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41.
42.
The combining site of the dinitrophenyl-binding immunoglobulin A myeloma protein MOPC 315 总被引:7,自引:6,他引:1 下载免费PDF全文
Steven K. Dower Simon Wain-Hobson Peter Gettins David Givol W. Roland C. Jackson Stephen J. Perkins Christopher A. Sunderland Brian J. Sutton Carolyn E. Wright Raymond A. Dwek 《The Biochemical journal》1977,165(2):207-223
Magnetic-resonance techniques are used to refine the model of the combining site of the Fv fragment of the dinitrophenyl-binding mouse myeloma protein MOPC 315 constructed by Padlan, Davies, Pecht, Givol & Wright (1976) (Cold Spring Harbor Symp. Quant. Biol. 41, in the press). Light-absorption studies indicate a dinitrophenyl–tryptophan interaction in the Fv fragment of the type occurring in free solution. The Dnp-aspartate–tryptophan complex is therefore used as a starting point for the n.m.r. (nuclear-magnetic-resonance) analysis of the dinitrophenyl–Fv fragment interaction. Ring-current calculations are used to determine the geometry of the complex. The specificity of complex-formation between dinitrophenyl and tryptophan is confirmed by the lack of ring-current shifts of the dinitrophenyl resonances when tryptophan is replaced by any other aromatic amino acid. Proton n.m.r. difference spectra (at 270MHz), resulting from the addition of a variety of haptens to the Fv fragment, show that the combining site is highly aromatic in nature. Calculations on the basis of ring-current shifts define the geometry of the combining site, which involves a dinitrophenyl ring in van der Waals contact with four aromatic amino acid residues on the protein. The observation of a nuclear Overhauser effect on the H(3) resonance of the dinitrophenyl ring provides additional constraints on the relative geometry of the H(3) proton and an aromatic amino acid residue on the Fv fragment. The specificity of the Fv fragment for dinitrophenyl ligands arises from a stacking interaction of the dinitrophenyl ring with tryptophan-93L, in an `aromatic box' of essentially tryptophan-93L, phenylalanine-34H and tyrosine-34L; asparagine-36L and tyrosine-34L also contribute by forming hydrogen bonds with the nitro groups on the dinitrophenyl ring. The n.m.r. results also confirm that the antibody–hapten reaction may be visualized as a single encounter step. An Appendix shows the method of calculation of ring currents for the four aromatic amino acids and their use in calculating structures. 相似文献
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Amino-terminal sequences of the L, M, and H subunits of reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26 总被引:7,自引:0,他引:7
We have determined the sequence of the 25-28 amino-terminal residues of the three subunits, L, M, and H, of the membrane-bound reaction center protein of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26. The sequences are as follows: L, H2N-Ala-Leu-Leu-Ser-Phe-Glu-Arg-Lys-Tyr-Arg- Val-Pro-Gly-Gly-Thr-Leu-Val-Gly-Gly-Asn-Leu-Phe-Asp-Phe-(His)-Val-; M, H2N-Ala-Glu-Tyr-Gln-Asn-Ile-Phe-Ser-Gln-Val-Gln-Val-Arg-Gly-Pro-Ala-Asp-Leu-Gly-Met-Thr-Glu-Asp-Val-Asn-Leu-Ala-Asn-; H, H2N-Met-Val-Gly-Val-Thr-Ala-Phe-Gly-Asn-Phe-Asp-Leu-Ala-Ser-Leu-Ala-Ile-Tyr-Ser-Phe-Trp-Ile-Phe-Leu-Ala-X-Leu-Ile-. The H sequence, especially after the aspartyl residue at position 11, is rich in hydrophobic residues, consistent with the possibility that this section of the polypeptide chain is located within the membrane. The L sequence is hydrophilic near the amino terminus and then becomes moderately hydrophobic. The M sequence is of average polarity. 相似文献
46.
Neutralization efficacy of Dey-Engley medium in testing of contact lens disinfecting solutions 总被引:2,自引:0,他引:2
A quantitative assay for the demonstration of neutralizer efficacy was developed to monitor contact lens disinfecting solutions. Adequate neutralization of disinfecting agents is essential to the accurate determination of disinfecting activity with time. This method employed the recovery of small numbers of micro-organisms from neutralizing medium containing a disinfectant. A statistical estimation of significance between treatments demonstrated that Dey-Engley medium (DE; Difco) was generally effective when tested as an agar growth medium with several bacterial test organisms. DE medium from another vendor was less effective, underscoring the need for laboratory quality control and monitoring. DE agar (Difco) adequately neutralized all solutions tested at a 1:20 dilution. The solutions included those containing Dymed (polyaminopropyl biguanide, 0.00005%), chlorhexidine (0.005%), Polyquad (0.001%), chlorhexidine (0.005%) and thimerosal (BP, 0.001%), thimerosal (BP, 0.002%) and Tris(2-hydroxyethyl) tallow ammonium chloride (0.013%), and a solution preserved with 115 ppm benzalkonium chloride (BAK). A modification of this medium was developed which retained virtually all of the neutralizing efficacy for the solutions tested while allowing the use of automated testing procedures. 相似文献
47.
Genetic analysis of potassium transport loci in Escherichia coli: evidence for three constitutive systems mediating uptake potassium. 总被引:14,自引:9,他引:5 下载免费PDF全文
D C Dosch G L Helmer S H Sutton F F Salvacion W Epstein 《Journal of bacteriology》1991,173(2):687-696
The analysis of mutants of Escherichia coli that require elevated concentrations of K+ for growth has revealed two new genes, trkG, near minute 30 within the cryptic rac prophage, and trkH, near minute 87, the products of which affect constitutive K+ transport. The analysis of these and other trk mutations suggests that high rates of transport, previously considered to represent the activity of a single system, named TrkA, appear to be the sum of two systems, here named TrkG and TrkH. Each of these two is absolutely dependent on the product of the trkA gene, a cytoplasmic protein associated with the inner membrane (D. Bossemeyer, A. Borchard, D. C. Dosch, G. C. Helmer, W. Epstein, I. R. Booth, and E. P. Bakker, J. Biol. Chem. 264:16403-16410, 1989). The TrkH system is also dependent on the products of the trkH and trkE genes, while the TrkG system is also dependent on the product of the trkG gene and partially dependent on the product of the trkE gene. It is suggested that the trkH and trkG products are membrane proteins that form the transmembrane path for the K+ movement of the respective systems. Two mutations altering the trkA product reduce the affinity for K+ of both TrkG and TrkH, indicating that changes in peripheral protein can alter the conformation of the sites at which K+ is bound prior to transport. The TrkD system has a relatively modest rate of transport, is dependent solely on the product of the trkD gene, and is the sole saturable system for Cs+ uptake in this species (D. Bossemeyer, A. Schl?sser, and E. P. Bakker, J. Bacteriol. 171:2219-2221, 1989). 相似文献
48.
Operation Everest II: metabolic and hormonal responses to incremental exercise to exhaustion. 总被引:5,自引:0,他引:5
P M Young J R Sutton H J Green J T Reeves P B Rock C S Houston A Cymerman 《Journal of applied physiology》1992,73(6):2574-2579
The reasons for the reduced exercise capacities observed at high altitudes are not completely known. Substrate availability or accumulations of lactate and ammonium could have significant roles. As part of Operation Everest II, peak oxygen uptakes were determined in five normal male volunteers with use of progressively increasing cycling work loads at ambient barometric pressures of 760, 380, and 282 Torr. Decrements from sea level (SL) to 380 and 282 Torr occurred in peak power output (19 and 47%), time to exhaustion (19 and 48%), and oxygen uptake (41 and 61%), respectively. Arterial saturations after exhaustive exercise were decreased to 63% at 380 Torr and 39% at 282 Torr. At 380 and 282 Torr, postexercise plasma concentrations of glucose and free fatty acids were not increased, whereas plasma glycerol concentrations were decreased relative to SL (145 +/- 24 microM at 380 Torr and 77 +/- 10 microM at 282 Torr vs. 213 +/- 24 microM at SL). Preexercise plasma insulin concentrations were elevated at both 380 and 282 Torr (87 +/- 16 pM at 380 Torr and 85 +/- 18 pM at 282 Torr vs. 41 +/- 30 pM at SL). In general, postexercise concentrations of plasma catecholamines were decreased at altitude compared with SL. Preexercise lactate and ammonium concentrations were not different at any simulated altitude. From these data neither substrate availability nor metabolic product accumulation limited exercise capacity at extreme simulated altitude. 相似文献
49.
Dissolved and suspended mercury. species in the Wabigoon River (Ontario,Canada): seasonal and regional variations 总被引:1,自引:1,他引:0
Jackson T. A. Parks J. W. Jones P. D. Woychuk R. N. Sutton J. A. Hollinger J. D. 《Hydrobiologia》1982,91(1):473-487
Seasonal and regional variations in the speciation, sediment-water partitioning, and dynamics of mercury (Hg) were studied
at selected sites along the Hg-polluted Wabigoon River, and at unpolluted headwater and tributary sites, during April–September,
1979. ‘Dissolved’ and ‘particulate’ forms of Hg in the water were separated by continuous-flow centrifugation in the field.
The Hg and other pollutants such as wood chips and salt had been discharged from a chlor-alkali plant and paper mill at Dryden,
Ontario.
Concentrations and loadings of particulate methyl mercury (CH3Hg+) and total particulate Hg (and loadings of total ‘dissolved’ Hg) were greatest during the spring flood (April-May) owing
to accelerated resuspension and transport of sediments. Concentrations of ‘dissolved’ CH3Hg+, however, were highest in the summer (July–September), probably reflecting stimulation of microbial methylating activity
by elevated temperatures, together with factors such as reduced levels of metal-scavenging particulates and minimal dilution
by runoff. Total dissolved Hg concentrations were relatively high in September at polluted sites only, possibly because of
desorption from sediments due to elevated concentrations of Cl− ions. Loadings of dissolved CH3Hg+ tended to be high in the summer but were generally depressed (suggesting sorption by suspended particles) during the major
spring-flood episode in May.
During July–August dissolved CH3Hg+ was a function of total dissolved Hg, suggesting rapid biomethylation of desorbed inorganic Hg; but in general dissolved
and suspended CH3Hg+ levels depended on environmental variables and were unrelated to total Hg concentrations. In the summer only, total dissolved
Hg was a function of dissolved Cl−. Hg species in particulates were associated with sulfides, hydrated Fe and Mn oxides, organic matter (notably high molecular
weight humic and humic-Fe components), and selenium (Se); but CH3Hg+ and total Hg differed in their specific preferences for binding agents, implying that binding sites discriminate between
CH3Hg+ and Hg2+ ions. CH3Hg+ was associated with sulfide and (in the spring only) with Fe oxides, whereas total Hg was associated with organic matter
and Se and with DTPA- and NaOH-extractable Fe in the spring but with Mn oxide and NaOH-extractable organics in the summer.
Sulfides were most abundant in May, indicating that they were eroded from bottom sediments, but Fe and Mn oxides were most
abundant in the summer, probably owing to activities of filamentous iron bacteria and other micro-organisms.
Particulate Hg was 98–100% nonextractable by mild solvents such as Ca acetate, CaCl2, dilute acetic acid, and (at polluted sites only) DTPA solutions, suggesting that the particulate Hg mobilized in the spring
may not be readily available to organisms; association with Se and high molecular weight humic matter also supports this hypothesis.
Hg probably becomes more bio-available in the summer, as suggested by the upsurge in dissolved CH3Hg+ and total dissolved Hg levels, and by increases in the solubility of particulate Hg in acetic acid, DTPA, H2O2, and NaOH solutions, as well as an increase in the relative importance of lower molecular weight fractions of NaOH-extractable
Hg (in September).
Regional variations in Hg speciation and partitioning reflected a gradient in sediment composition from wood chips near Dryden
to silt-clay mud further downstream. Hg in silt-clay mud relatively far (> 35 km) downstream from the source of pollution
or in unpolluted areas appeared to be more readily solubilized by Cl− ions or chelators such as DTPA, more readily methylated (as indicated by downstream increases in dissolved CH3Hg+ levels and CH3Hg+/total Hg ratios), and was to a greater degree organically bound (H2O2-extractable), and thus was probably more bio-available, than Hg in wood-chip deposits. Possible explanations include weaker
binding of Hg by the mud, the more finely divided state of the mud, and improved microbial growth at lower concentrations
of toxic pollutants. Owing to enrichment in sulfides and Fe oxides, resuspended wood-chip sediments were especially efficient
scavengers of CH3Hg+.
The results indicate that in any pollution abatement plan aimed at lowering the Hg levels in the biota of lakes fed by the
Wabigoon River, immobilization, removal, or detoxification of dissolved as well as particulate forms of Hg in the river would
probably have to be considered. Possibly, Hg species could be ‘scrubbed’ from the river water by increasing the suspended
load and by sedimentation and treatment with Hg-binding agents in special receiving basins. 相似文献
50.