全文获取类型
收费全文 | 5540篇 |
免费 | 538篇 |
国内免费 | 1篇 |
出版年
2022年 | 42篇 |
2021年 | 87篇 |
2020年 | 46篇 |
2019年 | 44篇 |
2018年 | 70篇 |
2017年 | 57篇 |
2016年 | 129篇 |
2015年 | 184篇 |
2014年 | 201篇 |
2013年 | 248篇 |
2012年 | 312篇 |
2011年 | 324篇 |
2010年 | 230篇 |
2009年 | 200篇 |
2008年 | 324篇 |
2007年 | 286篇 |
2006年 | 262篇 |
2005年 | 254篇 |
2004年 | 290篇 |
2003年 | 275篇 |
2002年 | 299篇 |
2001年 | 75篇 |
2000年 | 47篇 |
1999年 | 69篇 |
1998年 | 86篇 |
1997年 | 60篇 |
1996年 | 52篇 |
1995年 | 49篇 |
1994年 | 55篇 |
1993年 | 57篇 |
1992年 | 57篇 |
1991年 | 40篇 |
1990年 | 54篇 |
1989年 | 42篇 |
1988年 | 40篇 |
1987年 | 57篇 |
1986年 | 35篇 |
1985年 | 55篇 |
1984年 | 48篇 |
1983年 | 67篇 |
1982年 | 90篇 |
1981年 | 72篇 |
1980年 | 64篇 |
1979年 | 43篇 |
1978年 | 51篇 |
1977年 | 41篇 |
1976年 | 44篇 |
1975年 | 43篇 |
1974年 | 42篇 |
1973年 | 43篇 |
排序方式: 共有6079条查询结果,搜索用时 15 毫秒
991.
Amar S. Prashad Daniel Wang Joan Subrath Biqi Wu Melissa Lin Mei-Yi Zhang Natasha Kagan Julie Lee Xiaoke Yang Agnes Brennan Divya Chaudhary Xin Xu Louis Leung Jack Wang Diane H. Boschelli 《Bioorganic & medicinal chemistry letters》2009,19(19):5799-5802
We previously reported that a 3-pyridinecarbonitrile analog with a furan substituent at C-5 and a 4-methylindol-5-ylamino substituent at C-4, 1, was a potent inhibitor of PKCθ (IC50 = 4.5 nM). Replacement of the C-5 furan ring of 1 with bicyclic heteroaryl rings, led to compounds with significantly improved potency against PKCθ. Analog 6b with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-1-benzofuran-2-yl group at C-5 had an IC50 value of 0.28 nM for the inhibition of PKCθ. 相似文献
992.
993.
Maheshinie Rajapaksha Jack F. Eichler Jan Hajduch David E. Anderson Kenneth L. Kirk James G. Bann 《Protein science : a publication of the Protein Society》2009,18(1):17-23
The binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin‐like protease on the cell surface followed by the formation of a donut‐shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1‐dimensional 13C‐edited 1H NMR that low pH induces dissociation of the Von‐Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon‐13 labeled, 2‐fluorohistidine labeled CMG2, consistent with studies showing that protonation of His‐121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface. 相似文献
994.
Maobing Tu Xiao Zhang Mike Paice Paul McFarlane Jack N. Saddler 《Biotechnology progress》2009,25(4):1122-1129
The effects of surfactants addition on enzymatic hydrolysis and subsequent fermentation of steam exploded lodgepole pine (SELP) and ethanol pretreated lodgepole pine (EPLP) were investigated in this study. Supplementing Tween 80 during cellulase hydrolysis of SELP resulted in a 32% increase in the cellulose‐to‐glucose yield. However, little improvement was obtained from hydrolyzing EPLP in the presence of the same amount of surfactant. The positive effect of surfactants on SELP hydrolysis led to an increase in final ethanol yield after the fermentation. It was found that the addition of surfactant led to a substantial increase in the amount of free enzymes in the 48 h hydrolysates derived from both substrates. The effect of surfactant addition on final ethanol yield of simultaneous saccharification and fermentation (SSF) was also investigated by using SELP in the presence of additional furfural and hydroxymethylfurfural (HMF). The results showed that the surfactants slightly increased the conversion rates of furfural and HMF during SSF process by Saccharomyces cerevisiae. The presence of furfural and HMF at the experimental concentrations did not affect the final ethanol concentration either. The strategy of applying surfactants in cellulase recycling to reduce enzyme cost is presented. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
995.
How tightly land plants are adapted to the gravitational force (g) prevailing on Earth has been of interest because unlike many other environmental factors, g presents as a constant force. Ontogeny of mature angiosperms begins with an embryo that is formed after tip growth by a pollen
tube delivers the sperm nucleus to the egg. Because of the importance to plant fitness, we have investigated how gravity affects
these early stages of reproductive development. Arabidopsis thaliana (L.) Heynh. plants were grown for 13 days prior to being transferred to growth chambers attached to a large diameter rotor,
where they were continuously exposed to 2-g or 4-g for the subsequent 11 days. Plants began flowering 1 day after start of the treatments, producing hundreds of flowers for
analysis of reproductive development. At 4-g, Arabidopsis flowers self-pollinated normally but did not produce seeds, thus derailing the entire life cycle. Pollen viability and stigma
esterase activity were not compromised by hypergravity; however, the growth of pollen tubes into the stigmas was curtailed
at 4-g. In vitro pollen germination assays showed that 4-g average tube length was less than half that for 1-g controls. Closely related Brassica rapa L., which produces seeds at 4-g, required forces in excess of 6-g to slow in vitro tube growth to half that at 1-g. The results explain why seed production is absent in Arabidopsis at 4-g and point to species differences with regard to the g-sensitivity of pollen tube growth. 相似文献
996.
Jennifer L. Lowell Nathan Gordon Dale Engstrom Jack A. Stanford William E. Holben James E. Gannon 《Microbial ecology》2009,58(3):611-620
The Nyack floodplain is located on the Middle Fork of the Flathead River, an unregulated, pristine, fifth-order stream in
Montana, USA, bordering Glacier National Park. The hyporheic zone is a nutritionally heterogeneous floodplain component harboring
a diverse array of microbial assemblages essential in fluvial biogeochemical cycling, riverine ecosystem productivity, and
trophic interactions. Despite these functions, microbial community structure in pristine hyporheic systems is not well characterized.
The current study was designed to assess whether physical habitat heterogeneity within the hyporheic zone of the Nyack floodplain
was sufficient to drive bacterial β diversity between three different hyporheic flow path locations. Habitat heterogeneity
was assessed by measuring soluble reactive phosphorous, nitrate, dissolved organic carbon, dissolved oxygen, and soluble total
nitrogen levels seasonally at surface water infiltration, advection, and exfiltration zones. Significant spatial differences
were detected in dissolved oxygen and nitrate levels, and seasonal differences were detected in dissolved oxygen, nitrate,
and dissolved organic carbon levels. Denaturing gradient gel electrophoresis (DGGE) and cell counts indicated that bacterial
diversity increased with abundance, and DGGE fingerprints covaried with nitrate levels where water infiltrated the hyporheic
zone. The ribosomal gene phylogeny revealed that hyporheic habitat heterogeneity was sufficient to drive β diversity between
bacterial assemblages. Phylogenetic (P) tests detected sequence disparity between the flow path locations. Small distinct
lineages of Firmicutes, Actinomycetes, Planctomycetes, and Acidobacteria defined the infiltration zone and α- and β-proteobacterial lineages delineated the exfiltration and advection zone communities.
These data suggest that spatial habitat heterogeneity drives hyporheic microbial community development and that attempts to
understand functional differences between bacteria inhabiting nutritionally heterogeneous hyporheic environments might begin
by focusing on the biology of these taxa. 相似文献
997.
998.
Cryptosporidium parvum, an apicomplexan parasite transmitted via animal fecal wastes, is the causative agent of cryptosporidiosis. Clones were selected from 2 synthetic na?ve human single-chain variable fragment (scFv) phagemid libraries that bound to the recombinant P23 protein of C. parvum. Panning the Tomlinson I and J phagemid libraries resulted in 6 distinct clones. Two clones had full-length scFv sequences, while the remaining clones were either truncated or missing a section of the heavy chain. Despite these differences, all clones were able to detect both native C. parvum proteins and recombinant P23. None of the selected clones cross-reacted with Escherichia coli, Streptococcus pyogenes, Listeria monocytogenes, Bacillus cereus, Giardia lamblia (cysts or trophozoites), or with S16, another dominant surface antigen on C. parvum sporozoites. Clones expressed as the scFv-gIIIp fusion construct in soluble form detected C. parvum. Panning from na?ve libraries is a useful method for isolation and identification of recombinant antibodies that have the potential for use in pathogen detection and immunotherapy. 相似文献
999.
Copper is an essential co-factor in many important physiological processes, but at elevated levels it is toxic to cells. Thus at both the organism and cellular level mechanisms have evolved to finely tune copper homeostasis. The protein responsible for copper entry from the circulation in most human cells is hCTR1, a small protein (190 amino acid residues) that functions as a trimer in the plasma membrane. In the present work we employ cell surface biotinylation and isotopic copper uptake studies of overexpressed hCTR1 in HEK293 cells to examine the acute (minutes) response of hCTR1 to changes in extracellular copper. We show that within 10 min of exposure to copper at 2.5 μm or higher, plasma membrane hCTR1 levels are reduced (by ∼40%), with a concomitant reduction in copper uptake rates. We are unable to detect any degradation of internalized hCTR1 in the presence of cycloheximide after up to 2 h of exposure to 0–100 μm copper. Using a reversible biotinylation assay, we quantified internalized hCTR1, which increased upon the addition of copper and corresponded to the hCTR1 lost from the surface. In addition, when extracellular copper is then removed, internalized hCTR1 is promptly (within 30 min) recycled to the plasma membrane. We have shown that in the absence of added extracellular copper, there is a small but detectable amount of internalized hCTR1 that is increased in the presence of copper. Similar studies on endogenous hCTR1 show a cell-specific response to elevated extracellular copper. Copper-dependent internalization and recycling of hCTR1 provides an acute and reversible mechanism for the regulation of cellular copper entry.Copper is an essential micronutrient and plays an important function as a co-factor for a number of cellular processes including oxidative phosphorylation, free radical detoxification, neurotransmitter synthesis, iron metabolism, and maturation of connective tissue (1). Copper in excess of cellular requirements is toxic; therefore cells have developed sophisticated mechanisms for regulating copper acquisition and secretion, thus maintaining a critical copper homeostasis (2, 3). In eukaryotes a family of transporters known as the copper transporter (Ctr) proteins mediate cellular copper uptake (4). Ctr proteins are integral membrane proteins that are structurally conserved with three membrane-spanning domains and a number of methionine rich motifs in the N terminus (5). They contain a sequence of conserved cysteine and histidine residues at or close to the C terminus and are predominantly located at the plasma membrane (6). In the yeast, Saccharomyces cerevisiae, the first high affinity copper transporters, yCtr1 and yCtr3, were identified (7, 8), and this facilitated the identification of the human copper transporter gene, hCTR1,2 by functional complementation of yeast high affinity copper uptake mutant, ctr1 (9). The mouse CTR1 is 92% identical to hCTR1 (10), and the deletion of mCTR1 results in early embryonic lethality, suggesting an essential role for the high affinity copper transporter in mammalian growth and development (11).hCTR1 has 190 amino acid residues, three membrane-spanning domains, an extracellular N terminus (of 66 amino acids), a large cytoplasmic loop (of 46 amino acid residues), and a short C-terminal tail (of 15 amino acids) and has been shown to form stable dimers and trimers (12–14). The hCTR1 protein has been shown in 64Cu uptake experiments to mediate copper transport with a Km of 1–5 μm and is thought to transport the reduced form, Cu(I) (12, 13, 15). The extracellular N terminus has both N- and O-linked glycosylation at residues Asn15 and Thr27, respectively (12, 16, 17), and contains two histidine-rich regions and two methionine motifs that are thought to function in copper binding/sensing. Recent studies showed that mutation or deletion of the methionine residues closest to the first transmembrane domain (Met43 and Met45) and the conserved methionine residues in the second transmembrane domain (Met150 and Met154) had a large inhibitory effect on 64Cu uptake (18, 19). Mutational analysis provided no evidence for the tight binding of copper at any specific residues, and it was proposed that hCTR1 provided a pore for the permeation of copper across the membrane (18). Structural confirmation of such a mechanism was provided in the low resolution structure obtained by cryo-electron microscopy studies on recombinant protein (20, 21).Considerable progress has been made in understanding the biochemical, structure-functional, and molecular aspects of hCTR1-mediated copper transport, although many questions remain unanswered (22). It is also important to determine whether or not hCTR1 has a regulatory role preventing the accumulation of toxic levels of copper and maintaining cellular copper homeostasis. Previous reports on whether or not hCTR1 is involved in an acute response to elevated copper have been somewhat controversial. It has been reported that elevated extracellular copper (1–100 μm) stimulates rapid endocytosis and degradation of hCTR1-Myc-tagged protein in HEK293 cells (23), but also high copper levels had no effect on endogenous hCTR1 localization in both HeLa and Caco-2 cells (14). In a study of overexpressed hCTR1 in insect cells, no evidence was seen of internalization in response to elevated copper (24). Imaging studies have shown that the cellular location of hCTR1 varies among cell lines, CTR1 in MDCK and HEK293 cells resides mainly at the plasma membrane (13, 15, 23, 24). Endogenous hCTR1 is located in cytoplasmic vesicular compartments in HeLa, Caco-2, and HepG2 cell lines with some plasma membrane staining in Caco-2 (14). In intestinal sections, basolateral and subapical staining is seen (15).Previous studies (see above) have utilized internalization of prebound antibody (23) or imaging methods (14) to characterize the response of hCTR1 to elevated copper. In the present work we employed HEK cells overexpressing hCTR1 and used cell surface biotinylation, a sensitive and quantitative measure of CTR1 at the cell surface (15, 17). We have combined this with measurements of hCTR1-mediated 64Cu uptake as a functional measure of plasma membrane hCTR1 levels. We find that a fraction (∼40%) of hCTR1 is rapidly internalized in the presence of elevated copper and that there is a concomitant reduction in the hCTR1-mediated copper uptake rate. The internalized transporter is not degraded and can be detected in the cytosol. On removal of extracellular copper, the transporter is recycled promptly to the plasma membrane. Internalization of endogenous CTR1 is also observed in MDCK and HepG2 cells, and no reduction is seen in T47D cells. This is, to our knowledge, the first such report of copper-dependent recycling of hCTR1 in response to copper and represents an acute regulatory mechanism that reversibly modulates cellular copper entry. 相似文献
1000.
Julia Pimkina Olivier Humbey Jack T. Zilfou Michal Jarnik Maureen E. Murphy 《The Journal of biological chemistry》2009,284(5):2803-2810
The ARF tumor suppressor controls a well-described p53/Mdm2-dependent
oncogenic stress checkpoint. In addition, ARF has recently been shown to
localize to mitochondria, and to induce autophagy; however, this has never
before been demonstrated for endogenous ARF, and the molecular basis for this
activity of ARF has not been elucidated. Using an unbiased mass
spectrometry-based approach, we show that mitochondrial ARF interacts with the
Bcl2 family member Bcl-xl, which normally protects cells from autophagy by
inhibiting the Beclin-1/Vps34 complex, which is essential for autophagy. We
find that increased expression of ARF decreases Beclin-1/Bcl-xl complexes in
cells, thereby providing a basis for ARF-induced autophagy. Our data also
indicate that silencing p53 leads to high levels of ARF and increased
autophagy, thereby providing a possible basis for the finding by others that
p53 inhibits autophagy. The combined data support the premise that ARF induces
autophagy in a p53-independent manner in part by virtue of its interaction
with Bcl-xl.The ARF tumor suppressor, p14ARF in humans and p19ARF
in mouse, is a critical growth suppressor that is up-regulated by chronic
mitogenic signals and localizes predominantly to the nucleolus. At the
nucleolus and in the nucleoplasm, ARF can exert both p53-dependent and
-independent growth suppressive function, by virtue of interaction with and
inhibition of MDM2, nucleophosmin, E2F-1, CtBP, c-Myc, as well as others (see
Ref. 1 for review). Recently, a
small molecular weight variant of ARF, generated by translation from an
internal methionine, has been discovered to localize primarily to mitochondria
and to induce autophagy (2).
More recently, another group has shown that full-length ARF, in addition to
the small molecular weight variant, can likewise induce autophagy
(3). However, neither of these
studies revealed a mechanism whereby ARF induces autophagy.Autophagy is an evolutionarily conserved homeostatic process whereby
cytosolic components are targeted for removal or turnover in membrane-bound
compartments (autophagosomes) that fuse with the lysosome (for review see Ref.
4). This process regulates the
turnover of damaged organelles and long-lived proteins that are too large to
be delivered to the proteasome. Autophagy occurs constitutively at low levels
and is greatly induced during period of metabolic stress, where
lysosome-mediated digestion of sequestered molecules serves to release free
amino acids and ATP to fuel the continued survival of the cell.Several genes are implicated in the control of autophagy. Perhaps most
notable of these is Beclin-1, which is an evolutionarily-conserved mediator of
autophagy, with structural similarity to the yeast autophagy gene Apg6/Vps30.
Beclin-1 is a component of the class III PI3 kinase complex that includes
Vps34; this complex regulates the formation and nucleation of autophagosomes,
and the regulation of the activity of this complex is tightly regulated. For
example, Beclin-1 possesses a BH3 domain that interacts with the BH3 binding
groove of certain members of the Bcl-2 family, including Bcl-2, Bcl-xl, Bcl-w,
and to a lesser extent, Mcl-1
(5–9).
Binding of Bcl-2 family members to Beclin-1 inhibits autophagy, possibly by
decreasing the kinase activity of the Beclin/Bcl-2/Vps34 complex
(5) or by negatively regulating
Beclin-1 oligomerization (10).
The interaction between Beclin-1 and Bcl-2 family members is also regulated;
for example, BH3-only proteins can bind directly to Bcl-2 family members and
disrupt complex formation with Beclin-1
(11). Additionally,
phosphorylation of Bcl-2 by Jun-N-terminal kinase (JNK) can interfere with its
ability to bind to Beclin-1
(12). In all cases,
dissociation of the Beclin-1/Bcl-2 complex is associated with induction of
autophagy.In this report we confirm the findings of others that a fraction of ARF
protein localizes to mitochondria and can induce autophagy. We show for the
first time that endogenous ARF, up-regulated in non-transformed cells by
oncogenes, is capable of inducing autophagy, and further that silencing of p53
is sufficient to de-repress ARF and induce autophagy. We report the
identification of Bcl-xl as a mitochondrial ARF-binding protein, and show that
ARF-mediated autophagy is enhanced in cells with Bcl-xl silenced. Finally, we
show that ARF can reduce complex formation between Bcl-xl and Beclin-1. These
data offer the first mechanistic insights into ARF-mediated autophagy. They
also point to ARF as a novel regulator of Beclin/Bcl-xl complex formation. 相似文献