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111.
Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences. 相似文献
112.
113.
Atomic force microscopy of uncoated plasmid DNA: nanometer resolution with only nanogram amounts of sample. 总被引:1,自引:0,他引:1
M Q Li H G Hansma J Vesenka G Kelderman P K Hansma 《Journal of biomolecular structure & dynamics》1992,10(3):607-617
Reproducible, high-contrast, nanometer-resolution AFM images of uncoated plasmid DNA can be obtained with nanogram quantities of DNA with the help of two advances in sample preparation: (1) Heating a DNA solution at 35 degrees C for 10 to 20 minutes before deposition on mica helps separate and spread the DNA, and (2) Using 5 microliter drops of the heated DNA solution in the concentration range of 2 to 10 nanogram/microliter in contact with a specially prepared mica surface for 5 to 10 minutes gives optimal coverage with only nanograms of DNA. 相似文献
114.
p140trk mRNA marks NGF-responsive forebrain neurons: evidence that trk gene expression is induced by NGF. 总被引:18,自引:0,他引:18
D M Holtzman Y Li L F Parada S Kinsman C K Chen J S Valletta J Zhou J B Long W C Mobley 《Neuron》1992,9(3):465-478
Nerve growth factor (NGF) appears to act as a neurotrophic factor for basal forebrain and caudate-putamen cholinergic neurons. The mechanism by which NGF transduces its signal in these neurons is yet to be defined. Recent data indicate that the product of the trk gene, p140trk, is a critical component of the NGF receptor. Herein, we show that p140trk mRNA is highly restricted in its distribution in the adult rat forebrain, that it is present in cholinergic neurons, and that most if not all cholinergic neurons contain p140trk mRNA. Furthermore, induction of trk expression by NGF suggests that neurotrophin-mediated up-regulation of their receptor tyrosine kinases is an important feature of their actions and that neurotrophins may regulate the activity of responsive neurons through increasing the level of their receptors. 相似文献
115.
Systemic hypertension of mild to moderate degree is often associated with obesity. The hypothesis is that over-eating leads to increased sympathetic activity targeted at the peripheral vasculature as well as other tissues in an attempt (that in many cases may be futile) to stimulate facultative thermogenesis and burn-off the excess energy. This hypothesis represents an important modification of one proposed by Landsberg and is supported by: 1) recent observations that carbohydrate feeding to humans specifically increases muscle sympathetic vasoconstrictor activity in the peroneal nerve, and 2) studies with animal models in which active vasoconstriction in the limbs and elsewhere is associated with marked increases in oxygen consumption (energy expenditure). 相似文献
116.
The distribution of holly ( Ilex aquifolium ) and its habitat preferences indicate a sensitivity to low temperature, particularly when exposed to high light. Experiments were conducted to determine whether photoinhibition of photosynthesis occurs in holly leaves in the field in United Kingdom during the winter. Photosynthetic efficiency was assessed in holly leaves that were exposed to or shaded from direct sunlight using measurements of photosynthetic oxygen evolution and chlorophyll fluorescence. Field measurements were conducted over 3 weeks during January and February. Correlation of the measurements of photosynthetic efficiency with weather conditions indicated that holly was suffering photoinhibition, particularly in leaves exposed to direct sunlight. Controlled environment studies demonstrated that exposure of leaves to low temperature and high light resulted in reductions in photosynthetic efficiency; however, leaves recovered rapidly when exposed to a higher temperature and reduced light level. 相似文献
117.
Tomizawa Ken-ichi; Ito Naoko; Komeda Yoshibumi; Uyeda Taro Q. P.; Takio Koji; Furuya Masaki 《Plant & cell physiology》1991,32(1):95-102
The pea phytochrome I (PI) cDNA clone, pPP1001, was expressedin E. coli. The plasmid pPP1001 contains pea PI cDNA which coversthe entire coding region with the Shine-Dalgarno consensus sequencejoined upstream of the cDNA in an expression vector pNUT6. ThepPP1001 transformants formed typical inclusion bodies when culturedat 32?C. However, when cultured at 37?C or in the presence ofisopropyl-ß-D-thiogalactopyranoside (IPTG) at 32?C,the bacteria lysed before inclusion body formation. Immuno-stainingwith anti-PI monoclonal antibody, mAP5, of transformants fixedby cold methanol showed that stainable materials were distributedin whole cytoplasmic region. When the inclusion bodies wereobserved clearly, the regions corresponding to the inclusionbodies became difficult to stain. Western blot analysis, however,showed that a ca. 100 kDa PI polypeptide was detected in thefraction from inclusion bodies and a ca. 90 kDa PI polypeptidefrom the soluble fraction. The amino acid sequence analysisof purified 100 kDa PI sample indicated that its amino terminusis blocked. However, minor signals in one experiment yieldeda sequence corresponding to the expected amino terminus of peaPI except for the initiation methionine. One of the anti-peaPI monoclonal antibodies, mAP9, that recognizes the near N-terminusof pea phytochrome was reactive to the 100 kDa polypeptide. (Received June 22, 1990; Accepted November 18, 1990) 相似文献
118.
The Sequence of Change within the Photosynthetic Apparatus of Wheat following Short-Term Exposure to Ozone 总被引:30,自引:1,他引:29 下载免费PDF全文
The basis of inhibition of photosynthesis by single acute O3 exposures was investigated in vivo using analyses based on leaf gas exchange measurements. The fully expanded second leaves of wheat plants (Triticum aestivum L. cv Avalon) were fumigated with either 200 or 400 nanomoles per mole O3 for between 4 and 16 hours. This reduced significantly the light-saturated rate of CO2 uptake and was accompanied by a parallel decrease in stomatal conductance. However, the stomatal limitation, estimated from the relationship between CO2 uptake and the internal CO2 concentration, only increased significantly during the first 8 hours of exposure to 400 nanomoles per mole O3; no significant increase occurred for any of the other treatments. Analysis of the response of CO2 uptake to the internal CO2 concentration implied that the predominant factor responsible for the reduction in light-saturated CO2 uptake was a decrease in the efficiency of carboxylation. This was 58 and 21% of the control value after 16 hours at 200 and 400 nanomoles per mole O3, respectively. At saturating concentrations of CO2, photosynthesis was inhibited by no more than 22% after 16 hours, indicating that the capacity for regeneration of ribulose bisphosphate was less susceptible to O3. Ozone fumigations also had a less pronounced effect on light-limited photosynthesis. The maximum quantum yield of CO2 uptake and the quantum yield of oxygen evolution showed no significant decline after 16 hours with 200 nanomoles per mole O3, requiring 8 hours at 400 nanomoles per mole O3 before a significant reduction occurred. The photochemical efficiency of photosystem II estimated from the ratio of variable to maximum chlorophyll fluorescence and the atrazine-binding capacity of isolated thylakoids demonstrated that photochemical reactions were not responsible for the initial inhibition of CO2 uptake. The results suggest that the apparent carboxylation efficiency appears to be the initial cause of decline in photosynthesis in vivo following acute O3 fumigation. 相似文献
119.
Effect of light/dark cycles on expression of nitrate assimilatory genes in maize shoots and roots 总被引:8,自引:2,他引:6 下载免费PDF全文
The level of nitrate reductase (NR) and nitrite reductase (NiR) varied in both shoot and root tissue from nitrate-fed Zea mays L. grown under a 16-hour light/8-hour dark regime over a 10-day period postgermination, with peak activity occurring in days 5 to 6. To study the effect of different light regimes on NR and NiR enzyme activity and mRNA levels, 6-day-old plants were grown in the presence of continuous KNO3 (10 millimolar). Both shoot NRA and mRNA varied considerably, peaking 4 to 8 hours into the light period. Upon transferring plants to continuous light, the amplitude of the peaks increased, and the peaks moved closer together. In continuous darkness, no NR mRNA or NR enzyme activity could be detected by 8 hours and 12 hours, respectively. In either a light/dark or continuous light regime, root NRA and mRNA did not vary substantially. However, when plants were placed in continuous darkness, both declined steadily in the roots, although some remained after 48 hours. Although there was no obvious cycling of NiR enzyme activity in shoot tissue, changes in mRNA mimicked those seen for NR mRNA. The expression of NR and NiR genes is affected by the light regime adopted, but light does not have a direct effect on the expression of these genes. 相似文献
120.
The trk tyrosine protein kinase mediates the mitogenic properties of nerve growth factor and neurotrophin-3. 总被引:45,自引:0,他引:45
C Cordon-Cardo P Tapley S Q Jing V Nanduri E O'Rourke F Lamballe K Kovary R Klein K R Jones L F Reichardt 《Cell》1991,66(1):173-183
The product of the trk proto-oncogene encodes a receptor for nerve growth factor (NGF). Here we show that NGF is a powerful mitogen that can induce resting NIH 3T3 cells to enter S phase, grow in semisolid medium, and become morphologically transformed. These mitogenic effects are absolutely dependent on expression of gp140trk receptors, but do not require the presence of the previously described low affinity NGF receptor. gp140trk also serves as a receptor for the related factor neurotrophin-3 (NT-3), but not for brain-derived neurotrophic factor. Both NGF and NT-3 induce the rapid phosphorylation of gp140trk receptors and the transient expression of c-Fos proteins. However, NT-3 appears to elicit more limited mitogenic responses than NGF. These results indicate that the product of the trk proto-oncogene is sufficient to mediate signal transduction processes induced by NGF and NT-3, at least in proliferating cells. 相似文献