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61.
Herein we report the design of a direct and continuous fluorometric assay for determining tissue transglutaminase (TGase) activity. The progress of the TGase-catalyzed reaction of 4-(N-carbobenzoxy-l-phenylalanylamino)-butyric acid coumarin-7-yl ester was monitored as an increase of fluorescence (lambda(exc) 330 nm, lambda(em) 460 nm) due to the release of 7-hydroxycoumarin. Using this assay, we determined the K(m) of two acceptor substrates, N-acetyl-L-lysine methyl ester and aminoacetonitrile. We also determined the K(m) of 4-(N-carbobenzoxy-L-phenylalanylamino)-butyric acid coumarin-7-yl ester for its TGase-mediated hydrolysis and for its enzymatic reaction with the acyl acceptor substrates N-acetyl-L-lysine methyl ester and aminoacetonitrile. We ascertained that the fluorescent substrate was selective toward tissue TGase by testing it with different enzymes, namely microbial transglutaminase (mTGase), Factor XIIIa, papain, and gamma-glutamyl transpeptidase. 4-(N-carbobenzoxyglycinylamino)-butyric acid coumarin-7-yl ester, lacking the benzyl side chain, was also found to be an efficient fluorogenic substrate of tissue TGase. Finally, we have shown that this method is applicable to 96-well microtiter plate format. 相似文献
62.
Toyoshima Y Karas M Yakar S Dupont J Lee Helman LeRoith D 《The Journal of biological chemistry》2004,279(24):25898-25904
Insulin-like growth factor-I (IGF-I) receptors and insulin receptors belong to the same subfamily of receptor tyrosine kinases and share a similar set of intracellular signaling pathways, despite their distinct biological actions. In the present study, we evaluated T cell death-associated gene 51 (TDAG51), which we previously identified by cDNA microarray analysis as a gene specifically induced by IGF-I. We characterized the signaling pathways by which IGF-I induces TDAG51 gene expression and the functional role of TDAG51 in IGF-I signaling in NIH-3T3 (NWTb3) cells, which overexpress the human IGF-I receptor. Treatment with IGF-I increased TDAG51 mRNA and protein levels in NWTb3 cells. This effect of IGF-I was specifically mediated by the IGF-IR, because IGF-I did not induce TDAG51 expression in NIH-3T3 cells overexpressing a dominant-negative IGF-I receptor. Through the use of specific inhibitors of various protein kinases, we found that IGF-I induced TDAG51 expression via the p38 MAPK pathway. The ERK, JNK, and phosphatidylinositol 3-kinase pathways were not involved in IGF-I-induced regulation of TDAG51. To assess the role of TDAG51 in IGF-I signaling, we used small interfering RNA (siRNA) expression vectors directed at two different target sites to reduce the level of TDAG51 protein. In cells expressing these siRNA vectors, TDAG51 protein levels were decreased by 75-80%. Furthermore, TDAG51 siRNA expression abolished the ability of IGF-I to rescue cells from serum starvation-induced apoptosis. These findings suggest that TDAG51 plays an important role in the anti-apoptotic effects of IGF-I. 相似文献
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Paget C Ivanov S Fontaine J Renneson J Blanc F Pichavant M Dumoutier L Ryffel B Renauld JC Gosset P Gosset P Si-Tahar M Faveeuw C Trottein F 《The Journal of biological chemistry》2012,287(12):8816-8829
Invariant natural killer T (iNKT) cells are non-conventional lipid-reactive αβ T lymphocytes that play a key role in host responses during viral infections, in particular through the swift production of cytokines. Their beneficial role during experimental influenza A virus (IAV) infection has recently been proposed, although the mechanisms involved remain elusive. Here we show that during in vivo IAV infection, mouse pulmonary iNKT cells produce IFN-γ and IL-22, a Th17-related cytokine critical in mucosal immunity. Although permissive to viral replication, IL-22 production by iNKT cells is not due to IAV infection per se of these cells but is indirectly mediated by IAV-infected dendritic cells (DCs). We show that activation of the viral RNA sensors TLR7 and RIG-I in DCs is important for triggering IL-22 secretion by iNKT cells, whereas the NOD-like receptors NOD2 and NLRP3 are dispensable. Invariant NKT cells respond to IL-1β and IL-23 provided by infected DCs independently of the CD1d molecule to release IL-22. In vitro, IL-22 protects IAV-infected airway epithelial cells against mortality but has no role on viral replication. Finally, during early IAV infection, IL-22 plays a positive role in the control of lung epithelial damages. Overall, IAV infection of DCs activates iNKT cells, providing a rapid source of IL-22 that might be beneficial to preserve the lung epithelium integrity. 相似文献
66.
Andrews MJ Clase JA Bar G Tricarico G Edwards PJ Brys R Chambers M Schmidt W MacLeod A Hirst K Allen V Birault V Le J Harris J Self A Nash K Dixon G 《Bioorganic & medicinal chemistry letters》2012,22(6):2266-2270
MAPKAPK5 has been proposed to play a role in regulation of matrix metalloprotease expression and so to be a potential target for intervention in rheumatoid arthritis. We present here the identification of a series of compounds against this target which are effective in both biochemical and cell assays. The expansion of the series is described, along with early SAR and pharmacokinetics for some representative compounds. 相似文献
67.
Martin AR Katz IM Terzibachi K Gouinaud L Caillibotte G Texereau J 《Biomedical engineering online》2012,11(1):27
ABSTRACT: BACKGROUND: Expiratory time constants are used to quantify emptying of the lung as a whole, and emptying of individual lung compartments. Breathing low-density helium/oxygen mixtures may modify regional time constants so as to redistribute ventilation, potentially reducing gas trapping and hyperinflation for patients with obstructive lung disease. In the present work, bench and mathematical models of the lung were used to study the influence of heterogeneous patterns of obstruction on compartmental and whole-lung time constants. METHODS: A two-compartment mechanical test lung was used with the resistance in one compartment held constant, and a series of increasing resistances placed in the opposite compartment. Measurements were made over a range of lung compliances during ventilation with air or with a 8/22% mixture of helium/oxygen. The resistance imposed by the breathing circuit was assessed for both gases. Experimental results were compared with predictions of a mathematical model applied to the test lung and breathing circuit. In addition, compartmental and whole-lung time constants were compared with those reported by the ventilator. RESULTS: Time constants were greater for larger minute ventilation, and were reduced by substituting helium/oxygen in place of air. Notably, where time constants were long due to high lung compliance (i.e. low elasticity), helium/oxygen improved expiratory flow even for a low level of resistance representative of healthy, adult airways. In such circumstances, the resistance imposed by the external breathing circuit was significant. Mathematical predictions were in agreement with experimental results. Time constants reported by the ventilator were wellcorrelated with those determined for the whole-lung and for the low-resistance compartment, but poorly correlated with time constants determined for the high-resistance compartment. CONCLUSIONS: It was concluded that breathing a low-density gas mixture, such as helium/oxygen, can improve expiratory flow from an obstructed lung compartment, but that such improvements will not necessarily affect time constants measured by the ventilator. Further research is required to determine if alternative measurements made at the ventilator level are predictive of regional changes in ventilation. It is anticipated that such efforts will be aided by continued development of mathematical models to include pertinent physiological and pathophysiological phenomena that are difficult to reproduce in mechanical test systems. 相似文献
68.
Young MA Larson DE Sun CW George DR Ding L Miller CA Lin L Pawlik KM Chen K Fan X Schmidt H Kalicki-Veizer J Cook LL Swift GW Demeter RT Wendl MC Sands MS Mardis ER Wilson RK Townes TM Ley TJ 《Cell Stem Cell》2012,10(5):570-582
To assess the genetic consequences of induced pluripotent stem cell (iPSC) reprogramming, we sequenced the genomes of ten murine iPSC clones derived from three independent reprogramming experiments, and compared them to their parental cell genomes. We detected hundreds of single nucleotide variants (SNVs) in every clone, with an average of 11 in coding regions. In two experiments, all SNVs were unique for each clone and did not cluster in pathways, but in the third, all four iPSC clones contained 157 shared genetic variants, which could also be detected in rare cells (<1 in 500) within the parental MEF pool. These data suggest that most of the genetic variation in iPSC clones is not caused by reprogramming per se, but is rather a consequence of cloning individual cells, which "captures" their mutational history. These findings have implications for the development and therapeutic use of cells that are reprogrammed by any method. 相似文献
69.
John Gatfield Celia Mueller Grandjean Thomas Sasse Martine Clozel Oliver Nayler 《PloS one》2012,7(10)
Two endothelin receptor antagonists (ERAs), bosentan and ambrisentan, are currently approved for the treatment of pulmonary arterial hypertension (PAH), a devastating disease involving an activated endothelin system and aberrant contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC). The novel ERA macitentan has recently concluded testing in a Phase III morbidity/mortality clinical trial in PAH patients. Since the association and dissociation rates of G protein-coupled receptor antagonists can influence their pharmacological activity in vivo, we used human PASMC to characterize inhibitory potency and receptor inhibition kinetics of macitentan, ambrisentan and bosentan using calcium release and inositol-1-phosphate (IP1) assays. In calcium release assays macitentan, ambrisentan and bosentan were highly potent ERAs with Kb values of 0.14 nM, 0.12 nM and 1.1 nM, respectively. Macitentan, but not ambrisentan and bosentan, displayed slow apparent receptor association kinetics as evidenced by increased antagonistic potency upon prolongation of antagonist pre-incubation times. In compound washout experiments, macitentan displayed a significantly lower receptor dissociation rate and longer receptor occupancy half-life (ROt1/2) compared to bosentan and ambrisentan (ROt1/2∶17 minutes versus 70 seconds and 40 seconds, respectively). Because of its lower dissociation rate macitentan behaved as an insurmountable antagonist in calcium release and IP1 assays, and unlike bosentan and ambrisentan it blocked endothelin receptor activation across a wide range of endothelin-1 (ET-1) concentrations. However, prolongation of the ET-1 stimulation time beyond ROt1/2 rendered macitentan a surmountable antagonist, revealing its competitive binding mode. Bosentan and ambrisentan behaved as surmountable antagonists irrespective of the assay duration and they lacked inhibitory activity at high ET-1 concentrations. Thus, macitentan is a competitive ERA with significantly slower receptor dissociation kinetics than the currently approved ERAs. Slow dissociation caused insurmountable antagonism in functional PASMC-based assays and this could contribute to an enhanced pharmacological activity of macitentan in ET-1-dependent pathologies. 相似文献
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