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991.
Petra Hänzelmann Jan U. Dahl Jochen Kuper Alexander Urban Ursula Müller‐Theissen Silke Leimkühler Hermann Schindelin 《Protein science : a publication of the Protein Society》2009,18(12):2480-2491
Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple‐domain rhodanese‐like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well‐characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N‐terminal inactive rhodanese‐like domain. Phylogenetic analysis reveals that YnjE triple‐domain homologs can be found in a variety of other γ‐proteobacteria, in addition, some single‐, tandem‐, four and even six‐domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3‐mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C‐terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent. 相似文献
992.
Petra Obexer Judith Hagenbuchner Martina Rupp Christina Salvador Markus Holzner Martin Deutsch Verena Porto Reinhard Kofler Thomas Unterkircher Michael J. Ausserlechner 《The Journal of biological chemistry》2009,284(45):30933-30940
Loss of CDKN2A/p16INK4A in hematopoietic stem cells is associated with enhanced self-renewal capacity and might facilitate progression of damaged stem cells into pre-cancerous cells that give rise to leukemia. This is also reflected by the frequent loss of the INK4A locus in acute lymphoblastic T-cell leukemia. T-cell acute lymphoblastic leukemia cells designed to conditionally express p16INK4A arrest in the G0/G1 phase of the cell cycle and show increased sensitivity to glucocorticoid- and tumor necrosis factor receptor superfamily 6-induced apoptosis. To investigate the underlying molecular mechanism for increased death sensitivity, we interfered with specific steps of apoptosis signaling by expression of anti-apoptotic proteins. We found that alterations in cell death susceptibility resulted from changes in the composition of pro- and anti-apoptotic BCL2 proteins, i.e. repression of MCL1, BCL2, and PMAIP1/Noxa and the induction of pro-apoptotic BBC3/Puma. Interference with Puma induction by short hairpin RNA technology or retroviral expression of MCL1 or BCL2 significantly reduced both glucocorticoid- and FAS-induced cell death in p16INK4A-reconstituted leukemia cells. These results suggest that Puma, in concert with MCL1 and BCL2 repression, critically mediates p16INK4A-induced death sensitization and that in human T-cell leukemia the deletion of p16INK4A confers apoptosis resistance by shifting the balance of pro- and anti-apoptotic BCL2 proteins toward apoptosis protection. 相似文献
993.
Fourcade S Ruiz M Camps C Schlüter A Houten SM Mooyer PA Pàmpols T Dacremont G Wanders RJ Giròs M Pujol A 《American journal of physiology. Endocrinology and metabolism》2009,296(1):E211-E221
Peroxisomes are essential organelles exerting key functions in fatty acid metabolism such as the degradation of very long-chain fatty acids (VLCFAs). VLCFAs accumulate in X-adrenoleukodystrophy (X-ALD), a disease caused by deficiency of the Abcd1 peroxisomal transporter. Its closest homologue, Abcd2, exhibits a high degree of functional redundancy on the catabolism of VLCFA, being able to prevent X-ALD-related neurodegeneration in the mouse. In the search for specific roles of Abcd2, we screened fatty acid profiles in organs and primary neurons of mutant knockout mice lacking Abcd2 in basal conditions and under dietary challenges. Our results indicate that ABCD2 plays a role in the degradation of long-chain saturated and omega9-monounsaturated fatty acids and in the synthesis of docosahexanoic acid (DHA). Also, we demonstrated a defective VLCFA beta-oxidation ex vivo in brain slices of Abcd1 and Abcd2 knockouts, using radiolabeled hexacosanoic acid and the precursor of DHA as substrates. As DHA levels are inversely correlated with the incidence of Alzheimer's and several degenerative conditions, we suggest that ABCD2 may act as modulator/modifier gene and therapeutic target in rare and common human disorders. 相似文献
994.
Matthew T Cabeen Godefroid Charbon Waldemar Vollmer Petra Born Nora Ausmees Douglas B Weibel Christine Jacobs‐Wagner 《The EMBO journal》2009,28(9):1208-1219
The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament‐like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics of cell wall insertion to produce curved growth. Our study suggests that bacteria may use the cytoskeleton for mechanical control of growth to alter morphology. 相似文献
995.
Petra Behnen Alexander Scholten Nina Rätscho Karl-Wilhelm Koch 《Journal of biological inorganic chemistry》2009,14(1):89-99
Guanylate cyclase activating proteins (GCAPs) serve as neuronal Ca2+-sensor proteins in vertebrate rod and cone photoreceptor cells. Zebrafish express in their retina a variety of six different
GCAPs, of which four are specific for cone cells. One isoform, zGCAP4, is mainly expressed in double cones and long single
cones. We cloned the zGCAP4 gene, purified non-myristoylated and myristoylated forms of the protein after heterologous expression
in Escherichia coli and studied its properties: zGCAP4 was a strong activator of membrane-bound guanylate cyclases from bovine and zebrafish
retina, showing half-maximal activation at 520–570 nM free Ca2+ concentration. Furthermore, the Ca2+-sensitive activation properties of non-myristoylated and myristoylated zGCAP4 were similar, indicating no influence of the
myristoyl moiety on Ca2+-sensor function. Myristoylated zGCAP4 showed low affinity for membranes and did not exhibit a Ca2+–myristoyl switch, a feature typical of some but not all neuronal Ca2+-sensor proteins. However, tryptophan fluorescence studies and Ca2+-dependent differences in protease accessibility revealed Ca2+-induced conformational changes in myristoylated and non-myristoylated zGCAP4, indicating the operation as a Ca2+ sensor. Thus, expression and biochemical properties of zGCAP4 are in agreement with its function as an efficient Ca2+-sensitive regulator of guanylate cyclase activity in cone vision. 相似文献
996.
Marcus Franz Karin Spiegel Claudia Umbreit Petra Richter Carolina Codina-Canet Angela Berndt Annelore Altendorf-Hofmann Sven Koscielny Peter Hyckel Hartwig Kosmehl Ismo Virtanen Alexander Berndt 《Histochemistry and cell biology》2009,131(5):651-660
Snail is a regulator of epithelial–mesenchymal transition (EMT) and considered crucial to carcinoma metastasis, myofibroblast
transdifferentiation, and fibroblast activation. To investigate the role of Snail in oral squamous cell carcinoma (OSCC),
its immunohistochemical expression was analysed in 129 OSCC samples and correlated to nodal metastasis, histological grade,
E-cadherin, and alpha smooth-muscle-actin (αSMA). The results were compared to findings in 23 basal cell carcinomas (BCC).
Additionally, the influence of TGFβ1 and EGF on Snail, E-cadherin, vimentin, and αSMA expression was analysed in two OSCC
cell lines. As a result, Snail-positive cells were mainly found in the stroma of the OSCC invasive front without statistically
significant correlation to histological grade or nodal metastasis. Snail was co-localised to αSMA but not to E-cadherin or
cytokeratin and showed a significant correlation to the loss of membranous E-cadherin. All BCCs were Snail negative. In OSCC
culture, the growth-factor-mediated EMT-like phenomenon was accompanied by αSMA down-regulation. In summary, Snail expression
in OSCC is a stromal phenomenon associated with the myofibroblast phenotype and not related to growth-factor-mediated transdifferentiation
of the carcinoma cells themselves. Consequently, Snail immunohistochemistry cannot contribute to the prediction of the metastatic
potential. Furthermore, stromal Snail expression is suggested to be the result of mutual paracrine interaction of fibro-/myofibroblasts
and dedifferentiated carcinoma cells leading to the generation of a special type of carcinoma-associated fibroblasts.
M. Franz and K. Spiegel have contributed equally to the study. 相似文献
997.
The structure and functional role of the dimeric external stalk of FoF1-ATP synthases have been very actively researched over the last years. To understand the function, detailed knowledge of the structure and protein packing interactions in the dimer is required. In this paper we describe the application of structural prediction and molecular modeling approaches to elucidate the structural packing interaction of the cyanobacterial ATP synthase external stalk. In addition we present biophysical evidence derived from ESR spectroscopy and site directed spin labeling of stalk proteins that supports the proposed structural model. The use of the heterodimeric bb′ dimer from a cyanobacterial ATP synthase (Synechocystis sp. PCC 6803) allowed, by specific introduction of spin labels along each individual subunit, the evaluation of the overall tertiary structure of the subunits by calculating inter-spin distances. At defined positions in both b and b′ subunits, reporter groups were inserted to determine and confirm inter-subunit packing. The experiments showed that an approximately 100 residue long section of the cytoplasmic part of the bb′-dimer exists mostly as an elongated α-helix. The distant C-terminal end of the dimer, which is thought to interact with the δ-subunit, seemed to be disordered in experiments using soluble bb′ proteins. A left-handed coiled coil packing of the dimer suggested from structure prediction studies and shown to be feasible in molecular modeling experiments was used together with the measured inter-spin distances of the inserted reporter groups determined in ESR experiments to support the hypothesis that a significant portion of the bb′ structure exists as a left-handed coiled coil. 相似文献
998.
Petra Seemann Christine Gernert Susanne Schmitt Dietrich Mebs Ute Hentschel 《Antonie van Leeuwenhoek》2009,96(4):405-411
Palytoxin (PTX), one of the most potent and chemically complex marine toxins, is predominantly found in zoanthid corals and
sporadically in dinoflagellates. Its biosynthesis and metabolic pathways are largely unknown. However, the widespread occurrence
of the toxin in phylogenetically distinct marine organisms is consistent with its production by microorganisms and subsequent
accumulation in the food chain. To investigate a possible microbial origin, bacteria from two zoanthid corals (Palythoa caribaeorum, Zoanthus pulchellus) and one sponge (Neofibularia nolitangere) were isolated. More than 250 bacteria were screened for hemolysis using a newly developed PTX-screening assay of which 7%
showed PTX-like hemolytic activity. 16S rRNA gene sequencing revealed that these bacterial isolates belonged to strains of
Bacillus cereus group (n = 11) as well as the genera Brevibacterium (n = 4) and Acinetobacter (n = 2). The results indicate the presence of Na+/K+-ATPase toxins and possibly PTX in hemolytic bacteria from P. caribaeorum. 相似文献
999.
1000.
Marcelo E. Guerin Devinder Kaur B. S. Somashekar Sara Gibbs Petra Gest Delphi Chatterjee Patrick J. Brennan Mary Jackson 《The Journal of biological chemistry》2009,284(38):25687-25696
Phosphatidyl-myo-inositol mannosides (PIMs) are key glycolipids of the mycobacterial cell envelope. They are considered not only essential structural components of the cell but also important molecules implicated in host-pathogen interactions. Although their chemical structures are well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still incomplete. Here we show for the first time that although both mannosyltransferases PimA and PimB′ (MSMEG_4253) recognize phosphatidyl-myo-inositol (PI) as a lipid acceptor, PimA specifically catalyzes the transfer of a Manp residue to the 2-position of the myo-inositol ring of PI, whereas PimB′ exclusively transfers to the 6-position. Moreover, whereas PimB′ can catalyze the transfer of a Manp residue onto the PI-monomannoside (PIM1) product of PimA, PimA is unable in vitro to transfer Manp onto the PIM1 product of PimB′. Further assays using membranes from Mycobacterium smegmatis and purified PimA and PimB′ indicated that the acylation of the Manp residue transferred by PimA preferentially occurs after the second Manp residue has been added by PimB′. Importantly, genetic evidence is provided that pimB′ is an essential gene of M. smegmatis. Altogether, our results support a model wherein Ac1PIM2, a major form of PIMs produced by mycobacteria, arises from the consecutive action of PimA, followed by PimB′, and finally the acyltransferase MSMEG_2934. The essentiality of these three enzymes emphasizes the interest of novel anti-tuberculosis drugs targeting the initial steps of PIM biosynthesis.PIMs3 are unique mannolipids found in abundant quantities in the inner and outer membranes of the cell envelope of Mycobacterium spp. and a few other actinomycetes.4 They are based on a phosphatidyl-myo-inositol (PI) lipid anchor carrying one to six Manp residues and up to four acyl chains (for review see Refs. 1, 2). Based on a conserved mannosyl-PI anchor, they are also thought to be the precursors of the two major mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM) (1, 2). PIMs, LM, and LAM are considered not only essential structural components of the mycobacterial cell envelope (3–6), but also important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy (1).Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still fragmentary. According to the currently accepted model, the biosynthetic pathway is initiated by the transfer of two Manp residues and a fatty acyl chain to PI in the cytoplasmic leaflet of the plasma membrane. Based on genetic and biochemical evidence, Korduláková et al. (5) identified PimA (MSMEG_2935 in Mycobacterium smegmatis mc2155) as the enzyme that catalyzes the first mannosylation step of the pathway transferring a Manp residue most likely to the 2-position of the myo-inositol (myo-Ins) ring of PI. In contrast, the identity of PimB′, the enzyme responsible for the transfer of the second Manp to the 6-position of the myo-Ins ring of PIM1, still remains controversial. The Rv0557 protein of Mycobacterium tuberculosis H37Rv (PimB; MSMEG_1113 in M. smegmatis mc2155) was originally characterized as PimB′ (7). However, the lack of an Rv0557 ortholog in the genome of Mycobacterium leprae and the fact that the disruption of this gene in M. tuberculosis Erdman did not significantly affect the biosynthesis of PIMs suggest that compensatory activities exist in the bacterium or that Rv0557 serves another primary function (8, 9). Somewhat supporting the latter hypothesis, the ortholog of Rv0557 in Corynebacterium glutamicum (NCgl0452, renamed mgtA) was implicated in the mannosylation of a novel glycolipid (1,2-di-O-C16/C18:1-(α-d-mannosyl)-(1→4)-(α-d-glucopyranosyluronic acid)-(1→3)-glycerol), and Rv0557 from M. tuberculosis was reported to functionally complement for this enzyme in a C. glutamicum knock-out mutant (10). However, to our knowledge this mannosylated glycolipid has never been reported in mycobacteria, and it remains unclear whether PimB serves a similar physiological function in Mycobacterium spp.More recently, Lea-Smith et al. (11) have shown that the biosynthesis of Ac1PIM2 from Ac1PIM1 in C. glutamicum is catalyzed by NCgl2106 (Cg-PimB′). Disruption of the NCgl2106 gene totally abolished Ac1PIM2 production in the mutant, arguing against the existence of a compensatory activity associated with the corynebacterial PimB enzyme. Although Ac1PIM2 production in Cg-pimB′ and Cg-pimB′/Cg-pimB knock-out mutants was restored upon complementation with the M. tuberculosis Rv2188c gene (11, 12), direct evidence that Rv2188c carried out the same physiological function in mycobacteria has been lacking. Moreover, in light of the recent work by Torrelles et al. (9) showing an involvement of pimB (Rv0557) in the synthesis of LM and LAM in M. tuberculosis Erdman and of the demonstrated relaxed substrate specificity of the M. tuberculosis PimB (Rv0557) and PimB′ (Rv2188c) enzymes expressed in C. glutamicum (12), whether or not pimB and pimB′ could compensate for one another in mycobacteria remained open to speculation.Both PIM1 and PIM2 can be acylated with palmitate at position 6 of the Manp residue transferred by PimA by the acyltransferase MSMEG_2934 (orthologous to Rv2611c from M. tb) to form Ac1PIM1 and Ac1PIM2, respectively (13). Ac1PIM2 can further be acylated at position 3 of the myo-Ins ring by an as yet unidentified acyltransferase to yield Ac2PIM2. Importantly, Ac1PIM2 and Ac2PIM2 are among the most abundant forms of PIMs found in mycobacteria and are considered both metabolic end products and intermediates in the biosynthesis of more polar forms of PIMs (PIM5 and PIM6), LM, and LAM.In this work, clear evidence is provided that PimB′ (MSMEG_4253 in M. smegmatis mc2155) is the α-ManT responsible for the biosynthesis of PIM2 from PIM1 in mycobacteria and that no other ManT can compensate for a deficiency in this enzyme in M. smegmatis. Like PimA (5), PimB′ is essential to the growth of M. smegmatis. Cell-free assays using purified PimA and PimB′ and M. smegmatis membrane preparations provide new insights into the sequential events leading to the synthesis of the early forms of PIMs in mycobacteria. 相似文献