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101.
Twerenbold D Netuschill A de Rooij N Luginbuhl P Gerbe D Gritti D Gonin Y Rossel F Vuilleumier JL 《Proteomics》2002,2(4):436-440
The launch of molecules from liquid surfaces in a time of flight mass spectrometer has been investigated using different sample preparation techniques, and by exposing the liquid samples to two different laser wavelengths, 337 nm from a N2 ultraviolet laser and 10.6 microm from a CO2 infrared laser. The molecules were detected with cryodetectors measuring the energy of the individual molecules. We present insulin and lysozyme results from samples introduced into the vacuum through a micromachined silicon injector, and from samples consisting of a glycerol droplet deposited directly on the sample holder at the high voltage stage of the ion optics. 相似文献
102.
Biessen EA Sliedregt-Bol K 'T Hoen PA Prince P Van der Bilt E Valentijn AR Meeuwenoord NJ Princen H Bijsterbosch MK Van der Marel GA Van Boom JH Van Berkel TJ 《Bioconjugate chemistry》2002,13(2):295-302
In this study, we present the design and synthesis of an antisense peptide nucleic acid (asPNA) prodrug, which displays an improved biodistribution profile and an equally improved capacity to reduce the levels of target mRNA. The prodrug, K(GalNAc)(2)-asPNA, comprised of a 14-mer sequence complementary to the human microsomal triglyceride transfer protein (huMTP) gene, conjugated to a high-affinity tag for the hepatic asialoglycoprotein receptor (K(GalNAc)(2)). The prodrug was avidly bound and rapidly internalized by HepG2s. After iv injection into mice, K(GalNAc)(2)-asPNA accumulated in the parenchymal liver cells to a much greater extent than nonconjugated PNA (46% +/- 1% vs 3.1% +/- 0.5% of the injected dose, respectively). The prodrug was able to reduce MTP mRNA levels in HepG2 cells by 35-40% (P < 0.02) at 100 nM in an asialoglycoprotein receptor- and sequence-dependent fashion. In conclusion, hepatocyte-targeted PNA prodrugs combine a greatly improved tropism with an enhanced local intracellular availability and activity, making them attractive therapeutics to lower the expression level of hepatic target genes such as MTP. 相似文献
103.
104.
Sources of nitrate in rivers draining sixteen watersheds in the northeastern U.S.: Isotopic constraints 总被引:7,自引:4,他引:3
Mayer Bernhard Boyer Elizabeth W. Goodale Christine Jaworski Norbert A. van Breemen Nico Howarth Robert W. Seitzinger Sybil Billen Gilles Lajtha Kate Nadelhoffer Knute Van Dam Douwe Hetling Leo J. Nosal Miloslav Paustian Keith 《Biogeochemistry》2002,(1):171-197
The feasibility of using nitrogen and oxygenisotope ratios of nitrate (NO3
–) forelucidating sources and transformations ofriverine nitrate was evaluated in a comparativestudy of 16 watersheds in the northeastern U.S.A. Stream water was sampled repeatedly at theoutlets of the watersheds between January andDecember 1999 for determining concentrations,15N values, and 18Ovalues of riverine nitrate.In conjunction with information about land useand nitrogen fluxes,15Nnitrate and18Onitrate values providedmainly information about sources of riverinenitrate. In predominantly forested watersheds,riverine nitrate had mean concentrations ofless than 0.4 mg NO3
–-N L–1,15Nnitrate values of lessthan +5, and 18Onitratevalues between +12 and +19. This indicatesthat riverine nitrate was almost exclusivelyderived from soil nitrification processes withpotentially minor nitrate contributions fromatmospheric deposition in some catchments. Inwatersheds with significant agricultural andurban land use, concentrations of riverinenitrate were as high as 2.6 mg NO3
–-NL–1 with 15Nnitratevalues between +5 and +8 and18Onitrate values generallybelow +15. Correlations between nitrateconcentrations, 15Nnitratevalues, and N fluxes suggest that nitrate inwaste water constituted a major, and nitrate inmanure a minor additional source of riverinenitrate. Atmospheric nitrate deposition ornitrate-containing fertilizers were not asignificant source of riverine nitrate inwatersheds with significant agricultural andurban land use. Although complementary studiesindicate that in-stream denitrification wassignificant in all rivers, the isotopiccomposition of riverine nitrate sampled at theoutlet of the 16 watersheds did not provideevidence for denitrification in the form ofelevated 15Nnitrate and18Onitrate values. Relativelylow isotopic enrichment factors for nitrogenand oxygen during in-stream denitrification andcontinuous admixture of nitrate from theabove-described sources are thought to beresponsible for this finding. 相似文献
105.
Hsu ST Breukink E de Kruijff B Kaptein R Bonvin AM van Nuland NA 《Biochemistry》2002,41(24):7670-7676
Nisin is an example of type-A lantibiotics that contain cyclic lanthionine rings and unusual dehydrated amino acids. Among the numerous pore-forming antimicrobial peptides, type-A lantibiotics form an unique family of post-translationally modified peptides. Via the recognition of cell wall precursor lipid II, nisin has the capacity to form pores against Gram-positive bacteria with an extremely high activity in the nanomolar (nM) range. Here we report a high-resolution NMR spectroscopy study of nisin/lipid II interactions in SDS micelles as a model membrane system in order to elucidate the mechanism of molecular recognition at residue level. The binding to lipid II was studied through (15)N-(1)H HSQC titration, backbone amide proton temperature coefficient analysis, and heteronuclear (15)N[(1)H]-NOE relaxation dynamics experiments. Upon the addition of lipid II, significant changes were monitored in the N-terminal part of nisin. An extremely low amide proton temperature coefficient (Delta delta/Delta T) was found for the amide proton of Ala3 (> -0.1 ppb/K) in the complex form. This suggests tight hydrogen bonding and/or isolation from the bulk solvent for this residue. Large chemical shift perturbations were also observed in the first two rings. In contrast, the C-terminal part of nisin was almost unaffected. This part of the molecule remains flexible and solvent-exposed. On the basis of our results, a multistep pore-forming mechanism is proposed. The N-terminal part of nisin first binds to lipid II, and a subsequent structural rearrangement takes place. The C-terminal part of nisin is possibly responsible for the activation of the pore formation. In light of the emerging antibiotic resistance problems, an understanding of the specific recognition mechanism of nisin with lipid II at the residue specific level may therefore aid in the development of novel antibiotics. 相似文献
106.
Hammes F Boon N de Villiers J Verstraete W Siciliano SD 《Applied and environmental microbiology》2003,69(8):4901-4909
During a study of ureolytic microbial calcium carbonate (CaCO(3)) precipitation by bacterial isolates collected from different environmental samples, morphological differences were observed in the large CaCO(3) crystal aggregates precipitated within bacterial colonies grown on agar. Based on these differences, 12 isolates were selected for further study. We hypothesized that the striking differences in crystal morphology were the result of different microbial species or, alternatively, differences in the functional attributes of the isolates selected. Sequencing of 16S rRNA genes showed that all of the isolates were phylogenetically closely related to the Bacillus sphaericus group. Urease gene diversity among the isolates was examined by using a novel application of PCR-denaturing gradient gel electrophoresis (DGGE). This approach revealed significant differences between the isolates. Moreover, for several isolates, multiple bands appeared on the DGGE gels, suggesting the apparent presence of different urease genes in these isolates. The substrate affinities (K(m)) and maximum hydrolysis rates (V(max)) of crude enzyme extracts differed considerably for the different strains. For certain isolates, the urease activity increased up to 10-fold in the presence of 30 mM calcium, and apparently this contributed to the characteristic crystal formation by these isolates. We show that strain-specific calcification occurred during ureolytic microbial carbonate precipitation. The specificity was mainly due to differences in urease expression and the response to calcium. 相似文献
107.
Optimizing expression and purification from cell culture medium of trispecific recombinant antibody derivatives 总被引:3,自引:0,他引:3
Willems A Leoen J Schoonooghe S Grooten J Mertens N 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,786(1-2):161-176
Antibody fragments offer the possibility to build multifunctional manifolds tailored to meet a large variety of needs. We optimized the production of a manifold consisting of one (bibody) or two (tribody) single-chain variable fragments coupled to the C-terminus of Fab chains. Different strong mammalian promoters were compared and the influence of expression media on production and recovery was investigated. Since the physical and chemical nature of these molecules largely depends on the nature of the antibody building blocks incorporated, a generally applicable process for the purification of recombinant antibody derivatives from serum containing mammalian cell culture medium was designed. To this end we compared protein L, hydroxyapatite, immobilized metal affinity chromatography, cation-exchange chromatography and hydrophobic charge induction chromatography. 相似文献
108.
Localization, dynamics, and function of survivin revealed by expression of functional survivinDsRed fusion proteins in the living cell 总被引:12,自引:0,他引:12
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Temme A Rieger M Reber F Lindemann D Weigle B Diestelkoetter-Bachert P Ehninger G Tatsuka M Terada Y Rieber EP 《Molecular biology of the cell》2003,14(1):78-92
Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein. 相似文献
109.
Saeij JP Groeneveld A Van Rooijen N Haenen OL Wiegertjes GF 《Diseases of aquatic organisms》2003,57(1-2):67-75
Carp Cyprinus carpio macrophages were depleted by intraperitoneal (i.p.) injection of clodronate-liposomes for the in vivo study of the effect of macrophage depletion on the resistance of carp to infection with blood flagellate parasites. Clodronate released inside the cell induces apoptosis of (murine) macrophages. Following i.p. injection of carp with liposomes alone, but not with Trypanoplasma borreli, neutrophilic granulocytes rapidly migrated from the head kidney to the peritoneal cavity. The majority of liposomes in the peritoneal cavity were not taken up by newly arrived neutrophilic granulocytes, however, but by resident macrophages. After 2 i.p. injections of clodronate-liposomes, the percentage of macrophages present in the peritoneal cavity was significantly reduced, as evaluated by flow cytometry. Macrophage-depleted carp that were infected i.p. with T. borreli suffered from high mortality. However, these fish did not show lethal parasitaemia but did show clear bacteraemia. Macrophage-depleted carp that were infected i.p. with Trypanosoma carassii showed a minor increase in parasitaemia. In addition, macrophage-depleted carp, immune to T. borreli as a result of having survived a prior infection, remained immune to i.p. reinfection with T. borreli. Succesful depletion of peritoneal macrophages seemed to have a minor effect on the resistance of carp against blood flagellates. However, carp macrophages are essential as a first line of defence against (bacterial) infection. 相似文献
110.
Kaneko H Nakamura T Lindemann B 《American journal of physiology. Cell physiology》2001,280(6):C1387-C1393
Inwardly directed Ca(2+)-dependent chloride currents are thought to prolong and boost the odorant-induced transient receptor currents in olfactory cilia. Cl(-) inward current, of course, requires a sufficiently high intracellular Cl(-) concentration ([Cl(-)](i)). In previous measurements using a fluorescent Cl(-) probe, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), [Cl(-)](i) of newt olfactory cells was estimated to be only 40 mM. This low value led us to reexamine the [Cl(-)](i) by an improved procedure. When isolated rat olfactory neurons were bathed in Tyrode's solution (150 mM Cl(-)) at room temperature, the [Cl(-)] was 81.5 +/- 13.5 mM (mean +/- SE) in the tip of the dendrite (olfactory knob) and 81.8 +/- 10.2 mM (mean +/- SE) in the soma. The corresponding Cl(-) equilibrium potentials were -15.4 and -15.3 mV, respectively. Therefore, at resting potentials in the range of -90 to -50 mV, Cl(-) currents are predicted to be inward and capable of contributing to the depolarization induced by odorants. Yet, if the cell was depolarized beyond -15 mV, somal Cl(-) currents would be outward and facilitate repolarization during excitation. The measured [Cl(-)] in soma and knob are of interest, because in the cilia the chloride content may be expected to equilibrate with that of the knob in the resting state. They provide a starting point for the decrease in ciliary [Cl(-)] predicted to occur during transduction. 相似文献