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61.
K. Sonntag J. Schwinde A. A. de Graaf A. Marx B. J. Eikmanns W. Wiechert H. Sahm 《Applied microbiology and biotechnology》1995,44(3-4):489-495
The carbon flux distribution in the central metabolism of Corynebacterium glutamicum was studied in batch cultures using [1-13C]- and [6-13C]glucose as substrate during exponential growth as well as during overproduction of l-lysine and l-glutamate. Using the 13C NMR data in conjunction with stoichiometric metabolite balances, molar fluxes were quantified and normalised to the glucose uptake rate, which was set to 100. The normalised molar flux via the hexose monophosphate pathway was 40 during exponential growth, whereas it was only 17 during l-glutamate production. During l-lysine production, the normalised hexose monophosphate pathway flux was elevated to 47. Thus, the carbon flux via this pathway correlated with the NADPH demand for bacterial growth and l-lysine overproduction. The normalised molar flux in the tricarboxylic acid cycle at the level of 2-oxoglutarate dehydrogenase was 100 during exponential growth and 103 during l-lysine secretion. During l-glutamate formation, the normalised flux through the tricarboxylic acid cycle was reduced to 60. In contrast to earlier NMR studies with C. glutamicum, no significant activity of the glyoxylate pathway could be detected. All experiments indicated a strong in vivo flux from oxaloacetate back to phosphoenolpyruvate and/or pyruvate, which might be due to phosphoenolpyruvate carboxykinase activity in C. glutamicum. 相似文献
62.
snRNP Sm proteins share two evolutionarily conserved sequence motifs which are involved in Sm protein-protein interactions. 总被引:3,自引:2,他引:1
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H Hermann P Fabrizio V A Raker K Foulaki H Hornig H Brahms R Lührmann 《The EMBO journal》1995,14(9):2076-2088
The spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 share eight proteins B', B, D1, D2, D3, E, F and G which form the structural core of the snRNPs. This class of common proteins plays an essential role in the biogenesis of the snRNPs. In addition, these proteins represent the major targets for the so-called anti-Sm auto-antibodies which are diagnostic for systemic lupus erythematosus (SLE). We have characterized the proteins F and G from HeLa cells by cDNA cloning, and, thus, all human Sm protein sequences are now available for comparison. Similar to the D, B/B' and E proteins, the F and G proteins do not possess any of the known RNA binding motifs, suggesting that other types of RNA-protein interactions occur in the snRNP core. Strikingly, the eight human Sm proteins possess mutual homology in two regions, 32 and 14 amino acids long, that we term Sm motifs 1 and 2. The Sm motifs are evolutionarily highly conserved in all of the putative homologues of the human Sm proteins identified in the data base. These results suggest that the Sm proteins may have arisen from a single common ancestor. Several hypothetical proteins, mainly of plant origin, that clearly contain the conserved Sm motifs but exhibit only comparatively low overall homology to one of the human Sm proteins, were identified in the data base. This suggests that the Sm motifs may also be shared by non-spliceosomal proteins. Further, we provide experimental evidence that the Sm motifs are involved, at least in part, in Sm protein-protein interactions. Specifically, we show by co-immunoprecipitation analyses of in vitro translated B' and D3 that the Sm motifs are essential for complex formation between B' and D3. Our finding that the Sm proteins share conserved sequence motifs may help to explain the frequent occurrence in patient sera of anti-Sm antibodies that cross-react with multiple Sm proteins and may ultimately further our understanding of how the snRNPs act as auto-antigens and immunogens in SLE. 相似文献
63.
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
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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
64.
65.
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
66.
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
67.
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
68.
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
69.
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
70.
Determination of betaxolol, a new beta-blocker, by gas chromatography mass spectrometry: application to pharmacokinetic studies 总被引:1,自引:0,他引:1
Betaxolol, a beta selective adrenoceptor antagonist recently approved for the treatment of hypertension, was determined by monitoring in chemical ionization mode with ammonia the [MH]+ ions of the trimethylsilyl derivatives of the drug and of its internal standard [2H5)betaxolol). Its pharmacokinetic profile obtained following administration of a 20 mg oral dose was characterized by a half-life of 22 h and a bioavailability of 85%. The main acid metabolite formed by elimination of the isopropylamino group may also be determined as the methyl TMS derivative but methylation with BF3-methanol should be used with caution since it may induce the opening of the cyclopropyl group. The routine electron capture determination procedure was compared to this mass spectrometric method and an excellent correlation was found (r = 0.9974). Both procedures have the same sensitivity (1 ng ml-1). Finally it was observed that under electron impact mode betaxolol trimethylsilyl side chain rearranged to lose TMS-O-CH=CH2; this elimination was confirmed by deuterium labelling studies. 相似文献