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991.
Characterization of the biosynthetic pathway of glucosylglycerate in the archaeon Methanococcoides burtonii 下载免费PDF全文
Costa J Empadinhas N Gonçalves L Lamosa P Santos H da Costa MS 《Journal of bacteriology》2006,188(3):1022-1030
The pathway for the synthesis of the organic solute glucosylglycerate (GG) is proposed based on the activities of the recombinant glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from Methanococcoides burtonii. A mannosyl-3-phosphoglycerate phosphatase gene homologue (mpgP) was found in the genome of M. burtonii (http://www.jgi.doe.gov), but an mpgS gene coding for mannosyl-3-phosphoglycerate synthase (MpgS) was absent. The gene upstream of the mpgP homologue encoded a putative glucosyltransferase that was expressed in Escherichia coli. The recombinant product had GpgS activity, catalyzing the synthesis of glucosyl-3-phosphoglycerate (GPG) from GDP-glucose and d-3-phosphoglycerate, with a high substrate specificity. The recombinant MpgP protein dephosphorylated GPG to GG and was also able to dephosphorylate mannosyl-3-phosphoglycerate (MPG) but no other substrate tested. Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from Thermus thermophilus, Pyrococcus horikoshii, and "Dehalococcoides ethenogenes." GpgS had maximal activity at 50 degrees C. The maximal activity of GpgP was at 50 degrees C with GPG as the substrate and at 60 degrees C with MPG. Despite the similarity of the sugar donors GDP-glucose and GDP-mannose, the enzymes for the synthesis of GPG or MPG share no amino acid sequence identity, save for short motifs. However, the hydrolysis of GPG and MPG is carried out by phosphatases encoded by homologous genes and capable of using both substrates. To our knowledge, this is the first report of the elucidation of a biosynthetic pathway for glucosylglycerate. 相似文献
992.
Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection 总被引:4,自引:0,他引:4
Parfenova Helena; Basuroy Shyamali; Bhattacharya Sujoy; Tcheranova Dilyara; Qu Yan; Regan Raymond F.; Leffler Charles W. 《American journal of physiology. Cell physiology》2006,290(5):C1399
In cerebral circulation, epileptic seizures associated with excessive release of the excitatory neurotransmitter glutamate cause endothelial injury. Heme oxygenase (HO), which metabolizes heme to a vasodilator, carbon monoxide (CO), and antioxidants, biliverdin/bilirubin, is highly expressed in cerebral microvessels as a constitutive isoform, HO-2, whereas the inducible form, HO-1, is not detectable. Using cerebral vascular endothelial cells from newborn pigs and HO-2-knockout mice, we addressed the hypotheses that 1) glutamate induces oxidative stress-related endothelial death by apoptosis, and 2) HO-1 and HO-2 are protective against glutamate cytotoxicity. In cerebral endothelial cells, glutamate (0.12.0 mM) increased formation of reactive oxygen species, including superoxide radicals, and induced major keystone events of apoptosis, such as NF-B nuclear translocation, caspase-3 activation, DNA fragmentation, and cell detachment. Glutamate-induced apoptosis was greatly exacerbated in HO-2 gene-deleted murine cerebrovascular endothelial cells and in porcine cells with pharmacologically inhibited HO-2 activity. Glutamate toxicity was prevented by superoxide dismutase, suggesting apoptotic changes are oxidative stress related. When HO-1 was pharmacologically upregulated by cobalt protoporphyrin, apoptotic effects of glutamate in cerebral endothelial cells were completely prevented. Glutamate-induced reactive oxygen species production and apoptosis were blocked by a CO-releasing compound, CORM-A1 (50 µM), and by bilirubin (1 µM), consistent with the antioxidant and cytoprotective roles of the end products of HO activity. We conclude that both HO-1 and HO-2 have anti-apoptotic effects against oxidative stress-related glutamate toxicity in cerebral vascular endothelium. Although HO-1, when induced, provides powerful protection, HO-2 is an essential endogenous anti-apoptotic factor against glutamate toxicity in the cerebral vascular endothelium. endothelium; carbon monoxide; bilirubin; injury; reactive oxygen species; heme oxygenase 相似文献
993.
de Almeida Silva V Sayoko Takata C Sant'Anna OA Carlos Lopes A Soares de Araujo P Helena Bueno da Costa M 《Journal of liposome research》2006,16(3):215-227
The Dtxd (Diphtheria toxoid) was the first antigen encapsulated within liposomes, their adjuvant properties were discovered (their capacity to enhance the vaccine immunogenicity). The point here is not to propose a new method to prepare this lipossomal vaccine. The central idea is to give new dresses for old vaccines by using classical and well-established liposome preparation method changing only the encapsulation pH and the immunization protocol.The most appropriate method of Dtxd encapsulation within liposome was based on lipid film hydration in 100 mM citrate buffer, pH 4.0. This was accompanied by changes on protein hydrophobicity, observed by CD and fluorescence spectroscopies. Whenever the Dtxd exposed its hydrophobic residues at pH 4.0, it interacted better with the lipossomal (observed by electrophoretic mobility) film than when its hydrophobic residues were buried (pH 9.0). The Dtxd partition coefficient in Triton-X114 and the acrylamide fluorescence quenching were also pH dependent. Both were bigger at pH 4.0 than at pH 9.0. The relationship protein structure and lipid interaction was pH dependent and now it can be easily maximized to enhance encapsulation of antigens in vaccine development.Mice were primed with formulations containing 5 mug of Dtxd within liposomes prepared in pH 4.0 or 7.0 or 9.0. The boosters were done 38 or 138 days after the first immunization. The IgM produced by immediate response of all lipossomal formulations were higher than the control (free protein). The response patterns and the immune maturity were measured by IgG1 and IgG2a titrations. The IgG1 titers produced by both formulations at pH 4.0 and 7.0 were at least 22 higher than those produced by mice injected lipossomal formulation at pH 9.0. When the boosters were done, 138 days after priming the mice produced a IgG2a titer of 29 and the group that received the booster 30 days after priming produced a titer of 25. The strongest antibody production was the neutralizing antibody (245 higher than the control) produced by those mice injected with lipossomal formulation at pH 4.0 with the booster done 138 days after priming. The simple change on lipossomal pH formulation and timing of the booster enhanced both antibody production and selectivity. 相似文献
994.
Pääkkönen K Tossavainen H Permi P Rakkolainen H Rauvala H Raulo E Kilpeläinen I Güntert P 《Proteins》2006,64(3):665-672
F-spondin is a protein mainly associated with neuronal development. It attaches to the extracellular matrix and acts in the axon guidance of the developing nervous system. F-spondin consists of eight domains, six of which are TSR domains. The TSR domain family binds a wide range of targets. Here we present the NMR solution structures of TSR1 and TSR4. TSR domains have an unusual fold that is characterized by a long, nonglobular shape, consisting of two beta-strands and one irregular extended strand. Three disulfide bridges and stack of alternating tryptophan and arginine side-chains stabilize the structure. TSR1 and TSR4 structures are similar to each other and to the previously determined TSR domain X-ray structures from another protein, TSP, although TSR4 exhibits a mobile loop not seen in other structures. 相似文献
995.
Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse 总被引:1,自引:0,他引:1
Bjarnadóttir TK Gloriam DE Hellstrand SH Kristiansson H Fredriksson R Schiöth HB 《Genomics》2006,88(3):263-273
Understanding differences in the repertoire of orthologous gene pairs is vital for interpretation of pharmacological and physiological experiments if conclusions are conveyed between species. Here we present a comprehensive dataset for G protein-coupled receptors (GPCRs) in both human and mouse with a phylogenetic road map. We performed systematic searches applying several search tools such as BLAST, BLAT, and Hidden Markov models and searches in literature data. We aimed to gather a full-length version of each human or mouse GPCR in only one copy referring to a single chromosomal position. Moreover, we performed detailed phylogenetic analysis of the transmembrane regions of the receptors to establish accurate orthologous pairs. The results show the identity of 495 mouse and 400 human functional nonolfactory GPCRs. Overall, 329 of the receptors are found in one-to-one orthologous pairs, while 119 mouse and 31 human receptors originate from species-specific expansions or deletions. The average percentage similarity of the orthologue pairs is 85%, while it varies between the main GRAFS families from an average of 59 to 94%. The orthologous pairs for the lipid-binding GPCRs had the lowest levels of conservation, while the biogenic amines had highest levels of conservation. Moreover, we searched for expressed sequence tags (ESTs) and identified more than 17,000 ESTs matching GPCRs in mouse and human, providing information about their expression patterns. On the whole, this is the most comprehensive study of the gene repertoire that codes for human and mouse GPCRs. The datasets are available for downloading. 相似文献
996.
Chemical shift assignment of methyl-containing residues is essential in protein NMR spectroscopy, as these residues are abundant
in protein interiors and provide the vast majority of long-range NOE connectivities for structure determination. These residues
also constitute an integral part of hydrophobic cavities, the surroundings for many enzymatic reactions. Here we present a
powerful strategy for the assignment of methyl-containing residues in a uniformly 13C/15N double labeled protein sample. The approach is based on novel four-dimensional HCCmHm-TOCSY experiments, two of them utilizing
gradient selection and sensitivity enhancement in all three indirectly detected dimensions. Regardless of the number of dimensions,
the proposed experiments can be executed using only one transient per FID, providing outstanding resolution and sensitivity.
A complete assignment of the 51 methyl-containing residues in the 16 kDa Mus musculus coactosin was accomplished using a four-dimensional HCCmHm-TOCSY spectrum recorded in 16 hours. 相似文献
997.
Sauerzapfe B Krenek K Schmiedel J Wakarchuk WW Pelantová H Kren V Elling L 《Glycoconjugate journal》2009,26(2):141-159
Poly-N-acetyllactosamine (poly-LacNAc) structures have been identified as important ligands for galectin-mediated cell adhesion
to extra-cellular matrix (ECM) proteins. We here present the biofunctionalization of surfaces with poly-LacNAc structures
and subsequent binding of ECM glycoproteins. First, we synthesized β-GlcNAc glycosides carrying a linker for controlled coupling
onto chemically functionalized surfaces. Then we produced poly-LacNAc structures with defined lengths using human β1,4-galactosyltransferase-1
and β1,3-N-acetylglucosaminyltransferase from Helicobacter pylori. These compounds were also used for kinetic characterization of glycosyltransferases and lectin binding assays. A mixture
of poly-LacNAc-structures covalently coupled to functionalized microtiter plates were identified for best binding to our model
galectin His6CGL2. We further demonstrate for the first time that these poly-LacNAc surfaces are suitable for further galectin-mediated
binding of the ECM glycoproteins laminin and fibronectin. This new technology should facilitate cell adhesion to biofunctionalized
surfaces by imitating the natural ECM microenvironment.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
998.
C. Christian Johansson Suzanne Egyházi Giuseppe Masucci Helena Harlin Dimitrios Mougiakakos Isabel Poschke Bo Nilsson Liss Garberg Rainer Tuominen Diana Linden Marianne Frostvik Stolt Johan Hansson Rolf Kiessling 《Cancer immunology, immunotherapy : CII》2009,58(7):1085-1094
Purpose New prognostic markers are needed for malignant melanoma. Inducible nitric oxide synthase (iNOS) and cyclooxygenase type 2
(COX-2) have been described to correlate with progression of melanoma. Moreover, activating mutations in BRAF/NRAS oncogenes
are often detected in melanoma. The BRAF/NRAS mutation status and expression of COX-2 and iNOS were examined to compare their
prognostic value for overall survival (OS) in stage III malignant cutaneous melanoma.
Experimental design The expression of iNOS and COX-2 in metastatic lymph nodes from 21 rapidly progressing (OS from date of diagnosis of stage
III disease ≤14 months) and 17 slowly progressing (OS ≥60 months) stage III cutaneous melanoma patients was examined by immunohistochemistry.
The presence of BRAF/NRAS mutations was analyzed using direct DNA sequencing. χ2 exact trend test and logistic regression analysis were used for statistical analysis.
Results Both iNOS (P = 0.002) and COX-2 (P = 0.048) alone significantly predicted OS. The BRAF/NRAS mutation status did not significantly differ between patient groups,
although iNOS significantly (P = 0.013) correlated with BRAF mutation frequency. Furthermore, the odds ratio (OR) with respect to OS of iNOS (OR = 10.4)
was higher than that of COX-2 (OR = 5.6) and was stable in the multivariate analysis of OS together with disease stage IIIB/C,
ulceration, number of metastatic lymph nodes, and Breslow tumor thickness.
Conclusion Our data show that iNOS is an independent and stronger prognostic factor for OS in stage III malignant cutaneous melanoma
than COX-2. 相似文献
999.
Maria A.F. Vera-DiVaio Antônio C.C. Freitas Helena C. Castro Sérgio de Albuquerque Lucio M. Cabral Carlos R. Rodrigues Magaly G. Albuquerque Rita C.A. Martins Maria G.M.O. Henriques Luiza R.S. Dias 《Bioorganic & medicinal chemistry》2009,17(1):295-302
Chagas disease (American trypanosomiasis) is one of the most important parasitic diseases with serious social and economic impacts mainly on Latin America. This work reports the synthesis, in vitro trypanocidal evaluation, cytotoxicity assays, and molecular modeling and SAR/QSAR studies of a new series of N-phenylpyrazole benzylidene-carbohydrazides. The results pointed 6k (X = H, Y = p-NO2, pIC50 = 4.55 M) and 6l (X = F, Y = p-CN, pIC50 = 4.27 M) as the most potent derivatives compared to crystal violet (pIC50 = 3.77 M). The halogen-benzylidene-carbohydrazide presented the lowest potency whereas 6l showed the most promising profile with low toxicity (0% of cell death). The best equation from the 4D-QSAR analysis (Model 1) was able to explain 85% of the activity variability. The QSAR graphical representation revealed that bulky X-substituents decreased the potency whereas hydrophobic and hydrogen bond acceptor Y-substituents increased it. 相似文献
1000.
Vinícius R. Campos Paula A. Abreu Helena C. Castro Carlos R. Rodrigues Alessandro K. Jordo Vitor F. Ferreira Maria C.B.V. de Souza Fernanda da C. Santos Laura A. Moura Thaisa S. Domingos Carla Carvalho Eldio F. Sanchez Andr L. Fuly Anna C. Cunha 《Bioorganic & medicinal chemistry》2009,17(21):7429-7434
The current treatment used against envenomation by Lachesis muta venom still presents several side effects. This paper describes the synthesis, pharmacological and theoretical evaluations of new 1-arylsulfonylamino-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid ethyl esters (8a–f) tested against the hemolytic profile of the L. muta snake venom. Their structures were elucidated by one- and two-dimensional NMR techniques (1H, APT, HETCOR 1JCH and nJCH, n = 2, 3) and high-resolution electrospray ionization mass spectrometry. The series of triazole derivatives significantly neutralized the hemolysis induced by L. muta crude venom presenting a dose-dependent inhibitory profile (IC50 = 30−83 μM) with 1-(4′-chlorophenylsulfonylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid ethyl ester (8e) being the most potent compound. The theoretical evaluation revealed the correlation of the antiophidian profile with the coefficient distribution and density map of the Highest Occupied Molecular Orbitals (HOMO) of these molecules. The elucidation of this new series may help on designing new and more efficient antiophidian molecules. 相似文献