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171.
A. Shahar S. Reuveny M. Zhang A. Espinosa de los Monteros J. de Vellis A. Shainberg 《Cytotechnology》1992,9(1-3):107-115
Dispersed neuronal and muscular elements from fetal or neonatal origin, can organize and mature in culture when grown on positively
charged cylindrical microcarriers (MCS), to a stage which simulatein vivo maturation. Cells arrange themselves on the MCS to form aggregates which remain floating in the nutrient medium. In such
a tridimensional organization, the neuronal tissue is capable of regenerating a network of nerve fibers which establish synapse
interconnections and undergo myelination. Oligodendrocytes organize on MCS in a tridimensional pattern and produce extensive
myelin-like membranes. Myoblasts in MC-cultures fuse into polynucleated myotubes which become striated and contract spontaneously.
Creatine kinase and acetylcholine receptor (AChR) are formed during myogenesis in similar quantities in MC-cultures and in
monolayers. When both neuronal and muscle tissues are prepared from the same fetus (autologous nerve-muscle co-cultures) and
are cultured on MCS, they interconnect to form neuro-muscular junctions. Cells from both tissues, exhibit better differentiation,
for longer periods in MC-cultures than they do in monolayers. The floating functional entities are easy to sample and can
be harvested for ultrastructural, immunocytochemical and biochemical analysis. In addition, MC-cultures can be used as a good
tool for the study of acute and chronic exposures to toxicological agents, as well as for implantation into demyelinated,
injured or dystrophic tissues. In this case the MCS in the implanted entities will serve as identifiable markers. 相似文献
172.
Many N2-fixing organisms can turn off nitrogenase activity in the presence of NH4
+ and turn it on again when the NH4
+ is exhausted. One of the most interesting systems for accomplishing this is by covalent modification of one subunit of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). The system can be reactivated when NH4
+ is exhausted, by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the inactivating group. It is fascinating that some species of the genusAzospirillum possess the DRAT and DRAG systems (A. lipoferum andA. brasilense), whereasA. amazonense in the same genus lacks DRAT and DRAG.A. amazonense responds to NH4
+ but does not exhibit modification of dinitrogenase reductase characteristic of the action of DRAT. However, it has been possible to clone DRAT and DRAG and to introduce them intoA. amazonense, whereupon they become functional in this organism. The DRAT and DRAG system does not appear to function inAcetobacter diazotrophicus, an organism isolated from sugar cane, that fixes N2 at a pH as low as 3.0.A. diazotrophicus does show a rather sluggish response to NH4
+. A level of about 10 M NH4
+ is required to switch off the system. The response to NH4
+ is influenced by the dissolved oxygen concentration (DOC) as has been reported forAzospirillum sp. A DOC in equilibrium with 0.1 to 0.2 kPa O2 seems optimal for the response inA. diazotrophicus. 相似文献
173.
174.
Oxidation of dimethyl sulfide byPseudomonas acidovorans DMR-11 isolated from peat biofilter 总被引:1,自引:0,他引:1
Summary
Pseudomonas acidovorans DMR-11, capable of oxidizing dimethyl sulfide (DMS), was isolated from peat biofilter. DMS as a sole carbon or energy source was not degraded, but it was co-degraded in the medium containing organic carbon sources. The removal rate of DMS in heat-treated glucose medium was 1.12×10–17 mole/h cell at 30 °C. Dimethyl sulfoxide (DMSO) was the only product of DMS oxidation and was formed stoichiometrically. DMS was reversibly evolved in excess of DMSO. The cell free extract of strain DMR-11 oxidized DMS in presence of NADPH. 相似文献
175.
Regardless of the field of application, the raison d'etre of transgenic animals is to study gene regulation and function. With increasing frequency, mammalian genes are being isolated with no concomitant knowledge of their function. The human genome mapping initiative will undoubtedly produce a cornucopia of such genes. While the merit of taking a transgenic route to study genes of unknown function is axiomatic, the choices of strategies for gene regulation in vivo may not be fully appreciated. This review will address two main points: first, the targeted and regulated expression of genes, and second, the structural and functional ablation of genes. 相似文献
176.
Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent, additive effects on both structure and stability. 总被引:5,自引:1,他引:4 下载免费PDF全文
X. J. Zhang W. A. Baase B. W. Matthews 《Protein science : a publication of the Protein Society》1992,1(6):761-776
In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within alpha-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the alpha-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions. All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest (< or = 0.4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133-->Ala made alpha-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino- and carboxy-terminal domains changed by 3.6 degrees. This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change. 相似文献
177.
Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport. 总被引:3,自引:3,他引:0 下载免费PDF全文
R. A. Binnie H. Zhang S. Mowbray M. A. Hermodson 《Protein science : a publication of the Protein Society》1992,1(12):1642-1651
Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically. 相似文献
178.
Complete enzymatic deglycosylation of native sex steroid-binding protein (SBP or SHBG) of human and rabbit plasma: effect on the steroid-binding activity. 下载免费PDF全文
P. H. Petra P. R. Griffin J. R. Yates rd K. Moore W. Zhang 《Protein science : a publication of the Protein Society》1992,1(7):902-909
An enzymatic procedure for the complete removal of the N-linked and O-linked oligosaccharide side chains of the sex steroid-binding proteins (SBP or SHBG) of human and rabbit plasma under native conditions is described. Deglycosylation was catalyzed by N-glycanase, neuraminidase, and O-glycanase and was monitored by SDS-PAGE, lectin blotting, and molecular weight analyses by electrospray mass spectrometry. Digestion of rabbit SBP with N-glycanase generated a major 39,777-Da protein and two minor ones of 39,389 and 39,545 Da. The molecular weight of the major protein agrees with the molecular weight calculated from the sequence of the sugar-free polypeptide monomer (39,769 Da: Griffin, P.R., Kumar, S., Shabanowitz, J., Charbonneau, H., Namkung, P.C., Walsh, K.A., Hunt, D.F., & Petra, P.H., 1989, J. Biol. Chem. 264, 19066-19075), whereas the other two are deglycosylated proteolytic cleavage products lacking the TQR and TQ sequences at the amino-terminus. The N- and O-linked side chains of human SBP were removed by sequential digestion with N-glycanase and neuraminidase/O-glycanase. A 38,771-Da protein was generated, which agrees well with the molecular weight of the sugar-free polypeptide monomer (Walsh, K.A., Titani, K., Kumar, S., Hayes, R., & Petra, P.H., 1986, Biochemistry 25, 7584-7590). N-deglycosylation of human and rabbit SBP has no effect on the steroid-binding activity, but removal of the O-linked side chains of N-deglycosylated human SBP results in an apparent 50% loss of steroid-binding activity and an increase in the Kd for the binding of 5 alpha-dihydrotestosterone from 0.3 mM to 0.9 nM.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
179.
180.