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71.
Luc De Vries Frdric Finana Frdric Cachoux Bernard Vacher Pierre Sokoloff Didier Cussac 《Cellular signalling》2010,22(1):158-165
We developed a cellular Bioluminescent Resonance Energy Transfer (BRET) assay based on the interaction of TrkB fused to Renilla luciferase with the intracellular adaptor protein Shc fused to Enhanced Yellow Fluorescent Protein (EYFP). The TrkB agonist Brain Derived Neurotrophic Factor (BDNF) induced a maximum BRET signal as of 10 min with an EC50 value of 1.4 nM, similar to the other endogenous agonists NT-3 and NT-4/5, 1.5 nM and 0.34 nM, respectively. Interestingly, measure of the BRET signal with increasing expression of Shc-EYFP, in the presence or absence of BDNF, suggested a conformational change of preformed TrkB/Shc complexes rather than Shc recruitment. Furthermore, the Y516F TrkB mutant deficient to bind Shc as well as the kinase-dead K572R TrkB mutant was unable to respond to BDNF and exhibited a lower basal BRET signal than that of the wild-type TrkB receptor, again suggesting a preformed complex with constitutive activity. The double YY706/707FF TrkB mutant in the kinase activation loop also showed reduced basal activity but surprisingly kept its capacity to enhance BDNF-induced interaction with Shc, though with less efficacy. The Trk selective kinase inhibitors K252a and BMS-9 blocked BDNF-induced BRET signal with similar potency (100–150 nM), the preferential c-Met inhibitor PF-2341006 being one order of magnitude less potent. Remarkably, in the absence of BDNF, K252a and BMS-9 also reduced basal activity to the level of the Y516F TrkB mutant, suggesting that these compounds were able to reduce the TrkB constitutive activity. BRET responses of mutants and to kinase inhibitors thus reveal a complex level of interaction between TrkB and Shc and suggest that this BRET assay could be of great utility to test blockers of TrkB signalling in a physiologically relevant context. 相似文献
72.
73.
Vacher H Romi-Lebrun R Crest M Masmejean F Bougis PE Darbon H Martin-Eauclaire MF 《Biochimica et biophysica acta》2003,1646(1-2):152-156
We deleted the two C-terminal residues of the scorpion toxin BmTx3, a peptidyl inhibitor of a transient A-type K(+) current in striatum neurons in culture, to assess their contribution to receptor recognition. The sBmTX3-delYP analog was shown to have a native-like structure in one-dimensional 1H-nuclear magnetic resonance (NMR) spectroscopy. We found that sBmTX3-delYP bound to its receptor less efficiently than the wild-type molecule (by a factor of about 10(5)) in binding assays with rat brain membranes, and that this molecule did not block the A-type K(+) current (at a concentration of 35 microM) in whole-cell patch clamp experiments with striatum neurons. Also, these results show that the A-type K(+) channel blocked by BmTX3 should have a canonical K(+) channel pore structure. 相似文献
74.
Hélène Vacher Meriem Alami Marcel Crest Lourival D Possani Pierre E Bougis Marie-France Martin-Eauclaire 《European journal of biochemistry》2002,269(24):6037-6041
A novel toxin, AmmTX3 (3823.5 Da), was isolated from the venom of the scorpion Androctonus mauretanicus. It showed 94% sequence homology with Aa1 from Androctonus australis and 91% with BmTX3 from Buthus martensi which, respectively, block A-type K+ current in cerebellum granular cells and striatum cultured neurons. Binding and displacement experiments using rat brain synaptosomes showed that AmmTX3 and Aa1 competed effectively with 125I-labelled sBmTX3 binding. They fully inhibited the 125I-labelled sBmTX3 binding (Ki values of 19.5 pm and 44.2 pm, respectively), demonstrating unambiguously that the three molecules shared the same target in rat brain. The specific binding parameters of 125I-labelled AmmTX3 for its site were determined at equilibrium (Kd = 66 pm, Bmax = 22 fmol per mg of protein). Finally, patch-clamp experiments on striatal neurons in culture demonstrated that AmmTX3 was able to inhibit the A-type K+ current (Ki = 131 nm). 相似文献
75.
Readthrough in vitro of the Qβ coat protein terminator codon UGA has been used as an assay for suppression by UGA-suppressor tRNATrp. When the tRNA is covalently crosslinked between 4-thiouracil(8) and cytosine(13) by irradiation at 334 nm, it is found that UGA suppression by this assay is reduced to the low level characteristic of the wild type tRNATrp. In contrast, crosslinking has little effect on incorporation of tryptophan in response to UGG codons. Thus, incorporation of tryptophan during translation of R17 messenger RNA is unaffected by photochemical crosslinking. Furthermore, dilution experiments using R17 mRNA in which tryptophan incorporation is dependent on precharged suppressor Trp-tRNA show that the crosslinked species competes well with non-irradiated tRNA. These results further emphasize the influence on tRNA-ribosome interactions of the region in tRNA around the dihydrouridine arm, where the mutation, in the suppressor is found and the photochemical crosslink is introduced. 相似文献
76.
Arianna Tavanti Lambert AM Hensgens Selene Mogavero László Majoros Sonia Senesi Mario Campa 《BMC microbiology》2010,10(1):203
Background
Candida parapsilosis is known to show limited genetic variability, despite different karyotypes and phenotypes have been described. To further investigate this aspect, a collection of 62 sensu strictu C. parapsilosis independent isolates from 4 geographic regions (Italy, n = 19; New Zealand, n = 15; Argentina, n = 14; and Hungary, n = 14) and different body sites (superficial and deep seated) were analysed for their genetic and phenotypic traits. Amplification fragment length polymorphism (AFLP) analysis was used to confirm species identification and to evaluate intraspecific genetic variability. Phenotypic characterisation included clinically relevant traits, such as drug susceptibility, in vitro biofilm formation and aspartyl protease secretion. 相似文献77.
78.
AM Fietta AM Bardoni R Salvini I Passadore M Morosini L Cavagna V Codullo E Pozzi F Meloni C Montecucco 《Arthritis research & therapy》2007,8(6):R160
Lung fibrosis is a major cause of mortality and morbidity in systemic sclerosis (SSc). However, its pathogenesis still needs
to be elucidated. We examined whether the alteration of certain proteins in bronchoalveolar lavage fluid (BALF) might have
a protective or a causative role in the lung fibrogenesis process. For this purpose we compared the BALF protein profile obtained
from nine SSc patients with lung fibrosis (SScFib+) with that obtained from six SSc patients without pulmonary fibrosis (SScFib-) by two-dimensional gel electrophoresis (2-DE). Only spots and spot-trains that were consistently expressed in a different
way in the two study groups were taken into consideration. In total, 47 spots and spot-trains, corresponding to 30 previously
identified proteins in human BALF, showed no significant variation between SScFib+ patients and SScFib- patients, whereas 24 spots showed a reproducible significant variation in the two study groups. These latter spots corresponded
to 11 proteins or protein fragments, including serum albumin fragments (13 spots), 5 previously recognized proteins (7 spots),
and 4 proteins (3 spots) that had not been previously described in human BALF maps, namely calumenin, cytohesin-2, cystatin
SN, and mitochondrial DNA topoisomerase 1 (mtDNA TOP1). Mass analysis did not determine one protein-spot. The two study groups
revealed a significant difference in BALF protein composition. Whereas levels of glutathione S-transferase P (GSTP), Cu–Zn
superoxide dismutase (SOD) and cystatin SN were downregulated in SScFib+ patients compared with SScFib- patients, we observed a significant upregulation of α1-acid glycoprotein, haptoglobin-α chain, calgranulin (Cal) B, cytohesin-2,
calumenin, and mtDNA TOP1 in SScFib+ patients. Some of these proteins (GSTP, Cu–Zn SOD, and cystatin SN) seem to be involved in mechanisms that protect lungs
against injury or inflammation, whereas others (Cal B, cytohesin-2, and calumenin) seem to be involved in mechanisms that
drive lung fibrogenesis. Even if the 2-DE analysis of BALF did not provide an exhaustive identification of all BALF proteins,
especially those of low molecular mass, it allows the identification of proteins that might have a role in lung fibrogenesis.
Further longitudinal studies on larger cohorts of patients will be necessary to assess their usefulness as predictive markers
of disease. 相似文献
79.
Background
Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed to characterize regulatory elements and nutritional requirements that control the PA catabolic genes in B. cenocepacia K56-2. 相似文献80.
Cherrad S Girard V Dieryckx C Gonçalves IR Dupuy JW Bonneu M Rascle C Job C Job D Vacher S Poussereau N 《Metallomics : integrated biometal science》2012,4(8):835-846
Although essential in many cellular processes, metals become toxic when they are present in excess and constitute a global environmental hazard. To overcome this stress, fungi have evolved several mechanisms at both intracellular and extracellular levels. In particular, fungi are well known for their ability to secrete a large panel of proteins. However, their role in the adaptation of fungi to metal toxicity has not yet been investigated. To address this question, here, the fungus Botrytis cinerea was challenged to copper, zinc, nickel or cadmium stress and secreted proteins were collected and separated by 2D-PAGE. One hundred and sixteen spots whose volume varied under at least one tested condition were observed on 2D gels. Densitometric analyses revealed that the secretome signature in response to cadmium was significantly different from those obtained with the other metals. Fifty-five of these 116 spots were associated with unique proteins and functional classification revealed that the production of oxidoreductases and cell-wall degrading enzymes was modified in response to metals. Promoter analysis disclosed that PacC/Rim101 sites were statistically over-represented in the upstream sequences of the 31 genes corresponding to the varying unique spots suggesting a possible link between pH regulation and metal response in B. cinerea. 相似文献