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51.
Christiane Liers Caroline Bobeth Marek Pecyna René Ullrich Martin Hofrichter 《Applied microbiology and biotechnology》2010,85(6):1869-1879
The jelly fungus Auricularia auricula-judae produced an enzyme with manganese-independent peroxidase activity during growth on beech wood (∼300 U l−1). The same enzymatic activity was detected and produced at larger scale in agitated cultures comprising of liquid, plant-based
media (e.g. tomato juice suspensions) at levels up to 8,000 U l−1. Two pure peroxidase forms (A. auricula-judae peroxidase (AjP I and AjP II) could be obtained from respective culture liquids by three chromatographic steps. Spectroscopic
and electrophoretic analyses of the purified proteins revealed their heme and peroxidase nature. The N-terminal amino acid
sequence of AjP matched well with sequences of fungal enzymes known as “dye-decolorizing peroxidases”. Homology was found
to the N-termini of peroxidases from Marasmius scorodonius (up to 86%), Thanatephorus cucumeris (60%), and Termitomyces albuminosus (60%). Both enzyme forms catalyzed not only the conversion of typical peroxidase substrates such as 2,6-dimethoxyphenol and
2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) but also the decolorization of the high-redox potential dyes Reactive Blue 5
and Reactive Black 5, whereas manganese(II) ions (Mn2+) were not oxidized. Most remarkable, however, is the finding that both AjPs oxidized nonphenolic lignin model compounds (veratryl
alcohol; adlerol, a nonphenolic β-O-4 lignin model dimer) at low pH (maximum activity at pH 1.4), which indicates a certain ligninolytic activity of dye-decolorizing
peroxidases. 相似文献
52.
53.
Use of the additive main effects and multiplicative interaction model in QTL mapping for adaptation in barley 总被引:4,自引:0,他引:4
Ignacio Romagosa Steven E. Ullrich Feng Han Patrick M. Hayes 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1996,93(1-2):30-37
The additive main effects and multiplicative interaction (AMMI) model has emerged as a powerful analytical tool for genotype x environment studies. The objective of the present study was to assess its value in quantitative trait locus (QTL) mapping. This was done through the analysis of a large two-way table of genotype-by-environment data of barley (Hordeum vulgare L.) grain yields, where the genotypes constituted a genetic population suitable for mapping studies. Grain yield data of 150 doubled haploid lines derived from the Steptoe x Morex cross, and the two parental lines, were taken by the North American Barley Genome Mapping Project (NABGMP) at 16 environments throughout the barley production areas of the USA and Canada. Four regions of the genome were responsible for most of the differential genotypic expression across environments. They accounted for approximately 50% of the genotypic main effect and 30% of the genotype x environment interaction (GE) sums of squares. The magnitude and sign of AMMI scores for genotypes and sites facilitate inferences about specific interactions. The parallel use of classification (cluster analysis of environments) and ordination (principal component analysis of GE matrix) techniques allowed most of the variation present in the genotype x environment matrix to be summarized in just a few dimensions, specifically four QTLs showing differential adaptation to four clusters of environments. Thus, AMMI genotypic scores, when the genotypes constituted a population suitable for QTL mapping, could provide an adequate way of resolving the magnitude and nature of QTL x environment interactions.Ignacio Romagosa was on sabbatical leave from the University of Lleida and the Institut de Recerca i Tecnologia Agroalimentàries, Lleida, Spain, when this study was conducted 相似文献
54.
In contrast to signal generation and transmission, the mechanisms and molecules that negatively regulate receptor tyrosine kinase (RTK) signaling are poorly understood. Here we characterize Mig-6 as a novel negative feedback regulator of the epidermal growth factor receptor (EGFR) and potential tumor suppressor. Mig-6 was identified in a yeast two-hybrid screen with the kinase active domain of the EGFR as bait. Upon EGF stimulation Mig-6 binds to the EGFR involving a highly acidic region between amino acids 985-995. This interaction is kinase activity-dependent, but independent of tyrosine 992. Mig-6 overexpression results in reduced activation of the mitogenactivated protein kinase ERK2 in response to EGF, but not FGF or PDGF, stimulation and in enhanced receptor internalization without affecting the rate of degradation. The induction of Mig-6 mRNA expression in response to EGF, but not FGF, indicates the existence of a negative regulatory feedback loop. Consistent with these findings, a possible role as tumor suppressor is indicated by Mig-6-mediated inhibition of EGFR overexpression-induced transformation of Rati cells. 相似文献
55.
Michniewicz A Ullrich R Ledakowicz S Hofrichter M 《Applied microbiology and biotechnology》2006,69(6):682-688
Cerrena unicolor secreted two laccase isoforms with different characteristics during the growth in liquid media. In a synthetic low-nutrient
nitrogen glucose medium (Kirk medium), high amounts of laccase (4,000 U l−1) were produced in response to Cu2+. Highest laccase levels (19,000 U l−1) were obtained in a complex tomato juice medium. The isoforms (Lacc I, Lacc II) were purified to homogeneity with an overall
yield of 22%. Purification involved ultrafiltration and Mono Q separation. Lacc I and II had M
w of 64 and 57 kDa and pI of 3.6 and 3.7, respectively. Both isoforms had an absorption maximum at 608 nm but different pH optima and thermal stability.
Optimum pH ranged from 2.5 to 5.5 depending on the substrate. The pH optima of Lacc II were always higher than those of Lacc
I. Both laccases were stable at pH 7 and 10 but rapidly lost activity at pH 3. Their temperature optimum was around 60°C,
and at 5°C they still reached 30% of the maximum activity. Lacc II was the more thermostable isoform that did not lose any
activity during 6 months storage at 4°C. Kinetic constants (K
m, k
cat) were determined for 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol and syringaldazine. 相似文献
56.
Kluge MG Ullrich R Scheibner K Hofrichter M 《Applied microbiology and biotechnology》2007,75(6):1473-1478
Agrocybe aegerita peroxidase (AaP) is a versatile heme-thiolate protein that can act as a peroxygenase and catalyzes, among other reactions,
the hydroxylation of aromatic rings. This paper reports a rapid and selective spectrophotometric method for directly detecting
aromatic hydroxylation by AaP. The weakly activated aromatic compound naphthalene served as the substrate that was regioselectively
converted into 1-naphthol in the presence of the co-substrate hydrogen peroxide. Formation of 1-naphthol was followed at 303 nm
(ɛ
303 = 2,010 M−1 cm−1), and the apparent Michaelis–Menten (K
m) and catalytic (k
cat) constants for the reaction were estimated to be 320 μM and 166 s−1, respectively. This method will be useful in screening of fungi and other microorganisms for extracellular peroxygenase activities
and in comparing and assessing different catalytic activities of haloperoxidase–peroxygenases. 相似文献
57.
Salonikidis PS Niebert M Ullrich T Bao G Zeug A Richter DW 《The Journal of biological chemistry》2011,286(26):23419-23431
Ratiometric measurements with FRET-based biosensors in living cells using a single fluorescence excitation wavelength are often affected by a significant ion sensitivity and the aggregation behavior of the FRET pair. This is an important problem for quantitative approaches. Here we report on the influence of physiological ion concentration changes on quantitative ratiometric measurements by comparing different FRET pairs for a cAMP-detecting biosensor. We exchanged the enhanced CFP/enhanced YFP FRET pair of an established Epac1-based biosensor by the fluorophores mCerulean/mCitrine. In the case of enhanced CFP/enhanced YFP, we showed that changes in proton, and (to a lesser extent) chloride ion concentrations result in incorrect ratiometric FRET signals, which may exceed the dynamic range of the biosensor. Calcium ions have no direct, but an indirect pH-driven effect by mobilizing protons. These ion dependences were greatly eliminated when mCerulean/mCitrine fluorophores were used. For such advanced FRET pairs the biosensor is less sensitive to changes in ion concentration and allows consistent cAMP concentration measurements under different physiological conditions, as occur in metabolically active cells. In addition, we verified that the described FRET pair exchange increased the dynamic range of the FRET efficiency response. The time window for stable experimental conditions was also prolonged by a faster biosensor expression rate in transfected cells and a greatly reduced tendency to aggregate, which reduces cytotoxicity. These properties were verified in functional tests in single cells co-expressing the biosensor and the 5-HT(1A) receptor. 相似文献
58.
Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases. 总被引:101,自引:0,他引:101
E Y Skolnik B Margolis M Mohammadi E Lowenstein R Fischer A Drepps A Ullrich J Schlessinger 《Cell》1991,65(1):83-90
A novel method has been developed to allow cloning of protein targets for receptors with tyrosine kinase activity. By utilizing the carboxy-terminal tail of EGF receptor (EGFR) as a probe to screen lambda gt11 expression libraries, several EGFR-binding proteins have been cloned; two have been analyzed and contain unique SH2 and SH3 domains. One gene (GRB-1) has been fully sequenced, is expressed in various tissues and cell lines, and has a molecular mass of 85 kd. Interestingly, GRB-1 encodes the human counterpart of the PI3 kinase-associated protein p85. Advantages of this technique include the ease of cloning tyrosine kinase receptor targets present at low levels and the ability to identify proteins that are related in their capacity to bind activated receptors but contain no significant DNA sequence homology. This method, termed CORT (for cloning of receptor targets), offers a general approach for the identification and cloning of various receptor targets. 相似文献
59.
L. A. Marquez-Cedillo P. M. Hayes A. Kleinhofs W. G. Legge B. G. Rossnagel K. Sato S. E. Ullrich D. M. Wesenberg 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2001,103(4):625-637
A better understanding of the genetics of complex traits, such as yield, may be achieved by using molecular tools. This study
was conducted to estimate the number, genome location, effect and allele phase of QTLs determining agronomic traits in the
two North American malting barley (Hordeum vulgare L.) quality variety standards. Using a doubled haploid population of 140 lines from the cross of two-rowed Harrington×six-rowed
Morex, agronomic phenotypic data sets from nine environments, and a 107-marker linkage map, we performed QTL analyses using
simple interval mapping and simplified composite interval mapping procedures. Thirty-five QTLs were associated, either across
environments or in individual environments, with five grain and agronomic traits (yield, kernel plumpness, test weight, heading
date, and plant height). Significant QTL×environment interaction was detected for all traits. These interactions resulted
from both changes in the magnitude of response and changes in the sign of the allelic effect. QTLs for multiple traits were
coincident. The vrs1 locus on chromosome 2 (2H), which determines inflorescence row type, was coincident with the largest-effect QTL determining
four traits (yield, kernel plumpness, test weight, and plant height). QTL analyses were also conducted separately for each
sub-population (six-rowed and two-rowed). Seven new QTLs were detected in the sub-populations. Positive transgressive segregants
were found for all traits, but they were more prevalent in the six-rowed sub-population.QTL analysis should be useful for
identifying candidate genes and introgressing favorable alleles between germplasm groups.
Received: 18 August 2000 / Accepted: 15 December 2000 相似文献
60.
Characterization and mutational analysis of three allelic lsc genes encoding levansucrase in Pseudomonas syringae 下载免费PDF全文
In the plant pathogen Pseudomonas syringae pv. glycinea PG4180 and other bacterial species, synthesis of the exopolysaccharide levan is catalyzed by the extracellular enzyme levansucrase. The results of Southern blotting and PCR analysis indicated the presence of three levansucrase-encoding genes in strain PG4180: lscA, lscB, and lscC. In this study, lscB and lscC were cloned from a genomic library of strain PG4180. Sequence analysis of the two lsc genes showed that they were virtually identical to each other and highly similar to the previously characterized lscA gene. lscA and lscC had a chromosomal location, whereas lscB resided on an indigenous plasmid of PG4180. Mutants with impaired expression of individual lsc genes and double mutants were generated by marker exchange mutagenesis. Determination of levansucrase activities in these mutants revealed that the lscB gene product was secreted but not that of lscA or lscC. Our results indicated that lscB and lscC but not lscA contributed to periplasmic levan synthesis of PG4180. The lscB lscC double mutant was completely defective in levan formation and could be complemented by either lscB or lscC. Our data suggested a compartment-specific localization of two lsc gene products, with LscB being the secreted, extracellular enzyme and LscC being the predominantly periplasmic levansucrase. Results of Western blot analyses indicated that lscA was not expressed and that lscA was not associated with levansucrase activities in any particular protein fraction. LscA could be detected in PG4180 only when transcribed from the vector-borne P(lac) promoter. PCR screening in various P. syringae strains with primers derived from the three characterized lsc genes demonstrated the presence of multiple Lsc isoenzymes in other P. syringae pathovars. 相似文献