全文获取类型
收费全文 | 27396篇 |
免费 | 2534篇 |
国内免费 | 9篇 |
专业分类
29939篇 |
出版年
2023年 | 129篇 |
2022年 | 326篇 |
2021年 | 638篇 |
2020年 | 394篇 |
2019年 | 453篇 |
2018年 | 514篇 |
2017年 | 491篇 |
2016年 | 811篇 |
2015年 | 1306篇 |
2014年 | 1462篇 |
2013年 | 1686篇 |
2012年 | 2225篇 |
2011年 | 2262篇 |
2010年 | 1453篇 |
2009年 | 1199篇 |
2008年 | 1820篇 |
2007年 | 1696篇 |
2006年 | 1541篇 |
2005年 | 1523篇 |
2004年 | 1536篇 |
2003年 | 1317篇 |
2002年 | 1165篇 |
2001年 | 310篇 |
2000年 | 219篇 |
1999年 | 265篇 |
1998年 | 273篇 |
1997年 | 177篇 |
1996年 | 187篇 |
1995年 | 153篇 |
1994年 | 167篇 |
1993年 | 152篇 |
1992年 | 165篇 |
1991年 | 121篇 |
1990年 | 115篇 |
1989年 | 100篇 |
1988年 | 103篇 |
1987年 | 104篇 |
1986年 | 82篇 |
1985年 | 88篇 |
1984年 | 113篇 |
1983年 | 116篇 |
1982年 | 99篇 |
1981年 | 77篇 |
1980年 | 87篇 |
1979年 | 63篇 |
1978年 | 74篇 |
1977年 | 54篇 |
1976年 | 72篇 |
1975年 | 46篇 |
1974年 | 51篇 |
排序方式: 共有10000条查询结果,搜索用时 29 毫秒
991.
Alexei Kharitonenkov John M. Beals Radmila Micanovic Beth A. Strifler Radhakrishnan Rathnachalam Victor J. Wroblewski Shun Li Anja Koester Amy M. Ford Tamer Coskun James D. Dunbar Christine C. Cheng Christopher C. Frye Thomas F. Bumol David E. Moller 《PloS one》2013,8(3)
Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7–14 days resulted in a 25–50% lowering of plasma glucose coupled with a 10–30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans. 相似文献
992.
Jürgen Schymeinsky Hannah Mayer Christopher Tomsic Cornelia Tilp John D. Schuetz Yunhai Cui Lutz Wollin Florian Gantner Klaus J. Erb 《PloS one》2013,8(4)
The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4−/− and Mrp4+/+ mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4−/− and Mrp4+/+ mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4+/+ or Mrp4−/− mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure. 相似文献
993.
Gabrielle Richard Ashley J. Meyers Michael D. McLean Mehdi Arbabi-Ghahroudi Roger MacKenzie J. Christopher Hall 《PloS one》2013,8(7)
Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. 相似文献
994.
Perrine Pelosse Christopher M. Kribs-Zaleta Marine Ginoux Jorge E. Rabinovich Sébastien Gourbière Frédéric Menu 《PloS one》2013,8(8)
Insects are known to display strategies that spread the risk of encountering unfavorable conditions, thereby decreasing the extinction probability of genetic lineages in unpredictable environments. To what extent these strategies influence the epidemiology and evolution of vector-borne diseases in stochastic environments is largely unknown. In triatomines, the vectors of the parasite Trypanosoma cruzi, the etiological agent of Chagas’ disease, juvenile development time varies between individuals and such variation most likely decreases the extinction risk of vector populations in stochastic environments. We developed a simplified multi-stage vector-borne SI epidemiological model to investigate how vector risk-spreading strategies and environmental stochasticity influence the prevalence and evolution of a parasite. This model is based on available knowledge on triatomine biodemography, but its conceptual outcomes apply, to a certain extent, to other vector-borne diseases. Model comparisons between deterministic and stochastic settings led to the conclusion that environmental stochasticity, vector risk-spreading strategies (in particular an increase in the length and variability of development time) and their interaction have drastic consequences on vector population dynamics, disease prevalence, and the relative short-term evolution of parasite virulence. Our work shows that stochastic environments and associated risk-spreading strategies can increase the prevalence of vector-borne diseases and favor the invasion of more virulent parasite strains on relatively short evolutionary timescales. This study raises new questions and challenges in a context of increasingly unpredictable environmental variations as a result of global climate change and human interventions such as habitat destruction or vector control. 相似文献
995.
Dickinson RJ Delavaine L Cejudo-Marín R Stewart G Staples CJ Didmon MP Trinidad AG Alonso A Pulido R Keyse SM 《The Journal of biological chemistry》2011,286(44):38018-38026
MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo. 相似文献
996.
997.
In 2004, we evaluated the relative performance of 8-unit Lindgren multiple-funnel (funnel), Intercept panel (panel), and Colossus pipe (pipe) traps, baited with ethanol and ac-pinene lures, in catching saproxylic beetles (Coleoptera) in pine stands in northern Florida and western South Carolina. Panel traps were as good as, if not better than, funnel and pipe traps for catching Cerambycidae. In particular, more Monochamus titillator (F.) were captured in panel traps than in pipe and funnel traps. Of three species of Buprestidae captured in our study, most Buprestis lineata F. were caught in panel traps, whereas most Acmaeodera tubulus (F.) were caught in funnel traps. Catches of Chalcophora virginiensis Drury and the root-feeding weevils Hylobius pales Herbst an dPachylobius picivorus LeConte (Curculionidae) were unaffected by trap type. Among bark beetles (Curculionidae: Scolytinae), catches of Ips grandicollis (Eichhoff) were unaffected by trap type, whereas most Dendroctonus terebrans (Olivier) were caught in panel traps, most Hylastes salebrosus Eichhoff were caught in panel and pipe traps, and most Hylastes tenuis Eichhoff were caught in funnel traps. Among ambrosia beetles (Curculionidae: Scolytinae), panel traps caught the most Xyleborinus saxesenii (Ratzeburg), whereas pipe traps caught the most Xyleborus Eichhoff spp. More Xylosandrus crassiusculus (Motschulsky) and Dryoxylon onoharaensis (Murayama) were caught in panel and funnel traps than in pipe traps. Among bark beetle predators, more Platysoma Leach spp. (Histeridae) were caught in pipe and panel traps than in funnel traps, whereas most Lasconotus Erichson spp. (Zopheridae) were caught in funnel traps. Variation among trap performance for various species suggests that managers should consider more than one type of trap in their detection programs. 相似文献
998.
Weisberg E Ray A Nelson E Adamia S Barrett R Sattler M Zhang C Daley JF Frank D Fox E Griffin JD 《PloS one》2011,6(9):e25351
Objectives
Clinical responses achieved with FLT3 kinase inhibitors in acute myeloid leukemia (AML) are typically transient and partial. Thus, there is a need for identification of molecular mechanisms of clinical resistance to these drugs. In response, we characterized MOLM13 AML cell lines made resistant to two structurally-independent FLT3 inhibitors.Methods
MOLM13 cells were made drug resistant via prolonged exposure to midostaurin and HG-7-85-01, respectively. Cell proliferation was determined by Trypan blue exclusion. Protein expression was assessed by immunoblotting, immunoprecipitation, and flow cytometry. Cycloheximide was used to determine protein half-life. RT-PCR was performed to determine FLT3 mRNA levels, and FISH analysis was performed to determine FLT3 gene expression.Results and Conclusions
We found that MOLM13 cells readily developed cross-resistance when exposed to either midostaurin or HG-7-85-01. Resistance in both lines was associated with dramatically elevated levels of cell surface FLT3 and elevated levels of phosphor-MAPK, but not phospho-STAT5. The increase in FLT3-ITD expression was at least in part due to reduced turnover of the receptor, with prolonged half-life. Importantly, the drug-resistant phenotype could be rapidly reversed upon withdrawal of either inhibitor. Consistent with this phenotype, no significant evidence of FLT3 gene amplification, kinase domain mutations, or elevated levels of mRNA was observed, suggesting that protein turnover may be part of an auto-regulatory pathway initiated by FLT3 kinase activity. Interestingly, FLT3 inhibitor resistance also correlated with resistance to cytosine arabinoside. Over-expression of FLT3 protein in response to kinase inhibitors may be part of a novel mechanism that could contribute to clinical resistance. 相似文献999.
Rabbit anti-mouse-Ia serum was raised against Ia specificities present in CBAJH (H-2
k) serum. This xenogeneic antiserum was considered to react with similar specificities to those detected by mouse anti-Iak alloantisera and more evidence is now presented for this contention. By absorption, the xenogeneic antiserum was found to react with spleen, lymph node, bone marrow, and thymus, reactions similar to that found with the allogeneic anti-Iak antiserum. Furthermore, red cells, platelets, brain, kidney, and liver could not absorb the activity from the xenogeneic antiserum, demonstrating the selective tissue distribution of the antigens reactive with this serum. This reactive population was previously shown to consist of B cells and a subpopulation of T cells. In a backcross study of (C57BL/6 × A)F1 × C57BL/6, the rabbit anti-Ia and mouse anti-Ia reactions were found to segregate together, and some evidence for the genetic regulation of the expression of Ia specificities was also found. By direct testing, and by absorption testing using a number of strains, the xenogeneic antiserum was shown to contain high titers of antibody to Ia.1, 3, 7, 15, and 17; lower titers to Ia.19, and 22; little antibody to Ia.18, and no reaction for the private specificity Ia. 2, although the multiple absorptions required to define these specificities may have observed some reactions. The data indicate that the xenogeneic and allogeneic anti-Iak antisera recognize similar Ia determinants, which map to theLA, IE andIC subregions of theH-2 complex. These have been given the same specificity designation as the allogeneic specificities, but they are separately identified by a prime ('). 相似文献
1000.
Carol A. Sheppard Peter B. Simpson Alan H. Sharp Frederick C. Nucifora Christopher A. Ross †G. David Lange James T. Russell 《Journal of neurochemistry》1997,68(6):2317-2327
Abstract: We have examined the mechanisms that underlie Ca2+ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca2+ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca2+ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca2+ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP3 R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP3 Rs with Ca2+ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP3 Rs (InsP3 R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca2+ responses and InsP3 R2 immunofluorescence were compared in the same cell, domains of elevated Ca2+ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP3 R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca2+ release sites that support regenerative wave propagation include increased levels of InsP3 R2 expression. 相似文献