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111.
RNA-directed construction of structurally complex and active ligase ribozymes through recombination 总被引:1,自引:0,他引:1
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RNA-directed recombination can be used to catalyze a disproportionation reaction among small RNA substrates to create new combinations of sequences. But the accommodation of secondary and tertiary structural constraints in the substrates by recombinase ribozymes has not been explored. Here, we show that the Azoarcus group I intron can recombine oligoribonucleotides to construct class I ligase ribozymes, which are catalytically active upon synthesis. The substrate oligonucleotides, ranging in size from 58 to 104 nucleotides (nt), along with the 152-nt ligase ribozymes they reconstitute, can contain significant amounts of secondary structure. However, substrate recognition by the Azoarcus ribozyme depends on the existence of a single accessible CAU triplet for effective recombination. A biphasic temperature reaction profile was designed such that the sequential recombination/ligation events could take place in a thermocycler without human intervention. A temperature-dependent pH shift of the reaction buffer contributes to the success of the net reaction. When the substrate for the ligase ribozyme is introduced into the reaction mixture, as much as 11% can be observed being converted to product by the recombined ligase in the same reaction vessel. Recombination followed by ligation can also occur under isothermal conditions at 37 degrees C. Tertiary structure formation of the ligase upon construction can provide some protection from cleavage by the Azoarcus ribozyme when compared to the constituent substrates. These data suggest that RNA-directed recombination can, in fact, articulate complex ribozymes, and that there are logical rules that can guide the optimal placement of the CAU recognition sequence. 相似文献
112.
Wang GT Wang S Gentles R Sowin T Maring CJ Kempf DJ Kati WM Stoll V Stewart KD Laver G 《Bioorganic & medicinal chemistry letters》2005,15(1):125-128
(+/-)-(2R,3R,5R)-[2-(1'-S-acetamido-3'-methyl)butyl-3-methoxycarbonyl]tetrahydrofuran-5-carboxylic acid (9) and (+/-)-(2R,3R,5R)-[2-(1'-S-acetamido-3'-methyl)butyl-3-(4'-imidazolyl)]tetrahydrofuran 5-carboxylic acid (14) were synthesized as inhibitors of influenza neuraminidase (NA). Both compounds 9 and 14 inhibit influenza NA A with an IC(50) of about 0.5 microM and NA B with an IC(50) of 1.0 microM. 相似文献
113.
Ecogeographic size variations have been documented in some but not all sifakas. Few morphometric or body weight data have been available for two critically endangered subspecies of diademed sifakas: Perrier's sifakas (Propithecus diadema perrieri) and silky sifakas (Propithecus diadema candidus). The objectives of our study were to determine size variations in sifakas and if these variations are related to resource quality and/or resource seasonality. P. d. perrieri and P. d. candidus were captured, weighed, and measured in northern Madagascar. Body weights and morphometrics were compared with other subspecies of diademed sifakas and indris (Indri indri). Differences in body weights and morphometrics between taxa are particularly pronounced for P. d. perrieri compared to P. d. diadema, P. d. edwardsi, and I. indri. Most morphometrics varied in comparisons between P. d. candidus and the other Indriidae (P. d. diadema, P. d. edwardsi, and I. indri). Average body size in sifakas is positively correlated with annual rainfall and negatively correlated with length of dry season. Sifaka body size is not correlated with protein-to-fiber ratios. Thus, size variations in sifakas are related to resource seasonality rather than resource quality. The relationships between the temporal availability of food resources and sifaka body size reflect complex and regionally varying causalities. Detailed, longitudinal information on the ecological factors underlying food selection and nutrient requirements in sifakas are needed to determine the relationship between ecogeographic variables and body size in sifakas. 相似文献
114.
Drosophila muscle regulation characterized by electron microscopy and three-dimensional reconstruction of thin filament mutants
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Cammarato A Hatch V Saide J Craig R Sparrow JC Tobacman LS Lehman W 《Biophysical journal》2004,86(3):1618-1624
Wild-type and mutant thin filaments were isolated directly from "myosinless" Drosophila indirect flight muscles to study the structural basis of muscle regulation genetically. Negatively stained filaments showed tropomyosin with periodically arranged troponin complexes in electron micrographs. Three-dimensional helical reconstruction of wild-type filaments indicated that the positions of tropomyosin on actin in the presence and absence of Ca(2+) were indistinguishable from those in vertebrate striated muscle and consistent with a steric mechanism of regulation by troponin-tropomyosin in Drosophila muscles. Thus, the Drosophila model can be used to study steric regulation. Thin filaments from the Drosophila mutant heldup(2), which possesses a single amino acid conversion in troponin I, were similarly analyzed to assess the Drosophila model genetically. The positions of tropomyosin in the mutant filaments, in both the Ca(2+)-free and the Ca(2+)-induced states, were the same, and identical to that of wild-type filaments in the presence of Ca(2+). Thus, cross-bridge cycling would be expected to proceed uninhibited in these fibers, even in relaxing conditions, and this would account for the dramatic hypercontraction characteristic of these mutant muscles. The interaction of mutant troponin I with Drosophila troponin C is discussed, along with functional differences between troponin C from Drosophila and vertebrates. 相似文献
115.
Lobo RA Bélisle S Creasman WT Frankel NR Goodman NE Hall JE Ivey SL Kingsberg S Langer R Lehman R McArthur DB Montgomery-Rice V Notelovitz M Packin GS Rebar RW Rousseau M Schenken RS Schneider DL Sherif K Wysocki S 《MedGenMed : Medscape general medicine》2006,8(2):1 p preceding 35
Many physicians remain uncertain about prescribing hormone therapy for symptomatic women at the onset of menopause. The American Society for Reproductive Medicine (ASRM) convened a multidisciplinary group of healthcare providers to discuss the efficacy and risks of hormone therapy for symptomatic women, and to determine whether it would be appropriate to treat women at the onset of menopause who were complaining of menopausal symptoms. MAJOR FINDINGS: Numerous controlled clinical trials consistently demonstrate that hormone therapy, administered via oral, transdermal, or vaginal routes, is the most effective treatment for vasomotor symptoms. Topical vaginal formulations of hormone therapy should be preferred when prescribing solely for the treatment of symptoms of vulvar and vaginal atrophy. Data from the Women's Health Initiative indicate that the overall attributable risk of invasive breast cancer in women receiving estrogen plus progestin was 8 more cases per 10,000 women-years. No increased risk for invasive breast cancer was detected for women who never used hormone therapy in the past or for those receiving estrogen only. Hormone therapy is not effective for the treatment of cardiovascular disease and that the risk of cardiovascular disease with hormone therapy is principally in older women who are considerably postmenopause. CONCLUSIONS: Healthy symptomatic women should be offered the option of hormone therapy for menopausal symptoms. Symptom relief with hormone therapy for many younger women (at the onset of menopause) with menopausal symptoms outweighs the risks and may provide an overall improvement in quality of life. Hormone therapy should be individualized for symptomatic women. This involves tailoring the regimen and dose to individual needs. 相似文献
116.
Lam C Waldhorn R Toner E Inglesby TV O'Toole T 《Biosecurity and bioterrorism : biodefense strategy, practice, and science》2006,4(4):384-390
Alternative care facilities (ACFs) have been widely proposed in state, local, and national pandemic preparedness plans as a way to address the expected shortage of available medical facilities during an influenza pandemic. These plans describe many types of ACFs, but their function and roles are unclear and need to be carefully considered because of the limited resources available and the reduced treatment options likely to be provided in a pandemic. Federal and state pandemic plans and the medical literature were reviewed, and models for ACFs being considered were defined and categorized. Applicability of these models to an influenza pandemic was analyzed, and recommendations are offered for future ACF use. ACFs may be best suited to function as primary triage sites, providing limited supportive care, offering alternative isolation locations to influenza patients, and serving as recovery clinics to assist in expediting the discharge of patients from hospitals. 相似文献
117.
118.
Recombination during the PCR amplification of DNA templates can be a serious problem for those seeking to genotype heterogeneous populations, yet a boon to those seeking to enhance variation during in vitro evolution. Here, the extent to which PCR generates chimeric full-length products was estimated using a powerful restriction fragment-length polymorphism (RFLP) assay involving the use of fluorescently labeled PCR primers. Three different RNA-encoding DNA templates were assayed: (i) one for a group I ribozyme, (ii) one for a 16S ribosomal RNA (rRNA), and (iii) one for a messenger RNA (mRNA). In all cases, the observed frequency of chimeric PCR products exceeded 20%, and longer templates appear to produce more chimeric products. Although two of these templates have the potential to form secondary structures during the PCR, this tendency does not seem to heighten recombination frequency. These results corroborate previous studies that show that the production of chimeras can be best attenuated to a certain extent by varying the extension times in PCR. 相似文献
119.
Hillberg L Zhao Rathje LS Nyåkern-Meazza M Helfand B Goldman RD Schutt CE Lindberg U 《European journal of cell biology》2006,85(5):399-409
This paper shows that high-molecular-weight tropomyosins (TMs), as well as shorter isoforms of this protein, are present in significant amounts in lamellipodia and filopodia of spreading normal and transformed cells. The presence of TM in these locales was ascertained by staining of cells with antibodies reacting with endogenous TMs and through the expression of hemaglutinin- and green fluorescent protein-tagged TM isoforms. The observations are contrary to recent reports suggesting the absence of TMs in regions,where polymerization of actin takes place, and indicate that the view of the role of TM in the formation of actin filaments needs to be significantly revised. 相似文献
120.
Characterization of the pilin ortholog of the Helicobacter pylori type IV cag pathogenicity apparatus, a surface-associated protein expressed during infection 总被引:2,自引:0,他引:2
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Andrzejewska J Lee SK Olbermann P Lotzing N Katzowitsch E Linz B Achtman M Kado CI Suerbaum S Josenhans C 《Journal of bacteriology》2006,188(16):5865-5877
The Helicobacter pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) involved in host interaction and pathogenicity. Previously, seven cag PAI proteins were identified as homologs of Agrobacterium tumefaciens Vir proteins, which form a paradigm T4SS. The T pilus composed of the processed VirB2 pilin is an external structural part of the A. tumefaciens T4SS. In H. pylori, cag-dependent assembly of pili has not been observed so far, nor has a pilin (VirB2) ortholog been characterized. We have here identified, using a motif-based search, an H. pylori cag island protein (HP0546) that possesses sequence and predicted structural similarities to VirB2-like pilins of other T4SSs. The HP0546 protein displays interstrain variability in its terminal domains. HP0546 was expressed as a FLAG-tagged fusion protein in Escherichia coli, A. tumefaciens, and H. pylori and was detected as either two or three bands of different molecular masses in the insoluble fraction, indicating protein processing. As reported previously, isogenic H. pylori mutants in the putative cag pilin gene had reduced abilities to induce cag PAI-dependent interleukin-8 secretion in gastric epithelial cells. Fractionation analysis of H. pylori, using a specific antiserum raised against an N-terminal HP0546 peptide, showed that the protein is partially surface exposed and that its surface localization depended upon an intact cag system. By immunoelectron microscopy, HP0546 was localized in surface appendages, with surface exposure of an N-terminal epitope. Pronounced strain-to-strain variability of this predicted surface-exposed part of HP0546 indicates a strong selective pressure for variation in vivo. 相似文献