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891.
Functional studies of the PI(3)-kinase signalling pathway employing synthetic and expressed siRNA 总被引:17,自引:3,他引:14 下载免费PDF全文
Czauderna F Fechtner M Aygün H Arnold W Klippel A Giese K Kaufmann J 《Nucleic acids research》2003,31(2):670-682
RNA interference (RNAi) is a RNA-mediated sequence-specific gene silencing mechanism. Recently, this mechanism has been used to down-regulate protein expression in mammalian cells by applying synthetic- or vector-generated small interfering RNAs (siRNAs). However, for the evaluation of this new knockdown technology, it is crucial to demonstrate biological consequences beyond protein level reduction. Here, we demonstrate that this new siRNA-based technology is suitable to analyse protein functions using the phosphatidylinositol (PI) 3-kinase signal transduction pathway as a model system. We demonstrate stable and transient siRNA-mediated knockdown of one of the PI 3-kinase catalytic subunits, p110β, which leads to inhibition of invasive cell growth in vitro as well as in a tumour model system. Importantly, this result is consistent with loss-of-function phenotypes induced by conventional RNase H-dependent antisense molecules or treatment with the PI 3-kinase inhibitor LY294002. RNAi knockdown of the downstream kinases Akt1 and Akt2 does not reduce cell growth on extracellular matrix. Our data show that synthetic siRNAs, as well as vector-based expression of siRNAs, are a powerful new tool to interfere with signal transduction processes for the elucidation of gene function in mammalian cells. 相似文献
892.
Oberle S Abate A Grosser N Hemmerle A Vreman HJ Dennery PA Schneider HT Stalleicken D Schröder H 《Experimental biology and medicine (Maywood, N.J.)》2003,228(5):529-534
Pentaerithrityl tetranitrate (PETN) is a long-acting donor of nitric oxide (NO) and has recently been characterized as an antianginal agent that, in contrast with other nitric acid esters, does not induce oxidative stress and is therefore free of tolerance. Moreover, animal experiments have revealed that PETN actively reduces oxygen radical formation in vivoand specifically prevents atherogenesis and endothelial dysfunction. Because heme oxygenase-1 (HO-1) has been described as an antiatherogenic and cytoprotective gene in the endothelium, our aim was to investigate the effect of the active PETN metabolite pentaerithrityl trinitrate (PETriN) on HO-1 expression and catalytic activity in endothelial cells. Endothelial cells derived from human umbilical vein were incubated with PETriN (0.01-1 mM) for 8 hr. PETriN increased HO-1 mRNA and protein levels in a concentration-dependent fashion up to 3-fold over basal levels. Elevation of HO-1 protein was accompanied by a marked increase in catalytic activity of the enzyme as reflected by enhanced formation of both carbon monoxide and the endogenous antioxidant, bilirubin. Pretreatment of endothelial cells with PETriN or bilirubin at low micromolar concentrations protected endothelial cells from hydrogen peroxide-mediated toxicity. HO-1 induction and endothelial protection by PETriN were not mimicked by isosorbide dinitrate, another long-acting nitrate. The present study demonstrates that the active PETN metabolite, PETriN, stimulates mRNA and protein expression as well as enzymatic activity of the antioxidant defense protein, HO-1, in endothelial cells. Increased HO-1 expression and ensuing formation of bilirubin and carbon monoxide may contribute to and explain the specific antioxidant and antiatherogenic actions of PETN. 相似文献
893.
Gebert A al-Samir K Werner K Fassbender S Gebhard A 《Histochemistry and cell biology》2000,113(5):389-399
Brush cells are specialised epithelial cells that are assumed to represent chemoreceptors of the digestive tract. They comprise a small population of the epithelial cells lining the intestine, possess a unique ultrastructure and, in many aspects, resemble the receptor cells of taste buds. To characterise glycoconjugates possibly involved in a sensory function, we investigated brush cells in the small intestine of three species using lectin histochemistry in confocal light and thin-section electron microscopy. Brush cells of rats were selectively labelled by the sialic acid-specific lectin Maackia amurensis agglutinin, those of guinea-pigs by the D-galactose-specific lectin Bandeiraea simplicifolia agglutinin, isolectin B4 and those of mice by the L-fucose-specific lectin Ulex europaeus agglutinin lectin I. Lectin binding sites were consistently located in the glycocalyx of the apical membrane and in that of cytoplasmic vesicles. In vivo lectin labelling revealed that the glycoconjugates of the apical membrane are accessible under physiological conditions, that brush cells do not endocytose and that they probably possess a high membrane turnover rate. The results show that specialisations exist in the composition of glycoconjugates forming the glycocalyx of brush cells in all species investigated. The presence of brush cell-specific glycoconjugates would be in accordance with the current hypothesis of a receptive function of brush cells. Differences in the specific glycosylation patterns among rats, guinea-pigs and mice indicate that species-specific adaptations exist. 相似文献
894.
Solute mobility in cuticular membranes (CMs) of 14 plant species (Citrus aurantium L., Citrus grandis L., Hedera helix L., Ilex aquifolium L., Ilex paraguariensis St.-Hil., Malus domestica Borkh. cv. Golden Delicious, Populus alba L., Prunus laurocerasus L., Pyrus communis L. cv. Bartlett, Conference and Gellerts Butterbirne, Pyrus pyrifolia (Burm. f.) Nakai, Schefflera actinophylla (Endl.) Harms and Strophanthus gratus Baill.) was measured over the temperature range 25–55 °C. The five organic model compounds differed in size (130–349 cm3 mol−1) and cuticle/water partition coefficient (18–108). For all individual CMs (n = 297), the data were plotted according to the thermodynamic relationship between the preexponential factor (which is proportional
to entropy) of the Arrhenius equation and the activation energy (enthalpy) of diffusion (E
D
). A strict linear correlation was obtained, providing evidence that the five compounds diffused along the same lipophilic
diffusion path in all plant species tested. Extracting cuticular waxes from CMs of four plant species (Hedera, Pyrus, Schefflera and Strophanthus) had no effect on the slope of the plot but a parallel displacement towards higher entropy was observed with these polymer
matrix (MX) membranes. This displacement is interpreted as a temperature-independent tortuosity factor directly related to
entropy. The influence of the plasticiser tributyl phosphate on solute mobility at various temperatures was measured for CM
and MX membranes. The plasticiser increased solute mobility and E
D
was reduced drastically for both membrane types. This plasticiser effect was almost completely reversible, when tributyl
phosphate was desorbed from the membranes. For both, plasticised CM and MX, the thermodynamic correlation exists whereby all
data points lie on the same line. The data are used to characterise the lipophilic pathway across plant cuticles in terms
of the free-volume theory.
Received: 14 December 1999 / Accepted: 31 March 2000 相似文献
895.
Osterloh A Kalinke U Weiss S Fleischer B Breloer M 《The Journal of biological chemistry》2007,282(7):4669-4680
Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation of an efficient immune response. In this study we show that Hsp60 mediates immune stimulation by different mechanisms, dependent and independent of lipopolysaccharide (LPS). We have demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific interferon (IFN) gamma release in T cells. Here we show that in contrast to LPS Hsp60 induces IFNalpha production in professional APC. Neutralization of IFNalpha as well as the absence of functional IFNalphabeta receptor on APC and T cells interfered with Hsp60-mediated IFNgamma secretion in antigen-dependent T cell activation, strongly suggesting that IFNalpha represents one factor contributing to Hsp60-specific immune stimulation. On the other hand, we show that Hsp60 bound to the cell surface of APC colocalizes with the LPS co-receptor CD14 and LPS binding sites. Hsp60 specifically binds bacterial LPS and both molecules synergistically enhanced IL-12p40 production in APC and IFNgamma release in antigen-dependent T cell activation. This effect was Hsp60-specific and dependent on LPS-binding by Hsp60. Furthermore, we show that Hsp60 exclusively binds to macrophages and DC but not to T or B lymphocytes and that both, T cell stimulation by Hsp60 as well as Hsp60/LPS complexes, strictly depends on the presence of professional APC and is not mediated by B cells. Taken together, our data support an extension of the concept of Hsp60 as an endogenous danger signal: besides its function as a classical danger signal indicating unplanned tissue destruction to the innate immune system, in the incident of bacterial infection extracellular Hsp60 may bind LPS and facilitate microbe recognition by lowering the threshold of pathogen-associated molecular pattern (PAMP) detection and enhancing Toll-like receptor (TLR) signaling. 相似文献
896.
Tanneberger K Kirchberger J Bär J Schellenberger W Rothemund S Kamprad M Otto H Schöneberg T Edelmann A 《The Journal of biological chemistry》2007,282(32):23687-23697
Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of alpha subunits and alpha/beta subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, alpha, beta, and gamma. The sequence of the gamma subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the gamma subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the gamma subunit is not required for enzymatic activity, the gamma subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and gamma subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the gamma subunit. Therefore, we suggest that the gamma subunit contributes to adaptation of P. pastoris to energy resources. 相似文献
897.
Association of Nef with p21-activated kinase 2 is dispensable for efficient human immunodeficiency virus type 1 replication and cytopathicity in ex vivo-infected human lymphoid tissue 总被引:1,自引:1,他引:0 下载免费PDF全文
Schindler M Rajan D Specht A Ritter C Pulkkinen K Saksela K Kirchhoff F 《Journal of virology》2007,81(23):13005-13014
Interaction of the human immunodeficiency virus type 1 (HIV-1) Nef protein with p21-activated kinase 2 (PAK2) has been proposed to play a role in T-cell activation, viral replication, apoptosis, and progression to AIDS. However, these hypotheses were based on results obtained using Nef mutants impaired in multiple functions. Recently, it was reported that Nef residue F191 is specifically involved in PAK2 binding. However, only a limited number of Nef activities were investigated in these studies. To further evaluate the role of F191 in Nef function and to elucidate the biological relevance of Nef-PAK2 interaction, we performed a comprehensive analysis of HIV-1 Nef mutants carrying F191H and F191R mutations. We found that the F191H mutation reduces and the F191R mutation disrupts the association of Nef with PAK2. Both mutants upregulated the major histocompatibility complex II (MHC-II)-associated invariant chain and downregulated CD4, MHC-I, and CD28, although with reduced efficiency for the latter. Furthermore, the F191H/R changes neither affected the levels of interleukin-2 receptor expression and apoptosis of HIV-1-infected primary T cells nor reduced Nef-mediated induction of NFAT. Unexpectedly, the F191H change markedly reduced and the F191R mutation disrupted the ability of Nef to enhance virion infectivity in P4-CCR5 indicator cells but not in TZM-bl cells or peripheral blood mononuclear cells. Most importantly, all HIV-1 Nef mutants replicated efficiently and caused CD4+ T-cell depletion in ex vivo-infected human lymphoid tissue. Altogether, our data show that the interaction of Nef with PAK2 does not play a major role in T-cell activation, viral replication, and apoptosis. 相似文献
898.
Response to commentary by Woinarski (Critical‐weight‐range marsupials in northern Australia are declining: a commentary on Fisher et al. (2014) ‘The current decline of tropical marsupials in Australia: is history repeating?’) 下载免费PDF全文
Diana O. Fisher Chris N. Johnson Michael J. Lawes Susanne A. Fritz Hamish McCallum Simon P. Blomberg Jeremy VanDerWal Brett Abbott Anke Frank Sarah Legge Mike Letnic Colette R. Thomas Alaric Fisher Iain J. Gordon Alex Kutt 《Global Ecology and Biogeography》2015,24(1):123-125
The recent commentary by Woinarski (2014, Global Ecology and Biogeography, doi: 10.1111/geb.12165) disagreed with our conclusions on the correlates of decline in the marsupials of tropical Australia (Fisher et al., 2014, Global Ecology and Biogeography, 23 , 181–190). We compared traits of species that were associated with range decline in southern and northern Australia. We found that habitat structure, climate and body size were correlated with range decline. In the north, declines of marsupials were most severe in savanna with moderate rainfall. In the south, the ranges of species in open habitat with very low rainfall have declined most. Also, the association between range decline and body mass differed between north and south: this is the main concern of Woinarski, who further disagreed with our choice of the Tropic of Capricorn as a boundary between north and south, our omission of rodents, how to treat timing of extinctions, and our inference that cats are major drivers of decline. We address these concerns in this response. 相似文献
899.
Zieseniss A Schroeder U Buchmeier S Schoenenberger CA van den Heuvel J Jockusch BM Illenberger S 《Cell and tissue research》2007,327(3):583-594
Raver1, a ubiquitously expressed protein, was originally identified as a ligand for metavinculin, the muscle-specific isoform
of the microfilament-associated protein vinculin. The protein resides primarily in the nucleus, where it colocalises and may
interact with polypyrimidine-tract-binding protein, which is involved in alternative splicing processes. During skeletal muscle
differentiation, raver1 translocates to the cytoplasm and eventually targets the Z-line of sarcomeres. Here, it colocalises
with metavinculin, vinculin and alpha-actinin, all of which have biochemically been identified as raver1 ligands. To obtain
more information about the potential role of raver1 in muscle structure and function, we have investigated its distribution
and fine localisation in mouse striated and smooth muscle, by using three monoclonal antibodies that recognise epitopes in
different regions of the raver1 protein. Our immunofluorescence and immunoelectron-microscopic results indicate that the cytoplasmic
accumulation of raver1 is not confined to skeletal muscle but also occurs in heart and smooth muscle. Unlike vinculin and
metavinculin, cytoplasmic raver1 is not restricted to costameres but additionally represents an integral part of the sarcomere.
In isolated myofibrils and in ultrathin sections of skeletal muscle, raver1 has been found concentrated at the I-Z-I band.
A minor fraction of raver1 is present in the nuclei of all three types of muscle. These data indicate that, during muscle
differentiation, raver1 might link gene expression with structural functions of the contractile machinery of muscle.
This work was supported by grants from the Swiss National Science Foundation and the M.E. Müller Foundation (to C.A.S.) and
the Deutsche Forschungsgemeinschaft (to S.I. and B.M.J.) and from the Fonds der Chemischen Industrie (to B.M.J.). A.Z. was
the recipient of a G. Lichtenberg fellowship, within an International Graduate College funded by the State of Lower Saxony,
Germany. 相似文献
900.
Kakinuma T Nadiminti H Lonsdorf AS Murakami T Perez BA Kobayashi H Finkelstein SE Pothiawala G Belkaid Y Hwang ST 《Cancer immunology, immunotherapy : CII》2007,56(7):1119-1131
Luciferase-transduced B16 murine melanoma cells (luc-B16) inoculated in ear skin do not form tumors but prevent tumor formation
by luc-B16 cells injected into the footpad. To determine the requirements for such immunity, we followed the fate of luc-B16
cells following ear injection. Surprisingly, small numbers of viable luc-B16 cells were detected in tumor-free mouse skin
for up to 60 days post-inoculation. After 1 week, the number of Foxp3+CD4+CD25+ T cells (along with foxp3 mRNA expression) increased rapidly in the injected ear skin. Residual tumor cells in ears were reduced in mice treated with
anti-CD25 mAb and in CD4-deficient mice, but increased in CD8-deficient mice. Strikingly, the loss of luc-B16 cells in the
ear skin, either spontaneously or following amputation of the injected ear, resulted in significantly enhanced tumor formation
by parental and luciferase-expressing B16 cells after footpad injection. These studies suggest that small numbers of tumor
cells (possibly regulated by CD4+CD25+ regulatory T cells expressing Foxp3) are required for effective host anti-tumor responses at alternate inoculation sites. 相似文献