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601.
602.
M Sakuma Y Shirai KI Yoshino M Kuramasu T Nakamura T Yanagita K Mizuno I Hide Y Nakata N Saito 《Molecular biology of the cell》2012,23(18):3707-3721
Using specific inhibitors, kinase-negative mutants, and small interfering RNA against protein kinase Cα (PKCα) or PKCβI, we find that PKCβI positively regulates degranulation in rat basophilic leukemia-2H3 cells, whereas PKCα negatively regulates degranulation. Mass spectrometric and mutagenic analyses reveal that PKCα phosphorylates cofilin at Ser-23 and/or Ser-24 during degranulation. Overexpression of a nonphosphorylatable form (S23,24A), but not that of a mutant-mimicking phosphorylated form (S23,24E), increases degranulation. Furthermore, the S23,24A mutant binds to F-actin and retains its depolymerizing and/or cleavage activity; conversely, the S23,24E mutant is unable to sever actin filaments, resulting in F-actin polymerization. In addition, the S23,24E mutant preferentially binds to the 14-3-3ζ protein. Fluorescence-activated cell sorting analysis with fluorescein isothiocyanate-phalloidin and simultaneous observation of degranulation, PKC translocation, and actin polymerization reveals that during degranulation, actin polymerization is dependent on PKCα activity. These results indicate that a novel PKCα-mediated phosphorylation event regulates cofilin by inhibiting its ability to depolymerize F-actin and bind to 14-3-3ζ, thereby promoting F-actin polymerization, which is necessary for cessation of degranulation. 相似文献
603.
Kazufumi Honda Takuji Okusaka Klaus Felix Shoji Nakamori Naohiro Sata Hideo Nagai Tatsuya Ioka Akihiko Tsuchida Takeshi Shimahara Masashi Shimahara Yohichi Yasunami Hideya Kuwabara Tomohiro Sakuma Yoshihiko Otsuka Norihito Ota Miki Shitashige Tomoo Kosuge Markus W. Büchler Tesshi Yamada 《PloS one》2012,7(10)
Background
Among the more common human malignancies, invasive ductal carcinoma of the pancreas has the worst prognosis. The poor outcome seems to be attributable to difficulty in early detection.Methods
We compared the plasma protein profiles of 112 pancreatic cancer patients with those of 103 sex- and age-matched healthy controls (Cohort 1) using a newly developed matrix-assisted laser desorption/ionization (oMALDI) QqTOF (quadrupole time-of-flight) mass spectrometry (MS) system.Results
We found that hemi-truncated apolipoprotein AII dimer (ApoAII-2; 17252 m/z), unglycosylated apolipoprotein CIII (ApoCIII-0; 8766 m/z), and their summed value were significantly decreased in the pancreatic cancer patients [P = 1.36×10−21, P = 4.35×10−14, and P = 1.83×10−24 (Mann-Whitney U-test); area-under-curve values of 0.877, 0.798, and 0.903, respectively]. The significance was further validated in a total of 1099 plasma/serum samples, consisting of 2 retrospective cohorts [Cohort 2 (n = 103) and Cohort 3 (n = 163)] and a prospective cohort [Cohort 4 (n = 833)] collected from 8 medical institutions in Japan and Germany.Conclusions
We have constructed a robust quantitative MS profiling system and used it to validate alterations of modified apolipoproteins in multiple cohorts of patients with pancreatic cancer. 相似文献604.
Production of scFv-conjugated affinity silk powder by transgenic silkworm technology 总被引:1,自引:0,他引:1
Sato M Kojima K Sakuma C Murakami M Aratani E Takenouchi T Tamada Y Kitani H 《PloS one》2012,7(4):e34632
Bombyx mori (silkworm) silk proteins are being utilized as unique biomaterials for medical applications. Chemical modification or post-conjugation of bioactive ligands expand the applicability of silk proteins; however, the processes are elaborate and costly. In this study, we used transgenic silkworm technology to develop single-chain variable fragment (scFv)-conjugated silk fibroin. The cocoons of the transgenic silkworm contain fibroin L-chain linked with scFv as a fusion protein. After dissolving the cocoons in lithium bromide, the silk solution was dialyzed, concentrated, freeze-dried, and crushed into powder. Immunoprecipitation analyses demonstrate that the scFv domain retains its specific binding activity to the target molecule after multiple processing steps. These results strongly suggest the promise of scFv-conjugated silk fibroin as an alternative affinity reagent, which can be manufactured using transgenic silkworm technology at lower cost than traditional affinity carriers. 相似文献
605.
Estrogen receptor α (ERα) can be phosphorylated at various residues, one of which is serine 212 in the DNA binding domain. The majority of human nuclear receptors conserves, as a motif, this serine residue within their DNA binding domain. Among these nuclear receptors, phosphorylation of the corresponding threonine 38 in the nuclear receptor CAR is essential for determining its activity [9]. Here, we have investigated the role of phosphorylated serine 212 in the regulation of ERα activity by comparing it with serine 236, another potential phosphorylation site within the DNA binding domain, and demonstrated that phosphorylation of serine 212 confers upon ERα a distinct activity regulating gene expression in Huh-7 cells. In Western blot analysis, wild type ERα and mutants ERα S212A, ERα S212D, ERα S236A and ERα S236D were equally expressed in the nucleus, thus indicating that phosphorylation does not determine nuclear localization of ERα. ERα S212D, but not ERα S236D, retained its capability of activating an ERE-reporter gene in luciferase assays. Similar results were also obtained for human ERβ; the ERβ S176D mutant retained its trans-activation activity, but the ERβ S200D mutant did not. cDNA microarray and Ingenuity Pathway Analysis, employed on Huh-7 cells ectopically expressing either ERα S212A or ERα S212D, revealed that phosphorylation of serine 212 enabled ERα to regulate a unique set of genes and cellular functions. 相似文献
606.
607.
Matsumoto Y Kawamura Y Nakai H Sugata K Yoshikawa A Ihira M Ohashi M Kato T Yoshikawa T 《Microbiology and immunology》2012,56(9):651-655
Severe pneumonia and leukocytosis are characteristic, frequently observed, clinical findings in pediatric patients with pandemic A/H1N1/2009 influenza virus infection. The aim of this study was to elucidate the role of cytokines and chemokines in complicating pneumonia and leukocytosis in patients with pandemic A/H1N1/2009 influenza virus infection. Forty‐seven patients with pandemic A/H1N1/2009 influenza virus infection were enrolled in this study. Expression of interleukin (IL)‐10 (P = 0.027) and IL‐5 (P = 0.014) was significantly greater in patients with pneumonia than in those without pneumonia. Additionally, serum concentrations of interferon‐γ (P = 0.009), tumor necrosis factor‐α (P = 0.01), IL‐4 (P = 0.024), and IL‐2 (P = 0.012) were significantly lower in pneumonia patients with neutrophilic leukocytosis than in those without neutrophilic leukocytosis. Of the five serum chemokine concentrations assessed, only IL‐8 was significantly lower in pneumonia patients with neutrophilic leukocytosis than in those without leukocytosis (P = 0.001). These cytokines and chemokines may play important roles in the pathogenesis of childhood pneumonia associated with A/H1N1/2009 influenza virus infection. 相似文献
608.
Takemoto Y Takanari H Honjo H Ueda N Harada M Kato S Yamazaki M Sakuma I Opthof T Kodama I Kamiya K 《American journal of physiology. Heart and circulatory physiology》2012,303(5):H578-H586
Spiral-wave (SW) reentry is a major organizing principle of ventricular tachycardia/fibrillation (VT/VF). We tested a hypothesis that pharmacological modification of gap junction (GJ) conductance affects the stability of SW reentry in a two-dimensional (2D) epicardial ventricular muscle layer prepared by endocardial cryoablation of Langendorff-perfused rabbit hearts. Action potential signals were recorded and analyzed by high-resolution optical mapping. Carbenoxolone (CBX; 30 μM) and rotigaptide (RG, 0.1 μM) were used to inhibit and enhance GJ coupling, respectively. CBX decreased the space constant (λ) by 36%, whereas RG increased it by 22-24% (n = 5; P < 0.01). During centrifugal propagation, there was a linear relationship between the wavefront curvature (κ) and local conduction velocity (LCV): LCV = LCV(0) - D·κ (D, diffusion coefficient; LCV(0), LCV at κ = 0). CBX decreased LCV(0) and D by 27 ± 3 and 57 ± 3%, respectively (n = 5; P < 0.01). RG increased LCV(0) and D by 18 ± 3 and 54 ± 5%, respectively (n = 5, P < 0.01). The regression lines with and without RG crossed, resulting in a paradoxical decrease of LCV with RG at κ > ~60 cm(-1). SW reentry induced after CBX was stable, and the incidence of sustained VTs (>30 s) increased from 38 ± 4 to 85 ± 4% after CBX (n = 18; P < 0.01). SW reentry induced after RG was characterized by decremental conduction near the rotation center, prominent drift and self-termination by collision with the anatomical boundaries, and the incidence of sustained VTs decreased from 40 ± 5 to 17 ± 6% after RG (n = 13; P < 0.05). These results suggest that decreased intercellular coupling stabilizes SW reentry in 2D cardiac muscle, whereas increased coupling facilitates its early self-termination. 相似文献
609.
Keisuke Yuzu Naoki Yamamoto Masahiro Noji Masatomo So Yuji Goto Tetsushi Iwasaki Motonari Tsubaki Eri Chatani 《Biophysical journal》2021,120(2):284-295
Amyloid fibrils are aberrant protein aggregates associated with various amyloidoses and neurodegenerative diseases. It is recently indicated that structural diversity of amyloid fibrils often results in different pathological phenotypes, including cytotoxicity and infectivity. The diverse structures are predicted to propagate by seed-dependent growth, which is one of the characteristic properties of amyloid fibrils. However, much remains unknown regarding how exactly the amyloid structures are inherited to subsequent generations by seeding reaction. Here, we investigated the behaviors of self- and cross-seeding of amyloid fibrils of human and bovine insulin in terms of thioflavin T fluorescence, morphology, secondary structure, and iodine staining. Insulin amyloid fibrils exhibited different structures, depending on species, each of which replicated in self-seeding. In contrast, gradual structural changes were observed in cross-seeding, and a new type of amyloid structure with distinct morphology and cytotoxicity was formed when human insulin was seeded with bovine insulin seeds. Remarkably, iodine staining tracked changes in amyloid structure sensitively, and singular value decomposition analysis of the ultraviolet-visible absorption spectra of the fibril-bound iodine has revealed the presence of one or more intermediate metastable states during the structural changes. From these findings, we propose a propagation scheme with multistep structural changes in cross-seeding between two heterologous proteins, which is accounted for as a consequence of the rugged energy landscape of amyloid formation. 相似文献
610.