全文获取类型
收费全文 | 7161篇 |
免费 | 640篇 |
国内免费 | 1篇 |
专业分类
7802篇 |
出版年
2024年 | 11篇 |
2023年 | 42篇 |
2022年 | 109篇 |
2021年 | 206篇 |
2020年 | 92篇 |
2019年 | 119篇 |
2018年 | 161篇 |
2017年 | 135篇 |
2016年 | 238篇 |
2015年 | 471篇 |
2014年 | 495篇 |
2013年 | 476篇 |
2012年 | 671篇 |
2011年 | 637篇 |
2010年 | 364篇 |
2009年 | 308篇 |
2008年 | 480篇 |
2007年 | 456篇 |
2006年 | 405篇 |
2005年 | 373篇 |
2004年 | 338篇 |
2003年 | 355篇 |
2002年 | 293篇 |
2001年 | 51篇 |
2000年 | 37篇 |
1999年 | 71篇 |
1998年 | 65篇 |
1997年 | 46篇 |
1996年 | 40篇 |
1995年 | 35篇 |
1994年 | 26篇 |
1993年 | 32篇 |
1992年 | 22篇 |
1991年 | 15篇 |
1990年 | 17篇 |
1989年 | 11篇 |
1988年 | 10篇 |
1987年 | 11篇 |
1986年 | 6篇 |
1985年 | 10篇 |
1984年 | 9篇 |
1983年 | 4篇 |
1982年 | 5篇 |
1981年 | 10篇 |
1980年 | 8篇 |
1978年 | 5篇 |
1977年 | 6篇 |
1975年 | 2篇 |
1974年 | 3篇 |
1973年 | 4篇 |
排序方式: 共有7802条查询结果,搜索用时 15 毫秒
51.
Critical but distinct roles for the pleckstrin homology and cysteine-rich domains as positive modulators of Vav2 signaling and transformation 下载免费PDF全文
Vav2, like all Dbl family proteins, possesses tandem Dbl homology (DH) and pleckstrin homology (PH) domains and functions as a guanine nucleotide exchange factor for Rho family GTPases. Whereas the PH domain is a critical positive regulator of DH domain function for a majority of Dbl family proteins, the PH domains of the related Vav and Vav3 proteins are dispensable for DH domain activity. Instead, Vav proteins contain a cysteine-rich domain (CRD) critical for DH domain function. We evaluated the contribution of the PH domain and the CRD to Vav2 guanine nucleotide exchange, signaling, and transforming activity. Unexpectedly, we found that mutations of the PH domain impaired Vav2 signaling, transforming activity, and membrane association. However, these mutations do not influence exchange activity on Rac and only slightly affect exchange on RhoA and Cdc42. We also found that the CRD was critical for the exchange activity in vitro and contributed to Vav2 membrane localization. Finally, we found that phosphoinositol 3-kinase activation synergistically enhanced Vav2 transforming and signaling activity by stimulating exchange activity but not membrane association. In conclusion, the PH domain and CRD are mechanistically distinct, positive modulators of Vav2 DH domain function in vivo. 相似文献
52.
Ben Khalaf N De Muylder G Ratnam J Kean-Hooi Ang K Arkin M McKerrow J Chenik M 《Journal of biomolecular screening》2011,16(5):545-551
The use of a high-throughput technique to perform a pilot screen for Leishmania major protein disulfide isomerase (LmPDI) inhibitors identification is reported. In eukaryotic cells, protein disulfide isomerase (PDI) plays a crucial role in protein folding by catalyzing the rearrangement of disulfide bonds in substrate proteins following their synthesis. LmPDI displays similar domain structure organization and functional properties to other PDI family members and is involved in Leishmania virulence. The authors used a method based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol. The screen of a small library of 1920 compounds was performed in a 384-well format and led to the identification of 27 compounds with inhibitory activity against LmPDI. The authors further tested the cytotoxicity of these compounds using Jurkat cells as well as their effect on Leishmania donovani amastigotes using high-content analysis. Results show hexachlorophene and a mixture of theaflavin monogallates inhibit Leishmania multiplication in infected macrophages derived from THP-1 cells, although the inhibitory effect on LmPDI enzymatic activity does not necessarily correlate with the antileishmanial activity. 相似文献
53.
Jaing C Gardner S McLoughlin K Mulakken N Alegria-Hartman M Banda P Williams P Gu P Wagner M Manohar C Slezak T 《PloS one》2008,3(5):e2163
Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples. 相似文献
54.
Bitinaite J Rubino M Varma KH Schildkraut I Vaisvila R Vaiskunaite R 《Nucleic acids research》2007,35(6):1992-2002
Here we report a PCR-based DNA engineering technique for seamless assembly of recombinant molecules from multiple components. We create cloning vector and target molecules flanked with compatible single-stranded (ss) extensions. The vector contains a cassette with two inversely oriented nicking endonuclease sites separated by restriction endonuclease site(s). The spacer sequences between the nicking and restriction sites are tailored to create ss extensions of custom sequence. The vector is then linearized by digestion with nicking and restriction endonucleases. To generate target molecules, a single deoxyuridine (dU) residue is placed 6–10nt away from the 5′-end of each PCR primer. 5′ of dU the primer sequence is compatible either with an ss extension on the vector or with the ss extension of the next-in-line PCR product. After amplification, the dU is excised from the PCR products with the USER enzyme leaving PCR products flanked by 3′ ss extensions. When mixed together, the linearized vector and PCR products directionally assemble into a recombinant molecule through complementary ss extensions. By varying the design of the PCR primers, the protocol is easily adapted to perform one or more simultaneous DNA manipulations such as directional cloning, site-specific mutagenesis, sequence insertion or deletion and sequence assembly. 相似文献
55.
James Skelton Michelle A. Jusino Paige S. Carlson Katherine Smith Mark T. Banik Daniel L. Lindner Jonathan M. Palmer Jiri Hulcr 《Molecular ecology》2019,28(22):4971-4986
A prevailing paradigm in forest ecology is that wood‐boring beetles facilitate wood decay and carbon cycling, but empirical tests have yielded mixed results. We experimentally determined the effects of wood borers on fungal community assembly and wood decay within pine trunks in the southeastern United States. Pine trunks were made either beetle‐accessible or inaccessible. Fungal communities were compared using culturing and high‐throughput amplicon sequencing (HTAS) of DNA and RNA. Prior to beetle infestation, living pines had diverse fungal endophyte communities. Endophytes were displaced by beetle‐associated fungi in beetle‐accessible trees, whereas some endophytes persisted as saprotrophs in beetle‐excluded trees. Beetles increased fungal diversity several fold. Over forty taxa of Ascomycota were significantly associated with beetles, but beetles were not consistently associated with any known wood‐decaying fungi. Instead, increasing ambrosia beetle infestations caused reduced decay, consistent with previous in vitro experiments that showed beetle‐associated fungi reduce decay rates by competing with decay fungi. No effect of bark‐inhabiting beetles on decay was detected. Platypodines carried significantly more fungal taxa than scolytines. Molecular results were validated by synthetic and biological mock communities and were consistent across methodologies. RNA sequencing confirmed that beetle‐associated fungi were biologically active in the wood. Metabarcode sequencing of the LSU/28S marker recovered important fungal symbionts that were missed by ITS2, though community‐level effects were similar between markers. In contrast to the current paradigm, our results indicate ambrosia beetles introduce diverse fungal communities that do not extensively decay wood, but instead reduce decay rates by competing with wood decay fungi. 相似文献
56.
Role of fungi in freshwater ecosystems 总被引:7,自引:0,他引:7
Michelle K.M. Wong Teik-Khiang Goh I. John Hodgkiss Kevin D. Hyde V. Mala Ranghoo Clement K.M. Tsui Wai-Hong Ho Wilson S.W. Wong Tsz-Kit Yuen 《Biodiversity and Conservation》1998,7(9):1187-1206
There are more than 600 species of freshwater fungi with a greater number known from temperate, as compared to tropical, regions. Three main groups can be considered which include Ingoldian fungi, aquatic ascomycetes and non-Ingoldian hyphomycetes, chytrids and, oomycetes. The fungi occurring in lentic habitats mostly differ from those occurring in lotic habitats. Although there is no comprehensive work dealing with the biogeography of all groups of freshwater fungi, their distribution probably follows that of Ingoldian fungi, which are either cosmopolitan, restricted to pantemperate or pantropical regions, or in a few cases, have a restricted distribution. Freshwater fungi are thought to have evolved from terrestrial ancestors. Many species are clearly adapted to life in freshwater as their propagules have specialised aquatic dispersal abilities. Freshwater fungi are involved in the decay of wood and leafy material and also cause diseases of plants and animals. These areas are briefly reviewed. Gaps in our knowledge of freshwater fungi are discussed and areas in need of research are suggested. 相似文献
57.
Carolina C. Lisboa Richard T. Conant Michelle L. Haddix Carlos Eduardo P. Cerri Carlos C. Cerri 《Ecosystems》2009,12(7):1212-1221
The effect of conversion from forest-to-pasture upon soil carbon stocks has been intensively discussed, but few studies focus
on how this land-use change affects carbon (C) distribution across soil fractions in the Amazon basin. We investigated this
in the 20 cm depth along a chronosequence of sites from native forest to three successively older pastures. We performed a
physicochemical fractionation of bulk soil samples to better understand the mechanisms by which soil C is stabilized and evaluate
the contribution of each C fraction to total soil C. Additionally, we used a two-pool model to estimate the mean residence
time (MRT) for the slow and active pool C in each fraction. Soil C increased with conversion from forest-to-pasture in the
particulate organic matter (>250 μm), microaggregate (53–250 μm), and d-clay (<2 μm) fractions. The microaggregate comprised
the highest soil C content after the conversion from forest-to-pasture. The C content of the d-silt fraction decreased with
time since conversion to pasture. Forest-derived C remained in all fractions with the highest concentration in the finest
fractions, with the largest proportion of forest-derived soil C associated with clay minerals. Results from this work indicate
that microaggregate formation is sensitive to changes in management and might serve as an indicator for management-induced
soil carbon changes, and the soil C changes in the fractions are dependent on soil texture. 相似文献
58.
Demographic and clinical variables affecting mid‐ to late‐life trajectories of plasma ceramide and dihydroceramide species 下载免费PDF全文
Michelle M. Mielke Veera Venkata Ratnam Bandaru Dingfen Han Yang An Susan M. Resnick Luigi Ferrucci Norman J. Haughey 《Aging cell》2015,14(6):1014-1023
It has been increasingly recognized at the basic science level that perturbations in ceramide metabolism are associated with the development and progression of many age‐related diseases. However, the translation of this work to the clinic has lagged behind. Understanding the factors longitudinally associated with plasma ceramides and dihydroceramides (DHCer) at the population level and how these lipid levels change with age, and by sex, is important for the clinical development of future therapeutics and biomarkers focused on ceramide metabolism. We, therefore, examined factors cross‐sectionally and longitudinally associated with plasma concentrations of ceramides and DHCer among Baltimore Longitudinal Study of Aging participants (n = 992; 3960 total samples), aged 55 years and older, with plasma at a mean of 4.1 visits (range 2–6). Quantitative analyses were performed on a high‐performance liquid chromatography‐coupled electrospray ionization tandem mass spectrometer. Linear mixed models were used to assess the relationships between plasma ceramide and DHCer species and demographics, diseases, medications, and lifestyle factors. Women had higher plasma concentrations of most ceramide and DHCer species and showed steeper trajectories of age‐related increases compared to men. Ceramides and DHCer were more associated with waist–hip ratio than body mass index. Plasma cholesterol and triglycerides, prediabetes, and diabetes were associated with ceramides and DHCer, but the relationship showed specificity to the acyl chain length and saturation. These results demonstrate the importance of examining the individual species of ceramides and DHCer, and of establishing whether intra‐individual age‐ and sex‐specific changes occur in synchrony to disease onset and progression. 相似文献
59.
Fu VX Schwarze SR Kenowski ML Leblanc S Svaren J Jarrard DF 《The Journal of biological chemistry》2004,279(50):52218-52226
The imprinted insulin-like growth factor-2 (IGF2) gene is an auto/paracrine growth factor expressed only from the paternal allele in adult tissues. In tissues susceptible to aging-related cancers, including the prostate, a relaxation of IGF2 imprinting is found, suggesting a permissive role for epigenetic alterations in cancer development. To determine whether IGF2 imprinting is altered in cellular aging and senescence, human prostate epithelial and urothelial cells were passaged serially in culture to senescence. Allelic analyses using an IGF2 polymorphism demonstrated a complete conversion of the IGF2 imprint status from monoallelic to biallelic, in which the development of senescence was associated with a 10-fold increase in IGF2 expression. As a mechanism, a 2-fold decrease in the binding of the enhancer-blocking element CCCTC-binding factor (CTCF) within the intergenic IGF2-H19 region was found to underlie this switch to biallelic IGF2 expression in senescent cells. This decrease in CTCF binding was associated with reduced CTCF expression in senescent cells. No de novo increases in methylation at the IGF2 CTCF binding site were seen. The forced down-regulation of CTCF expression using small interfering RNA in imprinted prostate cell lines resulted in an increase in IGF2 expression and a relaxation of imprinting. Our data suggest a novel mechanism for IGF2 imprinting regulation, that is, the reduction of CTCF expression in the control of IGF2 imprinting. We also demonstrate that altered imprinting patterns contribute to changes in gene expression in aging cells. 相似文献
60.
Lightbody KL Renshaw PS Collins ML Wright RL Hunt DM Gordon SV Hewinson RG Buxton RS Williamson RA Carr MD 《FEMS microbiology letters》2004,238(1):255-262
We have previously shown that the secreted M. tuberculosis complex proteins CFP-10 and ESAT-6 form a tight, 1:1 complex, which may represent their functional form. In the work reported here a combination of yeast two-hybrid and biochemical analysis has been used to characterise complex formation between two other pairs of CFP-10/ESAT-6 family proteins (Rv0287/Rv0288 and Rv3019c/Rv3020c) and to determine whether complexes can be formed between non-genome paired members of the family. The results clearly demonstrate that Rv0287/Rv0288 and Rv3019c/3020c form tight complexes, as initially observed for CFP-10/ESAT-6. The closely related Rv0287/Rv0288 and Rv3019c/Rv3020c proteins are also able to form non-genome paired complexes (Rv0287/Rv3019c and Rv0288/Rv3020c), but are not capable of binding to the more distantly related CFP-10/ESAT-6 proteins. 相似文献