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151.
Truhlar SM Cervantes CF Torpey JW Kjaergaard M Komives EA 《Protein science : a publication of the Protein Society》2008,17(9):1636-1639
Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific (15)N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific (15)N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The (15)N incorporation is determined by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the (15)N-amino acid prevents the scrambling of the (15)N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" (15)N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot of protein. These advantages make it an ideal way of determining optimal expression conditions for selectively labeled NMR samples. 相似文献
152.
To examine the effect of negatively charged steroidal amphiphiles on antimicrobial activity, two pairs of epimeric, dendritic tricarboxylato amphiphiles--4-(2-carboxyethyl)-4-[3-(5alpha-cholestan-3-yl)ureido]heptanedioic acid (1) and 4-(2-carboxyethyl)-4-[3-(5alpha-cholestan-3-yloxycarbonylmethyl)ureido]heptanedioic acid (2)--were synthesized. A broad antimicrobial screen of 11 microbes revealed that these amphiphiles only showed good activity against a methicillin-resistant isolate of Staphylococcus aureus (MRSA) and modest activity against an unrelated strain of S. aureus. The best activity, a minimal inhibitory concentration (MIC) of 27 microM, was found for the 3beta epimer of 1 against MRSA. 相似文献
153.
Botelho FM Bauer CM Finch D Nikota JK Zavitz CC Kelly A Lambert KN Piper S Foster ML Goldring JJ Wedzicha JA Bassett J Bramson J Iwakura Y Sleeman M Kolbeck R Coyle AJ Humbles AA Stämpfli MR 《PloS one》2011,6(12):e28457
Background
Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality worldwide. Despite this, the cellular and molecular mechanisms that contribute to COPD pathogenesis are still poorly understood.Methodology and Principal Findings
The objective of this study was to assess IL-1 α and β expression in COPD patients and to investigate their respective roles in perpetuating cigarette smoke-induced inflammation. Functional studies were pursued in smoke-exposed mice using gene-deficient animals, as well as blocking antibodies for IL-1α and β. Here, we demonstrate an underappreciated role for IL-1α expression in COPD. While a strong correlation existed between IL-1α and β levels in patients during stable disease and periods of exacerbation, neutrophilic inflammation was shown to be IL-1α-dependent, and IL-1β- and caspase-1-independent in a murine model of cigarette smoke exposure. As IL-1α was predominantly expressed by hematopoietic cells in COPD patients and in mice exposed to cigarette smoke, studies pursued in bone marrow chimeric mice demonstrated that the crosstalk between IL-1α+ hematopoietic cells and the IL-1R1+ epithelial cells regulates smoke-induced inflammation. IL-1α/IL-1R1-dependent activation of the airway epithelium also led to exacerbated inflammatory responses in H1N1 influenza virus infected smoke-exposed mice, a previously reported model of COPD exacerbation.Conclusions and Significance
This study provides compelling evidence that IL-1α is central to the initiation of smoke-induced neutrophilic inflammation and suggests that IL-1α/IL-1R1 targeted therapies may be relevant for limiting inflammation and exacerbations in COPD. 相似文献154.
Gap junctions are specialized cell-cell contacts that provide direct intercellular communication between eukaryotic cells. The tyrosine-sorting signal (YXX?), present at amino acids 286-289 of Cx43 (connexin43), has been implicated in the internalization of the protein. In recent years, ubiquitination of Cx43 has also been proposed to regulate gap junction intercellular communication; however, the underlying mechanism and molecular players involved remain elusive. In the present study, we demonstrate that ubiquitinated Cx43 is internalized through a mechanism that is independent of the YXX? signal. Indeed, expression of a Cx43-Ub (ubiquitin) chimaera was shown to drive the internalization of a mutant Cx43 in which the YXX? motif was eliminated. Immunofluorescence, cycloheximide-chase and cell-surface-protein biotinylation experiments demonstrate that oligomerization of Cx43-Ub into hemichannels containing wild-type Cx43 or mutant Cx43Y286A is sufficient to drive the internalization of the protein. Furthermore, the internalization of Cx43 induced by Cx43-Ub was shown to depend on its interaction with epidermal growth factor receptor substrate 15. 相似文献
155.
Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms 总被引:1,自引:0,他引:1
Agnetti G Maraldi T Fiorentini D Giordano E Prata C Hakim G Muscari C Guarnieri C Caldarera CM 《Life sciences》2005,78(3):264-270
Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6-3H]-glucose uptake was increased (57% above control levels, p < 0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19%, p < 0.001) followed also treatment with Gö6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31%, p < 0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process. 相似文献
156.
Soares JB Pimentel-Nunes P Afonso L Rolanda C Lopes P Roncon-Albuquerque R Gonçalves N Boal-Carvalho I Pardal F Lopes S Macedo G Lara-Santos L Henrique R Moreira-Dias L Gonçalves R Dinis-Ribeiro M Leite-Moreira AF 《Innate immunity》2012,18(5):700-708
We evaluated expression of TLR2, TLR4 and proinflammatory genes [NF-κB, TNF-α, cyclooxygenase-2 (COX-2)] in liver samples of patients in different stages of liver disease. Fifteen patients with unexplained transaminases elevation (reference group), 22 with viral chronic hepatitis (hepatitis group), 14 with virus-induced severe fibrosis/cirrhosis (cirrhosis group) and 10 with hepatocarcinoma (hepatocarcinoma group) were consecutively included in the study. Quantification of TLR2, TLR4, NF-κB, TNF-α and COX-2 mRNA was done by real-time RT-PCR and TLR2 and TLR4 protein expression was evaluated by immunohistochemistry. Compared with reference, TLR2 and TLR4 mRNA was increased in hepatitis (TLR2: 2.66?±?0.69; TLR4: 3.11?±?0.79; P?0.05) and cirrhosis (TLR2: 2.14?±?0.5; TLR4: 1.74?±?0.27; P?0.05) and decreased in hepatocarcinoma (TLR2: 0.48?±?0.15; TLR4: 0.54?±?0.10; P?0.05). This associated with increased TNF-α and COX-2 mRNA in hepatitis (TNF-α: 3.24?±?0.79; COX-2: 2.47?±?0.36; P?0.05) and cirrhosis (TNF-α: 1.73?±?0.28; COX-2: 1.8?±?0.35, P?0.05), whereas NF-κB mRNA was increased in hepatitis (2.42?±?0.31; P?0.05) and unchanged in cirrhosis (1.34?±?0.17; P?=?0.3). Hepatocarcinoma presented increased COX-2 mRNA (1.63?±?0.15; P?0.05) and maintained (at decreased levels) mRNA of NF-κB (0.52?±?0.12) and TNF-α (0.52?±?0.12; P?0.05, all genes). Immunohistochemistry confirmed increased expression of TLR2 and TLR4 in hepatitis and cirrhosis and maintained expression in hepatocarcinoma. Upregulation of TLR2, TLR4 and their proinflammatory mediators is associated with virus-induced hepatic IFC sequence. 相似文献
157.
Alessandra Fierabracci Carla Bizzarri Alessia Palma Annamaria Milillo Emanuele Bellacchio Marco Cappa 《Gene》2012
Background
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED) is an autosomal recessive disease due to mutations of the autoimmune regulator (AIRE) gene. Typical manifestations include candidiasis, Addison's disease, and hypoparathyroidism. Type 1 diabetes, alopecia, vitiligo, ectodermal dystrophy, celiac disease and other intestinal dysfunctions, chronic atrophic gastritis, chronic active hepatitis, autoimmune thyroid disorders, pernicious anemia and premature ovarian failure are other rare associated diseases although other conditions have been associated with APECED.Case presentation
What follows is the clinical, endocrinological and molecular data of a female APECED patient coming from Lithuania. The patient was affected by chronic mucocutaneous candidiasis, hypoparathyroidism and pre-clinical Addison's disease. Using direct sequencing of all the 14 exons of the AIRE gene in the patient's DNA, we identified in exon 6 the known mutation c.769 C>T (p.Arg257X) in compound heterozygosity with the newly discovered mutation c.1214delC (p.Pro405fs) in exon 10. The novel mutation results in a frameshift that is predicted to alter the sequence of the protein starting from amino acid 405 as well as to cause its premature truncation, therefore a non-functional Aire protein.Conclusions
A novel mutation has been described in a patient with APECED with classical clinical components, found in compound heterozygosity with the c.769 C>T variation. Expanded epidemiological investigations based on AIRE gene sequencing are necessary to verify the relevancy of the novel mutation to APECED etiopathogenesis in the Lithuanian population and to prove its diagnostic efficacy in association with clinical and immunological findings. 相似文献158.
159.
160.
Marinella Silva Laport Marilene Ramos da Silva Carla Costa Silva Maria do Carmo de Freire Bastos Marcia Giambiagi-deMarval 《Current microbiology》2003,46(5):0313-0317
We have characterized the heat-shock response of the nosocomial pathogen Enterococcus faecium. The growth of E. faecium cells was analyzed at different temperatures; little growth was observed at 50°C, and no growth at 52°C or 55°C. In agreement,
a marked decrease of general protein synthesis was observed at 52°C, and very light synthesis was detected at 55°C. The heat
resistance of E. faecium cells was analyzed by measuring the survival at temperatures higher than 52°C and, after 2 h of incubation, viable cells
were still observed at 70°C. By Western blot analysis, two heat-induced proteins were identified as GroEL (65 kDa) and DnaK
(75 kDa). Only one isoform for either GroEL or DnaK was found. The gene expression of these heat-shock proteins was also analyzed
by pulsed-labeled experiments. The heat-induced proteins showed an increased rate of synthesis during the first 5 min, reaching
the highest level of induction after 10 min and returning to the steady-state level after 20 min of heat treatment.
Received: 29 March 2002 / Accepted: 5 July 2002 相似文献