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951.
Penning TD Russell MA Chen BB Chen HY Desai BN Docter SH Edwards DJ Gesicki GJ Liang CD Malecha JW Yu SS Engleman VW Freeman SK Hanneke ML Shannon KE Westlin MM Nickols GA 《Bioorganic & medicinal chemistry letters》2004,14(6):1471-1476
We describe a series of conformationally-restricted cinnamic acid peptidomimetics as well as several cinnamic acid isosteres, including 3-phenylpropionic acids, 2-amino-3-phenylpropionic acids, phenoxyacetic acids and 2-phenylcyclopropylcarboxylic acids. Several analogues demonstrated low to sub-nanomolar potencies against alpha(v)beta(3) and greater than 200-fold selectivity against the other beta(3) integrin alpha(IIb)beta(3). In whole 293 cells, many of these analogues also showed modest selectivity against other alpha(v) integrins such as alpha(v)beta(1) and alpha(v)beta(5). These compounds were synthesized from readily available starting materials using either Heck or Mitsunobu coupling conditions. 相似文献
952.
Fritzen M Flores MP Reis CV Chudzinski-Tavassi AM 《Biochemical and biophysical research communications》2005,333(2):517-523
A severe hemorrhagic syndrome produced by contact with Lonomia obliqua caterpillars has become epidemic in southern Brazil. A significant thrombin production with intense consumption of fibrinogen and high D-dimer production indicates a consumption coagulopathy and secondary fibrinolysis in patients. Lopap is a single-chain 69kDa serine protease isolated from the crude extract of L. obliqua bristles. Experiments in mice showed that the purified protein, similar to the crude extract, causes uncoagulable blood by fibrinogen depletion. In order to characterize the effects of Lopap on cells involved with hemostatic system, we performed experiments using human umbilical vein endothelial cells (HUVECs). Our results show that Lopap exerts a direct effect on endothelial cells by increasing the liberation of molecules involved in the regulation of vascular tone, inhibiting platelet activation and chemotaxis, apart from inducing the expression of cell adhesion molecules which participate in inflammatory responses. The release or new synthesis of mediators involved in coagulation as von Willebrand factor and tissue factor, or in fibrinolysis as tissue plasminogen activator, was not affected by Lopap. Also our results demonstrated that Lopap acts on cell survival of HUVECs, regulating the expression of molecules as NO and avoiding cell death. 相似文献
953.
Masip I Cortés N Abad MJ Guardiola M Pérez-Payá E Ferragut J Ferrer-Montiel A Messeguer A 《Bioorganic & medicinal chemistry》2005,13(6):1923-1929
Herein is reported the optimized solid-phase synthesis of a library of 5,120 trimeric N-alkylglycines (peptoids) using the positional scanning format and the submonomer strategy. Diversity at the N-terminal position was generated from 20 commercially available primary amines, whereas 16 primary amines were employed for the middle and C-terminal positions of the trimers. Formation of undesirable side-products observed in a previous library synthesis (Humet, M. et al. J. Comb. Chem. 2003, 5, 597-605) was averted by restricting the use of primary amines functionalized with tertiary amino groups to the third amination step. Screening of the new library for the identification of chemosensitizers yielded two peptoids, compounds 1 and 2, with potent in vitro activity as multidrug resistance (MDR) reversal agents. The structures of the lead peptoids are consistent with a pharmacophore model generated from the interaction of various known inhibitors with the MDR-implicated transmembrane glycoprotein P-gp. 相似文献
954.
In vitro and in vivo mutational analysis of the 3'-terminal regions of hepatitis e virus genomes and replicons
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Graff J Nguyen H Kasorndorkbua C Halbur PG St Claire M Purcell RH Emerson SU 《Journal of virology》2005,79(2):1017-1026
Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3'-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3'-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3'-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3' termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3' terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3'-terminal mutations in vivo. Incorporation of the 3'-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine. 相似文献
955.
The generalized transducing Salmonella bacteriophage ES18: complete genome sequence and DNA packaging strategy
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Casjens SR Gilcrease EB Winn-Stapley DA Schicklmaier P Schmieger H Pedulla ML Ford ME Houtz JM Hatfull GF Hendrix RW 《Journal of bacteriology》2005,187(3):1091-1104
The generalized transducing double-stranded DNA bacteriophage ES18 has an icosahedral head and a long noncontractile tail, and it infects both rough and smooth Salmonella enterica strains. We report here the complete 46,900-bp genome nucleotide sequence and provide an analysis of the sequence. Its 79 genes and their organization clearly show that ES18 is a member of the lambda-like (lambdoid) phage group; however, it contains a novel set of genes that program assembly of the virion head. Most of its integration-excision, immunity, Nin region, and lysis genes are nearly identical to those of the short-tailed Salmonella phage P22, while other early genes are nearly identical to Escherichia coli phages lambda and HK97, S. enterica phage ST64T, or a Shigella flexneri prophage. Some of the ES18 late genes are novel, while others are most closely related to phages HK97, lambda, or N15. Thus, the ES18 genome is mosaically related to other lambdoid phages, as is typical for all group members. Analysis of virion DNA showed that it is circularly permuted and about 10% terminally redundant and that initiation of DNA packaging series occurs across an approximately 1-kbp region rather than at a precise location on the genome. This supports a model in which ES18 terminase can move substantial distances along the DNA between recognition and cleavage of DNA destined to be packaged. Bioinformatic analysis of large terminase subunits shows that the different functional classes of phage-encoded terminases can usually be predicted from their amino acid sequence. 相似文献
956.
Brini M Manni S Pierobon N Du GG Sharma P MacLennan DH Carafoli E 《The Journal of biological chemistry》2005,280(15):15380-15389
Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca(2+) release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca(2+) pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca(2+) homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca(2+) revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca(2+) transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca(2+) derangements of different degrees account for the specific cellular phenotypes of the two disorders. 相似文献
957.
Cytometric bead array (CBA) for the measurement of cytokines in urine and plasma of patients undergoing renal rejection 总被引:4,自引:0,他引:4
Jiménez R Ramírez R Carracedo J Agüera M Navarro D Santamaría R Pérez R Del Castillo D Aljama P 《Cytokine》2005,32(1):45-50
Renal rejection is associated with an active immune response regulated by cytokines and in which immunocompetent cells are involved. Previous studies have measured high levels of cytokines in the urine and plasma in various renal dysfunction states. However, some methods used to measured cytokines hinder their use as a diagnostic tool in renal rejection. In this report, cytokine levels were determined in the plasma and urine of kidney transplant patients, with renal rejection and without it, using a cytometric bead array (CBA) technique. Concentrations of six human cytokines (IL-2, IL-4, IL-5, IL-10, TNF-alpha and INF-gamma) were established. Results show that patients who develop renal rejection presented high levels of IL-10 and IFN-gamma cytokines in plasma and urine compared to patients without renal rejection. The CBA technique displayed greater sensitivity in the determination of cytokines in urine than the conventional ELISA technique. Finally, when standard cytokines in plasma and in urine were compared, it was observed that, in plasma, levels of IL-4, IL-5, IL-10, TNF-alpha and IFN-gamma were detected, whereas in urine the levels detected were of IL-4, IL-5, IL-10 and IFN-gamma. These results indicate that the CBA assay is a sensitive method to measure cytokines in urine. In kidney transplant patients undergoing acute renal rejection, the presence of cytokines in urine reflects renal damage and could be a useful method in the diagnosis of renal rejection. 相似文献
958.
Schattner M Fritzen M Ventura Jde S de Albuquerque Modesto JC Pozner RG Moura-da-Silva AM Chudzinski-Tavassi AM 《Biological chemistry》2005,386(4):369-374
PIII snake venom metalloproteases (SVMPs) are metalloproteases structurally related to ADAMs (a disintegrin and metalloprotease human family of proteins). Berythractivase and jararhagin are PIII SVMPs with 69% homology that have different hemostatic properties. In order to clarify these differences and further characterize the biological effects of these proteins, we have analyzed the effect of both proteases on human umbilical-vein endothelial cell functions. We found that both proteins enhanced nitric oxide generation, prostacyclin production and interleukin-8 release. Berythractivase but not jararhagin increased the expression of decay accelerating factor. Jararhagin decreased cell viability in a concentration-dependent manner and induced cellular apoptosis, while berythractivase did not modulate cell survival. Our results show for the first time that, besides the known anti-aggregating or procoagulant effects of PIII SVMPs, these proteins trigger endothelial cell effector responses. Although structurally related, berythractivase and jararhagin induce a dissimilar generation and release of endothelial molecules that may account for their different hemorrhagic activity. 相似文献
959.
Nonviral genetic modification mediates effective transgene expression and functional RNA interference in human mesenchymal stem cells 总被引:6,自引:0,他引:6
Hoelters J Ciccarella M Drechsel M Geissler C Gülkan H Böcker W Schieker M Jochum M Neth P 《The journal of gene medicine》2005,7(6):718-728
BACKGROUND: Human mesenchymal stem cells (hMSC) are increasingly the focus of both basic and clinical research due to their ability to strike a balance between self-renewal and commitment to mesodermal differentiation. However, the promising therapeutic utility of hMSC in regenerative medical approaches requires detailed knowledge about their molecular characteristics. Therefore, genetic modification of hMSC provides a powerful tool to understand their complex molecular regulation mechanisms. METHODS: Here we describe a proof of concept approach of separate and combined gene transfer and gene silencing by nonviral DNA transfection of enhanced green fluorescent protein (EGFP) and EGFP-targeted small interfering RNAs (siRNAs) in hMSC. For optimization of nonviral DNA and siRNA transfer different liposomal-based transfection strategies were validated. RESULTS: The highest fraction of EGFP-expressing hMSC was obtained using Lipofectamine 2000 (50%) which also mediated the highest transfection rates of siRNAs into hMSC (>or=92%). Stably EGFP-expressing hMSC maintained their proliferation capacity paired with the ability to differentiate into different mesodermal lineages (bone, cartilage, and fat) without loss of transgene expression. Based on our nonviral nucleic acid delivery technique we showed efficient, functional, and long-term RNA interference (RNAi) in hMSC by gene specific knock-down of transiently and stably expressed EGFP (88-98%). CONCLUSIONS: This is the first demonstration of efficient nonviral transfer of both nucleic acids (DNA and siRNA) into hMSC, exhibiting the potential of targeted modification of hMSC. In particular, the combination of these techniques represents a powerful gene transfer/silencing strategy, thus facilitating detailed genetic approaches to study regulatory networks in stem cell differentiation processes. 相似文献
960.
Approximating identity-by-descent matrices using multiple haplotype configurations on pedigrees
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Identity-by-descent (IBD) matrix calculation is an important step in quantitative trait loci (QTL) analysis using variance component models. To calculate IBD matrices efficiently for large pedigrees with large numbers of loci, an approximation method based on the reconstruction of haplotype configurations for the pedigrees is proposed. The method uses a subset of haplotype configurations with high likelihoods identified by a haplotyping method. The new method is compared with a Markov chain Monte Carlo (MCMC) method (Loki) in terms of QTL mapping performance on simulated pedigrees. Both methods yield almost identical results for the estimation of QTL positions and variance parameters, while the new method is much more computationally efficient than the MCMC approach for large pedigrees and large numbers of loci. The proposed method is also compared with an exact method (Merlin) in small simulated pedigrees, where both methods produce nearly identical estimates of position-specific kinship coefficients. The new method can be used for fine mapping with joint linkage disequilibrium and linkage analysis, which improves the power and accuracy of QTL mapping. 相似文献