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71.
Roberta Pierattelli Lucia Banci D. L. Turner 《Journal of biological inorganic chemistry》1996,1(4):320-329
The chemical shifts of several 13C nuclei positioned α to the haems in oxidised cyanide complexes of horseradish peroxidase and lignin peroxidase are reported and analysed in terms
of π molecular orbitals with perturbed D4h symmetry. The additional contributions to the paramagnetic shifts of 13C nuclei in the vinyl groups which arise from conjugation with the porphyrin π molecular orbitals are discussed, and an empirical correction factor is derived from a number of other compounds which contain
haems b. The orbital mixing parameter which is obtained from the analysis of the experimental 13C shifts is compared with the orientation of the axial histidine ligands in X-ray structures of related compounds and found
to be close to the orientation of the normal to the histidine ring. Comparison with the magnetic axes determined by fitting
the dipolar shifts of several protons which have been assigned previously also shows close agreement with the negative in-plane
rotation of the magnetic y axis. It is therefore possible to obtain the approximate orientation of the magnetic axes from 13C resonances of the haem and hence to determine the dipolar shifts at any point in space with respect to the haem by using
these axes together with the anisotropy of the magnetic susceptibility, which can be obtained by extrapolation from EPR g values. Excellent agreement is found between dipolar shifts obtained by fitting an empirical magnetic susceptibility tensor
and predictions based on 13C NMR and EPR in the case of lignin peroxidase. The agreement is less good in the case of horseradish peroxidase, in which
the empirical magnetic z axis appears to be tilted significantly away from the haem normal, though this may be due in part to the lack of accurate
atomic coordinates. It is concluded that useful estimates of the magnetic susceptibility tensor may be obtained from 13C NMR and EPR studies even in large mammalian peroxidases for which no structural models are available.
Received: 27 December 1995 / Accepted: 17 April 1996 相似文献
72.
Alberto M. Martelli Lucia Manzoli Silvia Rubbini Anna Maria Billi Renato Bareggi Lucio Cocco 《Biology of the cell / under the auspices of the European Cell Biology Organization》1995,83(1):15-22
Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated. 相似文献
73.
A STAT factor mediates the sexually dimorphic regulation of hepatic cytochrome P450 3A10/lithocholic acid 6 beta-hydroxylase gene expression by growth hormone. 总被引:1,自引:0,他引:1
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Adult male rodents have a pulsatile profile of growth hormone (GH) release, whereas female rodents have a relatively steady-state pattern with uniform, albeit lower levels of GH. The expression of a number of sexually differentiated hepatic proteins is primarily determined by these plasma GH profiles and only secondarily regulated by gonadal hormones. An important subset of these sexually dimorphic proteins is cytochrome P450s. CYP3A10/6 beta-hydroxylase is a cytochrome P450 that catalyzes the 6 beta-hydroxylation of lithocholic acid. CYP3A10/6 beta-hydroxylase is expressed only in male hamsters; however, mimicking the male GH secretion pattern in females induces expression of the gene to male levels. Using chimeric CYP3A10/6 beta-hydroxylase promoter/luciferase reporter genes transfected into hamster primary hepatocytes, we have shown a GH-mediated induction of promoter activity. A combination of 5'-deletion constructs, heterologous promoter constructs, and specific mutagenesis was used to localize the DNA element involved in the GH-mediated regulation of CYP3A10/6 beta-hydroxylase promoter activity, which resembles a STAT binding site. Footprint and gel shift analyses confirmed that the expression of the protein binding to this site is regulated by GH and that the DNA-protein complex can be partially supershifted by anti-STAT-5 antibodies. This protein is 50% more abundant in male than in female hamster livers, is absent in hypophysectomized female livers, and is restored when hypophysectomized females are injected with GH in a manner that masculinizes female hamsters in terms of CYP3A10/6 beta-hydroxylase expression. The system characterized and described here is ideally suited for dissecting the molecular details governing the sexually dimorphic expression of liver-specific genes. 相似文献
74.
A. L. P. Freitas D. I. A. Teixeira F. H. F. Costa W. R. L. Farias A. S. C. Lobato A. H. Sampaio N. M. B. Benevides 《Journal of applied phycology》1997,9(6):495-501
Aqueous protein extracts from 30 Brazilian marine algae were examined for haemagglutinating activity using native and enzyme-treated
rabbit, chicken, sheep and human erythrocytes. Most extracts agglutinated at least one of the blood cells used. Sheep and
rabbit erythrocytes were more suitable for detection of the agglutinating activity. The minimum protein concentration necessary
to produce positive agglutination was usually lower with enzyme-treated erythrocytes than native ones. The five algal protein
extracts showing the greatest haemagglutination titre were tested for sugar-binding specificity. Only the activity present
in the green alga Cauler pacupressoides was inhibited by simple sugars and not by the glycoproteins tested. The activity of
the other four extracts was inhibited by at least one of the glycoproteins utilised.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
75.
Inês A. C. Pereira Jean LeGall António V. Xavier M. Teixeira 《Journal of biological inorganic chemistry》1997,2(1):23-31
The high-molecular-mass cytochromes c (Hmcs) from the sulfate-reducing bacteria Desulfovibrio gigas and Desulfovibrio vulgaris (Hildenborough) were found to be strongly bound to the cytoplasmic membrane. After detergent solubilization they were shown to be water soluble and to be similar to those previously isolated from the soluble fractions in terms of N-terminal sequence, molecular mass, UV-visible and EPR spectroscopies. In D. gigas, higher amounts of Hmc can be obtained from the membranes than from the soluble fraction. This enabled further characterization of both cytochromes. The apparent heme reduction potentials of both Hmcs, determined at pH 7.5 through visible and EPR redox titrations, span a large range of redox potentials, approximately between 0 and –280?mV, and can be roughly divided into three groups: four to five hemes have E 0s of –30?mV to –100?mV, three to four hemes have E 0s around –170?mV, and seven to eight hemes have a lower E 0 of –250 to –280?mV. Several of these redox potentials are strongly pH dependent. Mössbauer studies of oxidized and reduced D. vulgaris Hmc show that this protein contains two high-spin hemes in both oxidation states. The rate of reduction of both Hmcs with the periplasmic hydrogenases from the corresponding organisms is extremely slow. 相似文献
76.
Amicucci Antonella Rossi Ismaela Potenza Lucia Agostini Deborah Stocchi Vilberto 《Biotechnology Techniques》1997,11(3):149-154
Isolates of white truffles were identified as Tuber magnatum Pico species using a pair of primers selected from a sequence characterised amplified region (SCAR) and a specific random amplified polymorphic DNA (RAPD) marker. The present study reveals that PCR-fragment-pattern polymorphisms, the construction of probes and couples of primers from one or more of these polymorphic fragments may provide a useful and rapid tool for identifying species of ectomycorrhizal fungi in addition to conventional methods (morphological parameters). 相似文献
77.
A nuclear genetic lesion affecting Saccharomyces cerevisiae mitochondrial translation is complemented by a homologous Bacillus gene. 总被引:1,自引:0,他引:1
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S I Kim N Stange-Thomann O Martins K W Hong D Sll T D Fox 《Journal of bacteriology》1997,179(17):5625-5627
A novel Bacillus gene was isolated and characterized. It encodes a homolog of Saccharomyces cerevisiae Pet112p, a protein that has no characterized relative and is dispensable for cell viability but required for mitochondrial translation. Expression of the Bacillus protein in yeast, modified to ensure mitochondrial targeting, partially complemented the phenotype of the pet112-1 mutation, demonstrating a high degree of evolutionary conservation for this as yet unidentified component of translation. 相似文献
78.
In total, 86 enterococcal strains including representatives of most of the described species were tested for the ability to agglutinate human, sheep, and rabbit erythrocytes. Five strains did not react with any of the erythrocytes tested, and 81 (94.2%) strains agglutinated only rabbit erythrocytes. The hemagglutination titers ranged from 2 to 64. Loss of the hemagglutination activity was observed when rabbit erythrocytes were treated with trypsin or neuraminidase. Trypsin treatment of the bacterial suspensions also caused loss of the agglutination ability. On the other hand, heat treatment of bacterial suspensions increased the efficiency of the interactions, and higher titers were obtained. Assays for inhibition of hemagglutination were performed with -d-fucose, -d-galactose, -d-galactose, d-glucose, N-acetyl-galactosamine, N-acetyl-glucosamine, N-acetylneuraminic acid, N-acetylneuraminic acid-lactose, and fetuin. Only fetuin was able to inhibit the hemagglutination reactions. The results showed that hemagglutination properties are common to the different enterococcal species tested. They also suggest that enterococci possess hemagglutinins of proteic and non-proteic nature that are involved in the attachment to sialic acid-containing receptors on the surface of rabbit erythrocytes. 相似文献
79.
80.
Marcela F. Lopes Vânia Lúcia C. Merquior JoséMauro Peralta Lúcia M. Teixeira 《FEMS immunology and medical microbiology》1995,12(3-4):205-211
Abstract Exosubstances (cohemolysins) produced by Streptococcus agalactiae (CAMP-factor) and Streptococcus uberis (Uberis-factor) showing hemolytic synergism with β-lysin produced by Staphylococcus aureus were compared. Cohemolytic activity was evaluated in the supernatants of bacterial cultures, before and after ammonium sulfate precipitation. Sheep erythrocytes sensitized with β-lysin were used as substrate. The assays were performed in microtiter plates and results were expressed as cohemolytic units/ml. Maximum cohemolytic activity was detected, respectively, after 8 h and 14 h of growth in Columbia broth in S. uberis and S. agalactiae cultures. Cohemolytic activities of both microorganisms showed similarities when submitted to various physical and chemical treatments. They were significantly decreased by heating at 60°C and 100°C, or in presence of trypsin, and were abolished in the presence of Tween 20. Activities were found to be stable in crude supernatants and concentrated preparations maintained at −20°C for 3 months. Differences were related to levels of activity and kinetics of detection during the growth cycle. The results indicate the proteic nature, at least in part, of the Uberis factor. Analysis by PAGE in the presence or absence of SDS allowed us to correlate Uberis activity with a protein band with apparent molecular mass of 42 kDa, while CAMP activity was associated with a protein band of 27 kDa. 相似文献