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991.
Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli. The pressure sensitivity of the protoplasts was studied. Two different unit conductance mechanosensitive channels, 1100 ± 25 pS and 350 ± 14 pS in 400 mm symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically. The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca2+ and glutamate?. Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient. Both of the channels were voltage sensitive. Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization. The mech anosensitive channels were reversibly blocked by gadolinium ion. Also they could reversibly be inhibited by protons. Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly. This indicates a potential modulation of these channels by KefA. 相似文献
992.
Specific Activity of Brain Palmitoyl-CoA Pool Provides Rates of Incorporation of Palmitate in Brain Phospholipids in Awake Rats 总被引:4,自引:1,他引:3
Eric Grange Joseph Deutsch Quentin R. Smith Michael Chang Stanley I. Rapoport A. David Purdon 《Journal of neurochemistry》1995,65(5):2290-2298
Abstract: In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10-3 H]palmitate to maintain constant plasma specific activity. Animals were killed after 2–10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl-CoA, pools. [9,10-3 H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with >50% of brain tracer in choline-containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6–1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl-CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by ∼60-fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl-CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown. 相似文献
993.
This study addresses the ability of DNA fragments from various sources to mediate autonomous DNA replication in cultured Drosophila melanogaster cells. We created a series of plasmids containing genomic DNA fragments from the Ultrabithorax gene of Drosophila and tested them for autonomous replication after transfection into Schneider line 2 cells. We found that all plasmids containing Drosophila DNA fragments were able to replicate autonomously, as were plasmids containing random human and Escherichia coli genomic DNA fragments. Most of the plasmids were detectable 18 days after transfection in the absence of selection, suggesting that transfected DNA is maintained in Drosophila cells without rapid loss or degradation. The finding that all plasmids containing Drosophila, human, or bacterial DNA replicate autonomously in Drosophila cells suggests that the signals that direct autonomous replication in Drosophila contain a low degree of sequence specificity. A two-dimensional gel analysis of initiation on one of the plasmids was consistent with many dispersed initiation sites. Low sequence specificity and dispersed initiation sites also characterize autonomous replication in human cells and Senopus eggs and may be general properties of autonomous replication in animal cells. 相似文献
994.
995.
996.
Bernard N. Violand Michael R. Schlittler Kevin L. Duffin Christine E. Smith 《Journal of Protein Chemistry》1995,14(5):341-347
The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor. 相似文献
997.
The power of heteronuclear NMR spectroscopy to study macromoleculesand their complexes has been amply demonstrated over the last decade. Theobstacle to routinely applying these techniques to the study of DNA has beenthe synthesis of 13C,15N-labeled DNA. Here wepresent a simple and efficient method to generate isotope-labeled DNA forNMR studies that is as easy as that for isotope labeling of RNA. The methodwas used to synthesize a uniformly13 C,15N-labeled 32-nucleotide DNA that binds tohuman basic fibroblast growth factor with high affinity and specificity.Isotope-edited experiments were applied to the13 C,15N-labeled DNA bound to unlabeled protein,and the 13 C,15N-labeled DNA was also examined incomplex with 15N-labeled protein. The NMR experiments showthat the DNA adopts a well-defined stable structure when bound to theprotein, and illustrate the potential of13 C,15N-labeled DNA for structural studies ofDNA–protein complexes. 相似文献
998.
Timothy P. L. Smith Nestor Lopez-Corrales Michael D. Grosz Craig W. Beattie Steven M. Kappes 《Mammalian genome》1997,8(5):333-336
We report the placement of 34 new microsatellite (ms) markers, isolated from a lambda phage genomic clone library, on the
bovine genetic map by linkage to published markers. Five of these markers lie at or near the ends of linkage groups and are
used to establish chromosomal coverage and orientation. Fluorescence in situ hybridization (FISH) analysis demonstrates that
the linkage groups on the U.S. Meat Animal Research Center (MARC) map extend to the telomeric region of Chromosomes (Chrs)
7 and 10. Linkage groups on Chrs 4, 6, and 14 appear to be less inclusive.
Received: 23 September 1996 / Accepted: 28 December 1996 相似文献
999.
1000.
T. N. Ferraro G. T. Golden G. G. Smith N. J. Schork P. St. Jean C. Ballas H. Choi W. H. Berrettini 《Mammalian genome》1997,8(3):200-208
Mature DBA/2J (D2) mice are very sensitive to seizures induced by various chemical and physical stimuli, whereas C57BL/6J
(B6) mice are relatively seizure resistant. We have conducted a genome-wide search for quantitative trait loci (QTLs) influencing
the differential sensitivity of these strains to kainic acid (KA)-induced seizures by studying an F2 intercross population. Parental, F1, and F2 mice (8–10 weeks of age) were injected subcutaneously with 25 mg/kg of KA and observed for 3 h. Latencies to focal and generalized
seizures and status epilepticus were recorded and used to calculate an overall seizure score. Results of seizure testing indicated
that the difference in susceptibility to KA-induced seizures between D2 and B6 mice is a polygenic phenomenon with at least
65% of the variance due to genetic factors. First-pass genome screening (10-cM marker intervals) in F2 progeny (n = 257) documented a QTL of moderate effect on Chromosome (Chr) 1 with a peak LOD score of 5.5 (17% of genetic
variance explained) localized between D1Mit30 and D1Mit16. Provisional QTLs of small effect were detected on Chr 11 (D11Mit224–D11Mit14), 15 (D15Mit6–D15Mit46) and 18 (D18Mit9–D18Mit144). Multiple locus models generally confirmed the Mapmaker/QTL results and also provided evidence for another QTL on Chr 4
(D4Mit9). Multilocus analysis of seizure severity suggested that additional loci on Chrs 5 (D5Mit11), 7 (D7Mit66), and 15 (D15Nds2) might also contribute to KA-induced seizure response. Overall, our results document a complex genetic determinism for KA-induced
seizures in these mouse strains with contributions from as many as eight QTLs.
Received: 16 April 1996 / Accepted: 21 October 1996 相似文献