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961.
Three preparations of purified immunoglobulin (IgM) were isolated from serum of Atlantic halibut (Hippoglossus hippoglossus) by means of three different methods, and each of the three IgM preparations was used to produce a polyclonal rabbit anti-halibut IgM antiserum. One of the IgM preparations was employed in the characterisation of halibut serum immunoglobulin. Halibut IgM was shown to consist of two subunits, compatible with heavy (mu) and light (L) chains. A single mu chain at approximately 76 kDa, and six possible molecular weight (MW) variants of L chain were found (range approximately 25 to approximately 28.5 kDa). IgM was glycosylated on the heavy chain and N-linked carbohydrate constituted approximately 10.3% (w/w) of the total MW of IgM. The dominant form of non-reduced IgM had a MW of approximately 780 kDa, suggesting a tetrameric structure. Non-reduced IgM also showed a number of minor protein bands. Based on estimated MW, the relative carbohydrate content and the reactivity with all three anti-halibut IgM antisera, mono-, di- and trimeric redox forms of IgM were identified. The three antisera were characterised as to specificity and reactivity by means of enzyme linked immuno-sorbent assay (ELISA), crossed immuno-electrophoresis (CIE), and immunoblotting methods. The antisera showed a considerable diversity in their specificity to the suggested MW variants of halibut Ig light chain. A method for immunohistochemical detection of IgM in tissue was established. Protein A or protein G affinity for the IgM was not detectable. 相似文献
962.
Grånäs C Lundholt BK Loechel F Pedersen HC Bjørn SP Linde V Krogh-Jensen C Nielsen EM Praestegaard M Nielsen SJ 《Journal of biomolecular screening》2006,11(4):423-434
The RAS-mitogen-activated protein kinase (MAPK) signaling pathway has a central role in regulating the proliferation and survival of both normal and tumor cells. This pathway has been 1 focus area for the development of anticancer drugs, resulting in several compounds, primarily kinase inhibitors, in clinical testing. The authors have undertaken a cell-based, high-throughput screen using a novel ERF1 Redistribution assay to identify compounds that modulate the signaling pathway. The hit compounds were subsequently tested for activity in a functional cell proliferation assay designed to selectively detect compounds inhibiting the proliferation of MAPK pathway-dependent cancer cells. The authors report the identification of 2 cell membrane-permeable compounds that exhibit activity in the ERF1 Redistribution assay and selectively inhibit proliferation of MAPK pathway-dependent malignant melanoma cells at similar potencies (IC(50)=< 5 microM). These compounds have drug-like structures and are negative in RAF, MEK, and ERK in vitro kinase assays. Drugs belonging to these compound classes may prove useful for treating cancers caused by excessive MAPK pathway signaling. The results also show that cell-based, high-content Redistribution screens can detect compounds with different modes of action and reveal novel targets in a pathway known to be disease relevant. 相似文献
963.
Haste Andersen P Nielsen M Lund O 《Protein science : a publication of the Protein Society》2006,15(11):2558-2567
Discovery of discontinuous B-cell epitopes is a major challenge in vaccine design. Previous epitope prediction methods have mostly been based on protein sequences and are not very effective. Here, we present DiscoTope, a novel method for discontinuous epitope prediction that uses protein three-dimensional structural data. The method is based on amino acid statistics, spatial information, and surface accessibility in a compiled data set of discontinuous epitopes determined by X-ray crystallography of antibody/antigen protein complexes. DiscoTope is the first method to focus explicitly on discontinuous epitopes. We show that the new structure-based method has a better performance for predicting residues of discontinuous epitopes than methods based solely on sequence information, and that it can successfully predict epitope residues that have been identified by different techniques. DiscoTope detects 15.5% of residues located in discontinuous epitopes with a specificity of 95%. At this level of specificity, the conventional Parker hydrophilicity scale for predicting linear B-cell epitopes identifies only 11.0% of residues located in discontinuous epitopes. Predictions by the DiscoTope method can guide experimental epitope mapping in both rational vaccine design and development of diagnostic tools, and may lead to more efficient epitope identification. 相似文献
964.
Roussa E Bertram J Berge KE Labori KJ Thévenod F Raeder MG 《Histochemistry and cell biology》2006,125(4):419-428
The cholangiocytes lining the intrahepatic bile ducts modify the primary secretion from the hepatocytes. The cholangiocytes
secrete HCO3− into bile when stimulated with secretin in many species, including man. However, in rats, secretin stimulation neither affects
biliary HCO3− concentration nor bile flow, whereas following bile duct ligation (BDL) it induces hypercholeresis with significant increase
of NaHCO3 concentration. We hypothesized that BDL might affect the expression of cholangiocyte H+ transporters and thereby choleresis, and determined the expression and localization of the 31 kDa vacuolar type H+-ATPase (V-ATPase) subunit and of Na+/H+ exchanger NHE3 in the livers of control and BDL rats by real-time PCR, in situ hybridization, immunoblotting, and immunohistochemistry.
In controls, secretin had no effect on bile flow, whereas following BDL, secretin increased bile flow ∼threefold. V-ATPase
and NHE3 were expressed in control cholangiocytes showing intracellular and apical distribution, respectively. BDL significantly
up-regulated V-ATPase mRNA and protein expression and was associated with redistribution to the apical pole in ∼60% of the
cholangiocytes lining the small bile ductules. In contrast, NHE3 expression was significantly down-regulated by BDL at the
mRNA and protein level. The data demonstrate expression of V-ATPase in rat cholangiocytes. BDL-induced down-regulation of
NHE3 may contribute to a reduction of Na+ and HCO3− reabsorption and thus to their net secretion into bile. Apical localization of V-ATPase in cholangiocytes may indicate its
involvement in pH regulation and/or HCO3− salvage to compensate for NHE3 down-regulation in BDL. 相似文献
965.
To investigate substrate recruitment and transport across the Escherichia coli Ammonia transporter B (AmtB) protein, we performed molecular dynamics simulations of the AmtB trimer. We have identified residues important in recruitment of ammonium and intraluminal binding sites selective of ammonium, which provide a means of cation selectivity. Our results indicate that A162 guides translocation of an extraluminal ammonium into the pore lumen. We propose a mechanism for transporting the intraluminally recruited proton back to periplasm. Our mechanism conforms to net transport of ammonia and can explain why ammonia conduction is lost upon mutation of the conserved residue D160. We unify previous suggestions of D160 having either a structural or an ammonium binding function. Finally, our simulations show that the channel lumen is hydrated from the cytoplasmic side via the formation of single file water, while the F107/F215 stack at the inner-most part of the periplasmic vestibule constitutes a hydrophobic filter preventing AmtB from conducting water. 相似文献
966.
Thaysen-Andersen M Thøgersen IB Lademann U Offenberg H Giessing AM Enghild JJ Nielsen HJ Brünner N Højrup P 《Biochimica et biophysica acta》2008,1784(3):455-463
Cancer-induced alterations of protein glycosylations are well-known phenomena. Hence, the glycoprofile of certain glycoproteins can potentially be used as biomarkers for early diagnosis. However, there are a substantial number of candidates and the techniques for measuring their biomarker potential are limited, calling for new methods. Here, we have investigated the cancer marker potential of the glycoprofile of tissue inhibitor of metalloproteinase-1 (TIMP-1) using a method for comparative glycoprofiling. Glycoprofiles were obtained from plasma TIMP-1 of five healthy donors and five colorectal cancer (CRC) patients showing increased amounts of TIMP-1. Furthermore, the TIMP-1 glycoprofiles of media from two colon cancer cell lines (CCC) and a prostate cancer cell line were determined as disease references. TIMP-1 was purified from IgG-depleted samples using immuno affinity and gel electrophoresis and the glycoprofiling was performed using glycopeptide enrichment and mass spectrometry. The heterogeneous glycoprofiles of TIMP-1 were found to be highly conserved among the healthy donors, proving an ideal candidate marker and showed high reproducibility of the method. Numerous CCC-specific TIMP-1 glycans were observed illustrating cancer-induced changes. Unexpectedly, quantitation revealed that the glycoprofiles of healthy donors and CRC patients varied minimally. Considering the increased CRC TIMP-1 levels and the observed CCC-specific glycans, the lack of variation indicates that the increased amount of CRC TIMP-1 is not a direct product of the cancer cells. Hence, the TIMP-1 glycoprofile holds no biomarker potential for CRC when using plasma as the sample origin. This study clearly illustrates that the technique is capable of performing individualised site-specific glycan analysis and representing a new tool for biomarker investigation of low-abundant glycoproteins. 相似文献
967.
Manuela Panzacchi John D. C. Linnell Giulia Serrao Sveinung Eie Morten Odden John Odden Reidar Andersen 《Ecological Research》2008,23(5):889-896
Red fox Vulpes vulpes predation on roe deer Capreolus capreolus fawns has the potential to strongly affect prey population dynamics, but it is unclear whether this relationship is symmetrical
or not. We analysed the spring–summer diet of adult foxes and of their cubs in a fragmented agricultural area of southeastern
Norway, where a parallel study showed that the predator kills annually 25% of the radio-monitored roe deer fawns. The overall
diet was highly varied and was dominated by small mammals (33% volume), especially Microtus agrestis, and medium-large mammals (25%), largely represented by fawns. The frequency of occurrence (FO) of fawns in the diet of adult
foxes was highest in early spring, thus, supporting previous studies showing that the predator started actively hunting for
fawns from the very beginning of the birth season. During the summer, the FO of both fawns and small mammals markedly declined,
while that of berries and invertebrates increased. As expected for central-place foragers, cubs consumed a higher proportion
of large prey items compared to adults. In particular, 25% of scats from cubs—versus 9% from adults—contained roe deer remains,
suggesting a high profitability of fawns for vixens raising offspring. However, considering the wide food spectrum and the
availability of several large prey items in our study area, it seems unlikely that the importance of fawns to the diet and
population dynamics of red foxes could be as great as the impact of the predator on roe deer populations. This asymmetrical
relationship implies that there are unlikely to be any stabilising feedback mechanisms in the predator–prey relationship. 相似文献
968.
Marit Skog Eriksen Åsa Marie Espmark Trygve Poppe Bjarne Olai Braastad Ragnar Salte Morten Bakken 《Environmental Biology of Fishes》2008,81(1):87-99
Developmental stability reflects the degree to which phenotypic expression is unaffected by random accidents or developmental
noise. Developmental stability may be measured by phenodeviance or fluctuating asymmetry (FA), and estimation of developmental
stability has attracted substantial interest because it appears to represent a relatively simple method to identify sub lethal
stress exposure and to assess animal welfare. As a part of a long-term study, the work presented here primarily aimed to investigate
impacts on developmental instability in farmed salmon offspring ten months post hatch attributable to maternal cortisol administration
prior to spawning and mild hyperthermia exerted during incubation. Main results show that maternal cortisol enhancement increased
the level of FA in pectoral and pelvic fins, but did not affect the frequency of malformations in offspring. Mild hyperthermia
during incubation increased weight and fork length and also increased pelvic fin FA. Malformed fish were heavier and longer
than the normal ones, and pelvic fin asymmetry was positively related to condition factor. These results illustrate plausible
lasting impacts on offspring development due to the maternal endocrinological state at spawning and indicate that developmental
instability in farmed salmon juveniles may mirror aspects of the broodstock’s housing conditions. 相似文献
969.
Considerable work has been done on disentangling important factors determining early development in body size, yet our knowledge of the extent to which animals living under varying conditions can achieve catch-up growth for a bad start in life is limited. Here, we investigated how body mass at the age of 8 months influenced adult body mass in a moose Alces alces population living under excellent environmental conditions on the island of Vega in northern Norway. We also investigated if mother age and birth date effects on calf body mass persisted until adulthood. We show that neither males nor females were able to show compensatory growth before they reached adulthood, and that part of the variation in adult body mass may have been due to variation in mother age and date of birth. The pattern observed in males can be related to their growth strategy in relation to reproduction, while such results were not expected in females where size-dependent start of reproduction is likely to interact with body growth. We suggest that the good environmental conditions experienced on Vega led to females having small somatic costs of an early start of reproduction or that larger females were able to acquire more resources for growth than their smaller conspecifics. In both cases, females that postpone their first reproduction may not be able to achieve catch-up growth for their lower early body mass compared to females that start reproduction at an early age. Our results concur with previous studies indicating that compensatory growth is weak in moose, increasing the likelihood that variation in life history characters are also transferred between generations. 相似文献
970.
Nielsen MM Seo ES Bozonnet S Aghajari N Robert X Haser R Svensson B 《FEBS letters》2008,582(17):2567-2571
Certain starch hydrolases possess secondary carbohydrate binding sites outside of the active site, suggesting that multi-site substrate interactions are functionally significant. In barley alpha-amylase both Tyr380, situated on a remote non-catalytic domain, and Tyr105 in subsite -6 of the active site cleft are principal carbohydrate binding residues. The dual active site/secondary site mutants Y105A/Y380A and Y105A/Y380M show that each of Tyr380 and Tyr105 is important, albeit not essential for binding, degradation, and multiple attack on polysaccharides, while Tyr105 predominates in oligosaccharide hydrolysis. Additional delicate structure/function relationships of the secondary site are uncovered using Y380A/H395A, Y380A, and H395A AMY1 mutants. 相似文献