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991.
Yuan QA Robinson MK Simmons HH Russeva M Adams GP 《Cancer immunology, immunotherapy : CII》2008,57(3):367-378
While cell surface antigens represent the most common targets for antibody-based cancer therapy, isolation of new antibodies
specific for these targets from single-chain Fv phage display libraries has been hindered by limitations associated with traditional
selection techniques. Solid phase panning is often associated with conformational changes to the target protein due to its
immobilization on plastic tubes that can limit the ability of the isolated scFv to bind to conformational epitopes and solution
panning methods require the use of secondary tags that often mask desired sequences and create unintended epitopes. Commonly
utilized cell-based panning methods typically yield a panel of single-chain Fv (scFv) molecules that are specific for numerous
cell surface antigens, often obscuring the desired clones. Here, we describe a novel cell sorter-based system to isolate single-chain
Fv molecules specific for defined antigen targets expressed on stably-transformed mammalian cells. We employed these methods
to isolate promising scFv clones that bind specifically to the Müllerian inhibiting substance type II receptor, a cell surface
ovarian cancer antigen that has proven to be a difficult target for selection strategies. 相似文献
992.
G protein-coupled receptors (GPCRs) catalyze nucleotide release in heterotrimeric G proteins, the slow step in G protein activation. G i/o family proteins are permanently, cotranslationally myristoylated at the extreme amino terminus. While myristoylation of the amino terminus has long been known to aid in anchoring G i proteins to the membrane, the role of myristoylation with regard to interaction with activated receptors is not known. Previous studies have characterized activation-dependent changes in the amino terminus of Galpha proteins in solution [Medkova, M. (2002) Biochemistry 41, 9963-9972; Preininger, A. M. (2003) Biochemistry 42, 7931-7941], but changes in the environment of specific residues within the Galpha i1 amino terminus during receptor-mediated G i activation have not been reported. Using site-specific fluorescence labeling of individual residues along a stretch of the Galpha il amino terminus, we found specific changes in the environment of these residues upon interaction with the activated receptor and following GTPgammaS binding. These changes map to a distinct surface of the amino-terminal helix opposite the Gbetagamma binding interface. The receptor-dependent fluorescence changes are consistent with a myristoylated amino terminus in the proximity of the membrane and/or receptor. Myristoylation affects both the rate and intensity of receptor activation-dependent changes detected at several residues along the amino terminus (with no significant effect on the rate of receptor-mediated GTPgammaS binding). This work demonstrates that the myristoylated amino terminus of Galpha il proteins undergoes receptor-mediated changes during the dynamic process of G protein signaling. 相似文献
993.
Alex?Generous Molly?Thorson Jeff?Barcus Joseph?Jacher Marc?Busch Heidi?SleisterEmail author 《Virology journal》2014,11(1):228
Background
Influenza A viruses (IAVs) are important pathogens that affect the health of humans and many additional animal species. IAVs are enveloped, negative single-stranded RNA viruses whose genome encodes at least ten proteins. The IAV nucleoprotein (NP) is a structural protein that associates with the viral RNA and is essential for virus replication. Understanding how IAVs interact with host proteins is essential for elucidating all of the required processes for viral replication, restrictions in species host range, and potential targets for antiviral therapies.Methods
In this study, the NP from a swine IAV was cloned into a yeast two-hybrid “bait” vector for expression of a yeast Gal4 binding domain (BD)-NP fusion protein. This “bait” was used to screen a Y2H human HeLa cell “prey” library which consisted of human proteins fused to the Gal4 protein’s activation domain (AD). The interaction of “bait” and “prey” proteins resulted in activation of reporter genes.Results
Seventeen positive bait-prey interactions were isolated in yeast. All of the “prey” isolated also interact in yeast with a NP “bait” cloned from a human IAV strain. Isolation and sequence analysis of the cDNAs encoding the human prey proteins revealed ten different human proteins. These host proteins are involved in various host cell processes and structures, including purine biosynthesis (PAICS), metabolism (ACOT13), proteasome (PA28B), DNA-binding (MSANTD3), cytoskeleton (CKAP5), potassium channel formation (KCTD9), zinc transporter function (SLC30A9), Na+/K+ ATPase function (ATP1B1), and RNA splicing (TRA2B).Conclusions
Ten human proteins were identified as interacting with IAV NP in a Y2H screen. Some of these human proteins were reported in previous screens aimed at elucidating host proteins relevant to specific viral life cycle processes such as replication. This study extends previous findings by suggesting a mechanism by which these host proteins associate with the IAV, i.e., physical interaction with NP. Furthermore, this study revealed novel host protein-NP interactions in yeast.994.
Marcos F. DosSantos Ilkka K. Martikainen Thiago D. Nascimento Tiffany M. Love Misty D. DeBoer Heidi M. Schambra Marom Bikson Jon-Kar Zubieta Alexandre F. DaSilva 《PloS one》2014,9(7)
Transcranial Direct Current Stimulation (tDCS) is a method of non-invasive brain stimulation that has been frequently used in experimental and clinical pain studies. However, the molecular mechanisms underlying tDCS-mediated pain control, and most important its placebo component, are not completely established. In this pilot study, we investigated in vivo the involvement of the endogenous μ-opioid system in the global tDCS-analgesia experience. Nine healthy volunteers went through positron emission tomography (PET) scans with [11C]carfentanil, a selective μ-opioid receptor (MOR) radiotracer, to measure the central MOR activity during tDCS in vivo (non-displaceable binding potential, BPND) - one of the main analgesic mechanisms in the brain. Placebo and real anodal primary motor cortex (M1/2mA) tDCS were delivered sequentially for 20 minutes each during the PET scan. The initial placebo tDCS phase induced a decrease in MOR BPND in the periaqueductal gray matter (PAG), precuneus, and thalamus, indicating activation of endogenous μ-opioid neurotransmission, even before the active tDCS. The subsequent real tDCS also induced MOR activation in the PAG and precuneus, which were positively correlated to the changes observed with placebo tDCS. Nonetheless, real tDCS had an additional MOR activation in the left prefrontal cortex. Although significant changes in the MOR BPND occurred with both placebo and real tDCS, significant analgesic effects, measured by improvements in the heat and cold pain thresholds, were only observed after real tDCS, not the placebo tDCS. This study gives preliminary evidence that the analgesic effects reported with M1-tDCS, can be in part related to the recruitment of the same endogenous MOR mechanisms induced by placebo, and that such effects can be purposely optimized by real tDCS. 相似文献
995.
Mathieu Cayla Najma Rachidi Olivier Leclercq Dirk Schmidt-Arras Heidi Rosenqvist Martin Wiese Gerald F. Sp?th 《PLoS pathogens》2014,10(9)
Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage-specifically regulated, as its kinase activity increases during the promastigote to amastigote conversion. However, unlike canonical MAPKs that are activated by dual phosphorylation of the regulatory TxY motif in the activation loop, MPK10 activation is independent from the phosphorylation of the tyrosine residue, which is largely constitutive. Removal of the last 46 amino acids resulted in significantly enhanced MPK10 activity both for the recombinant and transgenic protein, revealing that MPK10 is regulated by an auto-inhibitory mechanism. Over-expression of this hyperactive mutant in transgenic parasites led to a dominant negative effect causing massive cell death during amastigote differentiation, demonstrating the essential nature of MPK10 auto-inhibition for parasite viability. Moreover, phosphoproteomics analyses identified a novel regulatory phospho-serine residue in the C-terminal auto-inhibitory domain at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10 as a potential signal sensor of the mammalian host environment, whose intrinsic pre-activated conformation is regulated by auto-inhibition. 相似文献
996.
Cheryl C. Y. Loh Rossarin Suwanarusk Yan Quan Lee Kitti W. K. Chan Kit-Ying Choy Laurent Rénia Bruce Russell Martin J. Lear Fran?ois H. Nosten Kevin S. W. Tan Larry M. C. Chow 《PloS one》2014,9(10)
Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQGREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQGREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC50s were determined. These data were compared with LynxTag-CQGREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC50 of chloroquine and, more weakly, a mutation in Pgh1, F1226Y. 相似文献
997.
Heidi Hopkins 《ZooKeys》2014,(384):1-256
The cockroach genus Arenivaga is revised. Forty-eight Arenivaga species are recognized with nine previously known species and 39 described as new including the following: A. pagana
sp. n., A. grandiscanyonensis
sp. n., A. haringtoni
sp. n., A. hopkinsorum
sp. n., A. umbratilis
sp. n., A. tenax
sp. n., A. impensa
sp. n., A. trypheros
sp. n., A. darwini
sp. n., A. nalepae
sp. n., A. sequoia
sp. n., A. mckittrickae
sp. n., A. gaiophanes
sp. n., A. belli
sp. n., A. estelleae
sp. n., A. delicata
sp. n., A. mortisvallisensis
sp. n., A. milleri
sp. n., A. pratchetti
sp. n., A. gumperzae
sp. n., A. rothi
sp. n., A. ricei
sp. n., A. adamsi
sp. n., A. nicklei
sp. n., A. akanthikos
sp. n., A. moctezuma
sp. n., A. paradoxa
sp. n., A. apaeninsula
sp. n., A. hebardi
sp. n., A. dnopheros
sp. n., A. aquila
sp. n., A. florilega
sp. n., A. galeana
sp. n., A. gurneyi
sp. n., A. pumila
sp. n., A. hypogaios
sp. n., A. diaphana
sp. n., A. nocturna
sp. n., A. alichenas
sp. n. All species are described or redescribed, major morphological features are illustrated, distributions are characterized, and the biology of the species is reviewed. A neotype series is designated for A. investigata Friauf & Edney. 相似文献
998.
Heidi Heim-Ballew Kristine N. Moody Michael J. Blum Peter B. McIntyre James D. Hogan 《Journal of fish biology》2020,96(2):456-468
We assessed the prevalence of life history variation across four of the five native amphidromous Hawai'ian gobioids to determine whether some or all exhibit evidence of partial migration. Analysis of otolith Sr.: Ca concentrations affirmed that all are amphidromous and revealed evidence of partial migration in three of the four species. We found that 25% of Lentipes concolor (n = 8), 40% of Eleotris sandwicensis (n = 20) and 29% of Stenogobius hawaiiensis (n = 24) did not exhibit a migratory life-history. In contrast, all individuals of Sicyopterus stimpsoni (n = 55) included in the study went to sea as larvae. Lentipes concolor exhibited the shortest mean larval duration (LD) at 87 days, successively followed by E. sandwicensis (mean LD = 102 days), S. hawaiiensis (mean LD = 114 days) and S. stimpsoni (mean LD = 120 days). These findings offer a fresh perspective on migratory life histories that can help improve efforts to conserve and protect all of these and other at-risk amphidromous species that are subject to escalating anthropogenic pressures in both freshwater and marine environments. 相似文献
999.
Heidi K. LaMoreaux George A. Brook John A. Knox 《Palaeogeography, Palaeoclimatology, Palaeoecology》2009,280(3-4):300-312
A 5.4 m peat core from the Sandy Run Creek (SRC) valley in the upper Coastal Plain of Georgia shows that before 30 ka the valley had an aggradational sandy floor with shallow swales and low ridges of 1–2 m amplitude suggesting a braided stream pattern and a low water table. The climate from 30–25 ka was cool and dry and the vegetation open grassland with stands of pine and spruce that produced few fires. At ca. 16 ka a warmer, wetter climate caused SRC to meander and incise the valley fill removing some previously deposited sediment at the site of our peat core so that sediments of 13 ka rest directly on sediments deposited at 25 ka. After ca. 13 ka higher groundwater levels initiated vertical accretion of floodplains allowing peat accumulation in shallow depressions across the valley. Pollen from the Younger Dryas period (ca. 13–11 ka) indicates a cool, moist environment of open oak woodland, mesic trees, riparian populations of alder, and reduced levels of pine. By the early to mid Holocene, tupelo and oak dominated over pine indicating wetter conditions, while sand stringers in peat record periodic heavy rains, an elevated water table, and intervals of substantial runoff. Cooling after ca. 4.5 ka brought drier and more variable conditions. Fires increased and southern pine replaced tupelo and oak. The absence of sand stringers in peat younger than 4.5 ka indicates fewer intense rainfall events. 相似文献
1000.