全文获取类型
收费全文 | 149272篇 |
免费 | 4938篇 |
国内免费 | 5344篇 |
专业分类
159554篇 |
出版年
2024年 | 143篇 |
2023年 | 813篇 |
2022年 | 1895篇 |
2021年 | 3142篇 |
2020年 | 2139篇 |
2019年 | 2566篇 |
2018年 | 13627篇 |
2017年 | 11953篇 |
2016年 | 9671篇 |
2015年 | 4272篇 |
2014年 | 4772篇 |
2013年 | 4855篇 |
2012年 | 9462篇 |
2011年 | 17260篇 |
2010年 | 14564篇 |
2009年 | 10599篇 |
2008年 | 12554篇 |
2007年 | 13816篇 |
2006年 | 2576篇 |
2005年 | 2436篇 |
2004年 | 2481篇 |
2003年 | 2451篇 |
2002年 | 1876篇 |
2001年 | 1178篇 |
2000年 | 1077篇 |
1999年 | 862篇 |
1998年 | 527篇 |
1997年 | 489篇 |
1996年 | 498篇 |
1995年 | 435篇 |
1994年 | 426篇 |
1993年 | 366篇 |
1992年 | 482篇 |
1991年 | 374篇 |
1990年 | 299篇 |
1989年 | 277篇 |
1988年 | 235篇 |
1987年 | 214篇 |
1986年 | 184篇 |
1985年 | 157篇 |
1984年 | 125篇 |
1983年 | 141篇 |
1982年 | 86篇 |
1981年 | 49篇 |
1980年 | 54篇 |
1979年 | 65篇 |
1976年 | 46篇 |
1974年 | 56篇 |
1972年 | 299篇 |
1971年 | 301篇 |
排序方式: 共有10000条查询结果,搜索用时 0 毫秒
991.
原位缺口平移标记断裂DNA技术及其在检测流行性出血热尸检和实验动物组织中细胞凋亡和早期死亡的应用 总被引:1,自引:0,他引:1
原位缺口平移技术(ISNT)已被用于检测细胞核中DNA断裂鉴别尸检组织中细胞的凋亡和坏死断裂、流行性出血热(EHF)组织中存在散在单个细胞变性死亡和灶性梗死样坏死,前者带有细胞凋亡的特征。本文以EHF肝脏和实验性病毒感染鼠脑组织为例,应用缺口平移法,在DNA聚合酶或Klenow酶的作用下,将地高辛标记的dUTP掺入合成到DNA的断裂部位,通过碱性磷酸酶标抗地高辛抗体免疫组化法显示细胞DNA的断裂,检测和鉴别细胞的凋亡和坏死。为分析死后解剖时间间隔及组织固定时间对该方法的影响,本文选用死后2~140h尸检、经常规固定石蜡包埋后存放10~35年的标本和在10%福尔马林固定了10~35年之后再进行常规处理的标本。实验时用蛋白酶K(PK)消化前后对比并分别在标记反应液中略去DNA聚合酶作为阴性对照,用DNA酶消化组织人为制造DNA缺日作为阳性对照。结果发现,未经PK消化的组织,仅灶性肝细胞核ISNT标记阳性,经PK消化后,散在的带有凋亡特征的肝细胞胞核也出现阳性,灶性肝细胞胞核标记染色增强,但无论是蛋白酶消化与否,明确梗死样坏死的肝细胞均不被标记。结果还发现,死后2~24h内尸检组织和长时间(10~34年)存放的石 相似文献
992.
Yang X Yu RH Calmettes C Moraes TF Schryvers AB 《The Journal of biological chemistry》2011,286(52):45165-45173
Gram-negative bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae, and Neisseriaceae families rely on an iron acquisition system that acquires iron directly from host transferrin (Tf). The process is mediated by a surface receptor composed of transferrin-binding proteins A and B (TbpA and TbpB). TbpA is an integral outer membrane protein that functions as a gated channel for the passage of iron into the periplasm. TbpB is a surface-exposed lipoprotein that facilitates the iron uptake process. In this study, we demonstrate that the region encompassing amino acids 7-40 of Actinobacillus pleuropneumoniae TbpB is required for forming a complex with TbpA and that the formation of the complex requires the presence of porcine Tf. These results are consistent with a model in which TbpB is responsible for the initial capture of iron-loaded Tf and subsequently interacts with TbpA through the anchor peptide. We propose that TonB binding to TbpA initiates the formation of the TbpB-TbpA complex and transfer of Tf to TbpA. 相似文献
993.
In this special issue of the Glycoconjugate Journal focusing on glycosciences and development, we summarize recent advances in our understanding of the role of mucin-type O-glycans
in development and disease. The presence of this widespread protein modification has been known for decades, yet identification
of its biological functions has been hampered by the redundancy and complexity of the enzyme family controlling the initiation
of O-glycosylation, as well as the diversity of extensions of the core sugar. Recent studies in organisms as diverse as mammals
and Drosophila have yielded insights into the function of this highly abundant and evolutionarily-conserved protein modification. Gaining
an understanding of mucin-type O-glycans in these diverse systems will elucidate crucial conserved processes underlying many
aspects of development and homeostasis. 相似文献
994.
Wen-Jie Ji Yong-Qiang Ma Xin Zhou Yi-Dan Zhang Rui-Yi Lu Zhao-Zeng Guo Hai-Ying Sun Dao-Chuan Hu Guo-Hong Yang Yu-Ming Li Lu-Qing Wei 《PloS one》2013,8(11)
Background
Recent experimental studies provide evidence indicating that manipulation of the mononuclear phagocyte phenotype could be a feasible approach to alter the severity and persistence of pulmonary injury and fibrosis. Mineralocorticoid receptor (MR) has been reported as a target to regulate macrophage polarization. The present work was designed to investigate the therapeutic potential of MR antagonism in bleomycin-induced acute lung injury and fibrosis.Methodology/Principal Findings
We first demonstrated the expression of MR in magnetic bead-purified Ly6G-/CD11b+ circulating monocytes and in alveolar macrophages harvested in bronchoalveolar lavage fluid (BALF) from C57BL/6 mice. Then, a pharmacological intervention study using spironolactone (20mg/kg/day by oral gavage) revealed that MR antagonism led to decreased inflammatory cell infiltration, cytokine production (downregulated monocyte chemoattractant protein-1, transforming growth factor β1, and interleukin-1β at mRNA and protein levels) and collagen deposition (decreased lung total hydroxyproline content and collagen positive area by Masson’ trichrome staining) in bleomycin treated (2.5mg/kg, via oropharyngeal instillation) male C57BL/6 mice. Moreover, serial flow cytometry analysis in blood, BALF and enzymatically digested lung tissue, revealed that spironolactone could partially inhibit bleomycin-induced circulating Ly6Chi monocyte expansion, and reduce alternative activation (F4/80+CD11c+CD206+) of mononuclear phagocyte in alveoli, whereas the phenotype of interstitial macrophage (F4/80+CD11c-) remained unaffected by spironolactone during investigation.Conclusions/Significance
The present work provides the experimental evidence that spironolactone could attenuate bleomycin-induced acute pulmonary injury and fibrosis, partially via inhibition of MR-mediated circulating monocyte and alveolar macrophage phenotype switching. 相似文献995.
Ujvarosi K Hunyadi J Nagy G Pocsi I Banfalvi G 《Apoptosis : an international journal on programmed cell death》2007,12(11):2089-2099
Exponentially growing human erythroleukemia K562 cells were permeabilized and the dose dependent decrease of DNA synthesis rate was measured after ultraviolet (UV B, 290 nm) irradiation. Cells were able to overcome 2 and 5 J/m2 UV doses, partial recovery was observed at 15 J/m2, while at high (25 J/m2) UV dose replicative DNA synthesis remained suppressed. K562 cells were subjected to synchronization prior to and after UV irradiation (24 J/m2) and 18 fractions were collected by centrifugal elutriation. Cell cycle analysis by flow cytometry did not show early apoptotic cells after UV irradiation. The gradual increase in DNA content typical for non-irradiated cells was contrasted by an early S phase block between 2.2 and 2.4 C-values after UV irradiation. Cell cycle dependent chromatin changes after ultraviolet irradiation were seen as a fine fibrillary network covering the mainly fibrous chromatin structures and incompletely folded primitive chromosomes. Based on observations after UV irradiation and on earlier results with cadmium treatment and gamma irradiation, we confirm that typical chromatin changes characteristic to genotoxic agents can be recognized and classified. 相似文献
996.
Micropropagation was assessed as an ex situ conservation strategy for the endangered Australian plant Pimelea spicata (Thymelaeaceae). Although regeneration of this species was achieved, several physiological problems were observed and examined. Explants of P. spicata had a higher multiplication rate on MS medium, than on ½ MS, but there was a significantly higher percentage of necrotic shoot tips on the higher salt medium. Increasing calcium concentration and gas exchange exacerbated shoot-tip necrosis. A number of hyperhydrated shoots were produced in all treatments, the cause of which could not be determined, although less hyperhydicity was observed in the ½ MS treatment. Shoots, rooted in vitro on ½ MS in the absence of plant growth regulators, were successfully acclimatised to greenhouse conditions, while direct rooting of microshoots using IBA gel treatment proved unsuccessful. This is the first report of tissue culture propagation of this endangered species. 相似文献
997.
Type A botulinum neurotoxin is one of the most lethal of the seven serotypes and is increasingly used as a therapeutic agent
in neuromuscular dysfunctions. Its toxic function is related to zinc-endopeptidase activity of the N-terminal light chain
(LC) on synaptosome-associated protein-25 kDa (SNAP-25) of the SNARE complex. To understand the determinants of substrate
specificity and assist the development of strategies for effective inhibitors, we used site-directed mutagenesis to investigate
the effects of 13 polar residues of the LC on substrate binding and catalysis. Selection of the residues for mutation was
based on a computational analysis of the three-dimensional structure of the LC modeled with a 17-residue substrate fragment
of SNAP-25. Steady-state kinetic parameters for proteolysis of the substrate fragment were determined for a set of 16 single
mutants. Of the mutated residues non-conserved among the serotypes, replacement of Arg-230 and Asp-369 by polar or apolar
residues resulted in drastic lowering of the catalytic rate constant (k
cat), but had less effect on substrate affinity (K
m). Substitution of Arg-230 with Lys decreased the catalytic efficiency (k
cat/K
m) by 50-fold, whereas replacement by Leu yielded an inactive protein. Removal of the electrostatic charge at Asp-369 by mutation
to Asn resulted in 140-fold decrease in k
cat/K
m. Replacement of other variable residues surrounding the catalytic cleft (Glu-54, Glu-63, Asn-66, Asp-130, Asn-161, Glu-163,
Glu-170, Glu-256), had only marginal effect on decreasing the catalytic efficiency, but unexpectedly the substitution of Lys-165
with Leu resulted in fourfold increase in k
cat/K
m. For comparison purposes, two conserved residues Arg-362 and Tyr-365 were investigated with substitutions of Leu and Phe,
respectively, and their catalytic efficiency decreased 140- and 10-fold, respectively, whereas substitution of the tyrosine
ring with Asn abolished activity. The altered catalytic efficiencies of the mutants were not due to any significant changes
in secondary or tertiary structures, or in zinc content and thermal stability. We suggest that, despite the large minimal
substrate size for catalysis, only a few non-conserved residues surrounding the active site are important to render the LC
competent for catalysis or provide conformational selection of the substrate. 相似文献
998.
Powdery mildew disease caused by Blumeria graminis f. sp. tritici (Bgt) is an economically important disease in wheat worldwide. The identification of germplasms resistant to the disease can not
only facilitate the breeding of resistant cultivars, but can also broaden the diversity of resistance genes. The Mexican M53
is a synthetic hexaploid wheat line developed at the International Maize and Wheat Improvement Center (CIMMYT) from the cross
between Triticum durum and Aegilops tauschii249. Infection of M53 with 15 different pathogen races revealed that the resistance in M53 was race-dependent and effective
against the majority of the tested Bgt races, including the race 15 predominant in the Beijing wheat growing area. Inoculation of the parents of M53 with the race
15 demonstrated that M53 and Ae. tauschii249 were resistant, whereas T. durum was susceptible. The inoculation of three segregating F2 populations developed from the crosses between M53 and three susceptible Chinese wheat cultivars with the race 15 showed
that the resistant gene in M53 segregated in a single dominant manner. Amplified fragment length polymorphism (AFLP) and simple
sequence repeat (SSR) markers were used to map the gene in a segregating F2 population consisting of 213 lines developed from the cross Wan7107 × M53. Two closely linked AFLP markers, Apm109 and Apm161, were identified to flank the gene with genetic distances of 1.0 cM and 3.0 cM, respectively. The recognized gene was assigned
to the long arm of chromosome 5D as determined by three linked SSR markers, Xwmc289b, Xgwm583, and Xgwm292, and by the physical mapping of Apm109 using Chinese Spring nullisomic–tetrasomic and ditelosomic stocks. The resistance gene identified in M53, temporarily designated
as Pm-M53, could be used in local wheat-breeding programs to improve powdery mildew resistance. 相似文献
999.
Ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex from Paracoccus denitrificans consists of only three polypeptide subunits (Yang, X., and Trumpower, B. L. (1986) J. Biol. Chem. 261, 12282-12289), whereas the analogous complexes of eukaryotic mitochondria consist of nine or more polypeptides (Schagger, H., Link, T. A., Engel, W. D., and von Jagow, G. (1986) Methods Enzymol. 126, 224-237). Using the purified three-subunit Paracoccus complex we have tested whether this simple cytochrome bc1 complex has the same electron transfer pathway and proton translocation activity as the bc1 complexes of mitochondria. Under presteady state conditions, the effects of inhibitors on reduction of cytochromes b and c1 by quinol and oxidant-induced reduction of cytochrome b indicate a cyclic electron transfer pathway and two routes of cytochrome b reduction in the three-subunit Paracoccus cytochrome bc1 complex. A novel method was developed to incorporate the cytochrome bc1 complex into liposomes with the detergent dodecyl maltoside. The enzyme reconstituted into liposomes translocated protons with an H+/2e value of 3.9. Carbonyl cyanide m-chlorophenylhydrazone eliminated proton translocation, while permitting the scalar release of protons from quinol, and thus reduced the H+/2e ratio to 2. These values agree with the predicted stoichiometries for proton translocation by a protonmotive Q cycle pathway. No inhibition of proton translocation by N',N'-dicyclohexylcarbodiimide was detected when the Paracoccus cytochrome bc1 complex was incubated with N',N'-dicyclohexylcarbodiimide before or after reconstitution into liposomes. Electron transfer in the three-subunit complex thus appears to occur by a protonmotive Q cycle pathway identical to that in mitochondrial cytochrome bc1 complexes. Only three polypeptides, cytochromes b, c1, and the Rieske iron-sulfur protein, are required for respiration and energy transduction in the cytochrome bc1 complex. The function of the supernumerary polypeptides in mitochondrial bc1 complexes is thus unclear. 相似文献
1000.
Xiangbin Xu Jufang Bian Songbai Liu Hongmiao Song Nongnong Shi Yuezhi Tao Huizhong Wang 《Molecular breeding : new strategies in plant improvement》2011,27(3):337-346
The PROMOTION OF CELL SURVIVAL 1 (PCS1) gene, encoding an aspartic protease, has an important role in determining the fate of cells in embryonic development and
reproduction processes in Arabidopsis. To explore the potential function of the PCS1 gene in generating reproductive sterility, we placed the PCS1 gene under the control of an 1,869-bp nucleotide sequence from the 3′ end of the second intron (AG-I) of Arabidopsis AGAMOUS and CaMV 35S (–60) minimal promoter [AG-I-35S (–60)::PCS1], and introduced it into tobacco. RT–PCR results demonstrated that the PCS1 gene driven by AG-I-35S (–60) chimeric promoter was expressed only in anthers and carpels in the reproductive tissues of transgenic tobacco. Compared to
wild-type plants, all AG-I-35S (–60) and AG-I-35S (–60)::PCS1 transgenic lines showed a normal phenotype throughout the vegetative growth phase. However, during the reproductive stage,
most AG-I-35S (–60)::PCS1 transgenic plant anthers displayed delayed dehiscence, failed dehiscence, petalody and hypoplasia, and the pollen grains
had different shapes and sizes with a distorted, shrunken, or collapsed morphology. Moreover, three transgenic lines, PCS1-1,
PCS1-3 and PCS1-4, showed higher sterility than wild-type and AG-I-35S (–60) transgenic plants, respectively. These results showed that the construct of AG-I-35S (–60)::PCS1 was partially effective at preventing seed set and provided a novel sterility strategy. 相似文献