified that protease-antiprotease imbalance and oxidative stress are critical pathways that exacerbate the pulmonary phenotypes of C57/BL6J-βENaC-Tg mice, consistent with the characteristics of human COPD/CF.

Random copolymers of poly(p-benzamide)s having a methyl-substituted tri(ethylene glycol) unit as a chiral side chain and a nonsubstituted tri(ethylene glycol) or branching alkyl unit as an achiral side chain were synthesized by copolymerization of N-substituted 4-aminobenzoic acid ester monomers with a base in the presence of an initiator. Copolymerizations of the chiral (S)-monomer with N-tri(ethylene glycol) achiral monomer and with the racemic monomer were carried out by the addition of a mixture of two monomers and an initiator to a solution of a base all at once, affording the corresponding random copolymers. On the other hand, random copolymerization of the chiral monomer with monomer having an achiral branching alkyl side chain required dropwise addition of the achiral monomer to a mixture of the chiral monomer, the initiator, and the base. These copolymers formed helical structures, but analysis of the CD spectra indicated the absence of cooperativity between the monomer units along the copolymers. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011

Toll-like receptor-2 (TLR2) is a pattern recognition receptor that senses many types of bacterial components and activates signaling pathways that induce inflammatory cytokines. A hyperresponsiveness to pathogens caused by increased expression of TLR2 triggers exaggeration of some inflammatory diseases. Here, we showed that curcumin, a well-known anti-inflammatory agent derived from the curry spice turmeric, inhibits TLR2 expression in various TLR2-expressing innate immune cell lines such as monocytic THP-1 cells, neutrophilic-differentiated HL-60 cells. Strong suppression of TLR2 gene expression was specifically observed at concentrations of curcumin in the range 40-100muM. Consistent with decreased expression of TLR2 mRNA, protein expression and ligand-responsiveness of TLR2 were markedly reduced by curcumin treatment. Moreover, curcumin-dependent down-regulation of TLR2 expression and function was also observed in primary peripheral blood monocytes (MC) and polymorphonuclear neutrophils (PMN). Finally, we determined the importance of curcumin-dependent radical generation for the suppressive effect of curcumin on TLR2 expression. Thus, our data demonstrate that curcumin inhibits TLR2 gene expression and function possibly via an oxidative process.

For this study, we applied a finite element-discrete element method (FE-DEM) to predict the tractive performance of an elastic iron wheel for mobile robots running on sand. We developed our program, where FEM is applied to a wheel and a subsurface soil layer, and where DEM is applied to the surface soil layer where the contact interaction between the tire and soil is greatest. The developed FE-DEM tool was found to have sufficient accuracy to predict the performance of an elastic wheel running on deformable soil. Moreover, parametric analysis was applied for those parameters of the rigidity of the tread part of the wheel, the surface soil layer thickness, and the lug height. The effect of rigidity of the tread part was negligible for 1-5 MPa. The surface soil layer thickness was found to have no significant effect on tractive performance. The lug height showed a strong effect on the tractive performance of the wheel, but the effect on net traction seemed to be degraded concomitantly with the increase in lug height.

The effect of amrinone on gas exchange and oxygen metabolism were evaluated in eight patients after cardiovascular surgery for cardiopulmonary bypass.Amrinone was adminisered without bolus infusion (infusion rate: 10μg·kg-1·min-1). Data were obtained 30 minutes and 90 minutes following amrinone administration and were compared to the control data. Values are described as mean±SD.Non-nutrient blood flow (NNBF) and conventional parameters for oxygen metabolism were used to evaluate regional blood flow. NNBF was calculated with Farrell's formula.Both the respiratory index (from 1.4±1.1 to 2.1±1.2 (30min), 2.4±1.5L·min-1·m-2 (90min)) and pulmonary shunt (from 10.1±to 11.6±6% (30m), 12.1±5% (90min)) significantly (p

Human α1-acid glycoprotein (hAGP) in serum functions as a carrier of basic drugs. In most individuals, hAGP exists as a mixture of two genetic variants, the F1*S and A variants, which bind drugs with different selectivities. We prepared a mutant of the A variant, C149R, and showed that its drug-binding properties were indistinguishable from those of the wild type. In this study, we determined the crystal structures of this mutant hAGP alone and complexed with disopyramide (DSP), amitriptyline (AMT), and the nonspecific drug chlorpromazine (CPZ). The crystal structures revealed that the drug-binding pocket on the A variant is located within an eight-stranded β-barrel, similar to that found in the F1*S variant and other lipocalin family proteins. However, the binding region of the A variant is narrower than that of the F1*S variant. In the crystal structures of complexes with DSP and AMT, the two aromatic rings of each drug interact with Phe-49 and Phe-112 at the bottom of the binding pocket. Although the structure of CPZ is similar to those of DSP and AMT, its fused aromatic ring system, which is extended in length by the addition of a chlorine atom, appears to dictate an alternative mode of binding, which explains its nonselective binding to the F1*S and A variant hAGPs. Modeling experiments based on the co-crystal structures suggest that, in complexes of DSP, AMT, or CPZ with the F1*S variant, Phe-114 sterically hinders interactions with DSP and AMT, but not CPZ. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Kinin release from guinea pig plasma high molecular weight kininogen (HMWK) induced by various microbial and mite proteases has been demonstrated previously (Molla, A., Yamamoto, T., Akaike, T., Miyoshi, S., and Maeda, H. (1989) J. Biol. Chem. 264, 10589-10594; Maruo, K., Akaike, T., Matsumura, Y., Kohmoto, S., Inada, Y., Ono, T., Arao, T., and Maeda, H. (1991) Biochim. Biophys. Acta 1074, 62-68). In this paper, we describe the effects of various microbial and mite proteases on low molecular weight kininogen (LMWK) and HMWK from human plasma. A protease from the house dust mite Dermatophagoides farinae (Df-protease) directly liberated kinin from both LMWK and HMWK to a significant degree. The Km, kcat, and kcat/Km values for kinin generation from LMWK were 3.24 microM, 0.61 s-1, and 1.9 x 10(5) M-1 x s-1, respectively, and those for kinin generation from HMWK were 0.56 microM, 0.12 s-1, and 2.1 x 10(5) M-1 x s-1, respectively; kcat/Km values for Df-protease were comparable with that for glandular kallikrein. In contrast, microbial proteases showed only weak kinin-releasing activity from both human plasma kininogens. Four of ten different microbial proteases liberated kinin from LMWK, and only serratial 56-kDa protease released kinin from HMWK. Furthermore, Df-protease markedly inactivated the thiol protease inhibitory activity of LMWK and HMWK, whereas all microbial proteases (as well as the endogenous protease trypsin) did not affect this inhibitory activity of both kininogens from human plasma.

Unglycosylated recombinant human alpha(1)-acid glycoprotein (hAGP) variants (rF1(*)S and rA) were prepared in an E. coli expression system using the Origami B strain and pET-3c vector. Thioredoxin was co-expressed to promote the appropriate folding of hAGP. SDS-PAGE under reducing conditions showed that rF1(*)S and rA migrate as single bands after purification. However, several bands derived from rA were observed under non-reducing conditions because of the high reactivity of a free cystein residue (C149). We therefore prepared a mutant of A variant (C149R-A), and confirmed that this mutant maintained homogeneity. Circular dichroism and intrinsic tryptophan fluorescence spectroscopic analyses indicated that rF1(*)S and C149R-A have almost the same conformational structures as F1(*)S and A purified from serum. Ligand binding experiments using propranolol as a F1(*)S ligand and disopyramide as an A specific ligand indicated that the capacity of rF1(*)S and C149R-A is equivalent to those ligands as well as F1(*)S and A from serum. These results suggest that the oligosaccharide moieties of hAGP have negligible effects on the structural and ligand binding properties of hAGP. Thus, rF1(*)S and C149R-A promise to be useful in studies on the drug binding sites of hAGP.

A newly developed sub-resolution assist feature (SRAF) placement technique with two-dimensional transmission cross coefficient (2D-TCC) is described in this paper. In SRAF placement with 2D-TCC, Hopkins' aerial image equation with four-dimensional TCC is decomposed into the sum of Fourier transforms of diffracted light weighted by 2D-TCC, introducing an approximated aerial image so as to place SRAFs into a given reticle layout. SRAFs are placed at peak positions of the approximated aerial image for enhanced resolution. Since the approximated aerial image can handle the full optical model, SRAFs can be automatically optimized to the given optical condition to generate the optimized reticle. The validity of this technique was confirmed by experiment using a Canon FPA6000-ES6a, 248 nm with a numerical aperture (NA) of 0.86. A binary reticle optimized by this technique with mild off-axis illumination was used in the experiment. Both isolated and dense 100 nm contacts (k 1 = 0.35) were simultaneously resolved with the aid of this technique.

A solution tool to optimize exposure tool functions has been developed. Shown are examples of pattern matching, maximizing ED-window or NILS considering several patterns simultaneously, and so on. From these results, the following conclusions have been derived. Pattern matching whose accuracy less than 1nm can be attained flexibly by tuning illumination. Ideal illumination for hole patterns through pitches are shown and the results are sensitive to setting of exposure latitude of ED-window. Evaluation conditions such as evaluated locations and their numbers have impact on the optimization results. An optimized illumination of a device pattern varies according to k1 factor. And it is important to apply OPC during illumination optimization in case of optimizing several patterns. A model of resist simulation created by an exposure condition should be available for various exposure conditions during optimization. For this purpose an image log slope and a pattern curvature have strong impact among various characteristics of optical image.

We investigated a relationship within zonal differences in DNA synthesis and in transglutaminase (TGase) activity between perivenous versus periportal regions of regenerating rat liver. Using the digitonin/collagenase perfusion technique, hepatocyte subpopulations were isolated from each region at various time points after partial hepatectomy. The amounts of DNA synthesis as well as the levels of TGase mRNA and activity in each subpopulation were measured. Although increased DNA synthesis was observed in both subpopulations with a peak at 24 h after partial hepatectomy, the amount of DNA synthesis in periportal hepatocytes (PPH) was twice as much as that in perivenous hepatocytes (PVH). In PVH, TGase activity peaked at 24 h after partial hepatectomy with a preceding increase in its mRNA expression at 12 h, whereas TGase activity in PPH at 24 h was one-half of that in PVH. As TGase is known to have a growth-arresting activity, our data indicate that relatively higher TGase activity in PVH at 24 h after partial hepatectomy might correlate with relatively lower DNA synthesis in this region compared to periportal region.