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931.
Lee HK Lee BH Dutta NK Seok SH Baek MW Lee HY Kim DJ Na YR Noh KJ Park SH Kariwa H Nakauchi M Mai le Q Heo SJ Park JH 《Journal of microbiology and biotechnology》2008,18(10):1717-1721
Severe acute respiratory syndrome (SARS) is a lifethreatening emerging respiratory disease caused by the coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is highly antigenic and may be a suitable candidate for diagnostic applications. We constructed truncated recombinant N proteins (N1 [1-422 aa], N2 [1- 109 aa], and N3 [110-422 aa]) and determined their antigenicity by Western blotting using convalescent SARS serum. The recombinants containing N1 and N3 reacted with convalescent SARS serum in Western blotting. However, the recombinant with N2 did not. In ELISA using N1 or N3 as the antigens, positive results were observed in 10 of 10 (100%) SARS-CoV-positive human sera. None of 50 healthy sera gave positive results in either assay. These data indicate that the ELISA using N1 or N3 has high sensitivity and specificity. These results suggest that the middle or C-terminal region of the SARS N protein is important for eliciting antibodies against SARS-CoV during the immune response, and ELISA reactions using N1 or N3 may be a valuable tool for SARS diagnosis. 相似文献
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936.
Shibasaki H Nakayama H Furuta T Kasuya Y Tsuchiya M Soejima A Yamada A Nagasawa T 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2008,870(2):164-169
Individual variability of the pharmacokinetics of prednisolone based on the unbound concentration in plasma is of significant clinical consideration. The unbound concentrations of prednisolone were measured in 10 patients with nephrotic syndrome, two patients with systemic lupus erythematosus, and one patient with dermatomyositis by examining protein bindings of prednisolone on one or more occasions during prednisolone treatment. In this study, plasma concentrations of prednisolone, prednisone, cortisol, and cortisone were simultaneously analyzed by GC-MS by using stable isotope-labeled internal standards. Equilibrium dialysis was employed to accurately estimate the unbound fractions of prednisolone in plasma. The unbound fraction of prednisolone changed depending on plasma total prednisolone concentration and plasma albumin concentration. The unbound fraction of prednisolone (Y) is calculated: Y=(-0.0101x' + 0.0736) x + 10.23, where x' is the plasma albumin concentration and x is the total prednisolone concentration. The estimated concentrations of unbound prednisolone by using the above equation were in good agreement with the measured concentrations of unbound prednisolone. Since the protein binding of prednisolone did not change in the presence of prednisone (114.0 ng/ml), it appeared that prednisone produced from the therapeutic dose of prednisolone did not affect the unbound fraction of prednisolone. 相似文献
937.
Post-translational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase
Ulrich M Petre A Youhnovski N Prömm F Schirle M Schumm M Pero RS Doyle A Checkel J Kita H Thiyagarajan N Acharya KR Schmid-Grendelmeier P Simon HU Schwarz H Tsutsui M Shimokawa H Bellon G Lee JJ Przybylski M Döring G 《The Journal of biological chemistry》2008,283(42):28629-28640
Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO(-/-), gp91phox(-/-), and NOS(-/-) mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils. 相似文献
938.
Taguchi H Planque S Sapparapu G Boivin S Hara M Nishiyama Y Paul S 《The Journal of biological chemistry》2008,283(52):36724-36733
Nucleophilic sites in the paired variable domains of the light and heavy chains (VL and VH domains) of Ig can catalyze peptide bond hydrolysis. Amyloid beta (Abeta)-binding Igs are under consideration for immunotherapy of Alzheimer disease. We searched for Abeta-hydrolyzing human IgV domains (IgVs) in a library containing a majority of single chain Fv clones mimicking physiological VL-VH-combining sites and minority IgV populations with nonphysiological structures generated by cloning errors. Random screening and covalent selection of phage-displayed IgVs with an electrophilic Abeta analog identified rare IgVs that hydrolyzed Abeta mainly at His14-Gln15. Inhibition of IgV catalysis and irreversible binding by an electrophilic hapten suggested a nucleophilic catalytic mechanism. Structural analysis indicated that the catalytic IgVs are nonphysiological structures, a two domain heterodimeric VL (IgVL2-t) and single domain VL clones with aberrant polypeptide tags (IgVL-t'). The IgVs hydrolyzed Abeta at rates superior to naturally occurring Igs by 3-4 orders of magnitude. Forced pairing of the single domain VL with VH or VL domains resulted in reduced Abeta hydrolysis, suggesting catalysis by the unpaired VL domain.Angstrom level amino acid displacements evident in molecular models of the two domain and unpaired VL domain clones explain alterations of catalytic activity. In view of their superior catalytic activity, the VL domain IgVs may help attain clearance of medically important antigens more efficiently than natural Igs. 相似文献
939.
Kanei-Ishii C Nomura T Takagi T Watanabe N Nakayama KI Ishii S 《The Journal of biological chemistry》2008,283(45):30540-30548
The c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling via a pathway involving TAK1 (transforming growth factor-beta-activated kinase 1), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). NLK directly binds to c-Myb, which results in the phosphorylation of c-Myb at multiple sites, and induces its ubiquitination and proteasome-dependent degradation. Here, we report that Fbxw7, the F-box protein of an SCF complex, targets c-Myb for degradation in a Wnt-1- and NLK-dependent manner. Fbxw7alpha directly binds to c-Myb via its C-terminal WD40 domain and induces the ubiquitination of c-Myb in the presence of NLK in vivo and in vitro. The c-Myb phosphorylation site mutant failed to interact with Fbxw7alpha, suggesting that the c-Myb/Fbxw7alpha interaction is enhanced by NLK phosphorylation of c-Myb. Treatment of M1 cells with Fbxw7 small interfering RNA (siRNA) rescued the Wnt-induced c-Myb degradation and also the Wnt-induced inhibition of cell proliferation. NLK bound to Cul1, a component of the SCF complex, while HIPK2 interacted with both Fbxw7alpha and Cul1, suggesting that both kinases enhance the c-Myb/SCF interaction. In contrast to c-Myb, the v-myb gene product (v-Myb) encoded by the avian myeloblastosis virus was resistant to NLK/Fbxw7alpha-induced degradation. Thus, Fbxw7 is an E3 ubiquitin ligase of c-Myb, and the increased c-Myb levels may contribute, at least partly, to transformation induced by mutation of Fbxw7. 相似文献
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