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121.
Binding and crosslinking of 125I-labeled recombinant human tumor necrosis factor to cell surface receptors 总被引:6,自引:0,他引:6
Highly purified recombinant human tumor necrosis factor (TNF) (molecular mass determined as 17 kilodaltons (kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as 36 kDa by Sephadex G-100 gel chromatography) was labeled with 125I to a specific activity of 5 microCi/micrograms without appreciable loss of activity. The binding of 125I-TNF to eighteen human and twelve animal cell lines was examined. The binding varied considerably among different cell lines. In most cell lines, the binding was inhibited up to greater than 90% by the addition of a 100-fold excess of unlabeled TNF. Some human and mouse cell lines showed no significant binding above background levels, suggesting that these cell lines had no receptors for TNF. Among the TNF receptor-positive cell lines, there was no direct correlation between the level of specific TNF binding and the level of sensitivity to the cytotoxic or cytostatic effect of TNF. Some cell lines were sensitive to TNF, whereas others were not affected at all by TNF. The TNF receptor-negative cell lines were also resistant to TNF. Therefore, although the existence of TNF receptor seems to be necessary, it does not alone determine cellular sensitivity to TNF. Scatchard analysis of the binding data revealed that human HeLa S3 and THP-1 had about 50,000 and 10,000 receptors/cell with a dissociation constant (KD) of 0.3-0.5 nM, respectively. Similarly, mouse L-929 and L-M cells had about 5,000 receptors/cell with KD of 3-5 nM. 125I-TNF bound to HeLa S3 cells was rapidly internalized at 37 degrees C, presumably by receptor-mediated endocytosis, and degraded to acid-soluble products. The turnover of TNF receptors on HeLA S3 cells seemed to be rapid, since the level of specific binding quickly decreased after treatment with 100 micrograms/ml of cycloheximide at 37 degrees C with a half-life of about 1.5 h. The crosslinking of the cell-bound 125I-TNF with the use of disuccinimidyl suberate yielded a complex of 105 kDa for HeLa S3 and THP-1 cells, and a complex of 100 kDa for U937 cells. The crosslinking was completely inhibited by the addition of a 100-fold excess of unlabeled TNF. Assuming that the complex was due to a one-to-one association of the dimeric form of TNF (34 kDa) with the receptor, we estimated the molecular size of the human TNF receptor to be 71 kDa for HeLa S3 and THP-1, and 66 kDa for U937. 相似文献
122.
Takuya Fujimoto Mamoru Tobisu Noriko Konishi Masaki Kawamura Norio Tada Terufumi Takagi Keiji Kubo 《Bioorganic & medicinal chemistry》2009,17(23):7993-8002
We have recently discovered imidazo[1,5-c]imidazol-3-one derivative 1 as a potent, selective, and orally bioavailable factor Xa (FXa) inhibitor. In this study, we have synthesized metabolites of 1 and evaluated their biological activities. As a result, we identified the active metabolites S-5 and 6 with a potent FXa inhibitory activity comparable to 1 and a favorable pharmacokinetic profile in monkeys. 相似文献
123.
We have amplified two cDNAs, coding for creatine kinases (CKs), from the skeletal muscle of sperm whale Physeter macrocephalus by PCR, and cloned these cDNAs into pMAL plasmid. These are the first CK cDNA and deduced amino acid sequences from cetaceans
to be reported. One of the two amino acid sequences is a cytoplasmic, muscle-type isoform (MCK), while the other was identified
as a sarcomeric, mitochondrial isoform (sMiCK) that included a mitochondrial targeting peptide. The amino acid sequences of
sperm whale MCK and sMiCK showed 94–96% sequence identity with corresponding isoforms of mammalian CKs, and all of the key
residues necessary for CK function were conserved. The phylogenetic analyses of vertebrate CKs with three independent methods
(neighbor-joining, maximum-likelihood and Bayes) supported the clustering of sperm whale MCK with Bos and Sus MCKs, in agreement with the contemporary view that these groups are closely related. Sperm whale MCK and sMiCK were expressed
in Escherichia coli as a fusion protein with maltose-binding protein, and the kinetic constants (K
m, K
d and k
cat) were determined for the forward reaction. Comparison of kinetic constants with those of human and mouse CKs indicated that
sperm whale MCK has a comparable affinity for creatine (K
mCr = 9.38 mM) to that of human MCK, and the sMiCK has two times higher affinity for creatine than the human enzyme. Both the
MCK and sMiCK of sperm whale display a synergistic substrate binding (K
d
/K
m = 3.1–7.8) like those of other mammalian CKs. 相似文献
124.
Leoratti FM Trevelin SC Cunha FQ Rocha BC Costa PA Gravina HD Tada MS Pereira DB Golenbock DT Antonelli LR Gazzinelli RT 《PLoS neglected tropical diseases》2012,6(6):e1710
Background
The activation of innate immune responses by Plasmodium vivax results in activation of effector cells and an excessive production of pro-inflammatory cytokines that may culminate in deleterious effects. Here, we examined the activation and function of neutrophils during acute episodes of malaria.Materials and Methods
Blood samples were collected from P. vivax-infected patients at admission (day 0) and 30–45 days after treatment with chloroquine and primaquine. Expression of activation markers and cytokine levels produced by highly purified monocytes and neutrophils were measured by the Cytometric Bead Assay. Phagocytic activity, superoxide production, chemotaxis and the presence of G protein-coupled receptor (GRK2) were also evaluated in neutrophils from malaria patients.Principal Findings
Both monocytes and neutrophils from P. vivax-infected patients were highly activated. While monocytes were found to be the main source of cytokines in response to TLR ligands, neutrophils showed enhanced phagocytic activity and superoxide production. Interestingly, neutrophils from the malaria patients expressed high levels of GRK2, low levels of CXCR2, and displayed impaired chemotaxis towards IL-8 (CXCL8).Conclusion
Activated neutrophils from malaria patients are a poor source of pro-inflammatory cytokines and display reduced chemotactic activity, suggesting a possible mechanism for an enhanced susceptibility to secondary bacterial infection during malaria. 相似文献125.
Enhancement of VOC without Loss of JSC in Organic Solar Cells by Modification of Donor/Acceptor Interfaces 下载免费PDF全文
Yufei Zhong Akira Tada Seiichiro Izawa Kazuhito Hashimoto Keisuke Tajima 《Liver Transplantation》2014,4(5)
Organic solar cells (OSCs) are promising low‐cost devices for generating electricity. In addition to fill factor, the short circuit current density (JSC) and the open circuit voltage (VOC) are two key factors that have critical influence on the device performance. The energy levels of the donor and acceptor materials are crucial for achieving a high JSC and VOC. However, the interfacial structures between the organic materials substantially affect the JSC and VOC through the energy of the charge transfer (CT) states and the charge separation and recombination reaction kinetics. Here, it is reported that separating the donor and acceptor layer in bilayer OSCs with a thin insulating layer increases the energy of the CT state by weakening the Coulomb interaction at the interface and this also suppresses photoinduced CT and recombination. Although these effects usually increase VOC and decrease JSC, the trade‐off is avoided by doping the insulating layer with a dye to utilize the energy transfer process. The increase in VOC without the reduction in JSC enhances the conversion efficiency of the OSCs by 30%. 相似文献
126.
Watanabe S Tada M Aizawa T Yoshida M Sugaya T Taguchi M Kouno T Nakamura T Mizuguchi M Demura M Hayakawa Y Kawano K 《Protein and peptide letters》2006,13(8):815-822
GBP, a small insect cytokine isolated from lepidopterans, has a variety of functions. We constructed a series of mutants focusing on the unstructured N-terminal residues of GBP by acetylation, deletion, and elongation in order to investigate the interaction between GBP and its receptor in plasmatocytes. The 1H NMR spectra showed no significant changes in the tertiary structures of these peptides, which indicated that all the mutants maintained their core beta-sheet structures. The deletion and acetylated mutants, 2-25GBP, Ac2-25GBP, and AcGBP, lost their activity. 2-25GBP was the strongest antagonist, while Ac2-25GBP and AcGBP were moderate. In contrast, the elongated mutants, (-1R)GBP, (-1A)GBP, and (-2G,-1R)GBP maintained their plasmatocyte-spreading activity. These results demonstrate the importance of the GBP N-terminal charged amine and length of N-terminal GBP-peptide backbone for plasmatocyte-spreading activity. Next, we analyzed other mutant peptides, 1-25(N2A)GBP and 2-25(N2A)GBP, focusing on Asn2. Surprisingly, 2-25(N2A)GBP had slight plasmatocyte-spreading activity, whereas 2-25GBP lost its activity. Finally, substituted mutant, F3AGBP, had neither plasmatocyte-spreading activity nor antagonistic activity. These results demonstrate the function of each N-terminal residue in the interaction between GBP and its receptor in plasmatocytes. 相似文献
127.
Several strategies for mapping ventricular outflow tract tachycardia have been reported as useful indices for differentiating between those originating from the right and the left side. Recently, tissue tracking imaging (TTI) has been demonstrated as a novel non-invasive modality for identifying the origin of outflow tract tachycardias. Tissue tracking imaging is an ultrasonographic technique that measures the myocardial motion amplitude towards the transducer in each region during systole, identifying regional myocardial displacement on the basis of myocardial velocities using color Doppler myocardial imaging principles. In this technique, the origin of the arrhythmia could be recognized as the site where the earliest color-coded signal (ECCS) appeared on the myocardium at the onset of the systole. In preliminary studies this modality was found to be useful in differentiating outflow tract ventricular tachycardias. ECCS was always found below or at the level of the pulmonary valve in all arrhythmias which could be ablated from the right ventricular outflow tract, while in those where the ECCS was above and close to the pulmonary valve it could be ablated from the left sinus of valsalva. These results indicate that TTI can provide detailed and accurate information on the arrhythmia origin of outflow tract tachycardia and may be useful for differentiating between an outflow tract tachycardia originating from the LV epicardium remote from the LSV and that from the LSV. Newer advances in echocardiographic technologies like high resolution, high frame rate real time three dimensional echocardiography with speckle tracking may further improve the precise localization of arrhythmias in the future. 相似文献
128.
129.
Johnson JA Hemnes AR Perrien DS Schuster M Robinson LJ Gladson S Loibner H Bai S Blackwell TR Tada Y Harral JW Talati M Lane KB Fagan KA West J 《American journal of physiology. Lung cellular and molecular physiology》2012,302(5):L474-L484
The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease. 相似文献
130.
Experiments were carried out in vitro to investigate whether the sera of several animals as well as albumins and peptides might act as attractants for larvae of Strongyloides ratti. Samples of sera from several mammal species were dialysed and the aliquots were further centrifuged using ultrafiltration cartridges to remove any remaining small molecules. Additional test substances included commercially obtained ovalbumin, rat and bovine serum albumins, polypeptides such as peptone, tryptone and tryptose, amino nitrogens, monosaccharides, and reduced glutathione (triaminopeptide). Larvae were strongly attracted to the dialysed mammalian sera, which mainly consisted of serum albumin and globulins. Ov- and serum albumins, and polypeptides also acted as attractants. On the other hand, reduced glutathione, 16 kinds of amino acids and four kinds of monosaccharides did not attract this nematode. 相似文献