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排序方式: 共有338条查询结果,搜索用时 34 毫秒
31.
Koduru S Vegiraju SR Nadimpalli SK von Figura K Pohlmann R Dennes A 《Development genes and evolution》2006,216(3):133-143
Mannose-6-phosphate receptors (MPRs) have been identified in a wide range of species from humans to invertebrates such as
molluscs. A characteristic of all MPRs is their common property to recognize mannose-6-phosphate residues that are labelling
lysosomal enzymes and to mediate their targeting to lysosomes in mammalian cells by the corresponding receptor proteins. We
present here the analysis of full-length sequences for MPR 46 from zebrafish (Danio rerio) and its functional analysis. This is the first non-mammalian MPR 46 to be characterised. The amino acid sequences of the
zebrafish MPR 46 displays 70% similarity to the human MPR 46 protein. In particular, all essential cysteine residues, the
transmembrane domain as well as the cytoplasmic tail residues harbouring the signals for endocytosis and Golgi-localizing,
γ-ear-containing, ARF-binding protein (GGA)-mediated sorting at the trans-Golgi network, are highly conserved. The zebrafish
MPR 46 has the arginine residue known to be essential for mannose-6-phosphate binding and other additional characteristic
residues of the mannose-6-phosphate ligand-binding pocket. Like the mammalian MPR 46, zebrafish MPR 46 binds to the multimeric
mannose-6-phosphate ligand phosphomannan and can rescue the missorting of lysosomal enzymes in mammalian MPR-deficient cells.
The conserved C-terminal acidic dileucine motif (DxxLL) in the cytoplasmic domain of zebrafish MPR 46 essential for the interaction
of the GGAs with the receptor domains interacts with the human GGA1-VHS domain. Interestingly, the serine residue suggested
to regulate the interaction between the tail and the GGAs in a phosphorylation-dependent manner is substituted by a proline
residue in fish.
Electronic Supplementary Material Supplementary material is available for this article at .
The zebrafish MPR 46 sequence data have been submitted to the GenBank database under accession no. DQ089037. 相似文献
32.
Koldzic-Zivanovic N Seitz PK Cunningham KA Thomas ML Hughes TK 《Cellular and molecular neurobiology》2006,26(4-6):977-985
1. Aim: The role of the serotonin transporter (SERT) is to remove serotonin (5-HT) from the synaptic space. In vitro studies have shown that 5-HT uptake via SERT is influenced by the availability of its substrate, 5-HT. We used RN46A cells, a line that expresses SERT, to investigate 5-HT regulation of 5-HT uptake and the intracellular signaling pathways involved. RN46A cells also express mRNAs for 5-HT receptors (5-HT1A, 5-HT1B, 5-HT2A, and 5-HT2C) and as cAMP and intracellular Ca2+ are modulated by different 5-HT receptors, we studied both pathways.2. Methods: 5-HT uptake was determined as imipramine-inhibitable uptake of [3H]5-HT, intracellular cAMP was measured by RIA and intracellular Ca2+ changes were determined using the ratiometric method of intracellular Ca2+ imaging.3. Results: For uptake experiments, cells were kept for 30 min either with or without 1 μM 5-HT in the medium before measuring uptake. Removal of 5-HT for 30 min significantly decreased [3H]5-HT uptake. The absence of 5-HT for 15 min failed to induce any changes in intracellular cAMP levels. Removal of 5-HT from the medium did not change intracellular Ca2+ levels either; however, adding 1 μM 5-HT after 5 min in 5-HT-free conditions rapidly increased intracellular Ca2+ levels in 50% of the cells. The remaining cells showed no changes in the intracellular Ca2+ levels.4. Conclusions: We have shown that in RN46A cells, that endogenously express SERT and mRNAs for several 5-HT receptors, changes in 5-HT levels influence 5-HT uptake rate as well as induce changes in intracellular Ca2+ levels. This suggests that 5-HT may utilize intracellular Ca2+ to regulate 5-HT uptake. 相似文献
33.
Friedman M Orlova A Johansson E Eriksson TL Höidén-Guthenberg I Tolmachev V Nilsson FY Ståhl S 《Journal of molecular biology》2008,376(5):1388-1402
The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (Kd = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, Kd, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (Kd = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection. 相似文献
34.
Carla Carluccio Francesco Salvatore Arianna Fornili 《Journal of biomolecular structure & dynamics》2016,34(3):497-507
The enzyme phenylalanine hydroxylase (PAH) is defective in the inherited disorder phenylketonuria. PAH, a tetrameric enzyme, is highly regulated and displays positive cooperativity for its substrate, Phe. Whether Phe binds to an allosteric site is a matter of debate, despite several studies worldwide. To address this issue, we generated a dimeric model for Phe–PAH interactions, by performing molecular docking combined with molecular dynamics simulations on human and rat wild-type sequences and also on a human G46S mutant. Our results suggest that the allosteric Phe-binding site lies at the dimeric interface between the regulatory and the catalytic domains of two adjacent subunits. The structural and dynamical features of the site were characterized in depth and described. Interestingly, our findings provide evidence for lower allosteric Phe-binding ability of the G46S mutant than the human wild-type enzyme. This also explains the disease-causing nature of this mutant. 相似文献
35.
Hiroyuki Kimura Saki Yamauchi Hidekazu Kawashima Kenji Arimitsu Yusuke Yagi Yuji Nakamoto Kaori Togashi Masahiro Ono Hideo Saji 《Bioorganic & medicinal chemistry letters》2018,28(17):2949-2952
The tripeptide formyl–Met–Leu–Phe (fMLF) is a prototype of N-formylated chemotactic peptides for neutrophils owing to its ability to bind and activate the G protein-coupled formyl peptide receptor (FPR). Here, we developed an 18F-labeled fMLF derivative targeting FPR as a positron emission tomography (PET) imaging probe for bacterial infections. The study demonstrates that the fMLF derivative fMLFXYk(FB)k (X?=?Nle) has a high affinity for FPR (Ki?=?0.62?±?0.13?nM). The radiochemical yield and purity of [18F]fMLFXYk(FB)k were 16% and >96%, respectively. The in vivo biodistribution study showed that [18F]fMLFXYk(FB)k uptake was higher in the bacterial infected region than in the non-infected region. We observed considerably higher infection-to-muscle ratio of 4.6 at 60?min after [18F]fMLFXYk(FB)k injection. Furthermore, small-animal PET imaging studies suggested that [18F]fMLFXYk(FB)k uptake in the bacterial infected region was clearly visualized 60?min after injection. 相似文献
36.
37.
Tove Olafsen Charlotte K. Munthe Lund Øyvind S. Bruland Inger Sandlie Terje E. Michaelsen 《Cancer immunology, immunotherapy : CII》1999,48(7):411-418
Osteosarcoma is the commonest malignant tumour of the bones. The presence of micrometastases at the time of primary diagnosis
is associated with poor prognosis. Despite developments in surgery and aggressive chemotherapy, about 50% of the patients
still succumb to the disease. Thus, there is a need to develop alternative treatment modalities. One such strategy is to use
antibodies with improved effector functions. The two monoclonal antibodies, TP-1 and TP-3, recognize a tumour-associated antigen
on human osteosarcoma cells. In the present study, we describe the cloning of the TP-1 variable genes, and the production
of complete chimeric mouse/human monoclonal antibodies. Constructs containing the constant genes from human IgG1, IgG3 or
a mutant IgG3 with a shortened hinge region, called m15, were expressed in the mouse myeloma cell line, NS0. The m15 mutant
has been shown to be very potent in triggering complement-mediated lysis. Our goal was to investigate whether this mutant
could overcome the complement protection on human osteosarcoma cells, which is generally present on all human cells. We found
that the target cells expressed several membrane-bound complement inhibitors, and that masking of these inhibitors rendered
the cells sensitive to lysis. The m15 mutant exhibited greater lytic activity than both IgG3 and IgG1, although it could not
cause extensive killing of the target cells alone.
Received: 21 January 1999 / Accepted: 3 June 1999 相似文献
38.
Serpil Sar?özkan Mustafa Numan Bucak Pürhan Barbaros Tuncer Ali Bilgen 《Cryobiology》2009,58(2):134-890
Oxidative stress significantly damages sperm functions such as motility, functional integrity, endogenous antioxidant enzyme activities and fertility due to lipid peroxidation induced by reactive oxygen species (ROS). The aim of this study was to determine the effects of antioxidants such as taurine and cysteine in Bioxcell® extender on standard semen parameters, fertilizing ability, lipid peroxidation (LPO) and antioxidant activities comprising reduced glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) after the cryopreservation/thawing of bull semen. Nine ejaculates for each bull were included in the study. Three groups, namely taurine (2 mM), cysteine (2 mM), and control, were designed to analyze the antioxidants in Bioxcell®. Insemination doses were processed so that each 0.25-ml straw contained 15 × 106 sperm.The addition of cysteine led to higher motility, compared to the other groups (P < 0.001). Cysteine showed a greater protective effect on the percentages of acrosome damage and total abnormalities in comparison to the other groups (P < 0.001). No significant differences were observed in hypo-osmotic swelling test (HOST), following supplementation with antioxidants during the freeze-thawing process. No significant difference was observed in non-return rates among groups. In biochemical assays, the additives did not show effectiveness on the elimination of malondialdehyde (MDA) formation and maintenance of GSH and GSH-Px activities, when compared to controls. CAT activity (35.1 ± 8.1 kU/g) was demonstrated to be significantly higher upon the addition of 2 mM taurine (P < 0.001), while the level of MDA increased, indicating oxidative stress in this group. SOD activity (21.4 ± 2.9 U/g protein) was significantly elevated in the group with cysteine, compared to the other groups (P < 0.001). 相似文献
39.
Anja W. Fjorback Patrick Pla Heidi K. Müller Frédéric Saudou Jens R. Nyengaard 《Biochemical and biophysical research communications》2009,380(4):724-728
The serotonin transporter is a member of the monoamine transporter family that also includes transporters of dopamine and norepinephrine. We have used sensitized acceptor emission fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) to study the oligomerization of SERT in HEK-MSR-239 cells, RN46A cells and in cultured hippocampal neurons. We were able to show identical FRET efficiencies in cell lines as well as in primary cultured hippocampal neurons, demonstrating that the oligomerization is cell type independent. The results obtained with both FRET approaches are very similar and furthermore, in agreement with previous results obtained by donor bleaching FRET microscopy. 相似文献
40.
Stephanie Fanucchi 《FEBS letters》2009,583(22):3557-3562
A novel survival role of focal adhesion kinase (FAK) that involves its nuclear translocation and direct association with p53 has been demonstrated. Here we examined the relationship between the p53/FAK interaction and Ser46 phosphorylation of p53 (p-p53Ser46) in the apoptotic regulation of human esophageal squamous cell carcinoma (HOSCC) cell lines, expressing either wild type (wt) p53 or mutant (mt) p53-R175H. In contrast to the wt p53 cell lines, the mt p53-R175H cell line was resistant to staurosporine (STS)-mediated detachment and caspase-3 activation. Furthermore, despite the resistance of mt p53-R175H to Ser46 phosphorylation, both wt and mt HOSCC cells translocate FAK into the nucleus and maintain the p53/FAK interaction post STS treatment. These findings provide unique insight into how tumor cells harboring the R175H mutant may resist chemotherapeutic intervention.