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941.
Jie Li Takahisa Kanekiyo Mitsuru Shinohara Yunwu Zhang Mary Jo LaDu Huaxi Xu Guojun Bu 《The Journal of biological chemistry》2012,287(53):44593-44601
Aggregation of amyloid-β (Aβ) peptides leads to synaptic disruption and neurodegeneration in Alzheimer disease (AD). A major Aβ clearance pathway in the brain is cellular uptake and degradation. However, how Aβ traffics through the endocytic pathway and how AD risk factors regulate this event is unclear. Here we show that the majority of endocytosed Aβ in neurons traffics through early and late endosomes to the lysosomes for degradation. Overexpression of Rab5 or Rab7, small GTPases that function in vesicle fusion for early and late endosomes, respectively, significantly accelerates Aβ endocytic trafficking to the lysosomes. We also found that a portion of endocytosed Aβ traffics through Rab11-positive recycling vesicles. A blockage of this Aβ recycling pathway with a constitutively active Rab11 mutant significantly accelerates cellular Aβ accumulation. Inhibition of lysosomal enzymes results in Aβ accumulation and aggregation. Importantly, apolipoprotein E (apoE) accelerates neuronal Aβ uptake, lysosomal trafficking, and degradation in an isoform-dependent manner with apoE3 more efficiently facilitating Aβ trafficking and degradation than apoE4, a risk factor for AD. Taken together, our results demonstrate that Aβ endocytic trafficking to lysosomes for degradation is a major Aβ clearance pathway that is differentially regulated by apoE isoforms. A disturbance of this pathway can lead to accumulation and aggregation of cellular Aβ capable of causing neurotoxicity and seeding amyloid. 相似文献
942.
Yin B Lee BS Yang-Iott KS Sleckman BP Bassing CH 《Journal of immunology (Baltimore, Md. : 1950)》2012,189(3):1372-1379
The ataxia telangiectasia mutated (ATM) kinase and H2AX histone tumor suppressor proteins are each critical for maintenance of cellular genomic stability and suppression of lymphomas harboring clonal translocations. ATM is the predominant kinase that phosphorylates H2AX in chromatin around DNA double-strand breaks, including along lymphocyte Ag receptor loci cleaved during V(D)J recombination. However, combined germline inactivation of Atm and H2ax in mice causes early embryonic lethality associated with substantial cellular genomic instability, indicating that ATM and H2AX exhibit nonredundant functions in embryonic cells. To evaluate potential nonredundant roles of ATM and H2AX in somatic cells, we generated and analyzed Atm-deficient mice with conditional deletion of H2ax in αβ T-lineage lymphocytes. Combined Atm/H2ax inactivation starting in early-stage CD4(-)/CD8(-) thymocytes resulted in lower numbers of later-stage CD4(+)/CD8(+) thymocytes, but led to no discernible V(D)J recombination defect in G1 phase cells beyond that observed in Atm-deficient cells. H2ax deletion in Atm-deficient thymocytes also did not affect the incidence or mortality of mice from thymic lymphomas with clonal chromosome 14 (TCRα/δ) translocations. Yet, in vitro-stimulated Atm/H2ax-deficient splenic αβ T cells exhibited a higher frequency of genomic instability, including radial chromosome translocations and TCRβ translocations, compared with cells lacking Atm or H2ax. Collectively, our data demonstrate that both redundant and nonredundant functions of ATM and H2AX are required for normal recombination of TCR loci, proliferative expansion of developing thymocytes, and maintenance of genomic stability in cycling αβ T-lineage cells. 相似文献
943.
Mark P. Wentland Rongliang Lou Qun Lu Yigong Bu Christoph Denhardt Jin Jin Rakesh Ganorkar Melissa A. VanAlstine Chengyun Guo Dana J. Cohen Jean M. Bidlack 《Bioorganic & medicinal chemistry letters》2009,19(8):2289-2294
A series of novel high affinity opioid receptor ligands have been made whereby the phenolic-OH group of nalbuphine, naltrexone methiodide, 6-desoxonaltrexone, hydromorphone and naltrindole was replaced by a carboxamido group and the furan ring was opened to the corresponding 4-OH derivatives. These furan ring ‘open’ derivatives display very high affinity for μ and κ receptors and much less affinity for δ. The observation that these target compounds have much higher receptor affinity than the corresponding ring ‘closed’ carboxamides significantly strengthens our underlying pharmacophore hypothesis concerning the bioactive conformation of the carboxamide group. 相似文献
944.
通过根癌农杆菌(含植物表达载体YXu55)介导的转化技术,将褪黑素生物合成酶-芳烷基胺N-乙酰转移酶(Arylalkylamine N-acetyltransferase,AANAT)与羟基吲哚O-甲基转移酶(Hydroxyindole O-methyltransferase,HIOMT)基因导入到烟草(秦烟95)中。对所获得的庆大霉素抗性烟草株系进行Southern blotting和RT-PCR分子生物学检测,结果表明,AANAT-HIOMT基因已成功地整合到烟草基因组中,并且可以在mRNA水平上进行转录。用反相高效液相色谱法(RP-HPLC)测定转化株系的褪黑素含量表明,转AANAT-HIOMT基因烟草株系的褪黑素含量均明显高于pZP122(不含AANAT和HIOMT基因的空白质粒)转基因株系和未转基因的对照植株,证明AANAT-HIOMT基因在转基因植株中的表达增强了褪黑素的合成能力。对不同株系抗氧化系统的部分指标进行了测定,并与其亲本对照植株比较,发现AANAT-HIOMT基因在转基因植物中的表达引起超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性增加,谷胱甘肽(GSH)浓度... 相似文献
945.
Rebecca A. Chanoux Bu Yin Karen A. Urtishak Amma Asare Craig H. Bassing Eric J. Brown 《The Journal of biological chemistry》2009,284(9):5994-6003
Chromosomal abnormalities are frequently caused by problems encountered
during DNA replication. Although the ATR-Chk1 pathway has previously been
implicated in preventing the collapse of stalled replication forks into
double-strand breaks (DSB), the importance of the response to fork collapse in
ATR-deficient cells has not been well characterized. Herein, we demonstrate
that, upon stalled replication, ATR deficiency leads to the phosphorylation of
H2AX by ATM and DNA-PKcs and to the focal accumulation of Rad51, a marker of
homologous recombination and fork restart. Because H2AX has been shown to play
a facilitative role in homologous recombination, we hypothesized that H2AX
participates in Rad51-mediated suppression of DSBs generated in the absence of
ATR. Consistent with this model, increased Rad51 focal accumulation in
ATR-deficient cells is largely dependent on H2AX, and dual deficiencies in ATR
and H2AX lead to synergistic increases in chromatid breaks and translocations.
Importantly, the ATM and DNA-PK phosphorylation site on H2AX
(Ser139) is required for genome stabilization in the absence of
ATR; therefore, phosphorylation of H2AX by ATM and DNA-PKcs plays a pivotal
role in suppressing DSBs during DNA synthesis in instances of ATR pathway
failure. These results imply that ATR-dependent fork stabilization and
H2AX/ATM/DNA-PKcs-dependent restart pathways cooperatively suppress
double-strand breaks as a layered response network when replication
stalls.Genome maintenance prevents mutations that lead to cancer and age-related
diseases. A major challenge in preserving genome integrity occurs in the
simple act of DNA replication, in which failures at numerous levels can occur.
Besides the mis-incorporation of nucleotides, it is during this phase of the
cell cycle that the relatively stable double-stranded nature of DNA is
temporarily suspended at the replication fork, a structure that is susceptible
to collapse into
DSBs.2 Replication
fork stability is maintained by a variety of mechanisms, including activation
of the ATR-dependent checkpoint pathway.The ATR pathway is activated upon the generation and recognition of
extended stretches of single-stranded DNA at stalled replication forks
(1-4).
Genome maintenance functions for ATR and orthologs in yeast were first
indicated by increased chromatid breaks in ATR-/- cultured cells
(5) and by the
“cut” phenotype observed in Mec1 (Saccharomyces
cerevisiae) and Rad3 (Schizosaccharomyces pombe) mutants
(6-9).
Importantly, subsequent studies in S. cerevisiae demonstrated that
mutation of Mec1 or the downstream checkpoint kinase Rad53 led to increased
chromosome breaks at regions of the genome that are inherently difficult to
replicate (10), and a
decreased ability to reinitiate replication fork progression following DNA
damage or deoxyribonucleotide depletion
(11-14).In vertebrates, similar replication fork stabilizing functions have been
demonstrated for ATR and the downstream protein kinase Chk1
(15-20).
Several possible mechanisms have been put forward to explain how ATR-Chk1 and
orthologous pathways in yeast maintain replication fork stability, including
maintenance of replicative polymerases (α, δ, and ε) at forks
(17,
21), regulation of branch
migrating helicases, such as Blm
(22-25),
and regulation of homologous recombination, either positively or negatively
(26-29).Consistent with the role of the ATR-dependent checkpoint in replication
fork stability, common fragile sites, located in late-replicating regions of
the genome, are significantly more unstable (5-10-fold) in the absence of ATR
or Chk1 (19,
20). Because these sites are
favored regions of instability in oncogene-transformed cells and preneoplastic
lesions (30,
31), it is possible that the
increased tumor incidence observed in ATR haploinsufficient mice
(5,
32) may be related to subtle
increases in genomic instability. Together, these studies indicate that
maintenance of replication fork stability may contribute to tumor
suppression.It is important to note that prevention of fork collapse represents an
early response to problems occurring during DNA replication. In the event of
fork collapse into DSBs, homologous recombination (HR) has also been
demonstrated to play a key role in genome stability during S phase by
catalyzing recombination between sister chromatids as a means to re-establish
replication forks (33).
Importantly, a facilitator of homologous recombination, H2AX, has been shown
to be phosphorylated under conditions that cause replication fork collapse
(18,
34).Phosphorylation of H2AX occurs predominantly upon DSB formation
(34-38)
and has been reported to require ATM, DNA-PKcs, or ATR, depending on the
context
(37-42).
Although H2AX is not essential for HR, studies have demonstrated that H2AX
mutation leads to deficiencies in HR
(43,
44), and suppresses events
associated with homologous recombination, such as the focal accumulation of
Rad51, BRCA1, BRCA2, ubiquitinated-FANCD2, and Ubc13-mediated chromatin
ubiquitination (43,
45-51).
Therefore, through its contribution to HR, it is possible that H2AX plays an
important role in replication fork stability as part of a salvage pathway to
reinitiate replication following collapse.If ATR prevents the collapse of stalled replication forks into DSBs, and
H2AX facilitates HR-mediated restart, the combined deficiency in ATR and H2AX
would be expected to dramatically enhance the accumulation of DSBs upon
replication fork stalling. Herein, we utilize both partial and complete
elimination of ATR and H2AX to demonstrate that these genes work cooperatively
in non-redundant pathways to suppress DSBs during S phase. As discussed, these
studies imply that the various components of replication fork protection and
regeneration cooperate to maintain replication fork stability. Given the large
number of genes involved in each of these processes, it is possible that
combined deficiencies in these pathways may be relatively frequent in humans
and may synergistically influence the onset of age-related diseases and
cancer. 相似文献
946.
947.
Tatsuya Inamura Yoshimi Mukai Akiko Maruyama Sachiko Ikenaga G.uili Li Xuemei Bu Yuhua Xiang Dakui Qin Takahisa Amano 《Plant and Soil》2009,315(1-2):195-209
The farm household responsibility system (FHRS) was adopted in Chinese rural areas during the economic reform in the early 1980s. Since then, many farm households have increased cropping intensity by using large quantities of nitrogen (N) fertilizers in their responsible fields to increase agricultural income. However, intensive cropping systems with low N input are still common in remote places of the southwestern region of China. Maintenance and improvement of soil quality in intensive cropping systems is critical for sustaining agricultural productivity and environmental quality for future generations. The effects of intensive cropping of vegetables on paddy rice (Oryza sativa L.) yield using small quantities of N fertilizers through N mineralization of paddy soil in irrigated rice-based multiple cropping systems were studied in 15 paddy fields in Sichuan Province, China for 3 years. Intensification of vegetable cropping with negative N balance and removal of vegetable crop residues has greatly decreased total N (TN) contents in paddy soil leading to low levels of effective cumulated soil temperature and thickness of plow layer. As a result, the N mineralization in paddy field during paddy rice growing period was decreased. In addition to the low levels of chemical fertilizer N input and residual mineral N input, the lower level of N mineralization in paddy fields and low N recovery efficiency decreased the amount of N accumulated in aboveground biomass of paddy rice at maturity, resulting in limited rice yields. The amount of mineralized N only correlated with TN (partial correlation analysis). Therefore, in paddy fields with very low N input, the N mineralization in paddy soil during the paddy rice-growing period was the major limiting factor affecting the total yield increases. In addition, a decline in soil fertility can be determined using TN as an indicator. To improve paddy rice yield and avoid soil deterioration, the development and adoption of rational soil management programs are needed. These include input of plant residues, conscientious soil tillage for the maintenance of soil temperature and thickness of the plow layer, and the split application of fertilizer for the improvement of N recovery efficiency. 相似文献
948.
Scaffolding proteins are molecular switches that control diverse signaling events. The scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF1) assembles macromolecular signaling complexes and regulates the macromolecular assembly, localization, and intracellular trafficking of a number of membrane ion transport proteins, receptors, and adhesion/antiadhesion proteins. NHERF1 begins with two modular protein-protein interaction domains—PDZ1 and PDZ2—and ends with a C-terminal (CT) domain. This CT domain binds to ezrin, which, in turn, interacts with cytosekeletal actin. Remarkably, ezrin binding to NHERF1 increases the binding capabilities of both PDZ domains. Here, we use deuterium labeling and contrast variation neutron-scattering experiments to determine the conformational changes in NHERF1 when it forms a complex with ezrin. Upon binding to ezrin, NHERF1 undergoes significant conformational changes in the region linking PDZ2 and its CT ezrin-binding domain, as well as in the region linking PDZ1 and PDZ2, involving very long range interactions over 120 Å. The results provide a structural explanation, at mesoscopic scales, of the allosteric control of NHERF1 by ezrin as it assembles protein complexes. Because of the essential roles of NHERF1 and ezrin in intracellular trafficking in epithelial cells, we hypothesize that this long-range allosteric regulation of NHERF1 by ezrin enables the membrane-cytoskeleton to assemble protein complexes that control cross-talk and regulate the strength and duration of signaling. 相似文献
949.
Brain amyloid-β (Aβ) peptide accumulation and aggregation are critical events in the pathogenesis of Alzheimer disease. Increasing evidence has demonstrated that LRP1 is involved in Alzheimer disease pathogenesis. The physiological ligands of LRP1, including apoE, play significant roles in the cellular clearance of Aβ. The receptor-associated protein (RAP) is a specialized chaperone for members of the low density lipoprotein receptor family. RAP shares structural and receptor-binding properties with apoE. Here, we show that RAP binds to both Aβ40 and Aβ42 in a concentration-dependent manner and forms complexes with them. Fluorescence-activated cell sorter analysis showed that RAP significantly enhances the cellular internalization of Aβ in different cell types, including brain vascular smooth muscle, neuroblastoma, glioblastoma, and Chinese hamster ovary cells. This effect of RAP was confirmed by fluorescence microscopy and enzyme-linked immunosorbent assay. RAP binds to both LRP1 and heparin; however, the ability of RAP to enhance Aβ cellular uptake was blocked by heparin and heparinase treatment but not by LRP1 deficiency. Furthermore, the effects of RAP were significantly decreased in heparan sulfate proteoglycan-deficient Chinese hamster ovary cells. Our findings reveal that RAP is a novel Aβ-binding protein that promotes cellular internalization of Aβ. 相似文献
950.
Isolation and biochemical characterization of two lipases from a metagenomic library of China Holstein cow rumen 总被引:1,自引:0,他引:1
Kailang Liu Dengpan Bu Chris McSweeney Dan Li 《Biochemical and biophysical research communications》2009,385(4):605-611
Two novel lipase genes RlipE1 and RlipE2 which encoded 361- and 265-amino acid peptides, respectively, were recovered from a metagenomic library of the rumen microbiota of Chinese Holstein cows. A BLAST search revealed a high similarity (90%) between RlipE2 and a carboxylesterase from Thermosinus carboxydivorans Nor1, while there was a low similarity (below 50%) between RlipE1 and other lipases. Phylogenetic analysis indicated that RlipE2 clustered with the lipolytic enzymes from family V while RlipE1 clustered with six other putative bacterial lipases which might constitute a new subfamily. The recombinant lipases were thermally unstable and retained 60% activity over a pH range of 6.5-8.5. Substrate specificity assay indicated that both enzymes had higher hydrolytic activity toward laurate (C12), palmitate (C16) and stearate (C18). The novel phylogenetic affiliation and high specificity of both enzymes for long-chain fatty acid make them interesting targets for manipulation of rumen lipid metabolism. 相似文献