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41.
Verónica Alvarez Maubecin Viviana N. Sanchez Marcelo D. Rosato Siri Bruce D. Cherksey Mutzuyuki Sugimori Rodolfo Llinás Osvaldo D. Uchitel 《Journal of neurochemistry》1995,64(6):2544-2551
Abstract: The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (ω-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and ω-agatoxin IVA]. K+-induced Ca2+ uptake by chicken synaptosomes was blocked by ω-conotoxin GVIA (IC50 = 250 nM). This toxin at 5 µM did not block Ca2+ entry into rat frontal cortex synaptosomes. FTX and ω-agatoxin IVA blocked Ca2+ uptake by rat synaptosomes (IC50 = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω-agatoxin IVA affected Ca2+ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC50 similar to the one obtained in rat preparations, whereas with ω-agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω-conotoxin GVIA was never greater than the one observed with ω-conotoxin GVIA. We also found that 60% of the Ca2+ uptake by rat and chicken synaptosomes was inhibited by ω-conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca2+ uptake in either the rat or the chicken. In conclusion, Ca2+ uptake by rat synaptosomes is potently inhibited by different P-type Ca2+ channel blockers, thus indicating that P-type channels are predominant in this preparation. In contrast, Ca2+ uptake by chicken synaptosomes is sensitive to ω-conotoxin GVIA, FTX, ω-agatoxin IVA, and ω-conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes. 相似文献
42.
Marcelo J. Yanovsky Jorge J. Casal Juan P. Luppi 《The Plant journal : for cell and molecular biology》1997,12(3):659-667
Phytochromes play a key role in the perception of light signals by plants. In this study, the three classical phytochrome action modes, i.e. very-low-fluence responses (VLFR), low-fluence responses (LFR) and high-irradiance responses (HIR), were genetically dissected using phyA and phyB mutants of Arabidopsis thaliana (respectively lacking phytochrome A or phytochrome B) and a polymorphism between ecotypes Landsberg erecta and Columbia. Seed germination and potentiation of greening, hypocotyl growth inhibition and cotyledon unfolding in etiolated seedlings of the ecotype Landsberg erecta showed biphasic responses to the calculated proportion of active phytochrome established by one light pulse or repeated light pulses. The first phase, i.e. the VLFR, was absent in the phyA mutant, normal in the phyB mutant (both in the Landsberg erecta background) and severely deficient in Columbia. The second phase, i.e. the LFR, was present in the phyA mutant, deficient in the phyB mutant and normal in Columbia. Under continuous far-red light, HIR of etiolated seedlings were absent in phyA and normal in phyB and Columbia. The segregation of VLFR in recombinant inbred lines derived from a cross between Landsberg erecta and Columbia was analysed by MAPMAKER/QTL. Two quantitative trait loci, one on chromosome 2 ( VLF1 ) and another on chromosome 5 ( VLF2 ), were identified as responsible for the polymorphism. Phytochrome A is proposed to initiate two transduction pathways, VLFR and HIR, involving different cells and/or different molecular steps. This is the first application of the analysis of quantitative trait loci polymorphic between ecotypes to dissect transduction chains of environmental signals. 相似文献
43.
Pedro J.I. Salas Marcelo L. Rodriguez Ana L. Viciana Dora E. Vega-Salas Hans-Peter Hauri 《The Journal of cell biology》1997,137(2):359-375
In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na+– K+ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.Cell polarity (asymmetry) is a broadly distributed and highly conserved feature of many different cell types, from prokaryotes to higher eukaryotes (Nelson, 1992). In multicellular organisms it is more conspicuous in, but not restricted to, neurons and epithelial cells. In the latter, the plasma membrane is organized in two different domains, apical and basolateral. This characteristic enables epithelia to accomplish their most specialized roles including absorption and secretion and, in general, to perform the functions of organs with an epithelial parenchyma such as the kidney, liver, intestine, stomach, exocrine glands, etc. (Simons and Fuller, 1985; Rodriguez-Boulan and Nelson, 1989).The acquisition and maintenance of epithelial polarity is based on multiple interrelated mechanisms that may work in parallel. Although the origin of polarization depends on the sorting of apical and basolateral membrane proteins at the trans-Golgi network (Simons and Wandinger-Ness, 1990), the mechanisms involved in the transport of apical or basolateral carrier vesicles, the specific fusion of such vesicles to the appropriate domain, and the retention of membrane proteins in their correct positions are also important (Wollner and Nelson, 1992). Various components of the cytoskeleton seem to be especially involved in these mechanisms (Mays et al., 1994). Among them, the microtubules, characteristically oriented in the apical–basal axis with their minus ends facing toward the apical domain, appear in a strategic position to transport carrier vesicles (Bacallao et al., 1989). This orientation is largely expected because of the apical distribution of centrioles and microtubule organizing centers in epithelial cells (Buendia et al., 1990). The molecular interactions responsible for that localization, however, are unknown.Actin is a widespread component of the membrane skeleton found under apical, lateral, and basal membranes in a nonpolarized fashion (Drenckhahn and Dermietzel, 1988; Vega-Salas et al., 1988). Actin bundling into microvillus cores in the presence of villin/fimbrin, on the other hand, is highly polarized to the apical domain (Ezzell et al., 1989; Louvard et al., 1992). In fact, different isoforms of plastins determine microvillus shape in a tissue-specific manner (Arpin et al., 1994b
). Why this arrangement is not found in other actin-rich regions of the cell is unclear (Louvard et al., 1992; Fath and Burgess, 1995).Fodrin, the nonerythroid form of spectrin, underlies the basolateral domain (Nelson and Veshnock, 1987a
,b) and is known to participate in the anchoring/retention of basolateral proteins (Drenckhahn et al., 1985; Nelson and Hammerton, 1989). Although different groups have found specific cytoskeletal anchoring of apical membrane proteins at the “correct” domain (Ojakian and Schwimmer, 1988; Salas et al., 1988; Parry et al., 1990), no specific apical counterpart of the basolateral fodrin cytoskeleton is known. This is especially puzzling since we showed that MDCK cells can maintain apical polarity in the absence of tight junctions, an indication that intradomain retention mechanisms are operational for apical membrane proteins (Vega-Salas et al., 1987a
).It is known that a network of intermediate filament (IF)1, the major component of the terminal web, bridges the desmosomes under the apical membrane in brush border cells (Franke et al., 1979; Hull and Staehelin, 1979; Mooseker, 1985), although no specific protein has been identified with this structure. The observation of a remarkable resistance to extractions of apical proteins anchored to cytoskeletal preparations (Salas et al., 1988) comparable to that of intermediate filaments, led us to the study of cytokeratins in polarized cells. We developed an antibody against a 53-kD intermediate filament protein in MDCK cells. This protein was found to be distributed exclusively to the apical domain and to form large (2,900 S) multi-protein complexes with apical plasma membrane proteins. Internal microsequencing of the 53-kD protein showed very high (95– 100%) homology with two polypeptides in the rod domain of cytokeratin 19 (CK19; Moll et al., 1982) a highly conserved and peculiar intermediate filament protein (Bader et al., 1986). A complete identification however, could not be achieved (Rodriguez et al., 1994). The present study was undertaken to establish that identity and to determine the possible functions of this apical membrane skeleton. Because cytokeratins have been poorly characterized in canine cells, and no cytokeratin sequences are available in this species, we decided to switch from MDCK cells to two human epithelial cell lines, CACO-2, an extensively studied model of epithelial polarization that differentiates in culture to form brush border containing cells (Pinto et al., 1983), and MCF-10A (Tait et al., 1990), a nontumorigenic cell line derived from normal mammary epithelia, as a model of nonbrush border cells.To assess possible functions of cytokeratin 19, we chose to selectively reduce its synthesis using anti-sense phosphorothioate oligodeoxy nucleotides, an extensively used approach in recent years (e.g., Ferreira et al., 1992 ; Hubber et al., 1993; Takeuchi et al., 1994). Although we could not achieve a complete knock out, the steady-state levels of cytokeratin 19 were decreased to an extent that enabled us to detect significant changes in the phenotype of CACO-2 and MCF-10A cells. 相似文献
44.
Characterization and regulation of the expression of FatB, an iron transport protein encoded by the pJM1 virulence plasmid 总被引:7,自引:0,他引:7
Luis A. Actis Marcelo E. Tolmasky Lidia M. Crosa Jorge H. Crosa 《Molecular microbiology》1995,17(1):197-204
The pJM1-encoded genes fatDCBA are essential for iron acquisition via the siderophore anguibactin. Sequence analysis indicated that the open reading frame corresponding to the fatB gene possesses domains that are characteristic of periplasmic proteins that bind the ferric siderophore. In this work, a monospecific antiserum against an oligopeptide containing the last 27 amino acids of the carboxy-terminal region from this open reading frame was used to demonstrate that fatB encodes a 35 kDa protein that is essential for iron transport. By using this antibody we were able to demonstrate that expression of the fatB gene is negatively regulated by the Fur protein at high iron concentrations. Conversely, its expression was positively regulated by the combined action of the AngR protein and products of the TAF region. FatB, the product of the fatB gene, is isolated with the membrane fraction. In accordance with these findings is the fact that the first 23 amino acid residues of this protein have the properties of a lipoprotein signal sequence. The lipoprotein nature of FatB is supported by the fact that treatment of Vibrio anguillarum cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 38 kDa pro-FatB precursor protein. 相似文献
45.
Barbara R. Hough-Evans Marcelo Jacobs-Lorena Michael R. Cummings Roy J. Britten Eric H. Davidson 《Genetics》1980,95(1):81-94
Comparative measurements are presented of the sequence complexity of the RNA stored in the eggs of two dipteran flies, Musca domestica and Drosophila melanogaster. The genome of Musca is about five times the size of the Drosophila genome and contains about 3.6 times as much single-copy sequence. As shown earlier, the interspersion of repetitive and single-copy sequence is of the short-period form in Musca, and is of the long-period form in Drosophila. The egg RNA complexities were determined by hybridization of excess RNA with radioactively labeled single-copy DNA. Complexity is expressed as the length (in nucleotides) of diverse single-copy sequence represented in the RNA. The complexity of the RNA of the Musca egg is about 2.4 x 107 nucleotides, and that of the Drosophila egg is about 1.2 x 107 nucleotides. The RNA of the Musca egg is similar to or very slightly lower in complexity than that of other egg RNAs, e.g., those of Xenopus and sea urchin. Compared to all previously measured egg RNAs, Drosophila egg RNA is low in sequence complexity. 相似文献
46.
Enrique J. del Pino Marcelo O. Cabada María C. Fonio 《Molecular reproduction and development》1981,4(2):97-104
Various plant lecins were employed in cell agglutination experiments to ascertain the presence of specific saccharides in the surface of B arenarum and L chaquensis spermatozoa. B arenarum spermatozoa were specifically agglutinated with Concanavalin A (Con A), phytohemagglutinin P (PHA-P), and wheat germ agglutinin (WGA), but not with soybean agglutinin (SBA). In contrast, L chaquensis spermatozoa were strongly agglutinated by SBA, WGA, and PHA-P. L chaquensis spermatozoa did not agglutinate with Con A even at high concentrations. Lectinmediated sperm agglutination was inhibited in the presence of specific lectinbinding sugars. Spermatozoa from both species were agglutinated randomly with all lectins suggesting a uniform distribution in the sperm surface of the lectinbinding saccharide ligands. B arenarum sperm agglutination induced by Con A is sensitive to temperature. B arenarum spermatozoa are more agglutinable at 24°C than at 4°C. These results suggest that lectin-binding site mobility is necessary for sperm agglutination. 相似文献
47.
Inhibition of protein tyrosine phosphatase activity by diamide is reversed by epidermal growth factor in fibroblasts. 总被引:1,自引:0,他引:1
Diamide (azodicarboxylic acid bis(dimethylamide] inhibits protein tyrosine phosphatase activity in fibroblasts without altering protein tyrosine kinase activity associated with the epidermal growth factor receptor. The loss of protein tyrosine phosphatase activity caused by diamide is reversed by 2-mercaptoethanol or epidermal growth factor. 相似文献
48.
We compared the rate of pollen tube growth following self- and cross-pollinations both among and within flowers of two clones of Dianthus chinensis L. For among-flower comparisons, both styles of a flower were pollinated with either self- or cross-pollen. Within-flower comparisons were made between the two styles of the same flower, one of which was self-pollinated and the other cross-pollinated. Comparisons between flowers indicated that self-pollen grew slower than cross-pollen in both clones. However, differences in the growth rate of pollen tubes from self- and cross-pollinations were greater when comparisons were made between the two styles of the same flower than when pollinations were made in different flowers. These results suggest the existence of interstyle interactions in pollen tube growth. 相似文献
49.
Cintia Camila Silva Angelieri Christine Adams-Hosking Katia Maria Paschoaletto Micchi de Barros Ferraz Marcelo Pereira de Souza Clive Alexander McAlpine 《PloS one》2016,11(1)
A mosaic of intact native and human-modified vegetation use can provide important habitat for top predators such as the puma (Puma concolor), avoiding negative effects on other species and ecological processes due to cascade trophic interactions. This study investigates the effects of restoration scenarios on the puma’s habitat suitability in the most developed Brazilian region (São Paulo State). Species Distribution Models incorporating restoration scenarios were developed using the species’ occurrence information to (1) map habitat suitability of pumas in São Paulo State, Southeast, Brazil; (2) test the relative contribution of environmental variables ecologically relevant to the species habitat suitability and (3) project the predicted habitat suitability to future native vegetation restoration scenarios. The Maximum Entropy algorithm was used (Test AUC of 0.84 ± 0.0228) based on seven environmental non-correlated variables and non-autocorrelated presence-only records (n = 342). The percentage of native vegetation (positive influence), elevation (positive influence) and density of roads (negative influence) were considered the most important environmental variables to the model. Model projections to restoration scenarios reflected the high positive relationship between pumas and native vegetation. These projections identified new high suitability areas for pumas (probability of presence >0.5) in highly deforested regions. High suitability areas were increased from 5.3% to 8.5% of the total State extension when the landscapes were restored for ≥ the minimum native vegetation cover rule (20%) established by the Brazilian Forest Code in private lands. This study highlights the importance of a landscape planning approach to improve the conservation outlook for pumas and other species, including not only the establishment and management of protected areas, but also the habitat restoration on private lands. Importantly, the results may inform environmental policies and land use planning in São Paulo State, Brazil. 相似文献
50.
Luiz Henrique Soares de Andrade Wilson Max Almeida Monteiro de Moraes Eduardo Hiroshi Matsuo Junior Elizabeth de Orleans Carvalho de Moura Hanna Karen Moreira Antunes Jairo Montemor Ednei Luiz Antonio Danilo Sales Bocalini Andrey Jorge Serra Paulo José Ferreira Tucci Patricia Chakur Brum Alessandra Medeiros 《Molecular and cellular biochemistry》2015,402(1-2):193-202