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941.
Martijn H. M. Holterman Markus Oggenfuss Sebastian Kiewnick 《Journal of Phytopathology》2012,160(2):59-66
In the detection of plant pests, speed and accuracy are vital. High‐resolution melting curve (HRMC) analysis was therefore evaluated as a new tool for the identification of root‐knot nematodes (Meloidogyne spp.). On the basis of the second intergenic spacer (IGS2) region of the ribosomal DNA cistron, Meloidogyne chitwoodi, M. fallax and M. hapla were successfully distinguished from each other and the group of the three tropical species, M. incognita, M. arenaria and M. javanica. Conversely, it was shown that the IGS2 region is not suitable for the tropical species M. enterolobii (senior synonym of M. mayaguensis) as the amplification of multiple fragments of different lengths prevented a reliable HRMC analysis. However, the obtained results provide a proof of principle that HRMC analysis can be a suitable single‐tube assay for fast and accurate root‐knot nematode identification. 相似文献
942.
Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis 总被引:1,自引:0,他引:1
The thermostability of microbial transglutaminase (MTG) of Streptomyces mobaraensis was further improved by saturation mutagenesis and DNA-shuffling. High-throughput screening was used to identify clones with increased thermostability at 55°C. Saturation mutagenesis was performed at seven "hot spots", previously evolved by random mutagenesis. Mutations at four positions (2, 23, 269, and 294) led to higher thermostability. The variants with single amino acid exchanges comprising the highest thermostabilities were combined by DNA-shuffling. A library of 1,500 clones was screened and variants showing the highest ratio of activities after incubation for 30 min at 55°C relative to a control at 37°C were selected. 116 mutants of this library showed an increased thermostability and 2 clones per deep well plate were sequenced (35 clones). 13 clones showed only the desired sites without additional point mutations and eight variants were purified and characterized. The most thermostable mutant (triple mutant S23V-Y24N-K294L) exhibited a 12-fold higher half-life at 60°C and a 10-fold higher half-life at 50°C compared to the unmodified recombinant wild-type enzyme. From the characterization of different triple mutants differing only in one amino acid residue, it can be concluded that position 294 is especially important for thermostabilization. The simultaneous exchange of amino acids at sites 23, 24, 269 and 289 resulted in a MTG-variant with nearly twofold higher specific activity and a temperature optimum of 55°C. A triple mutant with amino acid substitutions at sites 2, 289 and 294 exhibits a temperature optimum of 60°C, which is 10°C higher than that of the wild-type enzyme. 相似文献
943.
G Mandal M Sharma M Kruse C Sander-Juelch LA Munro Y Wang JV Vilg MJ Tamás H Bhattacharjee M Wiese R Mukhopadhyay 《Molecular microbiology》2012,85(6):1204-1218
Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defence against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen‐activated protein kinase, LmjMPK2. Leishmania parasites coexpressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo‐osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr‐197 and this phosphorylation requires LmjMPK2 activity. Lys‐42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild‐type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. Leishmania mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild‐type cells. This is the first report where a parasite aquaglyceroporin activity is post‐translationally modulated by a mitogen‐activated protein kinase. 相似文献
944.
Nawrotzki R Islinger M Vogel I Völkl A Kirsch J 《Histochemistry and cell biology》2012,137(4):471-482
Gephyrin is a scaffolding protein required for the accumulation of inhibitory neurotransmitter receptors at neuronal postsynaptic
membranes. In non-neuronal tissues, gephyrin is indispensible for the biosynthesis of molybdenum cofactor, the prosthetic
group of oxidoreductases including sulfite oxidase and xanthine oxidase. However, the molecular and cellular basis of gephyrin’s
non-neuronal function is poorly understood; in particular, the roles of its splice variants remain enigmatic. Here, we used
cDNA screening as well as Northern and immunoblot analyses to show that mammalian liver contains only a limited number of
gephyrin splice variants, with the C3-containing variant being the predominant isoform. Using new and established anti-gephyrin
antibodies in immunofluorescence and subcellular fractionation studies, we report that gephyrin localizes to the cytoplasm
of both tissue hepatocytes and cultured immortalized cells. These findings were corroborated by RNA interference studies in
which the cytosolic distribution was found to be abolished. Finally, by blue-native PAGE we show that cytoplasmic gephyrin
is part of a ~600 kDa protein complex of yet unknown composition. Our data suggest that the expression pattern of non-neuronal
gephyrin is simpler than indicated by previous evidence. In addition, gephyrin’s presence in a cytosolic 600 kDa protein complex
suggests that its metabolic and/or other non-neuronal functions are exerted in the cytoplasm and are not confined to a particular
subcellular compartment. 相似文献
945.
Cold-adapted ecosystems are often considered to be stable, species poor, and well protected. However, such ecosystems have
been identified as being especially sensitive to threats from global warming. Despite this, recent studies have found low
proportions of Red Listed species in these systems. In this study we explored the number of alpine species (dependent on alpine
habitats for their survival) and their Red List status in Sweden. We determined the proportion of Red Listed species and explored
discrepancies among different groups of organisms in terms of the proportion of Red Listed species and the criteria used for
Red Listing. We found a total of 389 alpine species in twelve analyzed species groups. The overall proportion of Red Listed
species was 29%, with 15% regarded as threatened. There were substantial differences among taxonomic groups with respect to
the proportion of Red Listed species. Among mammals 75% of the species are Red Listed, along with 63% of butterflies and 50%
of birds. In addition the single alpine dragonfly species and all three alpine stinging wasp species are also Red Listed.
Although beetles, bumblebees and grasshoppers are represented by a total of 17 alpine species, none are Red Listed. In contrast
to previous studies, our results show that the proportion of Red Listed species is high in alpine environments, indicating
that ecosystems found above the tree line are indeed threatened. No species in Sweden have been Red Listed on the basis of
the IUCN criterion E (unfavorable quantitative analysis), this is surprising since entire cold-adapted ecosystems are likely
to disappear in the future. We highlight the need for a better and more coordinated application of the IUCN criteria, as well
as a more stringent strategy to assess the extinction risks for alpine species, thus maintaining reliable Red Lists. 相似文献
946.
947.
Pothier J Riederer MA Peter O Leroy X Valdenaire A Gnerre C Fretz H 《Bioorganic & medicinal chemistry letters》2012,22(14):4660-4664
Hit-to-lead evolution of 2-(2-((2-(4-chlorophenoxy)ethyl)thio)-1H-benzo[d]imidazol-1-yl)acetic acid (1), discovered in a high-throughput screening campaign as a novel chemotype of CRTh2 receptor antagonist, is presented. SAR development as well as in vitro and in vivo DMPK properties of selected representatives of substituted 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetic acids are discussed. 相似文献
948.
Timing of reproduction and clutch size are important determinants of breeding success, especially in seasonal environments.
Several recent bird population studies have shown changes in breeding time and in natural selection on it. These changes have
often been linked with climate change, but few studies have investigated how the traits or natural selection are actually
connected with climatic factors. Furthermore, the effect of population density on selection has been rarely considered, despite
the potential importance of density in demographic processes. We studied variation in natural selection on laying date and
on clutch size in relation to measures of spring phenology and population density in a long-term study of pied flycatchers
in SW Finland. The phenological stage of the environment at mean egg-laying did not affect the direction of selection on either
laying date or on clutch size. There was, however, stronger selection for earlier laying date when the breeding density of
the population was high, suggesting that early breeding is not necessarily beneficial as such, but that its importance is
emphasized when high population density increases competition. In addition, early breeding was favoured when the pre-breeding
period was cool, which may indicate an increased advantage for the fittest individuals in harsher conditions. In the middle
of the twentieth century, there was selection for large clutch size, which subsequently ceased, along with an overall decrease
in recruit production. Our results indicate that attention should be paid to demographic factors such as breeding density
when studying natural selection and temporal changes in it. 相似文献
949.
950.