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81.
Mingone CJ Gupte SA Quan S Abraham NG Wolin MS 《Experimental biology and medicine (Maywood, N.J.)》2003,228(5):535-539
Heme is a co-factor required for the stimulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) and carbon monoxide, and sGC activation by these agents is inhibited by superoxide. Because heme promotes oxidant generation, we examined the influence of rat pulmonary microvascular endothelial cells (PMECs) with a stable human heme oxygenase-1 (HO-1) transfection and heme on oxidant generation and cGMP. Culture of PMEC with low serum heme decreased cGMP and the detection of peroxide with 10 microM 2',7'-dichlorofluorescin diacetate and increased HO-1 further decreased cGMP without altering the peroxide detection under these conditions. Under conditions where heme (30 microM) has been shown to stimulate cGMP production in PMECsby mechanisms involving NO and CO, heme increased the detection of peroxide in a PMEC-dependent manner and HO-1 transfection did not markedly alter the effects heme on peroxide detection. The addition of 1 microM catalase markedly inhibited the effects of heme on peroxide detection whereas increasing (0.1 mM ebselen) or decreasing (depleting glutathione with 7 mM diethylmaleate) rates of intracellular peroxide metabolism or inhibiting the biosynthesis of oxidants (with 10 microM diphenyliodonium or 0.1 mM nitro-L-arginine) had only modest effects. The detection of superoxide by 10 microM dihydroethidium from PMECs was not increased by exposure to heme. These actions of oxidant probes suggest that intracellular oxidants have a minimal influence on the response to heme. Thus, exposure of PMECs to heme causes a complex response involving an extracellular generation of peroxide-derived oxidant species, which do not appear to originate from increases in intracellular superoxide or peroxide. This enables heme and HO to regulate sGC through mechanisms involving NO and CO, which are normally inhibited by superoxide. 相似文献
82.
Nourizad N Ehn M Gharizadeh B Hober S Nyrén P 《Protein expression and purification》2003,27(2):4600-237
ATP-diphosphohydrolase (apyrase) catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di-phosphates in the presence of divalent cations. This enzyme has broad substrate specificity for nucleotides, which makes it an ideal enzyme for different biotechnical applications, such as DNA sequencing and platelet-aggregation inhibition. The only commercially available apyrase is isolated from potato tubers. To avoid batch-to-batch variations in activity and quality, we decided to produce a recombinant enzyme. The methylotrophic yeast Pichia pastoris was chosen as an eukaryotic expression host. The coding sequence of potato apyrase, without the signal peptide, was cloned into the YpDC541 vector to create a fusion with the alpha-mating secretion signal of Saccharomyces cerevisiae. The gene was placed under the control of the methanol-inducible alcohol oxidase promoter. The YpDC541-apyrase construct was integrated into P. pastoris strain SMD1168. Methanol induction resulted in secretion of apyrase to a level of 1mg/L. The biologically active recombinant apyrase was purified by hydrophobic interaction and ion exchange chromatography. According to SDS-PAGE and Western blot analysis, the purified enzyme showed to be hyperglycosylated. By enzymatic removal of N-glycans, a single band corresponding to a molecular mass of 48kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology. 相似文献
83.
Regeneration and transformation of Egyptian maize inbred lines via immature embryo culture and a biolistic particle delivery system 总被引:2,自引:0,他引:2
Hanaiya?A.?El-ItribyEmail author Shireen?K.?Assem Ebtissam?H.?A.?Hussein Fathy?M.?Abdel-Calil Magdy?A.?Madkour 《In vitro cellular & developmental biology. Plant》2003,39(5):524-531
Summary A regeneration system was developed for elite Egyptain maize inbred lines using immature embryos as explants. This system
proved to be highly genotype-dependent. Line Gz 643 was identified as the best line, revealing the highest regeneration frequency
(42.2%). Addition of l-proline and silver nitrate to culture media greatly enhanced the formation of embryogenic type II callus and the regenerability
of some of the tested lines. Transformation of the scutellar tissue of immature embryos from inbred line Gz643 was performed
with the particle delivery system using a single plasmid carrying both the GUS and Bar genes (pAB-6) or by co-transformation with two plasmids, pAct1-F (GUS) and pTW-a(Bar). Different transformation parameters were evaluated, i.e. ostomic treatment, acceleration pressure, and number of shots.
Osmotic treatment (0.25 M sorbitol + 0.25 M mannitol) along with the use of either acceleration pressure 1300 psi and one shot per plate (for co-transformation with
pAB-6) or 1100 psi and two shots per plate (for transformation with pAct1-F and pTW-a) gave the best results, as expressed
by the number of blue spots in the β-glucuronidase (GUS) assay. Stable transformation was confirmed in Ro transformed plants
by means of histochemical GUS assay and herbicide application. PCR and Southern blot analysis proved the integration of the
full-length genes in some of the transgenics. 相似文献
84.
85.
Background
Short tandem repeat polymorphisms (STRPs) are powerful tools for gene mapping and other applications. A STRP genome scan of 10 cM is usually adequate for mapping single gene disorders. However mapping studies involving genetically complex disorders and especially association (linkage disequilibrium) often require higher STRP density. 相似文献86.
Functional organization of adult motor cortex is dependent upon continued protein synthesis 总被引:5,自引:0,他引:5
Kleim JA Bruneau R Calder K Pocock D VandenBerg PM MacDonald E Monfils MH Sutherland RJ Nader K 《Neuron》2003,40(1):167-176
The functional organization of adult cerebral cortex is characterized by the presence of highly ordered sensory and motor maps. Despite their archetypical organization, the maps maintain the capacity to rapidly reorganize, suggesting that the neural circuitry underlying cortical representations is inherently plastic. Here we show that the circuitry supporting motor maps is dependent upon continued protein synthesis. Injections of two different protein synthesis inhibitors into adult rat forelimb motor cortex caused an immediate and enduring loss of movement representations. The disappearance of the motor map was accompanied by a significant reduction in synapse number, synapse size, and cortical field potentials and caused skilled forelimb movement impairments. Further, motor skill training led to a reappearance of movement representations. We propose that the circuitry of adult motor cortex is perpetually labile and requires continued protein synthesis in order to maintain its functional organization. 相似文献
87.
Chavante SF Santos EA Oliveira FW Guerrini M Torri G Casu B Dietrich CP Nader HB 《International journal of biological macromolecules》2000,27(1):49-57
With the aid of heparinase and heparitinases from Flavobacterium heparinum and 13C and IH NMR spectroscopy it was shown that the heparan sulphate isolated from the brine shrimp Artemia franciscana exhibits structural features intermediate between those of mammalian heparins and heparan sulphates. These include an unusually high degree of N-sulphation (with corresponding very low degree of N-acetylation), a relatively high content of iduronic acid residues (both unsulphated and 2-O-sulphated) and a relatively low degree of 6-O-sulphation of the glucosamine residues. The major sequences (glucuronic acid-->N-sulphated glucosamine and glucuronic acid-->N, 6-disulphated glucosamine) are most probably arranged in blocks. Although exhibiting negligible anticlotting activity in the APTT and anti-factor Xa assays the A. franciscana heparan sulphate has a high heparin cofactor-II activity (about 1/3 that of heparin). 相似文献
88.
89.
Vos J. van der Putten P.E.L. Hassan Hussein Muktar van Dam A.M. Leffelaar P.A. 《Plant and Soil》1998,201(1):149-155
Nitrogen catch crops help to reduce the loss of nitrogen from arable cropping systems during autumn and winter. The ability of catch crops to absorb nitrogen from the soil profile is affected by rate and depth of colonization of the soil by roots. The aim of the current work was to analyze total root length and root length density of catch crops in relation to above ground growth, nitrogen supply and crop species. In two field experiments roots were sampled with an auger. Experimental factors included crop species (winter rye, Secale cereale and forage rape, Brassica napus ssp. oleifera (Metzg.) Sinsk., or oil radish, Raphanus sativus spp. oleiferus (DC.) Metzg.), two sowing dates S1 and S2 (end of August and three weeks later) and two nitrogen treatments: N0, no nitrogen applied, and N1, nitrogen applied at non-limiting rate.The natural logarithm of the total root length, measured in the top 40 cm, L0–40 (km m-2), was linearly related to natural logarithm of the dry weight of the shoot, W (g m-2). There was no effect of species or sowing date on this relation. For a given W, N1 treatments showed lower values of L0–40 than N0 treatments. The decline in root length density, D (cm cm-3), with depth, X (cm), was described with the function ln D = ln D0 – qX, where D0 is the value of D at zero depth and q the linear coefficient. D0 was linearly related to L0–40, without effect of species, time of observation or N supply. The ratio D0/q, an estimate of the absolute root length, was 1.24 × L0–40.Together the relations enable estimates to be made of total root length and of root length distribution with depth using shoot dry weight of catch crops and its change with time as input. The generation of such estimates of root distribution is necessary for model studies in which the efficacy of catch crops to prevent N losses is evaluated in relation to sowing dates, distribution of N in the soil profile and the distribution of rainfall in the season. 相似文献
90.
Nader Rahimi Eric Tremblay Laura McAdam Anita Roberts Bruce Elliott 《In vitro cellular & developmental biology. Animal》1998,34(5):412-420
Summary We have developed an in vitro system to examine the influence of adipocytes, a major mammary stromal cell type, on the growth of a murine mammary carcinoma,
SP1. Previously, we have shown that 3T3-L1 adipocytes release a mitogenic factor, hepatocyte growth factor, which strongly
stimulates proliferation of SP1 cells. We now show that 3T3-L1 pre-adipocytes secrete active inhibitory molecules which inhibit
DNA synthesis in SP1 cells. In addition, latent inhibitory activity is present in conditioned media (CM) from both pre-adipocytes
and adipocytes, and is activated following acid treatment. CM also inhibited DNA synthesis in Mv1Lu wild type epithelial cells,
but not DR27 mutant epithelial cells which lack TGF-β type II receptor. Inhibitory activity of CMs was partially abrogated
by neutralizing anti-TGF-β1 and anti-TGF-β2 antibodies, and was removed following ultrafiltration through membranes of 10
000 Mr but not 30 000 Mr pore size. These results show that the inhibitory effect on DNA synthesis is mediated by TGF-β1-like and TGF-β2-like molecules.
In addition, acid-treated CM as well as purified TGF-β inhibited differentiation of pre-adipocytes. Untreated pre-adipocyte
CM, but not mature adipocyte CM, spontaneously inhibited adipocyte differentiation. Together, these findings indicate that
pre-adipocytes spontaneously activate their own secreted TGF-β, whereas mature adipocytes do not, and suggest that activation
of TGF-β has a potent negative regulatory effect on adipocyte differentiation and tumor growth. Thus, TGF-β may be an important
modulator of tumor growth and adipocyte differentiation via both paracrine and autocrine mechanisms. These findings emphasize
the importance of adipocyte-tumor interactions in the regulation of tumor microenvironment. 相似文献