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981.
Generation of mature fat pads in vitro and in vivo utilizing 3-D long-term culture of 3T3-L1 preadipocytes 总被引:1,自引:0,他引:1
Fischbach C Spruss T Weiser B Neubauer M Becker C Hacker M Göpferich A Blunk T 《Experimental cell research》2004,300(1):54-64
Tissue-inherent factors such as cell-cell and cell-extracellular matrix interactions are regarded to exert a potentially large impact on adipogenesis as well as on secretory functions of adipose tissue. However, an appropriate 3-D adipogenesis model useful for addressing such interactions is still lacking. In this study, using tissue-engineering techniques, we demonstrate for the first time the development of coherent fat pads consisting of unilocular signet-ring cells in vitro. The constructs were generated by differentiating 3T3-L1 preadipocytes on 3-D polymeric scaffolds for either 9, 21, or 35 days in vitro. Only long-term culture yielded uniform tissues histologically comparable to native fat. Light and scanning electron microscopy provided direct evidence of 3-D tissue coherence and cell-cell contact in a tissue context, which was in strong contrast to conventional 2-D monolayer culture. Further differences between the two culture systems included enhanced secretion of leptin in 3-D tissue culture and differences in laminin expression (mRNA and protein level). Increase of triglyceride content over culture time and mRNA expression of other adipocyte genes, such as PPARgamma and Glut-4, were found to be similar. Implantation of long-term differentiated tissue constructs in nude mice resulted in further development and maintenance of fat pads. The presented model system is suggested to contribute to a better understanding of adipose tissue development and function facilitating studies on tissue-inherent interactions in vitro and in vivo. 相似文献
982.
Monoamine oxidase B (MAO B) is an outer mitochondrial membrane protein that oxidizes arylalkylamine neurotransmitters and has been a valuable drug target for many neurological disorders. The 1.7 angstrom resolution structure of human MAO B shows the enzyme is dimeric with a C-terminal transmembrane helix protruding from each monomer and anchoring the protein to the membrane. This helix departs perpendicularly from the base of the structure in a different way with respect to other monotopic membrane proteins. Several apolar loops exposed on the protein surface are located in proximity of the C-terminal helix, providing additional membrane-binding interactions. One of these loops (residues 99-112) also functions in opening and closing the MAO B active site cavity, which suggests that the membrane may have a role in controlling substrate binding. 相似文献
983.
Meyer G Rodighiero S Guizzardi F Bazzini C Bottà G Bertocchi C Garavaglia L Dossena S Manfredi R Sironi C Catania A Paulmichl M 《FEBS letters》2004,560(1-3):45-50
A novel xyloglucan-specific endo-β-1,4-glucanase (XEG), xyloglucanase, with a molecular mass of 80 kDa and a pI of 4.8, was isolated from the fungus Geotrichum sp. M128. It was found to be an endoglucanase active toward xyloglucan and not active toward carboxymethylcellulose, Avicel, or barley 1,3-1,4-β-glucan. Analysis of the precise substrate specificity using various xyloglucan oligosaccharide structures revealed that XEG has at least four subsites (−2 to +2) and specifically recognizes xylose branching at the +1 and +2 sites. The full-length cDNA encoding XEG was cloned and sequenced. It consists of a 2436-bp open reading frame encoding a 776-amino acid protein. From its deduced amino acid sequence, XEG can be classified as a family 74 glycosyl hydrolase. The cDNA encoding XEG was then expressed in Escherichia coli, and enzymatically active recombinant XEG was obtained. 相似文献
984.
The molecular mechanisms that control P element transposition and determine its tissue specificity remain incompletely understood, although much information has been compiled about this element in the last decade. This review summarizes the currently available information about P element transposition, P-M hybrid dysgenesis and P cytotype features, P element-encoded repressors, and regulation of transposition. 相似文献
985.
Absence of the RGS9.Gbeta5 GTPase-activating complex in photoreceptors of the R9AP knockout mouse 总被引:1,自引:0,他引:1
Keresztes G Martemyanov KA Krispel CM Mutai H Yoo PJ Maison SF Burns ME Arshavsky VY Heller S 《The Journal of biological chemistry》2004,279(3):1581-1584
Timely termination of the light response in retinal photoreceptors requires rapid inactivation of the G protein transducin. This is achieved through the stimulation of transducin GTPase activity by the complex of the ninth member of the regulator of G protein signaling protein family (RGS9) with type 5 G protein beta subunit (Gbeta5). RGS9.Gbeta5 is anchored to photoreceptor disc membranes by the transmembrane protein, R9AP. In this study, we analyzed visual signaling in the rods of R9AP knockout mice. We found that light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or Gbeta5 knockout rods. This effect was a consequence of the complete absence of any detectable RGS9 from the retinas of R9AP knockout mice. On the other hand, the level of RGS9 mRNA was not affected by the knockout. These data indicate that in photoreceptors R9AP determines the stability of the RGS9.Gbeta5 complex, and therefore all three proteins, RGS9, Gbeta5 , and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP. 相似文献
986.
Brehmer D Gässler C Rist W Mayer MP Bukau B 《The Journal of biological chemistry》2004,279(27):27957-27964
The DnaK chaperone of Escherichia coli assists protein folding by an ATP-dependent interaction with short peptide stretches within substrate polypeptides. This interaction is regulated by the DnaJ and GrpE co-chaperones, which stimulate ATP hydrolysis and nucleotide exchange by DnaK, respectively. Furthermore, GrpE has been claimed to trigger substrate release independent of its role as a nucleotide exchange factor. However, we show here that GrpE can accelerate substrate release from DnaK exclusively in the presence of ATP. In addition, GrpE prevented the association of peptide substrates with DnaK through an activity of its N-terminal 33 amino acids. A ternary complex of GrpE, DnaK, and a peptide substrate could be observed only when the peptide binding to DnaK precedes GrpE binding. Furthermore, we demonstrate that GrpE slows down the release of a protein substrate, sigma(32), from DnaK in the absence of ATP. These findings suggest that the ATP-triggered dissociation of GrpE and substrates from DnaK occurs in a concerted fashion. 相似文献
987.
988.
Orsini F Migliaccio E Moroni M Contursi C Raker VA Piccini D Martin-Padura I Pelliccia G Trinei M Bono M Puri C Tacchetti C Ferrini M Mannucci R Nicoletti I Lanfrancone L Giorgio M Pelicci PG 《The Journal of biological chemistry》2004,279(24):25689-25695
P66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress. It functions as a downstream target of the tumor suppressor p53 and is indispensable for the ability of oxidative stress-activated p53 to induce apoptosis. The molecular mechanisms underlying the apoptogenic effect of p66Shc are unknown. Here we report the following three findings. (i) The apoptosome can be properly activated in vitro in the absence of p66Shc only if purified cytochrome c is supplied. (ii) Cytochrome c release after oxidative signals is impaired in the absence of p66Shc. (iii) p66Shc induces the collapse of the mitochondrial trans-membrane potential after oxidative stress. Furthermore, we showed that a fraction of cytosolic p66Shc localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Treatment of cells with ultraviolet radiation induced the dissociation of this complex and the release of monomeric p66Shc. We propose that p66Shc regulates the mitochondrial pathway of apoptosis by inducing mitochondrial damage after dissociation from an inhibitory protein complex. Genetic and biochemical evidence suggests that mitochondria regulate life span through their effects on the energetic metabolism (mitochondrial theory of aging). Our data suggest that mitochondrial regulation of apoptosis might also contribute to life span determination. 相似文献
989.
Nonenzymatic chromophore attachment in biliproteins: conformational control by the detergent Triton X-100 总被引:3,自引:0,他引:3
Zhao KH Zhu JP Song B Zhou M Storf M Böhm S Bubenzer C Scheer H 《Biochimica et biophysica acta》2004,1657(2-3):131-145
While chromophore attachment to alpha-subunits of cyanobacterial biliproteins has been studied in some detail, little is known about this process in beta-subunits. The ones of phycoerythrocyanin and C-phycocyanin each carry two phycocyanobilin (PCB) chromophores covalently attached to cysteins beta84 and beta155. The differential nonenzymatic reconstitution of PCB to the apoproteins, PecA, PecB, CpcA and CpcB, as well as to mutant proteins of the beta-subunits lacking either one of the two binding cysteins, was studied using overexpression of the respective genes. PCB adds selectively to Cys-84 of CpcA, CpcB, PecA, and PecB, but the bound chromophore has a nonnative configuration, and in the case of CpcA, is partly oxidized to mesobiliverdin (MBV). The oxidation is independent of thiols but can be suppressed by ascorbate. The addition to Cys-beta84 is suppressed in the presence of detergents like Triton X-100, in favor of an addition to Cys-beta155 yielding the correctly bound chromophore. Triton X-100 also inhibits oxidation of the chromophore during addition to CpcA. The effect of Triton X-100 was studied on the isolated components of the reconstitution system. Absorption, fluorescence and circular dichroism spectra indicate a major conformational change of the chromophore upon addition of the detergent, which probably controls the site selectivity of the addition reaction, and inhibits the oxidation of PCB to MBV. 相似文献
990.
Carelli V Rugolo M Sgarbi G Ghelli A Zanna C Baracca A Lenaz G Napoli E Martinuzzi A Solaini G 《Biochimica et biophysica acta》2004,1658(1-2):172-179
Leber's hereditary optic neuropathy (LHON) was the first maternally inherited disease to be associated with point mutations in mitochondrial DNA and is now considered the most prevalent mitochondrial disorder. The pathology is characterized by selective loss of ganglion cells in the retina leading to central vision loss and optic atrophy, prevalently in young males. The pathogenic mtDNA point mutations for LHON affect complex I with the double effect of lowering the ATP synthesis driven by complex I substrates and increasing oxidative stress chronically. In this review, we first consider the biochemical changes associated with the proton-translocating NADH-quinone oxidoreductase of mitochondria in cybrid cells carrying the most common LHON mutations. However, the LHON cybrid bioenergetic dysfunction is essentially compensated under normal conditions, i.e. in glucose medium, but is unrevealed by stressful conditions such as growing cybrids in glucose free/galactose medium, which forces cells to rely only on respiratory chain for ATP synthesis. In fact, the second part of this review deals with the investigation of LHON cybrid death pathway in galactose medium. The parallel marked changes in antioxidant enzymes, during the time-course of galactose experiments, also reveal a relevant role played by oxidative stress. The LHON cybrid model sheds light on the complex interplay amongst the different levels of biochemical consequences deriving from complex I mutations in determining neurodegeneration in LHON, and suggests an unsuspected role of bioenergetics in shaping cell death pathways. 相似文献