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991.
992.
Bibhudatta Mishra Mostafa Ghannad-Rezaie Jiaxing Li Xin Wang Yan Hao Bing Ye Nikos Chronis Catherine A. Collins 《Journal of visualized experiments : JoVE》2014,(84)
Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the ''larva chip''. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush. 相似文献
993.
Agrobacterium tumefaciens -mediated transformation of soybean [Glycine max (L.) Merrill. cv. Jack] using immature zygotic cotyledons was investigated to identify important factors that affected transformation
efficiency and resulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature
zygotic cotyledon size, Agrobacterium concentration during inoculation and co-culture and the selection regime. Our results showed that 8- to 10-mm zygotic cotyledons
exhibited a higher transformation rate, as indicated by transient GUS gene expression, whereas the smaller zygotic cotyledons,
at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon explants were found to have a
higher embryogenic potential. Analysis of Agrobacterium and immature cotyledon explant interactions involved two Agrobacterium concentrations for the inoculation phase and three co-culture regimes. No differences in explant survival or somatic embyogenic
potential were observed between the two Agrobacterium concentrations tested. Analysis of co-culture regimes revealed that the shorter co-culture times resulted in higher explant
survival and higher somatic embryo production on the explants, whereas the co-culture time of 4 days severely reduced survival
of the cotyledon explants and lowered their embryogenic potential. Analysis of selection regimes revealed that direct placement
of cotyledon explants on hygromycin 25 mg/l was detrimental to explant survival, whereas 10 mg/l gave continued growth and
subsequent somatic embryo development and plant regeneration. The overall transformation frequency in these experiments, from
initial explant to whole plant, was 0.03 %. Three fertile soybean plants were obtained during the course of these experiments.
Enzymatic GUS assays and Southern blot hybridizations confirmed the integration of T-DNA and expression of the GUS-intron
gene in the three primary transformants. Analysis of 48 progeny revealed that three copies of the transgene were inherited
as a single Mendelian locus.
Received: 6 December 1999 / Revised: 11 February 2000 / Accepted: 14 March 2000 相似文献
994.
Andrew Collins 《Free radical research》2000,32(4):333-341
We are attempting to resolve some of the problems encountered in measuring 8-hydroxy-2'-deoxyguanosine (8-oxodG) in human cellular DNA as a marker of oxidative stress. Samples of authentic 8-oxodG were distributed, and participating laboratories undertook to analyse this material within a specified period. Most HPLC procedures gave values for 8-oxodG within ±40% of the target, as did two of four GC-MS procedures, and both LC-MS-MS methods. Calf thymus DNA samples containing increasing amounts of 8-oxodG were also distributed for analysis. Fewer than half the procedures tested were able to detect the dose response; those that were successful tended to be procedures with low coefficients of variation. For the analysis of 8-oxodG in human cells, where it is likely to be present at much lower concentrations than in the calf thymus DNA, it is crucial to reduce analytical variation to a minimum; a coefficient of variation of less than 10% should be the aim, to give reasonable precision. HPLC with amperometric electrochemical detection is not recommended, as it is less sensitive than coulometric detection. Immunological detection, 32P-postlabelling and LC-MS-MS are alternative approaches to measurement of 8-oxodG in DNA that, on the grounds of precision and detection of dose response, cannot at present be recommended. 相似文献
995.
Jeffrey M. Collins Dean P. Jones Ashish Sharma Manoj Khadka Ken H. Liu Russell R. Kempker Brendan Prideaux Kristal Maner-Smith Nestani Tukvadze N. Sarita Shah James C. M. Brust Rafick-Pierre Skaly Neel R. Gandhi Henry M. Blumberg Eric A. Ortlund Thomas R. Ziegler 《PLoS pathogens》2021,17(9)
The metabolic signaling pathways that drive pathologic tissue inflammation and damage in humans with pulmonary tuberculosis (TB) are not well understood. Using combined methods in plasma high-resolution metabolomics, lipidomics and cytokine profiling from a multicohort study of humans with pulmonary TB disease, we discovered that IL-1β-mediated inflammatory signaling was closely associated with TCA cycle remodeling, characterized by accumulation of the proinflammatory metabolite succinate and decreased concentrations of the anti-inflammatory metabolite itaconate. This inflammatory metabolic response was particularly active in persons with multidrug-resistant (MDR)-TB that received at least 2 months of ineffective treatment and was only reversed after 1 year of appropriate anti-TB chemotherapy. Both succinate and IL-1β were significantly associated with proinflammatory lipid signaling, including increases in the products of phospholipase A2, increased arachidonic acid formation, and metabolism of arachidonic acid to proinflammatory eicosanoids. Together, these results indicate that decreased itaconate and accumulation of succinate and other TCA cycle intermediates is associated with IL-1β-mediated proinflammatory eicosanoid signaling in pulmonary TB disease. These findings support host metabolic remodeling as a key driver of pathologic inflammation in human TB disease. 相似文献
996.
997.
Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot filamentous fungus, Phanerochaete chrysosporium 总被引:15,自引:0,他引:15
An analysis of nucleotide sequences of two types of ligninase cDNAs isolated from the basidiomycete Phanerochaete chrysosporium, designated CLG4 and CLG5, are presented here. The amino acid sequences of the corresponding ligninase proteins, designated LG4 and LG5, respectively, have been deduced from the cDNA sequences. Mature ligninases LG4 and LG5 are preceded by leader sequences containing 28 and 27 amino acids (aa), respectively, and each contains 344 aa residues. The estimated Mrs of mature LG4 and LG5 are 36,540 and 36,607, respectively. Potential N-glycosylation site(s) with the general sequence Asn-X-Thr/Ser are found in both LG4 and LG5. Nucleotide sequence homology between the coding region of CLG4 and CLG5 is 71.5%, whereas the amino acid sequence homology between the two ligninases is 68.5%. The codon usage of ligninases is extremely biased in favor of codons rich in cytosine and guanine. Amino acid sequences of two tryptic peptides of ligninase H8 have exactly matching sequences in ligninase LG5. Also, the sequences of the oligodeoxynucleotide probes, which correspond to the sequences in the tryptic peptides of ligninase H8 and which were used in isolating the ligninase clones from the cDNA library, have exactly matching sequences in CLG5. The experimentally determined N-terminal sequence of purified ligninase H8 is found in the deduced N-terminal amino acid sequence of LG5. These results suggest that CLG5 encodes ligninase H8 and that CLG4 represents a related but different ligninase gene. 相似文献
998.
The anaesthetic complication malignant hyperpyrexia (MH) is due to an elevation of the myoplasmic Ca2+ concentration. Examination of calmodulin isolated from MH susceptible swine suggests that the disorder in calcium regulation in MH is not due to an abnormality in calmodulin. 相似文献
999.
Jiwen Liu Zice Fu An-Rong Li Michael Johnson Liusheng Zhu Andrew Marcus Jay Danao Tim Sullivan George Tonn Tassie Collins Julio Medina 《Bioorganic & medicinal chemistry letters》2009,19(17):5114-5118
The evaluation of the CXCR3 antagonist AMG 487 in clinic trials was complicated due to the formation of an active metabolite. In this Letter, we will discuss the further optimization of the quinazolinone series that led to the discovery of compounds devoid of the formation of the active metabolite that was seen with AMG 487. In addition, these compounds also feature increased potency and good pharmacokinetic properties. We will also discuss the efficacy of the lead compound 34 in a mouse model of cellular recruitment induced by bleomycin. 相似文献
1000.
Liqun Zhang Brian Button Sherif E. Gabriel Susan Burkett Yu Yan Mario H. Skiadopoulos Yan Li Dang Leatrice N. Vogel Tristan McKay April Mengos Richard C. Boucher Peter L. Collins Raymond J. Pickles 《PLoS biology》2009,7(7)
Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl− and Na+ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways. 相似文献