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41.
Vincenza Dolo Sandra D'Ascenzo Maurizio Sorice Antonio Pavan Mariateresa Sciannamblo Alessandro Prinetti Vanna Chigorno Guido Tettamanti Sandro Sonnino 《Glycoconjugate journal》2000,17(3-4):261-268
This paper is the first report on the use of the electron microscopy autoradiography technique to detect metabolically tritium labeled sphingolipids in intact cells in culture.To label cell sphingolipids, human fibroblasts in culture were fed by a 24 hours pulse, repeated 5 times, of 3×10–7 M [1-3H]sphingosine. [1-3H]sphingosine was efficently taken up by the cells and very rapidly used for the biosynthesis of complex sphingolipids, including neutral glycolipids, gangliosides, ceramide and sphingomyelin. The treatment with [1-3H]sphingosine did not induce any morphological alteration of cell structures, and well preserved cells, plasma membranes, and intracellular organelles could be observed by microscopy.Ultrathin sections from metabolic radiolabeled cells were coated with autoradiographic emulsion. One to four weeks of exposition resulted in pictures where the location of radioactive sphingolipids was evidenced by the characteristic appearance of silver grains as irregular coiled ribbons of metallic silver. Radioactive sphingolipids were found at the level of the plasma membranes, on the endoplasmic reticulum and inside of cytoplasmic vesicles. Thus, electron microscopy autoradiography is a very useful technique to study sphingolipid-enriched membrane domain organization and biosynthesis. 相似文献
42.
Sandro L. Pereira Mário Gr?os Ana Sofia Rodrigues Sandra I. Anjo Rui A. Carvalho Paulo J. Oliveira Ernest Arenas Jo?o Ramalho-Santos 《PloS one》2013,8(12)
The mitochondrion is emerging as a key organelle in stem cell biology, acting as a regulator of stem cell pluripotency and differentiation. In this study we sought to understand the effect of mitochondrial complex III inhibition during neuronal differentiation of mouse embryonic stem cells. When exposed to antimycin A, a specific complex III inhibitor, embryonic stem cells failed to differentiate into dopaminergic neurons, maintaining high Oct4 levels even when subjected to a specific differentiation protocol. Mitochondrial inhibition affected distinct populations of cells present in culture, inducing cell loss in differentiated cells, but not inducing apoptosis in mouse embryonic stem cells. A reduction in overall proliferation rate was observed, corresponding to a slight arrest in S phase. Moreover, antimycin A treatment induced a consistent increase in HIF-1α protein levels. The present work demonstrates that mitochondrial metabolism is critical for neuronal differentiation and emphasizes that modulation of mitochondrial functions through pharmacological approaches can be useful in the context of controlling stem cell maintenance/differentiation. 相似文献
43.
Ricardo D. Ekmay Catalina Salas Judy England Sandro Cerrate Craig N. Coon 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2013,164(1):38-43
A study was conducted to determine the changes that occur to proteolysis and related genes due to age, protein, and energy intake in high-yield broiler breeder hens (Gallus gallus). Cobb 700 broiler breeders were randomly assigned to one of six diets in a 2 × 3 factorial fashion. Two levels of energy (390 and 450 kcal/day) and three levels of protein (22, 24, and 26 g CP/day) were utilized. Protein turnover was determined in the left pectoralis at 22, 26, 31 and 44 weeks. Relative mRNA expression of calpain 2 (CAPN2), proteasome C2 subunit (PSMA1), and F box protein 32 (FBXO32) were determined via RT-PCR at 20, 25, and 44 weeks. Contrasts indicate fractional synthesis rate (FSR) and FBXO32 increase to a maximum at 25–26 weeks and a decrease thereafter. A significant drop in PSMA1 and FBXO32 was observed between 25 and 44 weeks and matched the decrease observed in FBR. No differences were detected in the levels of fractional synthesis and degradation, or the expression of CAPN2, PSMA1, and FBXO32, due to protein or energy intake. In summary, protein turnover was upregulated during the transition into sexual maturity and decreased thereafter. The observed changes in degradation appeared to be mediated by the ubiquitin–proteasome pathway. 相似文献
44.
Kerstin Vocke Kristin Dauner Anne Hahn Anne Ulbrich Jana Broecker Sandro Keller Stephan Frings Frank M?hrlen 《The Journal of general physiology》2013,142(4):381-404
Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca2+/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca2+/calmodulin, one at submicromolar Ca2+ concentrations and one in the micromolar Ca2+ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca2+/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca2+ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca2+ regulation in anoctamin Cl− channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types. 相似文献
45.
Casalino L Magnani D De Falco S Filosa S Minchiotti G Patriarca EJ De Cesare D 《Molecular biotechnology》2012,50(3):171-180
The use of Embryonic Stem Cells (ESCs) holds considerable promise both for drug discovery programs and the treatment of degenerative
disorders in regenerative medicine approaches. Nevertheless, the successful use of ESCs is still limited by the lack of efficient
control of ESC self-renewal and differentiation capabilities. In this context, the possibility to modulate ESC biological
properties and to obtain homogenous populations of correctly specified cells will help developing physiologically relevant
screens, designed for the identification of stem cell modulators. Here, we developed a high throughput screening-suitable
ESC neural differentiation assay by exploiting the Cell
maker robotic platform and demonstrated that neural progenies can be generated from ESCs in complete automation, with high standards
of accuracy and reliability. Moreover, we performed a pilot screening providing proof of concept that this assay allows the
identification of regulators of ESC neural differentiation in full automation. 相似文献
46.
Grasielle Pereira Jannuzzi Nicole de Araújo Souza Kátia Sanches Françoso Roney Henrique Pereira Raquel Possemozer Santos Gilberto Hideo Kaihami José Roberto Fogaça de Almeida Wagner Luiz Batista André Corrêa Amaral Andrea Queiroz Maranhão Sandro Rogério de Almeida Karen Spadari Ferreira 《Microbes and infection / Institut Pasteur》2018,20(1):48-56
Paracoccidioidomycosis (PCM) is a systemic mycosis with lymphatic dissemination that is caused by Paracoccidioides species. Treatment of PCM consists of chemotherapeutics such as itraconazole, trimethoprim, sulfamethoxazole or amphotericin B. However, several studies are aiming to develop therapeutic alternatives for the treatment of fungal infection using new molecules as adjuvants. The single-chain variable fragments (scFv) from an antibody that mimics the main fungal component incorporated within poly(lactide-co-glycolic) acid (PLGA) nanoparticles helped treat the fungal disease. After expressing the scFv in Picchia pastoris (P. pastoris), the recombinant molecules were coupled with PLGA, and the BALB/c mice were immunized before or after infection with yeast Paracoccidioides brasiliensis (P. brasiliensis). Our results showed decreased disease progression and decreased fungal burden. Taken together, our results showed an increased of IFN-γ and IL-12 cytokine production and an increased number of macrophages and dendritic cells in the pulmonary tissue of BALB/c mice treated with a high concentration of our molecule. Our data further confirm that the scFv plays an important role in the treatment of experimental PCM. 相似文献
47.
Ande SR Kommoju PR Draxl S Murkovic M Macheroux P Ghisla S Ferrando-May E 《Apoptosis : an international journal on programmed cell death》2006,11(8):1439-1451
L-amino acid oxidase (LAAO) from the Malayan pit viper induces both necrosis and apoptosis in Jurkat cells. Cell death by
necrosis is attributed to H2O2 produced by oxidation of α-amino acids. In the presence of catalase that effectively scavenges H2O2, a switch to apoptosis is observed. The major factors contributing to apoptosis are proposed to be: (i) generation of toxic
intermediates from fetal calf serum (ii) binding and internalization of LAAO. The latter process appears to be mediated by
the glycan moiety of the enzyme as desialylation reduces cytotoxicity. D-amino acid oxidase (DAAO), which catalyzes the same
reaction as LAAO but lacks glycosylation, triggers necrosis as a consequence of H2O2 production but not apoptosis in the presence of catalase. Thus induction of cell death by LAAO appears to involve both the
generation of H2O2 and the molecular interaction of the glycan moiety of the enzyme with structures at the cell surface.
S. R. Ande, P. R. Kommoju contributed equally to this work. 相似文献
48.
Chigorno V Giannotta C Ottico E Sciannamblo M Mikulak J Prinetti A Sonnino S 《The Journal of biological chemistry》2005,280(4):2668-2675
Human fibroblasts, rat neurons, and murine neuroblastoma cells, cultured in the presence of fetal calf serum, were fed with [1-(3)H]sphingosine to radiolabel sphingolipids. The fate of cell sphingolipids, the release of sphingolipids in the culture medium, the interaction of sphingolipids with the proteins and lipoproteins of fetal calf serum, and the fate of sphingolipids taken up by the cells were investigated. For this latter purpose, the culture medium containing radioactive sphingolipids was delivered to nonlabeled cells. The presence of tritium at position 1 of sphingosine allowed us to follow the extent of sphingolipid catabolism by measuring the production of radioactive phosphatidylethanolamine and proteins by recycling the radioactive ethanolamine formed during sphingosine catabolism and the production of tritiated water. We confirmed that in cells the recycling of sphingosine occurred to a high extent and that only a minor portion of cell sphingolipids was catabolized to the small fragments of ethanolamine and water. Cell sphingolipids were released in the culture medium, where they formed large lipoproteic aggregates at a rate of about 12% per day. Released sphingolipids were taken up by the cells and catabolized to the sphingosine and then to ethanolamine, and recycling of sphingosine was not observed. This suggests that in the presence of fetal calf serum in the culture medium, exogenous sphingolipids directly reach the lysosomes, were they are entirely catabolized. Thus, the trafficking of sphingolipids from cells to the extracellular environment and from this to other cells does not allow the modification of the plasma membrane composition. 相似文献
49.
Felipe G. Grazziotin Hussam Zaher Robert W. Murphy Gustavo Scrocchi Marco A. Benavides Ya‐Ping Zhang Sandro L. Bonatto 《Cladistics : the international journal of the Willi Hennig Society》2012,28(5):437-459
We present a phylogenetic analysis of the New World dipsadids based on an expanded data matrix that includes 246 terminal taxa including 196 dipsadids. The species are sampled for eight genes (12S, 16S, cytb, nd2, nd4, bdnf, c‐mos, rag2). The data are explored using two distinct optimality procedures—maximum parsimony and maximum likelihood—and two alignment strategies—dynamic homology and static homology. Two previously unsampled dipsadid genera, Sordellina and Rhachidelus, are now included in the analysis. The definitions of the genera, Erythrolamprus, Clelia, Hypsirhynchus, Philodryas and Phimophis, and the tribes Alsophiini, Echinantherini and Conophiini, are revised. In order to maintain monophyly, the genus Umbrivaga is synonymized with Erythrolamprus, and two new genera are erected to accommodate Phimophis iglesiasi and Clelia rustica, as well as their closely related species. The West Indian genera Schwartzophis, Darlingtonia, Antillophis and Ocyophis are resurrected. © The Willi Hennig Society 2012. 相似文献
50.