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61.
T Aoyama K Korzekwa K Nagata J Gillette H V Gelboin F J Gonzales 《European journal of biochemistry》1989,181(2):331-336
The modified vaccinia virus, T7-RNA-polymerase cDNA-expression system was used to express rat cytochrome P-450a. Various parameters such as host-cell type and density, and duration of infection were tested to optimize the level of expression of cytochrome P-450a enzyme activity. Cytochrome P-450a expressed from the cDNA sequence was exclusively incorporated into the membrane-containing portions of the cell lysates, as expected from its normal association in the liver endoplasmic reticulum. The enzyme displayed a carbon-monoxide-reduced-cytochrome-P-450a difference spectrum with a Soret maximum of 450 nm. Activity measurements revealed that cytochrome P-450a produced three metabolites of testosterone; 7 alpha-hydroxytestosterone and 6 alpha-hydroxytestosterone and delta 6-testosterone at a ratio of about 38:1:1. Under the appropriate conditions, the vaccinia-virus, T7-RNA-polymerase system produces high levels of a single form of cytochrome P-450 in cells that are virtually devoid of endogenous cytochrome P-450. Analysis of the cytochrome P-450 in its natural membrane-bound state, as opposed to artificial-lipid reconstitution studies of purified enzymes, allows accurate and confident measurements of substrate specificities. 相似文献
62.
Omi K Hachiya NS Tokunaga K Kaneko K 《Biochemical and biophysical research communications》2005,338(2):1229-1235
Huntingtin is a ubiquitously expressed cytoplasmic protein encoded by the Huntington disease (HD) gene, in which a CAG expansion induces an autosomal dominant progressive neurodegenerative disorder; however, its biological function has not been completely elucidated. Here, we report for the first time that short interfering RNA (siRNA)-mediated inhibition of endogenous Hdh (a mouse homologue of huntingtin) gene expression induced an aberrant configuration of the endoplasmic reticulum (ER) network in vitro. Studies using immunofluorescence microscopy with several ER markers revealed that the ER network appeared to be congregated in various types of cell lines transfected with siRNA directed against Hdh, but not with other siRNAs so far tested. Other subcellular organelles and structures, including the nucleus, Golgi apparatus, mitochondria, lysosomes, microtubules, actin cytoskeletons, cytoplasm, lipid rafts, and plasma membrane, exhibited normal configurations. Western blot analysis of cellular prion protein (PrP(C)) revealed normal glycosylation, which is a simple marker of post-translational modification in the ER and Golgi compartments, and immunofluorescence microscopy detected no altered subcellular distribution of PrP(C) in the post-ER compartments. Further investigation is required to determine whether the distorted ER network, i.e., loss of the huntingtin function, participates in the development of HD. 相似文献
63.
Mitochondrial ATP synthase (F(1)F(0)-ATPase) is regulated by an intrinsic ATPase inhibitor protein, IF(1). We previously found that six residues of the yeast IF(1) (Phe17, Arg20, Glu21, Arg22, Glu25, and Phe28) form an ATPase inhibitory site [Ichikawa, N. and Ogura, C. (2003) J. Bioenerg. Biomembr. 35, 399-407]. In the crystal structure of the F(1)/IF(1) complex [Cabezón, E. et al. (2003) Nat. Struct. Biol. 10, 744-750], the core residues of the inhibitory site interact with Arg408, Arg412 and Glu454 of the beta-subunit of F(1). In the present study, we examined the roles of the three beta residues by means of site-directed mutagenesis. A total of six yeast mutants were constructed: R408I, R408T, R412I, R412T, E454Q, and E454V. The betaArg412 and betaGlu454 mutants (R412I, R412T, E454Q, and E454V) could grow on a nonfermentable lactate medium, but the betaArg408 mutants (R408I and R408T) could not. The ATPase activity of isolated mitochondria was decreased in R412I, R412T, E454Q, and E454V mutant cells, and undetectable in R408I and R408T cells. The subunits of F(1) (alpha, beta, and gamma) were detected in mitochondria from each mutant on immunoblotting, and the F(1)F(0) complex was isolated from them. These results indicate that betaArg408 is essential not for assembly of the F(1)F(0) complex but for the catalytic activity of the enzyme. In the crystal structure of F(1), betaArg408 binds to alphaGlu399 in the alpha(DP)/beta(DP) pair and seems to be important for formation of the closed alpha(DP)/beta(DP) conformation. IF(1) seems to disrupt this alpha(DP)Glu399/beta(DP)Arg408 interaction by binding to beta(DP)Arg408, and to interfere with the change from the open alpha(DP)/beta(DP) conformation to the closed conformation that is required for catalysis by F(1)F(0)-ATPase. 相似文献
64.
Impacts of climate variability on tree demography in second growth tropical forests: the importance of regional context for predicting successional trajectories 下载免费PDF全文
María Uriarte Naomi Schwartz Jennifer S. Powers Erika Marín‐Spiotta Wenying Liao Leland K. Werden 《Biotropica》2016,48(6):780-797
Naturally regenerating and restored second growth forests account for over 70% of tropical forest cover and provide key ecosystem services. Understanding climate change impacts on successional trajectories of these ecosystems is critical for developing effective large‐scale forest landscape restoration (FLR) programs. Differences in environmental conditions, species composition, dynamics, and landscape context from old growth forests may exacerbate climate impacts on second growth stands. We compile data from 112 studies on the effects of natural climate variability, including warming, droughts, fires, and cyclonic storms, on demography and dynamics of second growth forest trees and identify variation in forest responses across biomes, regions, and landscapes. Across studies, drought decreases tree growth, survival, and recruitment, particularly during early succession, but the effects of temperature remain unexplored. Shifts in the frequency and severity of disturbance alter successional trajectories and increase the extent of second growth forests. Vulnerability to climate extremes is generally inversely related to long‐term exposure, which varies with historical climate and biogeography. The majority of studies, however, have been conducted in the Neotropics hindering generalization. Effects of fire and cyclonic storms often lead to positive feedbacks, increasing vulnerability to climate extremes and subsequent disturbance. Fragmentation increases forests’ vulnerability to fires, wind, and drought, while land use and other human activities influence the frequency and intensity of fire, potentially retarding succession. Comparative studies of climate effects on tropical forest succession across biogeographic regions are required to forecast the response of tropical forest landscapes to future climates and to implement effective FLR policies and programs in these landscapes. 相似文献
65.
Jeffrey?D.?HollandEmail author Lenore?Fahrig Naomi?Cappuccino 《Journal of Insect Conservation》2005,9(2):109-119
Reproductive rate has been suggested to have a positive effect on the amount of habitat loss a species can tolerate while emigration from habitat patches has been suggested to have both positive and negative effects. Forest fragmentation has been suggested to have negative effects on forest species. We determined the extinction threshold for 12 species of saproxylic (dead wood dependent) longhorned beetles (Coleoptera: Cerambycidae) using trap catch data from Ontario, Canada. We also determined the maximum egg production of each species and whether they were likely to move outside of forest patches. We found a strong negative relationship between reproductive rate and the minimum habitat amount required for species presence. This relationship is obscured if the scale of investigation is not appropriate for the study organism. As well, species caught moving outside forest habitat had lower extinction thresholds than species not caught moving outside forest but this was not significant after accounting for reproductive rate. Fragmentation did not have an effect on the minimum habitat requirements. These relationships can inform predictions of which species will be most affected by habitat loss. 相似文献
66.
Orfila C Sørensen SO Harholt J Geshi N Crombie H Truong HN Reid JS Knox JP Scheller HV 《Planta》2005,222(4):613-622
An insertion in the promoter of the Arabidopsis thaliana QUA1 gene (qua1-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhesion, particularly between epidermal cells in seedlings
and young leaves. This coincides with a reduction in the level of homogalacturonan epitopes and the amount of GalA in isolated
cell walls (Bouton et al., Plant Cell 14: 2577 2002). The present study was undertaken in order to investigate further the link between QUA1 and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild
type and qua1-1 mutant tissue. Relative to the wild type, homogalacturonan α-1-4-D-galacturonosyltransferase activity was consistently reduced in qua1-1 stems (by about 23% in microsomal and 33% in detergent-solubilized membrane preparations). Activities of β-1-4-D-xylan synthase, β-1-4-D-galactan synthase and β-glucan synthase II activities were also measured in microsomal membranes. Of these, only β-1-4-D-xylan synthase was affected, and was reduced by about 40% in qua1-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using
microscopy and cell wall composition analyses. Using in situ PCR techniques, QUA1 mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of
these tissues was disrupted and there was a modest reduction in homogalacturonan (JIM5) epitopes. This study demonstrates
a specific role for QUA1 in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role
of QUA1 in pectin and hemicellulose cell wall synthesis through affects on α-1,4-D-galacturonosyltransferase and β-1,4-D-xylan synthase activities. 相似文献
67.
Effective virtual screening relies on our ability to make accurate prediction of protein-ligand binding, which remains a great challenge. In this work, utilizing the molecular-mechanics Poisson-Boltzmann (or Generalized Born) surface area approach, we have evaluated the binding affinity of a set of 156 ligands to seven families of proteins, trypsin β, thrombin α, cyclin-dependent kinase (CDK), cAMP-dependent kinase (PKA), urokinase-type plasminogen activator, β-glucosidase A, and coagulation factor Xa. The effect of protein dielectric constant in the implicit-solvent model on the binding free energy calculation is shown to be important. The statistical correlations between the binding energy calculated from the implicit-solvent approach and experimental free energy are in the range of 0.56-0.79 across all the families. This performance is better than that of typical docking programs especially given that the latter is directly trained using known binding data whereas the molecular mechanics is based on general physical parameters. Estimation of entropic contribution remains the barrier to accurate free energy calculation. We show that the traditional rigid rotor harmonic oscillator approximation is unable to improve the binding free energy prediction. Inclusion of conformational restriction seems to be promising but requires further investigation. On the other hand, our preliminary study suggests that implicit-solvent based alchemical perturbation, which offers explicit sampling of configuration entropy, can be a viable approach to significantly improve the prediction of binding free energy. Overall, the molecular mechanics approach has the potential for medium to high-throughput computational drug discovery. 相似文献
68.
Polygyny does not explain the superior competitive ability of dominant ant associates in the African ant‐plant,Acacia (Vachellia) drepanolobium 下载免费PDF全文
John H. Boyle Dino J. Martins Julianne Pelaez Paul M. Musili Staline Kibet S. Kimani Ndung'u David Kenfack Naomi E. Pierce 《Ecology and evolution》2018,8(3):1441-1450
The Acacia drepanolobium (also known as Vachellia drepanolobium) ant‐plant symbiosis is considered a classic case of species coexistence, in which four species of tree‐defending ants compete for nesting space in a single host tree species. Coexistence in this system has been explained by trade‐offs in the ability of the ant associates to compete with each other for occupied trees versus the ability to colonize unoccupied trees. We seek to understand the proximal reasons for how and why the ant species vary in competitive or colonizing abilities, which are largely unknown. In this study, we use RADseq‐derived SNPs to identify relatedness of workers in colonies to test the hypothesis that competitively dominant ants reach large colony sizes due to polygyny, that is, the presence of multiple egg‐laying queens in a single colony. We find that variation in polygyny is not associated with competitive ability; in fact, the most dominant species, unexpectedly, showed little evidence of polygyny. We also use these markers to investigate variation in mating behavior among the ant species and find that different species vary in the number of males fathering the offspring of each colony. Finally, we show that the nature of polygyny varies between the two commonly polygynous species, Crematogaster mimosae and Tetraponera penzigi: in C. mimosae, queens in the same colony are often related, while this is not the case for T. penzigi. These results shed light on factors influencing the evolution of species coexistence in an ant‐plant mutualism, as well as demonstrating the effectiveness of RADseq‐derived SNPs for parentage analysis. 相似文献
69.
The effects of glucagon, 3′,5′-AMP, 3′,5′-GMP and dexamethasone on ion fluxes and transmembrane-potential changes were compared in perfused livers from normal and adrenalectomized rats. Glucagon and cyclic nucleotide administration resulted in a similar redistribution of Na+ and K+ and membrane hyperpolarization in both groups. Dexamethasone at a dose which restores the gluconeogenic response after adrenalectomy, had no effect on either the ion movements or membrane potential and did not alter the responses to cyclic nucleotides or glucagon in either normal or adrenalectomized rat livers. These results suggest that the permissive effect of glucacorticoids on gluconeogenesis might be related to an event following ion movement. 相似文献
70.
Analysis of Vibrio seventh pandemic island II and novel genomic islands in relation to attachment sequences among a wide variety of Vibrio cholerae strains 下载免费PDF全文
Tuan Hai Nguyen Tho Duc Pham Naomi Higa Hanako Iwashita Taichiro Takemura Makoto Ohnishi Kouichi Morita Tetsu Yamashiro 《Microbiology and immunology》2018,62(3):150-157