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81.
Transaldolase B of Escherichia coli K-12: cloning of its gene, talB, and characterization of the enzyme from recombinant strains. 总被引:2,自引:0,他引:2 下载免费PDF全文
A previously recognized open reading frame (T. Yura, H. Mori, H. Nagai, T. Nagata, A. Ishihama, N. Fujita, K. Isono, K. Mizobuchi, and A. Nakata, Nucleic Acids Res. 20:3305-3308) from the 0.2-min region of the Escherichia coli K-12 chromosome is shown to encode a functional transaldolase activity. After cloning of the gene onto high-copy-number vectors, transaldolase B (D-sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate dihydroxyacetone transferase; EC 2.2.1.2) was overexpressed up to 12.7 U mg of protein-1 compared with less than 0.1 U mg of protein-1 in wild-type homogenates. The enzyme was purified from recombinant E. coli K-12 cells by successive ammonium sulfate precipitations (45 to 80% and subsequently 55 to 70%) and two anion-exchange chromatography steps (Q-Sepharose FF, Fractogel EMD-DEAE tentacle column; yield, 130 mg of protein from 12 g of cell wet weight) and afforded an apparently homogeneous protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit size of 35,000 +/- 1,000 Da. As the enzyme had a molecular mass of 70,000 Da by gel filtration, transaldolase B is likely to form a homodimer. N-terminal amino acid sequencing of the protein verified its identity with the product of the cloned gene talB. The specific activity of the purified enzyme determined at 30 degrees C with the substrates fructose-6-phosphate (donor of C3 compound) and erythrose-4-phosphate (acceptor) at an optimal pH (50 mM glycylglycine [pH 8.5]) was 60 U mg-1.Km values for the substrates fructose-6-phosphate and erythrose-4-phosphate were determined at 1,200 and 90 microM, respectively. Kinetic constants for the other two physiological reactants, D,L-glyceraldehyde 3-phosphate (Km, 38 microM; relative activity [V(rel)], 8%) and sedoheptulose-7-phosphate (K(m), 285 microM; V(rel), 5%) were also determined. Fructose acted as a C(3) donor at a high apparent K(m) (>/=M) and with a V(rel) of 12%. The enzyme was inhibited by Tris-HCl, phosphate, or sugars with the L configuration at C(2) (L-glyceraldehyde, D-arabinose-5-phosphate). 相似文献
82.
83.
Hermann Neudeck Shiao Li Oei Beate Stiemer Hartmut Hopp Renat E Graf 《The Histochemical journal》1997,29(5):419-430
Recent immunocytochemical studies have shown that placental villous trophoblasts contain the high molecular weight cytokeratin
(CK) proteins 5/6 and 17. In the case of CK 17, trophoblastic immunostaining was positive in villi covered by fibrinoid. CKs
5/6 and 17 are expressed by hyperproliferative cells. The aim of this investigation was to examine the location of these CKs
in placental infarcts, known to be demarcated by fibrinoid and hyperproliferative trophoblasts. The results were compared
with those obtained by immunostaining against Ki-67, tenascin and α1-, α6- and β1- integrins, which are involved in cell proliferation,
differentiation and regenerative processes. Furthermore, the expression of the single CKs 7, 8, 10, 13, 14, 18 and 19 was
investigated by immunocytochemistry and immunoblotting. While low and high molecular weight CKs were present in villous and
extravillous trophoblasts, only low molecular weight CKs were detected in vascular and extravascular placental smooth muscle
cells. Placental infarcts revealed different immunoreactivities in the infarct margin and centre: high molecular CKs, tenascin,
Ki-67 and oncofoetal fibronectin predominated in the infarct margin, low molecular CKs, fibrin and integrins in the centre.
The expression of tenascin and a defined change in the expression of CK 17 indicates villous repair and hyperproliferative
mechanisms in placental infarcts.
This revised version was published online in November 2006 with corrections to the Cover Date. 相似文献
84.
Determination of apoplastic K+ in intact leaves by ratio imaging of PBFI fluorescence 总被引:2,自引:0,他引:2
The tetraammonium salt of the K+ binding fluorescent dye benzofuranisophthalate (PBFI) was used to investigate the influence ofpotassium nutrition (0.12.1 mol m3) on apoplasticK+ inVicia faba leaves by means of ratio imaging. As a referencethe infiltration-centrifugation method was used. Both methodsreflected the influence of K+ supply on apoplastic K+ concentration.The abaxial leaf side revealed significantly higher K+ concentrations(20-25 mol m3) than the adaxial side (58 mol m3).Application of CCCP led to an immediate increase in apoplasticK+ demonstrating the reliability of the PBFI method. Key words: Vicia faba, leaf, apoplast, K+, PBFI, ratio imaging, ratiometric fluorescence microscopy 相似文献
85.
The effect of ethanol on maxi Ca2+-activated K+ channels (BK channels) in GH3 pituitary tumor cells was investigated using single-channel recordings and focusing on intracellular
signal transduction. In outside-out patches, ethanol caused a transient concentration-dependent increase of BK-channel activity.
30 mm (1.4‰) ethanol significantly increased mean channel open time and channel open probability by 26.3 ± 9% and 78.8 ± 10%, respectively;
single-channel current amplitude was not affected by ethanol. The augmenting effect of ethanol was blocked in the presence
of protein kinase C (PKC) inhibitors staurosporine, bisindolylmaleimide, and PKC (19–31) pseudosubstrate inhibitor as well
as by AMP-PNP (5′-adenylylimidodiphosphate), a nonhydrolyzable ATP-analogue, but not by the phospholipase C blocker U-73122.
Phosphatase inhibitors microcystin-LR and okadaic acid promoted the ethanol effect. The blocking effect was released at higher
concentrations of ethanol (100 mm) suggesting a second site of action or a competition between blockers and ethanol. Our results suggest that the effect of
ethanol on BK-channels is mediated by PKC stimulation and phosphorylation of the channels which increases channel activity
and hence may influence action potentials duration and hormone secretion.
Received: 24 July 1996/Revised: 27 December 1996 相似文献
86.
Growth of Nitrosomonas europaea on hydroxylamine 总被引:2,自引:0,他引:2
Peter de Bruijn Astrid A. Van de Graaf Mike S.M. Jetten Lesley A. Robertson J. Gijs Kuenen 《FEMS microbiology letters》1995,125(2-3):179-184
Abstract Hydroxylamine is an intermediate in the oxidation of ammonia to nitrite, but until now it has not been possible to grow Nitrosomonas europaea on hydroxylamine. This study demonstrates that cells of N. europaea are capable of growing mixotrophically on ammonia and hydroxylamine. The molar growth yield on hydroxylamine (4.74 g mol−1 at a growth rate of 0.03 h−1 ) was higher than expected. Aerobically growing cells of N. europaea oxidized ammonia to nitrite with little loss of inorganic nitrogen, while significant inorganic nitrogen losses occurred when cells were growing mixotrophically on ammonia and hydroxylamine. In the absence of oxygen, hydroxylamine was oxidized with nitrite as electron acceptor, while nitrous oxide was produced. Anaerobic growth of N. europaea on ammonium, hydroxylamine and nitrite could not be observed at growth rates of 0.03 h−1 and 0.01 h−1 . 相似文献
87.
S. S. Wijmenga H. A. Heus H. A. E. Leeuw H. Hoppe M. van der Graaf C. W. Hilbers 《Journal of biomolecular NMR》1995,5(1):82-86
Summary A new 1H−13C−31P triple resonance experiment is described which allows unambigous sequential backbone assignment in 13C-labeled oligonucleotides via through-bond coherence transfer from 31P via 13C to 1H. The approach employs INEPT to transfer coherence from 31P to 13C and homonuclear TOCSY to transfer the 13C coherence through the ribose ring, followed by 13C to 1H J-cross-polarisation. The efficiencies of the various possible transfer pathways are discussed. The most efficient route
involves transfer of 31Pi coherence via C4′i and C4′i-1, because of the relatively large J′PC4 couplings involved. Via the homonuclear and heteronuclear mixing periods, the C4′i and C4′i-1 coherences are subsequently transferred to, amongst others, H1′i and H1′i-1, respectively, leading to a 2D 1H−31P spectrum which allows a sequential assignment in the 31P−1H1′ region of the spectrum, i.e. in the region where the proton resonances overlap least. The experiment is demonstrated on
a 13C-labeled RNA hairpin with the sequence 5′(GGGC-CAAA-GCCU)3′. 相似文献
88.
The fixation of trans-(NH3)2Cl2 Pt(II) to poly(I)·poly(C) at low rb (< 0.05) leads to the formation of two complexed species. The major species (ca. 82% of bound platinum) involves coordination of platinum to a single hypoxanthine base, while the other species involves coordination of two hypoxanthine bases, which are either far apart on the same strand or on separate poly(I) strands, to the platinum. These same two species are found after reaction with poly(I), as are two other species throughout the entire rb range studied (rb = 0–0.30). The latter two species are assigned to trans-Pt bound to two bases on a poly(I) strand with (a) one or (b) two free bases between the two bound bases. These two species, (a) and (b), account for ca. 35% of the bound platinum, although the 1:1 species remains dominant (ca. 55%). These two additional species are observed at high rb (>0.075) after reaction with poly(I)·poly(C) but as very minor species. They are formed by reaction with melted poly(I) loops. Also at high rb, we have observed a shifted cytidine H5 resonance arising from interaction of trans-Pt with a melted loop of poly(C). Most probably, this arises from an intramolecular poly(I) to poly(C) crosslink. Results from the reaction of trans-Pt with poly(C) are presented for comparison. 相似文献
89.
In gram-negative bacteria only few proteins are exported across both the cytoplasmic membrane and the outer membrane which
forms an extra barrier for protein excretion.
In this review we describe the mechanisms of production and export of two types of plasmid-encoded proteins inEscherichia coli. These proteins are the bacteriocin cloacin DF13 and the K88ab and K99 fimbrial subunits. Specific so-called helper proteins
located at different positions in the cell envelope play an essential role in the export of these proteins. The genetic organization,
subcellular location and functions of these helper proteins, as well as the effects of mutations and culture conditions on
the export of the proteins are described. Models for the export mechanisms are presented and future application possibilities
for engineering foreign protein excretion inE. coli with these export systems are discussed. 相似文献
90.
Abstract The present communication defines the conditions under which thioredoxin activates glutamine synthetase from Anabaena cylindrica . Effects are obtained at pH values around neutrality, and the activation is affected by Mg2+ in the assays. The thioredoxin systems from A. cylindrica and spinach are functionally interchangeable in the activation of glutamine synthetase. The enzyme is efficiently activated by thioredoxinm and also by thioredoxinf , but at much higher concentrations. Thioredoxinm has previously been shown to activate NADPH-dependent malate dehydrogenase and isocitrate dehydrogenase from cyanobacteria. It is speculated that thioredoxinm plays a role in the differentiation of vegetative cells to heterocysts. 相似文献