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981.
Oligosaccharyltransferase (OST) is an oligomeric protein complex which catalyses the transfer en bloc of Glc3-Man9-GlcNAc2 from Dol-PP to specific asparagine residues in the nascent polypeptide chain. In order to study the function of the pig enzyme subunits, we have cloned OST48, ribophorin I and ribophorin II and characterized these proteins after in vitro translation as well as after expression in COS-1 cells. The individual full-length cDNAs contained open reading frames (ORFs) encoding polypeptides with calculated molecular masses of 48.9[emsp4 ]kDa (OST48), 68.7[emsp4 ]kDa (ribophorin I) and 69.3[emsp4 ]kDa (ribophorin II), respectively. A Kyte and Doolittle hydrophobicity analysis revealed that OST48, ribophorin I and ribophorin II possess a type I membrane topology with the bulk of their polypeptide chains directed towards the ER-lumen. In contrast to OST48, ribophorin I and II contain, respectively, three or two potential N-glycosylation sites of the Asn-Xaa-Thr/Ser type; only one is found to function as the acceptor site in each protein.Transfection of COS-1 cells with vector constructs encoding either OST48, ribophorin I, or a ribophorin I variant tagged with a myc-peptide sequence, resulted in the over-expression of polypeptides whose molecular masses were similar to those calculated from the respective cDNA ORFs. None of these three polypeptides, or ribophorin II, were found to display OST activity when over-expressed alone. By contrast, a modest but reproducible 25% increase of activity was observed when OST48 together with ribophorin I, or OST48 and myc-tagged ribophorin I, were co-expressed, indicating that these two subunits are probably responsible for the catalytic activity in the hetero-oligomeric OST complex. The only modest over-expression of transferase activity suggests that either the dimeric enzyme complex is catalytically unstable, or that the OST48 and ribophorin I polypeptides are unable to fold properly when other subunit components of the hetero-oligomeric OST complex are lacking. OST48 as well as ribophorin I are expressed in COS-1 cells as ER-resident proteins. Whereas OST48 carries a double-lysine motif in the –3/–5 position of its cytosolic C-terminal domain, ribophorin I does not contain recognizable ER-retention information. Replacing the lysine residue in the –3 position by leucine resulted in plasma membrane expression of the OST48-Leu polypeptide, indicating that this sequence motif may be able to influence OST48 localisation. No cell surface staining was observed when OST48-Leu was co-expressed with ribophorin I. This suggests that localisation of OST48 in the ER is mediated by interaction with ribophorin I rather than by the double-lysine motif. 相似文献
982.
Ernst E 《BMJ (Clinical research ed.)》2000,321(7269):1133-1135
983.
984.
985.
The X-ray structure of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae
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Monzingo AF Breksa A Ernst S Appling DR Robertus JD 《Protein science : a publication of the Protein Society》2000,9(7):1374-1381
Eucaryotes possess one or more NADP-dependent methylene-THF dehydrogenases as part of multifunctional enzymes. In addition, yeast expresses an unusual monofunctional NAD-dependent enzyme, yMTD. We report X-ray structures for the apoenzyme and its complex with NAD+ at 2.8 and 3.0 A resolution, respectively. The protein fold resembles that seen for the human and Escherichia coli dehydrogenase/cyclohydrolase bifunctional enzymes. The enzyme has two prominent domains, with the active site cleft between them. yMTD has a noncanonical NAD-binding domain that has two inserted strands compared with the NADP-binding domains of the bifunctional enzymes. This insert precludes yMTD from dimerizing in the same way as the bifunctional enzymes. yMTD functions as a dimer, but the mode of dimerization is novel. It does not appear that the difference in dimerization accounts for the difference in cofactor specificity or for the loss of cyclohydrolase activity. These functional differences are probably accounted for by minor differences within the tertiary structure of the active site of the monomeric protein. 相似文献
986.
Ernst Freese 《Molecular & general genetics : MGG》1957,88(3):388-406
Summary In crosses involving three allelic pab markers of the same cistron, the recombination frequencies between any two of the markers were such, that a consistent order of the three markers could be established. In these crosses two closely linked markers (0,9% distance, on each side) were also present. The segregation of the pab independent types showed, with respect to the outside markers, a frequent appearance of all four types of outside marker combinations, which in the crossing-over theory would belong to one crossover between the pab markers and 0, 1, or 2 crossovers in the adjacent regions. This deviation from ordinary crossingover expectations, called correlation effect, is confined to very small dimensions of the genome. In the present case to regions of about 0,05% map units.-Inspite of this effect, which sometimes nearly equals out the four types of outside marker combinations, the two recombinant types were, in all present crosses, significantly different, such that the pab markers could be ordered with respect to the outside markers. This ordering is in agreement with that established independently from recombination frequencies. Thus the frequencies of the different types of outside marker combinations are in agreement with the assumption, that the linear genes are built lengthwise into the chromosomal thread, and in disagreement with the assumption that genes or even larger chromosomal parts are sidechains of the chromosome.For this work a subdividing technique has been used by which it was relatively easy to obtain the double mutants between close markers. The same technique can also be applied for the partial selection of double mutants of allelic markers, provided that relatively close markers are available. In determining the linkage relationships it has been found, that me-3 seems to involve a fairly large deletion.
Mit 8 Textabbildungen
Diese Arbeit wurde unterstützt durch Stipendium und Sachbeihilfe der Deutschen Forschungsgemeinschaft und durch ein Stipendium des Damon Runyon Memorial Funds for Cancer Research. Die experimentelle Arbeit wurde ermöglicht durch die Gastfreundlichkeit von Herrn Prof. J. Straub und die großzügige Unterstützung von Herrn Dr. C. Bresch (Botanisches Institut Köln). 相似文献
Mit 8 Textabbildungen
Diese Arbeit wurde unterstützt durch Stipendium und Sachbeihilfe der Deutschen Forschungsgemeinschaft und durch ein Stipendium des Damon Runyon Memorial Funds for Cancer Research. Die experimentelle Arbeit wurde ermöglicht durch die Gastfreundlichkeit von Herrn Prof. J. Straub und die großzügige Unterstützung von Herrn Dr. C. Bresch (Botanisches Institut Köln). 相似文献
987.
988.
Ernst M Kaup B Müller M Bringer-Meyer S Sahm H 《Applied microbiology and biotechnology》2005,66(6):629-634
A whole-cell biotransformation system for the reduction of prochiral carbonyl compounds, such as methyl acetoacetate, to chiral hydroxy acid derivatives [methyl (R)-3-hydroxy butanoate] was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. Alcohol dehydrogenase from Lactobacillus brevis catalyzes a highly regioselective and enantioselective reduction of several ketones or keto acid derivatives to chiral alcohols or hydroxy acid esters. The adh gene encoding for the alcohol dehydrogenase of L. brevis was expressed in E. coli. As expected, whole cells of the recombinant strain produced only low quantities of methyl (R)-3-hydroxy butanoate from the substrate methyl acetoacetate. Therefore, the fdh gene from Mycobacterium vaccae N10, encoding NAD+-dependent formate dehydrogenase, was functionally coexpressed. The resulting two-fold recombinant strain exhibited an in vitro catalytic alcohol dehydrogenase activity of 6.5 units mg–1 protein in reducing methyl acetoacetate to methyl (R)-3-hydroxy butanoate with NADPH as the cofactor and 0.7 units mg–1 protein with NADH. The in vitro formate dehydrogenase activity was 1.3 units mg–1 protein. Whole resting cells of this strain catalyzed the formation of 40 mM methyl (R)-3-hydroxy butanoate from methyl acetoacetate. The product yield was 100 mol% at a productivity of 200 mol g–1 (cell dry weight) min–1. In the presence of formate, the intracellular [NADH]/[NAD+] ratio of the cells increased seven-fold. Thus, the functional overexpression of alcohol dehydrogenase in the presence of formate dehydrogenase was sufficient to enable and sustain the desired reduction reaction via the relatively low specific activity of alcohol dehydrogenase with NADH, instead of NADPH, as a cofactor. 相似文献
989.
Consumption of herbal medicines is widespread and increasing. Harvesting from the wild, the main source of raw material, is causing loss of genetic diversity and habitat destruction. Domestic cultivation is a viable alternative and offers the opportunity to overcome the problems that are inherent in herbal extracts: misidentification, genetic and phenotypic variability, extract variability and instability, toxic components and contaminants. The use of controlled environments can overcome cultivation difficulties and could be a means to manipulate phenotypic variation in bioactive compounds and toxins. Conventional plant-breeding methods can improve both agronomic and medicinal traits, and molecular marker assisted selection will be used increasingly. There has been significant progress in the use of tissue culture and genetic transformation to alter pathways for the biosynthesis of target metabolites. Obstacles to bringing medicinal plants into successful commercial cultivation include the difficulty of predicting which extracts will remain marketable and the likely market preference for what is seen as naturally sourced extracts. 相似文献
990.
Nitrate and phosphate affect cultivability of cyanobacteria from environments with low nutrient levels 总被引:3,自引:0,他引:3
Ernst A Deicher M Herman PM Wollenzien UI 《Applied and environmental microbiology》2005,71(6):3379-3383
Nitrate and phosphate concentrations higher than those found in the natural environment slowed down growth of two strains of non-bloom-forming, phycoerythrin-rich Synechococcus spp. isolated from mesotrophic subalpine lakes. The results make clear why isolation of these picocyanobacteria in standard cultivation media was difficult. At low concentrations, closely related strains exhibited distinct growth characteristics with respect to these two nutrients, possibly explaining differences in their seasonal appearance in the natural environment. 相似文献