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981.
The time dependence of the human
1-antitrypsin polymerization process was studied by means of the intrinsic fluorescence stopped-flow technique as well as the fluorescence-quenching-resolved spectra (FQRS) method and native PAGE. The polymerization was induced by mild denaturing conditions (1 M GuHCl) and temperature. The data show that the dimer formation reaction under mild conditions was followed by an increase of fluorescence intensity. This phenomenon is highly temperature sensitive. The structure of
1-antitrypsin dimer resembles the conformation of antithrombin III dimer. In the presence of the denaturant the polymerization process is mainly limited to the dimer state. The
1-antitrypsin activity measurements confirm monomer-to-dimer transition under these conditions. These results are in contrast to the polymerization process induced by temperature, where the dimer state is an intermediate step leading to long-chain polymers. On the basis of stopped-flow and electrophoretic data it is suggested that both C-sheet as well as A-sheet mechanisms contribute to the polymerization process under mild conditions.Abbreviations GuHCL
guanidinium hydrochloride
- RSL
reactive site loop
- PAI-1
plasminogen activator inhibitor type 1
- AT III
antithrombin III
- FQRS
fluorescence quenching resolved spectra 相似文献
982.
Marianne Jegouic Valerij Ya. Grinberg André Guingant Thomas Haertlé 《Journal of Protein Chemistry》1996,15(6):501-509
Denaturation and aggregation of-lactalbumin at high pressure (up to 10 kbar, 1000 MPa) were studied by means of circular dichroism, gel-permeation chromatography, sodium dodecyl sulfate and gel electrophoresis. It was found that the unfolding of-lactalbumin at high pressure is reversible even in basic pH and at a protein concentration as large as 10%. In these conditions only a negligible fraction of the protein is denatured irreversibly and aggregates. The rate of aggregation of-lactalbumin at high pressure increases significantly in the presence of low-molecular reducing agents such as cysteine, 2-mercaptoethanol, and dithiothreitol. Maximal yield of-lactalbumin oligomerization (over 90%) was achieved in the presence of cysteine at the molar cysteine/protein ratioq=2 and atpH 8.5. Apparent molecular weight of the obtained oligomers was over 500 kDa. It was shown that the size distribution of oligomers can be modulated by varyingpH and reducing agent. The size distribution shifts in the direction of very large, poorly soluble particles whenpH decreases. Maximal content of the insoluble fraction (about 30%) can be reached at pH 5.5 when cysteine (q=2) is used as reducing agent. The oligomers of-lactalbumin are stabilized mainly by nonnative interchain disulfide bridges. Circular dichroism measurements point to an additional mechanism of cohesion of polypeptide chains in the oligomers, which is formation of intermolecular-sheets. 相似文献
983.
James M. Chen Spero Manolatos Paul W. Brandt-Rauf Randall B. Murphy Regina Monaco Matthew R. Pincus 《Journal of Protein Chemistry》1996,15(6):511-518
The three-dimensional structures of theras-p21 protein and its protein inhibitor, rap-1A, have been computed bound to theras-binding domain, RBD (residues 55–131), of theraf-p74 protein, a critical target protein ofras-p21 in theras-induced mitogenic signal transduction pathway. The coordinates of RBD have been reconstructed from the stereoview of an X-ray crystal structure of this domain bound to rap-1A and have been subjected to energy minimization. The energy-minimized structures of bothras- p21 and rap-1A, obtained in previous studies, have been docked against RBD, using the stereo figure of the RBD-rap-1A complex, based on a six-step procedure. The final energy-minimized structure of rap-1A-RBD is identical to the X-ray crystal structure. Comparison of theras-p21- and rap-1A-RBD complexes reveals differences in the structures of effector domains ofras-p21 and rap-1a, including residues 32–47, a domain that directly interacts with RBD, 60–66, 96–110, involved in the interaction ofras-p21 withjun kinase (JNK) andjun protein, and 115–126, involved in the interaction of p21 with JNK. The structure of the RBD remained the same in both complexes with the exception of small deviations in its-2 binding loop (residues 63–71) and residues 89–91, also involved in binding to rap-1A. The results suggest that the binding of these two proteins to RBD may allow them to interact with other cellular target proteins such as JNK andjun. 相似文献
984.
Paracrine functions of the coronary vascular endothelium 总被引:5,自引:0,他引:5
Ingrid Fleming Johann Bauersachs Rudi Busse 《Molecular and cellular biochemistry》1996,157(1-2):137-145
985.
Immaculada Martin Josep A. Villena Marta Giralt Roser Iglesias Teresa Mampel Octavi Vińas Francesc Villarroya 《Molecular and cellular biochemistry》1996,154(2):107-111
The action of thyroid hormones on the expression of the mitochondrial ATP synthase -subunit gene (ATPsyn) is controversial. We detected a binding site for the thyroid hormone receptor between-366 and-380 in the human ATPsyn gene by DNase I footprint analysis and band-shift assays. However, expression vectors in which the chloramphenicol acetyl transferase (CAT) reporter gene is driven by the 5 upstream region of ATPsyn gene were unresponsive to T3 when transiently transfected to HepG2 or GH4C1 cells. CAT constructs driven by the rat phosphoenolpyruvate carboxykinase (PEPCK) or the growth hormone (GH) promoters were stimulated several fold by T3 in parallel experiments. It is proposed that the biological effects of thyroid hormones on the ATPsyn expression occur through indirect mechanisms. 相似文献
986.
Mechanical effects of ET-1 in cardiomyocytes isolated from normal and heart-failed rabbits 总被引:1,自引:0,他引:1
Elizabeth J. Kelso Robert F. Geraghty Barbara J. McDermott Elisabeth R. Trimble D. Paul Nicholls Bernard Silke 《Molecular and cellular biochemistry》1996,157(1-2):149-155
Endothelin (ET-1) is found at elevated concentrations in the plasma of patients with heart failure and in animal models of cardiomyopathy. The peptide is a potent positive inotropic agent, the effects of which are mediated by increases in cytosolic Ca2+ in cardiomyocytes. The object of this study was to investigate at the cellular level, the actions of ET-1 on contractile function and on Ca2+ currents in heart-failed ventricular myocardium. Male New Zealand White rabbits (8 wks) were treated with twice weekly injections of epirubicin (4 mg/kg/wk, n=7) or with saline (n=7) for 6 wks, followed by a washout period of 2 wks. Ventricular cardiomyocytes were isolated from rabbit hearts using Langendorff perfusion with collagenase; contractile function was examined using a video microscopy method, and L-type Ca2+ currents were recorded using a whole-cell patch-clamp technique. ET-1 produced a concentration-dependent increase in contractile response (% increase from basal value) to a maximum at 1 nM ET-1 of 69 ± 11% (mean ± S.D.) in control cardiomyocytes and 33 ± 6% in heart-failed cells. However, there was no significant change in the EC50 obtained with ET-1 for healthy (0.31 ± 0.1 nM) and for failed cardiomyocytes (0.24 ± 0.1 nM). The effects of ET-1 on L-type Ca2+ channels were similar with a peak amplitude at 1 nM ET-1 of –3.26 ± 0.8 in control cardiomyocytes and –3.32 ± 0.9 nA in heart-failed cells. The attenuation of the contractile response to ET-1 in heart-failed cells may reflect a desensitization of ET receptors as a consequence of elevated circulating levels of ET and was not reflected by alteration of transmembrane Ca2+ conductance. It is probable, therefore, that multiple signalling pathways are involved in the actions of ET on ventricular myocardium.Recipient of Servier Investigator Award 相似文献
987.
Panagakos Fotinos S. Fernandez Cesar Kumar Suriender 《Molecular and cellular biochemistry》1996,159(1):81-84
The study, addressed to understand whether or not human platelets possess a unique thiol-oxidase whose activity could be modulated by signalling pathway initiated upon the activation of Receptor-Ck revealed the existence of disulphide-dependent oxidation within these cells and this phenomenon was regulated by Receptor-Ck-dependent generation of second messengers especially phosphatidic acid (PA); cAMP and cGMP. Purification of this activity revealed the existence of 47 kDa protein having thiol-oxidase activity. Keeping in view these results we propose that the existence of this novel 47 kDa Thiol-oxidase within human platelets may provide a crucial switch for the regulation of Receptor-Ck-dependent mevalonate pathway in human platelets. 相似文献
988.
The localization of the ai adrenoceptors (1-AR) in the heart tissues from rat and human and in the cultured heart cells from neonatal rats was studied by indirect immunofluorescence and postembedding electronmicroscopical immuno-gold technique. With antipeptide antibodies directed against the second extracellular loop of the human 1-AR (AS sequence 192–218), this receptor was found to be localized along the sarcolemma in both human and rat hearts. Similar localization sites were detected in cultivated rat neonatal cardiomyocytes. Beside the localization in cardiomyocytes, 1-AR were identified in endothelial cells of capillaries and smooth muscle cells of coronary vessels, in neuronal endings, in mast cells of cultivated heart cells but not, or in less amount in fibroblasts. Interestingly, in the right atrium of rat heart the localization of 1-AR was found to be near or on atrial natriuretic factor (ANF) granules, providing the basis for the -adrenergic influence on ANF release. The immunocytochemical studies further confirm and complete the findings known by using autoradiographic binding studies with specific ligands. 相似文献
989.
A systemic oxidative stress of HIV (+) individuals has been recognized from a low glutathione level and a high level of inflammatory cytokines such as TNF. Previously, we demonstrated that the catalase enzyme activity in HIV (+) population is significantly altered depending on the cell types; the level was significantly high in red blood cells while the enzymes in white blood cells were remarkably low (Res Commun Subs Abuse 16: 161–176, 1995). In this study, we further characterized the difference in RBC catalase molecules between HIV (+) and control population. We have found that RBC from HIV (+) population, whether they were asymptomatic or symptomatic, contained a significantly elevated catalase protein accompanied by the enzyme activities, and that the majority of the elevated protein were acidic pl of the molecules with an identical subunit mass of approximately 60 KDa. These results suggest that catalase is induced prior to and/or during erythroid differentiation lineage in HIV (+) population as a somatic defense to respond and compensate for a systemic oxidative stress and for an anemic condition. (Mol Cell Biochem 165: 77–81, 1996) 相似文献
990.
Kaumann Alberto J. Sanders Louise Lynham James A. Bartel Sabine Kuschel Meike Karczewski Peter Krause Ernst-Georg 《Molecular and cellular biochemistry》1996,163(1):113-123
Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both 1-adrenoceptors (1AR) and 2-adrenoceptors (2AR) mediate positive inotropic effects but that only 1AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both 1 AR and 2 AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of 1AR and 1/3 of 2AR, whether or not 2AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate 2AR, we used the 2AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t1:2 of relaxation) effects with EC50 values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the 2AR-selective antagonist ICI 118551 (50 nM) which reduced both EC50 values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC50 of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[125I] cyanopindolol with higher affinity for 2AR than for - 1 AR; the binding to 2AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both 1AR and 2AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both 1AR and 2AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation. 相似文献