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161.
Restrepo BI Gomez DI Shipley GL McCormick JB Fisher-Hoch SP 《Journal of microbiological methods》2006,67(2):220-229
A major challenge for tuberculosis control is mycobacterial detection in paucibacillary disease, particularly in pediatric, extrapulmonary and smear-negative pulmonary infections. We developed a simple and efficient DNA extraction and real-time quantitative PCR (qPCR) protocol for mycobacterial detection and quantification in paucibacillary specimens. The method was refined using an in vitro model mimicking blood specimens which are characterized by the presence of numerous qPCR inhibitors. Mycobacterial DNA detection in blood is of interest given the high sensitivity we previously reported using conventional PCR in blood of patients with tuberculosis lymphadenitis. Mechanical lysis of mycobacteria in the presence of an organic solvent provided the highest sensitivity. Mycobacterial DNA amplification was compromised when the human:bacterial genome ratio was at least 190:1. Separation of the specimen into bacterial- and host-rich fractions prior to DNA extraction improved mycobacterial DNA detection by 30%. Preliminary testing of our protocol in smear-negative, culture-positive specimens (gastric and lymph node aspirates, pleural and cerebrospinal fluid, and blood) confirmed the applicability of our technique to a range of paucibacillary specimens for the detection, quantification and speciation (M. tuberculosis versus M. avium) of mycobacteria, several weeks before culture results were available. Our protocol provides a novel, efficient and simple strategy to improve the performance of qPCR in paucibacillary specimens, including those with excess human DNA background. This tool is useful to study the pathophysiology of early pulmonary or occult tuberculosis, and for more rapid and accurate diagnosis in difficult to diagnose infections. 相似文献
162.
Almeida AJ Martins M Carmona JA Cano LE Restrepo A Leão C Rodrigues F 《Fungal genetics and biology : FG & B》2006,43(6):401-409
The present work focuses on the analysis of cell cycle progression of Paracoccidioides brasiliensis yeast cells under different environmental conditions. We optimized a flow cytometric technique for cell cycle profile analysis based on high resolution measurements of nuclear DNA. Exponentially growing cells in poor-defined or rich-complex nutritional environments showed an increased percentage of daughter cells in accordance with the fungus' multiple budding and high growth rate. During the stationary growth-phase cell cycle progression in rich-complex medium was characterized by an accumulation of cells with higher DNA content or pseudohyphae-like structures, whereas in poor-defined medium arrested cells mainly displayed two DNA contents. Furthermore, the fungicide benomyl induced an arrest of the cell cycle with accumulation of cells presenting high and varying DNA contents, consistent with this fungus' unique pattern of cellular division. Altogether, our findings seem to indicate that P. brasiliensis may possess alternative control mechanisms during cell growth to manage multiple budding and its multinucleate nature. 相似文献
163.
Dai Q Restrepo BI Porcella SF Raffel SJ Schwan TG Barbour AG 《Molecular microbiology》2006,60(6):1329-1343
The relapsing fever agent Borrelia hermsii undergoes multiphasic antigenic variation through gene conversion of a unique expression site on a linear plasmid by an archived variable antigen gene. To further characterize this mechanism we assessed the repertoire and organization of archived variable antigen genes by sequencing approximately 85% of plasmids bearing these genes. Most archived genes shared with the expressed gene a 相似文献
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165.
Landsliding is a natural process influencing montane ecosystems, particularly in areas with elevated rainfall and seismic activity. Yet, to date, little effort has been made to quantify the contribution of this process to land‐cover change. Focusing on the mountains of Mexico and Central America (M‐CA), we estimated the contribution of landsliding to land‐cover change at two scales. At the scale of M‐CA, we classified the terrain into major landforms and entered in a GIS historical data on earthquake‐ and rainfall‐triggered landslides. At the scale of the Sierra de Las Minas of Guatemala, we investigated Landsat TM data to map rainfall‐triggered landslides. During the past 110 yr, >136,200 ha of land in the mountains of M‐CA have been affected by landslides, which translates into disturbance rates exceeding 0.317 percent/century. In Sierra de Las Minas, rainfall associated with hurricane Mitch affected 1765 ha of forest, or equivalently, landslides triggered by storms of this magnitude transformed between 0.196 (return time of 500 yr) and 1.290 (return time of 75 yr) percent of forest/century. Although landsliding results in smaller rates of land‐cover change than deforestation, we hypothesize that it has a stronger impact on ecosystems, both in qualitative and quantitative terms, given its influence on vegetation and soil. Moreover, interactions between landsliding and deforestation may be altering the expression of this complex process such that the few protected areas in the mountains of M‐CA may represent the only possibility for the conservation of this process. 相似文献
166.
Carolina Gonzalez-Silva Jorge Vera María Rosa Bono Christian González-Billault Brooke Baxter Anne Hansen Robert Lopez Emily A. Gibson Diego Restrepo Juan Bacigalupo 《PloS one》2013,8(7)
The Ca2+-activated Cl− channel is considered a key constituent of odor transduction. Odorant binding to a specific receptor in the cilia of olfactory sensory neurons (OSNs) triggers a cAMP cascade that mediates the opening of a cationic cyclic nucleotide-gated channel (CNG), allowing Ca2+ influx. Ca2+ ions activate Cl− channels, generating a significant Cl− efflux, with a large contribution to the receptor potential. The Anoctamin 2 channel (ANO2) is a major constituent of the Cl− conductance, but its knock-out has no impairment of behavior and only slightly reduces field potential odorant responses of the olfactory epithelium. Likely, an additional Ca2+-activated Cl− channel of unknown molecular identity is also involved. In addition to ANO2, we detected two members of the ClCa family of Ca2+-activated Cl− channels in the rat olfactory epithelium, ClCa4l and ClCa2. These channels, also expressed in the central nervous system, may correspond to odorant transduction channels. Whole Sprague Dawley olfactory epithelium nested RT-PCR and single OSNs established that the mRNAs of both channels are expressed in OSNs. Real time RT-PCR and full length sequencing of amplified ClCa expressed in rat olfactory epithelium indicated that ClCa4l is the most abundant. Immunoblotting with an antibody recognizing both channels revealed immunoreactivity in the ciliary membrane. Immunochemistry of olfactory epithelium and OSNs confirmed their ciliary presence in a subset of olfactory sensory neurons. The evidence suggests that ClCa4l and ClCa2 might play a role in odorant transduction in rat olfactory cilia. 相似文献
167.
Synchronized firing of mitral cells (MCs) in the olfactory bulb (OB) has been hypothesized to help bind information together in olfactory cortex (OC). In this survey of synchronized firing by suspected MCs in awake, behaving vertebrates, we find the surprising result that synchronized firing conveys information on odor value ("Is it rewarded?") rather than odor identity ("What is the odor?"). We observed that as?mice learned to discriminate between odors, synchronous firing responses to the rewarded and unrewarded odors became divergent. Furthermore, adrenergic blockage decreases the magnitude of odor divergence of synchronous trains, suggesting that MCs contribute to decision-making through adrenergic-modulated synchronized firing. Thus, in the olfactory system information on stimulus reward is found in MCs one synapse away from the sensory neuron. 相似文献
168.
169.
The interplay between olfactory activity and cholinergic modulation remains to be fully understood. This report examines the pattern of cholinergic innervation throughout the murine main olfactory bulb across different developmental stages and in naris-occluded animals. To visualize the pattern of cholinergic innervation, we used a transgenic mouse model, which expresses a fusion of the microtubule-associated protein, tau, with green fluorescence protein (GFP) under the control of the choline acetyltransferase (ChAT) promoter. This tau-GFP fusion product allows for a remarkably vivid and clear visualization of cholinergic innervation in the main olfactory bulb (MOB). Interestingly, we find an uneven distribution of GFP label in the adult glomerular layer (GL), where anterior, medial, and lateral glomerular regions of the bulb receive relatively heavier cholinergic innervation than other regions. In contrast to previous reports, we find a marked change in the pattern of cholinergic innervation to the GL following unilateral naris occlusion between the ipsilateral and contralateral bulbs in adult animals. 相似文献
170.
Carla GS Saad Ana CM Ribeiro Julio CB Moraes Liliam Takayama Celio R Goncalves Marcelo B Rodrigues Ricardo M de Oliveira Clovis A Silva Eloisa Bonfa Rosa MR Pereira 《Arthritis research & therapy》2012,14(5):R216