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61.
62.
A light and electron microscopic procedure for sequential double antigen localization using diaminobenzidine and benzidine dihydrochloride 总被引:4,自引:0,他引:4
A I Levey J P Bolam D B Rye A E Hallanger R M Demuth M M Mesulam B H Wainer 《The journal of histochemistry and cytochemistry》1986,34(11):1449-1457
Very few double-antigen staining methods are available that are applicable to both light and electron microscopy. The objective of this study was to develop for localization of two neural antigens simultaneously a procedure which would be sensitive, simple to perform, offer permanent reaction products, and permit correlated light and ultrastructural analysis. The method employs sequential immunoperoxidase staining without antibody elution, in which the first sequence of antibodies is visualized with 3,3'-diaminobenzidine (DAB) and the second with benzidine dihydrochloride (BDHC). The DAB reaction product (brown and diffuse) was easily distinguishable from the BDHC deposit (blue, granular, and more electron-dense) by both light and electron microscopy. The procedure was used to simultaneously localize choline acetyltransferase-and either substance P or tyrosine hydroxylase in rat brain at both light and ultrastructural levels. Control experiments demonstrated the absence of both color mixing and antibody crossreactions, even when both primary antibodies were from the same species. This study demonstrates the usefulness of BDHC as a chromogen for immunoperoxidase staining either alone or in combination with DAB, and describes a double method which should have wide applicability for detailed studies of most pairs of antigens at both light and ultrastructural levels. 相似文献
63.
The enantioselective and diastereoselective resolutions of the stereoisomers of N alpha-aspartyl-phenylalanine 1-methyl ester (APME) have been accomplished on an HPLC chiral stationary phase based upon alpha-chymotrypsin (the ACHT-CSP) with observed enantioselectivities (alpha 1) for the DL-/LD-enantiomer of as high as 29.17 and for the DD-/LL-enantiomers of as high as 28.97. In addition, the effect on the chromatographic retention of the APME stereoisomers of the activity of the ACHT and the composition of the mobile phase--structure of the anionic component, molarity, and pH--have been studied. The results of this study suggest that the aspartyl moiety and/or the aspartyl-phenylalanine amide linkage play key roles in the observed enantioselectivity; the APME stereoisomers containing L-phenylalanine, i.e., DL- and LL-APME, bind at a different site in the ACHT molecule (the L-Phe site) than the APME stereoisomers containing D-phenylalanine (the D-Phe site); and the observed enantioselectivity is a measure of the difference in the binding affinities at the two sites rather than the consequence of differential affinities at a single site. 相似文献
64.
Anthony Yiu-Ho Woo Krzysztof Jozwiak Lawrence Toll Mary J. Tanga Joseph A. Kozocas Lucita Jimenez Ying Huang Ying Song Anita Plazinska Karolina Pajak Rajib K. Paul Michel Bernier Irving W. Wainer Rui-Ping Xiao 《The Journal of biological chemistry》2014,289(28):19351-19363
Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β2-adrenoceptor (β2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of β2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most β2-AR agonists cause both Gs and Gi activation, (R,R′)-fenoterol preferentially activates β2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on β2-AR essential for Gs-biased signaling. Following stimulation with a β2-AR-Gs-biased agonist (R,R′)-4′-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β2-AR. Interestingly, Y308F substitution on β2-AR enabled (R,R′)-4′-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering β2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of β2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism. 相似文献
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66.
BACKGROUND: rac-Fenoterol is a beta2-adrenoceptor agonist (beta2-AR) used in the treatment of asthma. It has two chiral centers and is marketed as a racemic mixture of R,R'- and S,S'-fenoterol (R-F and S-F). Here we report the separation of the R-F and S-F enantiomers and the evaluation of their binding to and activation of the beta2-AR. METHODS: R-F and S-F were separated from the enantiomeric mixture by chiral chromatography and absolute configuration determined by circular dichroism. Beta2-AR binding was evaluated using frontal affinity chromatography with a stationary phase containing immobilized membranes from HEK-293 cells that express human beta2-AR and standard membrane binding studies using the same membranes. The effect of R-F and S-F on cardiomyocyte contractility was also investigated using freshly isolated adult rat cardiomyocytes. RESULTS: Chiral chromatography of rac-fenoterol yielded separated peaks with an enantioselectivity factor of 1.21. The less retained peak was assigned the absolute configuration of S-F and the more retained peak R-F. Frontal chromatography using membrane-bound beta2-AR as the stationary phase and rac-3H-fenoterol as a marker ligand showed that addition of increasing concentrations of R-F to the mobile phase produced concentration-dependent decreases in rac-3H-fenoterol retention, while similar addition of S-F produced no change in rac-3H-fenoterol retention. The calculated dissociation constant of R-F was 472 nM and the number of available binding sites 176 pmol/column, which was consistent with the results from the membrane binding study 460 +/- 55 nM (R-F) and 109,000 +/- 10,400 nM (S-F). In the cardiomyocytes, R-F increased maximum contractile response from (265 +/- 11.6)% to (306 +/- 11.8)% of resting cell length (P < 0.05) and reduced EC50 from -7.0 +/- 0.270 to -7.1 +/- 0.2 log[M] (P < 0.05), while S-F had no significant effect. DISCUSSION: Previous studies have shown that rac-fenoterol acts as an apparent beta2-AR/G(s) selective agonist and fully restores diminished beta2-AR contractile response in cardiomyocytes from failing hearts of spontaneously hypertensive rats (SHR). Here we report the separation of the enantiomers of rac-fenoterol and that R-F is the active component of rac-fenoterol. Further evaluation of R-F will determine if it has enhanced selectivity and specificity for beta2-AR/G(s) activation and if it can be used in the treatment of congestive heart failure. 相似文献
67.
Krzysztof Jozwiak Anthony Yiu-Ho Woo Mary J. Tanga Lawrence Toll Lucita Jimenez Joseph A. Kozocas Anita Plazinska Rui-Ping Xiao Irving W. Wainer 《Bioorganic & medicinal chemistry》2010,18(2):728-736
Purpose: To use a previously developed CoMFA model to design a series of new structures of high selectivity and efficacy towards the β2-adrenergic receptor. Results: Out of 21 computationally designed structures 6 compounds were synthesized and characterized for β2-AR binding affinities, subtype selectivities and functional activities. Conclusion: the best compound is (R,R)-4-methoxy-1-naphthylfelnoterol with Kiβ2-AR = 0.28 μm, Kiβ1-AR/Kiβ2-AR = 573, EC50cAMP = 3.9 nm, EC50cardio = 16 nm. The CoMFA model appears to be an effective predictor of the cardiomocyte contractility of the studied compounds which are targeted for use in congestive heart failure. 相似文献
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69.
We present a method for modelling and simulating metabolic pathways in the cells (namely, the glycolysis and Krebs' cycle), using the discrete event system specification (DEVS) formalism. The hierarchical nature of DEVS makes it ideal for describing naturally hierarchical systems as the Cell, while its discrete-event approach improves performance due to the asynchronous nature of the events involved. DEVS time-based nature can adequately represent the timing of the chemical reactions. We show how this methodology enables creating a precise and easy way to model and simulate biological systems, including advanced visualisation of the experiments. The results presented, which focus on the simulation of the cellular metabolism pathways in mitochondria, show the potential of our approach. 相似文献
70.
Fabbri F Zoli W Carloni S Ulivi P Arienti C Brigliadori G Montanari M Tesei A Silvestrini R Amadori D 《Journal of cellular physiology》2011,226(11):3035-3042
The efficacy of therapy for hormone‐refractory prostate cancer (HRPC) is still unsatisfactory and new agents and therapeutic modalities are needed. The aims of the present work were to examine the in vitro activity and mechanisms of action of doxorubicin (DX), pegylated liposomal DX (PLDX), and non‐pegylated liposomal DX (NPLDX) in DU145 and taxane‐resistant DU145‐R HRPC cell lines. Drug activity and incorporation, apoptosis, and expression of cell death‐related markers were evaluated by SRB test, cytofluorimetric assays, and Western blot, respectively. Among the different DX formulations, NPLDX showed the highest cytotoxic activity in both cell lines, with more than 50% of apoptotic cells at only 1/10 of the plasma peak concentration after 72 h exposure. Anthracyclines, in particular NPLDX, were highly concentrated in the Golgi apparatus. Moreover, a significant increase was observed in the expression of CD95 receptor, GD3 ganglioside and, caspase‐2 and ‐8 active forms in both cell lines followed by caspase‐3 activation and mitochondrial membrane depolarization. The Golgi apparatus, probably acting as a stress sensor, intensified the conventional apoptotic mechanism induced by anthracyclines. Our data support the hypothesis that organelle‐dependent initiation of cell death other than that induced by mitochondria and nucleus is a research area worthy of pursuing and suggest that the Golgi apparatus could be an ideal target for anti‐cancer therapy. Of note, the activity of NLPDX in taxane‐resistant DU145‐R cells warrants further evaluation as second‐line treatment of advanced HRPC after taxane failure. J. Cell. Physiol. 226: 3035–3042, 2011. © 2011 Wiley‐Liss, Inc. 相似文献