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21.
Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity. 总被引:3,自引:0,他引:3 下载免费PDF全文
Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function. 相似文献
22.
Levin I Meiri G Peretz M Burstein Y Frolow F 《Protein science : a publication of the Protein Society》2004,13(6):1547-1556
Pseudomonas aeruginosa alcohol dehydrogenase (PaADH; ADH, EC 1.1.1.1) catalyzes the reversible oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones, using NAD as coenzyme. We crystallized the ternary complex of PaADH with its coenzyme and a substrate molecule and determined its structure at a resolution of 2.3 A, using the molecular replacement method. The PaADH tetramer comprises four identical chains of 342 amino acid residues each and obeys ~222-point symmetry. The PaADH monomer is structurally similar to alcohol dehydrogenase monomers from vertebrates, archaea, and bacteria. The stabilization of the ternary complex of PaADH, the coenzyme, and the poor substrate ethylene glycol (k(cat) = 4.5 sec(-1); Km > 200 mM) was due to the blocked exit of the coenzyme in the crystalline state, combined with a high (2.5 M) concentration of the substrate. The structure of the ternary complex presents the precise geometry of the Zn coordination complex, the proton-shuttling system, and the hydride transfer path. The ternary complex structure also suggests that the low efficiency of ethylene glycol as a substrate results from the presence of a second hydroxyl group in this molecule. 相似文献
23.
Deifilia Ahuatzi-chacón Guadalupe Ordorica-morales Nora Ruiz-ordaz Eliseo Cristiani-urbina Cleotilde Juárez-ramírez Juvencio Galíndez-mayer 《World journal of microbiology & biotechnology》2004,20(7):695-702
When Candida tropicalis was grown on phenol, catechol or resorcinol, the highest levels of specific activity of phenol hydroxylase (EC. 1.14.13.7) and catechol 1,2-dioxygenase (EC. 1.13.11.1) were attained with phenol. With the three aromatic compounds tested, the yeast cells exhibited sharp peaks of specific activity of both enzymes at particular incubation times. Phenol-induced cells containing high levels of both enzymes were capable of degrading rapidly and without delay 4-chlorophenol and 2,6-dichlorophenol, and to a lesser extend pentachlorophenol. However, the yeast could not grow on chlorophenols as major carbon and energy source. 相似文献
24.
P Galéra D Vivien S Pronost J Bonaventure F Rédini G Loyau J P Pujol 《Journal of cellular physiology》1992,153(3):596-606
25.
Fabio Orlandi Herminia Garcia-Mozo Carmen Galán Bruno Romano Consuelo Diaz de la Guardia Luis Ruiz Maria del Mar Trigo Eugenio Dominguez-Vilches Marco Fornaciari 《International journal of biometeorology》2010,54(2):151-163
The aim of this study was to investigate the main climatic and biological trends related to olive flowering in central-southern
Italy compared to those in Andalusia, Spain. Results since 1982 were compared for the two long-series monitoring areas of
Cordoba and Perugia, and since 1992–1999 for the short-series areas. The relationship between climatic trends and the biological
response of the olive, a widespread culture in the Mediterranean basin, were investigated. An aerobiological method involving
capturing pollen released into the atmosphere was utilised as a bioindicator of flowering phenology. The study results confirm
the strong relationship between flowering periods and spring temperature trends for the olive. Temperature during March, April
and May was the parameter most related to flowering date in the study areas, particularly in Italy. In some cases we found
a significant correlation between flowering and past autumn temperatures, probably due to their effect on floral bud dormancy
induction, but this phenomenon appeared to be of minor importance in the studied areas. The phenological trend results show
the continuous advance of flowering dates to the late 1990s, followed by a relatively stationary time series related to a
short-term temperature fluctuation in the Mediterranean area. This latter period probably represents a mesoscale event forced
by a macroscale event—the North Atlantic Oscillation. The results reveal that the trend towards increased temperatures, and
the consequent flowering advance of some species, indicated some years ago is nowadays not as clear as was expected and should
be confirmed over the next few years in the Mediterranean areas under investigation. 相似文献
26.
Methadone is a clinically used opioid agonist that is oxidatively metabolized by cytochrome P450 (CYP) isoforms to a stable metabolite, EDDP. Methadone is a chiral drug administered as the racemic mixture of (R)-(-)- and (S)-(+)-methadone, but (R)-methadone is the active isomer. The cytochrome P450 (CYP) isoform involved in methadone's metabolism is thought to be CYP3A4, but human drug-drug interaction studies are not consistent with this. The ability of the common human drug-metabolizing CYPs (obtained from baculovirus-infected insect cell supersomes) to generate 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrilidine (EDDP) from racemic methadone was examined and then determined if the CYP isoforms metabolized methadone stereoselectively. Only CYP2B6, 2C19, and 3A4 generated measurable EDDP from 1 microg/ml of racemic methadone. The hierarchy of EDDP generation was CYP2B6 > CYP2C19 >/= CYP3A4. At 10 microg/ml of methadone, CYP2C9 and CYP2D6 also generated EDDP, but in at least 10-fold lower quantities than CYP2B6. Michaelis-Menten kinetic data demonstrated that CYP2B6 had the highest V(max) (44 ng/min/10pmol) and the lowest K(m) (12.6 microg/ml) for EDDP formation of all the CYP isoforms. In human liver microsomes with high and low CYP2B6 expression but equivalent CYP3A4 expression, high CYP2B6 expression microsomes generated twice the amount of EDDP from 10 microg/ml of methadone than low CYP2B6 expression microsomes. When stereoselective metabolism of racemic methadone by CYP2B6, 2C19, and 3A4 was examined using an enantiospecific methadone assay, CYP2B6 preferentially metabolized (S)-methadone, CYP2C19 preferentially metabolized (R)-methadone, and CYP3A4 showed no preference. These data suggest that multiple CYPs metabolized methadone but CYP2B6 had the highest V(max)/K(m). In addition, only CYP2B6 and 2C19 showed stereoselective metabolism. Our data could explain why the plasma concentration ratio of R/S methadone is variable and why drugs that induce CYP2B6 such as nevirapine and efavirenz also induce methadone metabolism, while the CYP3A4 inducer rifabutin has no effect on methadone pharmacokinetics. 相似文献
27.
Srabasti Acharya Brian M. Safaie Piriya Wongkongkathep Magdalena I. Ivanova Aida Attar Frank-Gerrit Kl?rner Thomas Schrader Joseph A. Loo Gal Bitan Lisa J. Lapidus 《The Journal of biological chemistry》2014,289(15):10727-10737
Recent work on α-synuclein has shown that aggregation is controlled kinetically by the rate of reconfiguration of the unstructured chain, such that the faster the reconfiguration, the slower the aggregation. In this work we investigate this relationship by examining α-synuclein in the presence of a small molecular tweezer, CLR01, which binds selectively to Lys side chains. We find strong binding to multiple Lys within the chain as measured by fluorescence and mass-spectrometry and a linear increase in the reconfiguration rate with concentration of the inhibitor. Top-down mass-spectrometric analysis shows that the main binding of CLR01 to α-synuclein occurs at the N-terminal Lys-10/Lys-12. Photo-induced cross-linking of unmodified proteins (PICUP) analysis shows that under the conditions used for the fluorescence analysis, α-synuclein is predominantly monomeric. The results can be successfully modeled using a kinetic scheme in which two aggregation-prone monomers can form an encounter complex that leads to further oligomerization but can also dissociate back to monomers if the reconfiguration rate is sufficiently high. Taken together, the data provide important insights into the preferred binding site of CLR01 on α-synuclein and the mechanism by which the molecular tweezer prevents self-assembly into neurotoxic aggregates by α-synuclein and presumably other amyloidogenic proteins. 相似文献
28.
29.
Pontier SM Percherancier Y Galandrin S Breit A Galés C Bouvier M 《The Journal of biological chemistry》2008,283(36):24659-24672
Determining the role of lipid raft nanodomains in G protein-coupled receptor signaling remains fraught by the lack of assays directly monitoring rafts in native membranes. We thus combined extensive biochemical and pharmacological approaches to a nanoscale strategy based on bioluminescence resonance energy transfer (BRET) to assess the spatial and functional influence of cholesterol-rich liquid-ordered lipid nanodomains on beta(2) adrenergic receptor (beta(2)AR) signaling. The data revealed that whereas beta(2)AR did not partition within liquid-ordered lipid phase, a pool of G protein and adenylyl cyclase (AC) were sequestered in these domains. Destabilization of the liquid-ordered phase by cholesterol depletion led to a lateral redistribution of Galpha(s) and AC that favored interactions between the receptor and its signaling partners as assessed by BRET. This resulted in an increased basal and agonist-promoted beta(2)AR-stimulated cAMP production that was partially dampened as a result of constitutive protein kinase A-dependent phosphorylation and desensitization of the receptor. This restraining influence of nanodomains on beta(2)AR signaling was further substantiated by showing that liquid-ordered lipid phase stabilization using caveolin overexpression or increasing membrane cholesterol amount led to an inhibition of beta(2)AR-associated signaling. Given the emerging concept that clustering of receptors and effectors into signaling platforms contributes to the efficacy and selectivity of signal transduction, our results support a model whereby cholesterol-promoted liquid-ordered lipid phase-embedding G(s) and AC allows their lateral separation from the receptor, thus restraining the basal activity and controlling responsiveness of beta(2)AR signaling machinery within larger signaling platforms. 相似文献
30.
We developed a simple DNA elution method from agarose gels. After electrophoresis of DNA in an agarose gel, the DNA fragment to be recorved was excised out of gel with a scalpel. The excised gel was placed in the middle of small Parafilm piece, and the Parafilm was folded over the gel piece. Using the petriplate, or thumb, the gel piece was pressed between the Parafilm. Upon squeezing, the DNA inside of the gel gets extruded along with the buffer. The droplets were collected with a pipet. The DNA was then purified by conventional phenol: chloroform extraction method. Typical yields are greater than 50% as determined by UV absorbance. 相似文献