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101.
Carbon metabolism in Bradyrhizobium japonicum bacteroids 总被引:2,自引:0,他引:2
Timothy R. McDermott Stephen M. Griffith Carroll P. Vance Peter H. Graham 《FEMS microbiology letters》1989,63(4):327-340
Abstract Carbon metabolism in Bradyrhizobium japonicum bacteroids is reviewed. Additionally, the bacteroid tricarboxylic acid (TCA) cycle and its regulation under oxygen-limited conditions is considered, with emphasis on possible sites of TCA cycle rate-limiting reactions. Furthermore, we consider other adaptive pathways that may be employed by these organisms while in symbiosis. These pathways include: (1) a poly-β-hydroxy-butyrate shunt, (2) a malate-aspartate shuttle, (3) an α-ketoglutarate-glutamate shunt, (4) a modified dicarboxylic acid cycle, and (5) fermentation pathways leading to lactate, acetaldehyde and ethanol. The effects of oxygen limitation on host carbon metabolism are also considered briefly. 相似文献
102.
David G. Griffiths Michael D. Partis Perry Churchill Stephen C. Brenner Sidney Fleischer Roger J. Moore R. Brian Beechey 《Journal of bioenergetics and biomembranes》1990,22(5):691-707
A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane. 相似文献
103.
104.
A method for the purification of full-length nerve growth factor receptor (NGFRc) using membranes from three different cell lines was developed. We emphasized recovery of NGFRc that retained specific binding activity. Lipids were required to preserve binding activity during solubilization and throughout the purification procedure. Phosphatidylcholine was used for this purpose. Lectin affinity chromatography followed by high-resolution anion-exchange chromatography was used, and a 3000-fold increase in specific binding activity was obtained for NGFRc from human melanoma A875 membranes. Seven percent of the original binding activity was recovered as pure NGFRc. NGFRc binding activity eluted at 0.35 M NaCl in anion-exchange chromatography of solubilized A875, rat pheochromocytoma PC12, and human neuroblastoma MC-IXC membranes. Eight and three percent of the original binding activity were recovered as highly enriched NGFRc from membranes prepared from PC12 and MC-IXC cells, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of highly enriched, 125I-labeled NGFRc revealed several protein species. After chromatography, identification of proteins as NGFRc was verified both by immunoprecipitation using receptor-specific monoclonal antibodies and by covalent cross-linking to 125I-NGF using N-hydroxysuccinimidyl-4-azidobenzoate. Predominantly, NGFRc was recovered as a mixture of species of 80 and 160-180 kDa. Small amounts of larger species as well as smaller species were observed, consistent with minor amounts of receptor aggregation and proteolysis occurring during purification. 相似文献
105.
Michael A. Bowers Stephen F. Matter James L. Dooley Jr. Jennifer L. Dauten John A. Simkins 《Oecologia》1996,108(1):182-191
Habitat fragmentation involves a reduction in the effective area available to a population and the imposition of hard patch edges. Studies seeking to measure effects of habitat fragmentation have compared populations in fragments of different size to estimate and area effect but few have examined the effect of converting open populations to closed ones (an effect of edges). To do so requires a shift in spatial scope-from comparison of individual fragments to that of fragmented versus unfragmented landscapes. Here we note that large-scale, controlled studies of habitat fragmentation have rarely been performed and are needed. In making our case we develop a simple computer simulation model based on how individual animals with home ranges are affected by the imposition of habitat edges, and use it to predict population-level responses to habitat fragmentation. We then compare predictions of the model with results from a field experiment on Peromyscus and Microtus. Our model treats the case where home ranges/territories fall entirely within or partially overlap with that of sample areas in continuous landscapes, but are restricted to areas within habitat fragments in impacted landscapes. Results of the simulations demonstrate that the imposition of hard edges can produce different population abundances for similar-sized areas in continuous and fragmented landscapes. This edge effect is disproportionately greater in small than large fragments and for species with larger than smaller home ranges. These predictions were generally supported by our field experiment. We argue that large-scale studies of habitat fragmentation are sorely needed, and that control-experiment contrasts of fragmented and unfragmented microlandscapes provide a logical starting point. 相似文献
106.
James D. Pancook Jürgen C. Becker Stephen D. Gillies R. A. Reisfeld 《Cancer immunology, immunotherapy : CII》1996,42(2):88-92
A major problem in the treatment of solid tumors is the eradication of established, disseminated metastases. Here we describe
an effective treatment for established experimental hepatic metastases of human neuroblastoma in C. B.-17 scid/scid mice. This was accomplished with an antibody-cytokine fusion protein, combining the unique targeting ability of antibodies
with the multifunctional activity of cytokines. An anti-(ganglioside GD2) antibody (ch14.18) fusion protein with interleukin-2
(ch14.18-IL2), constructed by fusion of a synthetic sequence coding for human interleukin-2 (IL-2) to the carboxyl end of
the Cγ1 gene of ch14.18, was tested for its therapeutic efficacy against xenografted human neuroblastoma in vivo. The ch14.18-IL2
fusion protein markedly inhibited growth of established hepatic metastases in SCID (severe combined immunodeficiency) mice
previously reconstituted with human lymphokine-activated killer cells. Animals treated with ch14.18-IL2 showed an absence
of macroscopic liver metastasis. In contrast, treatment with combinations of ch14.18 and recombinant IL2 at dose levels equivalent
to the fusion protein only reduced the tumor load. Survival times of SCID mice treated with the fusion protein were more than
double that of control animals. These results demonstrate that an immunotherapeutic approach using a cytokine targeted by
an antibody to tumor sites is highly effective in eradicating the growth of established tumor metastases.
Received: 7 November 1995 / Accepted: 15 December 1995 相似文献
107.
Alessandro Pintar Meike Hensmann Kornelia Jumel Maureen Pitkeathly Stephen E. Harding Iain D. Campbell 《European biophysics journal : EBJ》1996,24(6):371-380
The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155–270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of1H-15N-correlated 2D HSQC,15N-edited 3D TOCSY-HMQC, and15N-edited 3D NOESY-HMQC spectra. By analysis of the -proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain.To investigate the internal dynamics of the protein backbone, T1 and T2 relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.Abbreviations SH
Src homology
- GST
glutathione-S-transferase
- IPTG
isopropyl--D-galactopyranoside
- DTT
dithiothreitol
- PMSF
phenyl-methyl-sulphonyl-fluoride
- TBS
50 mM Tris, 150 mM NaCl, 5 mM DTT, pH 8.0
- MWCO
molecular weight cut off
- NMR
nuclear magnetic resonance
- HSQC
heteronuclear single-quantum correlation
- NOESY
nuclear Overhauser effect spectroscopy 相似文献
108.
K. Harikrishna Rachanee Jampates-Beale Stephen B. Milligan Charles S. Gasser 《Plant molecular biology》1996,30(5):899-911
A gene (pMON9617; Chi2;1) identified by screening a tomato pistil cDNA library has been found to encode a protein similar in sequence to class II chitinases. Using a baculovirus expression system we show that the Chi2;1 protein is an active endochitinase. The Chi2;1 protein is most similar in sequence to a previously described stylar chitinase (SK2) isolated from potato. Both proteins lack the diagnostic N-terminal cysteine-rich regions and the C-terminal vacuolar targeting signals of class I chitinases and appear to define a novel type of class II endochitinases associated with flowers. Chi2;1 is expressed predominantly in floral organs and its expression within these organs is temporally regulated. The highest level of expression is found in the transmitting tissue of the style where Chi2;1 mRNA begins to accumulate just prior to anthesis. In vegetative tissue, low levels of Chi2;1 mRNA were detected, and these levels did not increase in response to wounding or treatment with ethephon. mRNA from Chi2;1 orthologs was not detected in most other angiosperms tested, even including some members of the Solanaceae, and it is therefore unlikely that Chi2;1 is essential for stylar function. A fragment containing 1.4 kilobase pairs of 5-flanking DNA from the Chi2;1 gene was shown to drive high-level expression of an attached reporter gene in the styles of transgenic tomatoes. This fragment has utility for engineering expression of other coding regions in styles. 相似文献
109.
110.