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301.
302.
Improved Sugar Conversion and Ethanol Yield for Forage Sorghum (Sorghum bicolor L. Moench) Lines with Reduced Lignin Contents 总被引:1,自引:0,他引:1
Bruce S. Dien Gautam Sarath Jeffrey F. Pedersen Scott E. Sattler Han Chen Deanna L. Funnell-Harris Nancy N. Nichols Michael A. Cotta 《Bioenergy Research》2009,2(3):153-164
Lignin is known to impede conversion of lignocellulose into ethanol. In this study, forage sorghum plants carrying brown midrib (bmr) mutations, which reduce lignin contents, were evaluated as bioenergy feedstocks. The near-isogenic lines evaluated were: wild type, bmr-6, bmr-12, and bmr-6 bmr-12 double mutant. The bmr-6 and bmr-12 mutations were equally efficient at reducing lignin contents (by 13% and 15%, respectively), and the effects were additive (27%) for the double mutant. Reducing lignin content was highly beneficial for improving biomass conversion yields. Sorghum biomass samples were pretreated with dilute acid and recovered solids washed and hydrolyzed with cellulase to liberate glucose. Glucose yields for the sorghum biomass were improved by 27%, 23%, and 34% for bmr-6, bmr-12, and the double mutant, respectively, compared to wild type. Sorghum biomass was also pretreated with dilute acid followed by co-treatment with cellulases and Saccharomyces cerevisiae for simultaneous saccharification and fermentation (SSF) into ethanol. Conversion of cellulose to ethanol for dilute-acid pretreated sorghum biomass was improved by 22%, 21%, and 43% for bmr-6, bmr-12, and the double mutant compared to wild type, respectively. Electron microscopy of dilute-acid treated samples showed an increased number of lignin globules in double-mutant tissues as compared to the wild-type, suggesting the lignin had become more pliable. The mutations were also effective for improving ethanol yields when the (degrained) sorghum was pretreated with dilute alkali instead of dilute acid. Following pretreatment with dilute ammonium hydroxide and SSF, ethanol conversion yields were 116 and 130 mg ethanol/g dry biomass for the double-mutant samples and 98 and 113 mg/g for the wild-type samples. 相似文献
303.
Adenovirus infection activates cellular DNA damage response and repair pathways. Viral proteins that are synthesized before viral DNA replication prevent recognition of viral genomes as a substrate for DNA repair by targeting members of the sensor complex composed of Mre11/Rad50/NBS1 for degradation and relocalization, as well as targeting the effector protein DNA ligase IV. Despite inactivation of these cellular sensor and effector proteins, infection results in high levels of histone 2AX phosphorylation, or γH2AX. Although phosphorylated H2AX is a characteristic marker of double-stranded DNA breaks, this modification was widely distributed throughout the nucleus of infected cells and was coincident with the bulk of cellular DNA. H2AX phosphorylation occurred after the onset of viral DNA replication and after the degradation of Mre11. Experiments with inhibitors of the serine-threonine kinases ataxia telangiectasia mutated (ATM), AT- and Rad3-related (ATR), and DNA protein kinase (DNA-PK), the kinases responsible for H2AX phosphorylation, indicate that H2AX may be phosphorylated by ATR during a wild-type adenovirus infection, with some contribution from ATM and DNA-PK. Viral DNA replication appears to be the stimulus for this phosphorylation event, since infection with a nonreplicating virus did not elicit phosphorylation of H2AX. Infected cells also responded to high levels of input viral DNA by localized phosphorylation of H2AX. These results are consistent with a model in which adenovirus-infected cells sense and respond to both incoming viral DNA and viral DNA replication.Cellular DNA damage response pathways protect and preserve the integrity of the genome. These pathways, which are activated in response to various forms of DNA damage, involve a number of proteins that participate in both DNA repair and cell cycle progression (62). The serine-threonine kinases ataxia telangiectasia mutated (ATM), AT- and Rad3-related (ATR), and DNA protein kinase (DNA-PK) are activated in response to distinct types of damage. The ATM pathway is activated primarily by double-stranded DNA breaks (4, 30). DNA-PK acts in conjunction with the DNA ligase IV/XRCC4 complex to mediate the ligation of double-stranded breaks through nonhomologous end joining (34). The ATR pathway can be activated in response to a wide range of genotoxic stresses, such as base or nucleotide excision, double-stranded breaks, or single-stranded breaks. Activation of ATR is generally thought to occur via the recognition of single-stranded tracks of DNA (63). Each of these pathways leads to the phosphorylation and activation of a number of cellular proteins such as the variant histone H2AX, checkpoint kinases 1 and 2 (Chk1 and Chk2), and Nijmegen break syndrome protein 1 (NBS1), among others (62). Signals transmitted by a cascade of phosphorylation events result in cell cycle arrest and the accumulation of repair protein complexes at sites of DNA damage.Upon recognition of a double-stranded DNA break by the cell, H2AX is phosphorylated on an extended C-terminal tail at serine 139 by the phosphatidylinositol 3-kinase (PI3K)-related kinases ATM, ATR, and DNA-PK (9, 41, 44, 58). Considered one of the earliest indications of a double-stranded DNA break, phosphorylated H2AX (γH2AX) acts as a scaffolding protein to which a number of DNA repair factors can dock to facilitate repair of the damaged DNA (36, 42, 53). Areas of phosphorylated H2AX, termed γH2AX foci, are enriched for proteins involved in both homologous recombination and nonhomologous end joining, such as NBS1, BRCA1 (42), and Mdc1 (24, 50).Although adenovirus is able to activate both ATM and ATR pathways (11), adenoviral proteins limit the extent and consequences of signaling through these pathways. The E1B-55K and E4orf6 proteins form an E3 ubiquitin ligase with the cellular proteins Cullin-5, elongins B and C, and Rbx1 (28, 43). This complex targets key cellular proteins involved in cellular response to DNA damage, including p53 (28, 43), Mre11 (51), and DNA ligase IV (3). The E4orf3 gene product targets cellular proteins central to both the cellular DNA damage response and the antiviral response. The E4orf3 protein of species C adenoviruses alters the localization of Mre11/Rad50/NBS1 (MRN) complex members within the nucleus to prevent association with centers of viral DNA replication and to ensure efficient viral DNA replication (17, 18, 52). In addition, these three viral early proteins direct members of the MRN complex (2, 35) and the single-stranded DNA-binding protein 2 (20) to cytoplasmic aggresomes, where these sequestered proteins are effectively inactivated. These viral activities, along with the inactivation of DNA-PK by E4orf3 and E4orf6 gene products (7), appear to prevent recognition of viral genomes by the MRN complex and prevent ligation of these genomes through nonhomologous end joining. In cells infected with a virus with E4 deleted, Mre11 physically binds to viral DNA in an NBS1-dependent manner and may prevent efficient genome replication (37). The overlapping means by which adenovirus disables the MRN complex and prevents DNA damage repair serves to illustrate the importance of this activity for a productive adenovirus infection. However, despite having DNA damage signaling and DNA repair pathways dismantled, adenovirus-infected cells exhibit some characteristic changes associated with DNA damage signaling events, such as the phosphorylation of H2AX (6, 15). Thus, it appears that adenovirus effectively inhibits DNA repair activity but may not fully suppress the early events of DNA damage signaling.The focus of the present study was to elucidate the activation of DNA damage signaling pathways revealed by phosphorylation of the variant histone H2AX during wild-type adenovirus infection and to determine what stage of the virus life cycle leads to this activation. We demonstrate that infected cells respond to viral genome replication with high levels of H2AX phosphorylation throughout the cell nucleus. This phosphorylation event is not localized to viral replication centers and does not appear to be concurrent with cellular double-stranded DNA breaks; rather, H2AX phosphorylation occurs coincident with the bulk of cellular chromatin. H2AX phosphorylation follows viral DNA replication and reaches peak levels after the degradation of the Mre11. In addition, we observed that infected cells can respond to both the presence of incoming viral genomes and genome replication by initiating H2AX phosphorylation. 相似文献
304.
305.
Shizhen Wang Yewande Alimi Ailing Tong Colin G. Nichols Decha Enkvetchakul 《The Journal of biological chemistry》2009,284(5):2854-2860
Potassium channels are tetrameric proteins that mediate
K+-selective transmembrane diffusion. For KcsA, tetramer stability
depends on interactions between permeant ions and the channel pore. We have
examined the role of pore blockers on the tetramer stability of KirBac1.1. In
150 mm KCl, purified KirBac1.1 protein migrates as a monomer
(∼40 kDa) on SDS-PAGE. Addition of Ba2+
(K1/2 ∼ 50 μm) prior to loading results
in an additional tetramer band (∼160 kDa). Mutation A109C, at a residue
located near the expected Ba2+-binding site, decreased tetramer
stabilization by Ba2+ (K1/2 ∼ 300
μm), whereas I131C, located nearby, stabilized tetramers in the
absence of Ba2+. Neither mutation affected Ba2+ block of
channel activity (using 86Rb+ flux assay). In contrast
to Ba2+, Mg2+ had no effect on tetramer stability (even
though Mg2+ was a potent blocker). Many studies have shown
Cd2+ block of K+ channels as a result of cysteine
substitution of cavity-lining M2 (S6) residues, with the implicit
interpretation that coordination of a single ion by cysteine side chains along
the central axis effectively blocks the pore. We examined blocking and
tetramer-stabilizing effects of Cd2+ on KirBac1.1 with cysteine
substitutions in M2. Cd2+ block potency followed an α-helical
pattern consistent with the crystal structure. Significantly, Cd2+
strongly stabilized tetramers of I138C, located in the center of the inner
cavity. This stabilization was additive with the effect of Ba2+,
consistent with both ions simultaneously occupying the channel:
Ba2+ at the selectivity filter entrance and Cd2+
coordinated by I138C side chains in the inner cavity.Potassium channels are expressed in many cell types and are key players in
a wide range of physiological processes. One subset of potassium channels, the
inward-rectifying potassium (Kir) channels, are functionally blocked by
cytosolic cations such as Mg2+ and polyamines and contribute to the
regulation of membrane excitability, cardiac rhythm, vascular tone, insulin
release, and salt flow across epithelia
(1–3).
There are seven subfamilies of eukaryotic Kir channel genes. Among them, Kir1
encodes weak rectifiers, whereas Kir2 and Kir5 encode strong rectifiers; Kir3
encodes G-protein-regulated channels; and Kir6 encodes ATP-sensitive channels
(4). Recently, a related
bacterial family of genes (KirBac) has been identified
(5,
6), and in 2003, the first
member (KirBac1.1) was crystallized
(7), providing a structural
model for eukaryotic channels.The crystal structure of KirBac1.1 revealed a tetrameric pore structure
similar to that seen in KcsA and a novel cytoplasmic domain
(7,
8). The selectivity filter of
both KirBac1.1 and KcsA consists of an extremely conserved pore loop followed
by a central cavity, forming a transmembrane ion-selective permeation pore
(7,
8). The linear arrangement of
five oxygen rings (four from carbonyl oxygens and one from a Thr side chain)
in the selectivity filter coordinates with ions, compensating for the energy
barrier caused by K+ dehydration, thereby facilitating the rapid
diffusion of K+ across the membrane
(8–12).
Two-thirds of the KirBac1.1 amino acid residues constitute the cytosolic
domain that is highly conserved among the Kir subfamilies and form the
cytosolic vestibule
(13–16),
which, together with the transmembrane pore, generates an 88-Å-long ion
conduction pore (7).The prototypic potassium channel KcsA exists very stably as a tetramer,
even in the harsh conditions of SDS-PAGE
(17). In addition to
protein-protein interaction between monomers, protein-lipid and protein-ion
interactions play important roles in stabilizing the KcsA tetramer
(17–20).
The selectivity filter of KcsA, coordinated with K+ ions, can serve
as a bridge between the four monomers to maintain the structure of the
selectivity filter and the tetrameric architecture of the channel as a whole
(11,
21). Blocking ions, such as
Ba2+, also act as strong stabilizers
(17). In the crystal structure
of KcsA, Ba2+ occupies a site equivalent to the S4
K+-binding site within the selectivity filter
(22). Other permeant ions
(Rb+, Cs+, Tl+, and
NH+4) and strong blockers (Sr2+) can also
contribute to the thermostability of the KcsA tetramer in SDS-PAGE
(17). In contrast, impermeant
ions such as Na+ and Li+ or weak blockers such as
Mg2+ tend to destabilize the KcsA tetramer
(17,
19).Like KcsA, KirBac1.1 purified using decylmaltoside or tridecylmaltoside is
active and presumably stable as a tetramer in mild detergent solutions.
However, in SDS-PAGE, KirBac1.1 migrates exclusively as a monomer
(23). Because KcsA and
KirBac1.1 are structurally similar in the transmembrane region of the pore, we
hypothesized that permeant and blocking ions would also affect KirBac1.1
tetramer stability in SDS-PAGE. In the present work, the effects of blocking
ions such as Ba2+ and Mg2+ on KirBac1.1 tetramer
stability were examined to provide insight to the physical nature of their
interaction with KirBac1.1, particularly in the selectivity filter and TM2
cavity. The data reveal important differences in the nature of the interaction
of Mg2+ and Ba2+ with the channel as well as provide
previously unavailable evidence for the nature of Cd2+ coordination
within the channel. 相似文献
306.
The lipid bilayer is a critical determinant of ion channel activity; however, efforts to define the lipid dependence of channel function have generally been limited to cellular expression systems in which the membrane composition cannot be fully controlled. We reconstituted purified human Kir2.1 and Kir2.2 channels into liposomes of defined composition to study their phospholipid dependence of activity using 86Rb+ flux and patch-clamp assays. Our results demonstrate that Kir2.1 and Kir2.2 have two distinct lipid requirements for activity: a specific requirement for phosphatidylinositol 4,5-bisphosphate (PIP2) and a nonspecific requirement for anionic phospholipids. Whereas we previously showed that PIP2 increases the channel open probability, in this work we find that activation by POPG increases both the open probability and unitary conductance. Oleoyl CoA potently inhibits Kir2.1 by antagonizing the specific requirement for PIP2, and EPC appears to antagonize activation by the nonspecific anionic requirement. Phosphatidylinositol phosphates can act on both lipid requirements, yielding variable and even opposite effects on Kir2.1 activity depending on the lipid background. Mutagenesis experiments point to the role of intracellular residues in activation by both PIP2 and anionic phospholipids. In conclusion, we utilized purified proteins in defined lipid membranes to quantitatively determine the phospholipid requirements for human Kir channel activity. 相似文献
307.
James D. Nichols Mark D. Koneff Patricia J. Heglund Melinda G. Knutson Mark E. Seamans James E. Lyons John M. Morton Malcolm T. Jones G. Scott Boomer Byron K. Williams 《The Journal of wildlife management》2011,75(1):6-18
Climate change and its associated uncertainties are of concern to natural resource managers. Although aspects of climate change may be novel (e.g., system change and nonstationarity), natural resource managers have long dealt with uncertainties and have developed corresponding approaches to decision-making. Adaptive resource management is an application of structured decision-making for recurrent decision problems with uncertainty, focusing on management objectives, and the reduction of uncertainty over time. We identified 4 types of uncertainty that characterize problems in natural resource management. We examined ways in which climate change is expected to exacerbate these uncertainties, as well as potential approaches to dealing with them. As a case study, we examined North American waterfowl harvest management and considered problems anticipated to result from climate change and potential solutions. Despite challenges expected to accompany the use of adaptive resource management to address problems associated with climate change, we conclude that adaptive resource management approaches will be the methods of choice for managers trying to deal with the uncertainties of climate change. © 2010 The Wildlife Society. 相似文献
308.
Design and synthesis of potent,orally bioavailable dihydroquinazolinone inhibitors of p38 MAP kinase 总被引:3,自引:0,他引:3
Stelmach JE Liu L Patel SB Pivnichny JV Scapin G Singh S Hop CE Wang Z Strauss JR Cameron PM Nichols EA O'Keefe SJ O'Neill EA Schmatz DM Schwartz CD Thompson CM Zaller DM Doherty JB 《Bioorganic & medicinal chemistry letters》2003,13(2):277-280
The development of potent, orally bioavailable (in rat) and selective dihydroquinazolinone inhibitors of p38alpha MAP kinase is described. These analogues are hybrids of a pyridinylimidazole p38alpha inhibitor reported by Merck Research Laboratories and VX-745. Optimization of the C-5 phenyl and the C-7 piperidinyl substituents led to the identification of 15i which gave excellent suppression of TNF-alpha production in LPS-stimulated whole blood (IC(50)=10nM) and good oral exposure in rats (F=68%, AUCn PO=0.58 microM h). 相似文献
309.
Laban U Kurrasch-Orbaugh D Marona-Lewicka D Nichols DE 《Bioorganic & medicinal chemistry letters》2001,11(6):793-795
Synthesis and biological evaluation of a novel fluorinated tryptamine analogue are described. This new compound 1-(4-fluoro-5-methoxyindol-3-yl)pyrrolidine (2) was found to be a potent serotonin 5-HT1A agonist. 相似文献
310.
Insulator function and topological domain border strength scale with architectural protein occupancy
Kevin Van Bortle Michael H Nichols Li Li Chin-Tong Ong Naomi Takenaka Zhaohui S Qin Victor G Corces 《Genome biology》2014,15(5):R82