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31.
C-terminal peptide identification by fast atom bombardment mass spectrometry. 总被引:1,自引:0,他引:1 下载免费PDF全文
A previously described technique [Rose, Simona, Offord, Prior, Otto & Thatcher (1983) Biochem. J. 215, 273-277] permits the identification of the C-terminal peptide of a protein as the only peptide that does not incorporate any 18O upon partial enzymic hydrolysis in 18O-labelled water. Formation of chemical derivatives followed by combined g.l.c.-m.s. was used in this earlier work. We now describe the isolation from protein digests, by reversed-phase h.p.l.c., of labelled and unlabelled polypeptides and their direct analysis by fast atom bombardment mass spectrometry. Under the conditions used, the 18O label is retained throughout the separation and analysis, thus permitting assignments of C-terminal peptides to be made. Enzyme-catalysed exchange of label into the terminal carboxy group was found to occur in some cases without hydrolysis of a peptide bond. This effect, which may be exploited to prepare labelled peptides, does not prevent application of the method (two separate digests must then be used). We have applied our method to the analysis of enzymic partial hydrolysates of glucagon, insulin and of several proteins produced by expression of recombinant DNA. 相似文献
32.
The hydrated volumes, Vh, of collagens extracted from various fish species were calculated by using the Simha-Einstein equation, and it was found that the hydration of warm-water fish collagen is greater than that of cold-water fish collagen (halibut). Although the intrinsic viscosities of warm-water fish (bigeye-tuna, carp and catfish) collagens are almost the same, the hydrated volume of bigeye-tuna collagen is approx. 1.5 and 3 times those of carp and catfish collagens respectively. The extent of hydration at 20 degrees C is in the following order: bigeye tuna greater than carp greater than catfish greater than halibut. The various thermodynamic activation parameters (delta G*, delta H* and delta S*) were calculated and it was found that they are useful for determining the exact denaturation temperature. It was calculated that the denaturation temperatures of halibut, bigeye-tuna, carp and catfish collagens are 17, 31, 32 and 26-30 degrees C respectively. The variations of hydration, intrinsic viscosity, denaturation temperature and the thermodynamic parameters with the variation of concentration of catfish collagen were also thoroughly examined. The change of thermodynamic parameters from coiled-coil to random-coil conformation upon denaturation of collagen were calculated from the amount of proline and hydroxyproline residues and compared with viscometric results. 相似文献
33.
Insulin proteinase liberates from glucagon a fragment known to have enhanced activity against Ca2+ + Mg2+-dependent ATPase. 下载免费PDF全文
K Rose L A Savoy A V Muir J G Davies R E Offord G Turcatti 《The Biochemical journal》1988,256(3):847-851
We find, contrary to previous reports, that substantial cleavage of glucagon by insulin proteinase occurs at only one region, namely the double-basic sequence -Arg17-Arg18-. Cleavage takes place almost exclusively between these two residues, liberating fragments glucagon-(1-17) and glucagon-(18-29). Others have shown that the fragment glucagon-(19-29) is 1000-fold more efficient compared with intact glucagon, at inhibiting the Ca2+-activated and Mg2+-dependent ATPase activity and the Ca2+ pump of liver plasma membranes. We show that this fragment is not liberated in detectable quantities by our insulin proteinase preparation. On the other hand, others have shown that glucagon-(18-29), though less active than glucagon-(19-29), was still 100-fold more active than glucagon itself in the above-mentioned system. Our observations represent the first demonstration of the release by insulin proteinase of a hormone fragment having enhanced activity, although it has yet to be shown that the activity of this fragment is important in vivo. Since the formation of glucagon-(19-29) from glucagon-(18-29) would involve merely removal of Arg18, a second enzyme might exist to provide the more active fragment. 相似文献
34.
A method is described for the preparation of polypeptides activated uniquely at the C-terminus. The polypeptide is incubated in a concentrated solution of an amino acid active ester, the latter having its amino group free but adequately protected by protonation. The amino acid ester is coupled via its amino group to the C-terminus of the polypeptide by enzymic catalysis (reverse proteolysis). The resulting polypeptide C-terminal active ester is then isolated and coupled to a suitable amino component (generally a polypeptide) in a subsequent chemical coupling. The method appears to be generally applicable; fragments of horse heart cytochrome c, and porcine insulin, are used as examples. Two new analogues of cytochrome c have been prepared by using this method, with yields of up to 60% in the final coupling. Scope and limitations of the method are discussed. 相似文献
35.
Sequence identity between an inverted repeat family of transposable elements in Drosophila and Caenorhabditis. 总被引:7,自引:5,他引:2 下载免费PDF全文
The Tc1-like transposable elements, originally described in Caenorhabditis elegans, have a much wider phylogenetic distribution than previously thought. In this paper, we demonstrate that Tc1 shares sequence identity in its open reading frame and terminal repeats with a new transposable element Barney (also known as TCb1-Transposon Caenorhabditis briggsae 1). Barney was detected and isolated by Tc1 hybridization from the closely related nematode species, Caenorhabditis briggsae. The conserved open reading frames of Tc1 and Barney share identity with a structurally similar family of elements named HB found in Drosophila melanogaster, after the introduction of 3 small centrally located deletions in HB1. These reading frames would code for proteins with 30% amino acid identity (42% when conservative changes are included). Tc1, Barney and HB1 contain highly conserved blocks of amino acids which are likely to be in the functional domains of the putative transposase. 相似文献
36.
R E Rose 《Nucleic acids research》1988,16(1):355
37.
38.
In vivo electrochemical studies of the effects of cocaine on dopamine nerve terminals in the rat neostriatum 总被引:3,自引:0,他引:3
In vivo electrochemical measurements, involving chronoamperometric recordings using monoamine-selective Nafion-coated electrodes, were used to study the effects of locally applied cocaine (50-500 micromolar barrel concentrations) on dopamine (DA) nerve terminals in the neostriatum of the anaesthetized rat. Local application of cocaine did not elicit detectable increases in basal levels of extracellular DA. However, locally applied cocaine significantly augmented the concentration of DA detected following a potassium (K+)-evoked depolarization. Data obtained with a new high-speed chronoamperometric recording technique further support that DA is the predominant species detected electrochemically following potassium-evoked depolarizations both before and after local application of cocaine. Unlike other locally applied uptake inhibitors that we have studied, cocaine failed to augment the time dynamics of released DA. In addition, large doses of the highest concentration of cocaine caused an attenuation of K+-evoked DA release, presumably due to cocaine's local anaesthetic properties. These data suggest that cocaine elevates synaptic levels of DA, but in a manner that is not identical to other potent monoamine uptake inhibitors. 相似文献
39.
Nichola J. K. Hole Edmundo Lamoyi Masanori Komatsu Nagaradona Harindranath Glendowlyn O. Young-Cooper Rose G. Mage 《Immunogenetics》1988,28(2):99-107
In order to investigate linkage, we used serum allotypes of the two rabbit C
isotypes and restriction fragment length polymorphisms (RFLPs) of the genes for V
, C
, and T-cell receptor C
. The inheritance of these genetic markers was studied through backcross and F2 matings. Southern analysis and hybridization of genomic DNA with a C probe detected a 5 kb Pst I fragment linked to expression of the K2bas1 allotype and the presence of the 1b
bas gene and a 6.6 kb Pst I fragment linked to the expression of the K1b9 allotype, the presence of the 2
bas2 gene and lack of expression of the K2bas1 allotype. A V probe detected a 1.3 kb Eco RI fragment linked to the presence of the 1b
bas gene and expression of the K2bas1 allotype. In contrast, the 9 or 14 kb Eco RI RFLP (C
a or C
b) detected with a Tcr
chain probe segregated independently from C allotypes and RFLPs. It has previously been found that C and C
are also unlinked in man, whereas in the mouse they are linked at a distance of 8 centimorgans. 相似文献
40.
The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5 non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus. 相似文献