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81.
We report here a new strategy for derivatizing peptides and proteins at the N-terminus. To achieve this, a nonnatural amino acid was charged onto a tRNA and then enzymatically transferred to a lysine (Lys) unit at the N-terminus of a peptide or a protein by using L/F-tRNA-protein transferase. By using the chemoenzymatic technique, beta-(2-quinolyl)-L-alanine, p-azido-L-phenylalanine, and p-acetyl-L-phenylalanine were introduced to the N-terminus. The latter two nonnatural amino acids possess bioorthogonal functional groups to which artificial tags can be introduced. Actually, a biotin tag was coupled to the bioorthogonal ketone group of acetylphenylalanine at the N-terminus of a peptide. N-terminal-specific biotinylation and fluorescence derivatization of the bioorthogonal azido-containing protein or peptide was also carried out based on a [3 + 2] cycloaddition. The enzymatic transfer of a nonnatural amino acid to the N-terminus of target peptides or proteins was also successfully achieved in the presence of other peptides or crude protein mixtures. 相似文献
82.
Elaboration of size and shape in multicellular organisms involves coordinated cell division and cell growth. In higher plants, continuity of cell layer structures exists from the shoot apical meristem (SAM), where organ primordia arise, to mature aboveground organs. To unravel the extent of inter-cell layer coordination during SAM and aboveground organ development, cell division in the epidermis was selectively restricted by expressing two cyclin-dependent kinase inhibitor genes, KRP1/ICK1 and KRP4, driven by the L1 layer-specific AtML1 promoter. The transgenes conferred reduced plant size with striking, distorted lateral organ shape. While epidermal cell division was severely inhibited with compensatory cell size enlargement, the underlying mesophyll/cortex layer kept normal cell numbers and resulted in small, packed cells with disrupted cell files. Our results demonstrate the autonomy of cell number checkpoint in the underlying tissues when epidermal cell division is restricted. Finally, the L1 layer-specific expression of both KRP1/ICK1 and KRP4 showed no effects on the structure and function of the SAM, suggesting that the effects of these cyclin-dependent kinase inhibitors are context dependent. 相似文献
83.
The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, “early neurogenesis” occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows “established neurogenesis,” in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mash1 at E10.5 in the Six1-deficient mice (Six1−/−). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1−/− at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1−/−. Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5. 相似文献
84.
Yoshizawa K Mishima Y Park SY Heddle JG Tame JR Iwahori K Kobayashi M Yamashita I 《Journal of biochemistry》2007,142(6):707-713
The denaturation of recombinant horse L-chain apoferritin (rLF), which is composed of 24 L-chain subunits, in acidic solution was studied. Using two rLF mutants, lacking four (Fer4) or eight (Fer8) N-terminal amino acid residues, the effect of N-terminal residues on the protein's stability was investigated. Of the two mutants and wild-type rLF, the tertiary and secondary structures of Fer8 were found to be most sensitive to an acidic environment. The Fer8 protein dissociated easily into subunit dimers at or below pH 2.0. Comparing the crystal structures of the mutant proteins, deletion of the N-terminal residues was found to result in fewer inter- and intra-subunit hydrogen bonds. The loss of these bonds is assumed to be responsible for lower endurance against acidic denaturation in N-terminus-deleted mutants. These results indicated that the inter- and intra-subunit hydrogen bonds of N-terminal residues affect the denaturation, especially oligomer formation of apoferritin subunits and will be of use in designing ferritin-based nanodevices. 相似文献
85.
Xie M Kobayashi I Kiyoshima T Yamaza H Honda JY Takahashi K Enoki N Akamine A Sakai H 《The Journal of biological chemistry》2007,282(32):23275-23283
We examined the functional implication of nucleolin in the mouse first molar development. Both the nucleolin mRNA and protein expressions were demonstrated in the odontogenic epithelial cells in the early stage and in the inner enamel epithelial layer in the late stage. The expression pattern of nucleolin corresponded to the proliferating cells in the tooth germ, thus showing that nucleolin could possibly be related to cell proliferation. No in situ signal of nucleolin was found in the primary enamel knot (PEK). Furthermore, nucleolin protein was demonstrated in the PEK by immunohistochemistry. The existence of nucleolin protein in the PEK may possibly be related to the apoptosis in the PEK cells. An inhibition assay using the hemagglutinating virus of Japan-liposome containing nucleolin antisense phosphorothioated oligonucleotide (AS S-ODN) in cultured mouse mandibles at embryonic day (E) 11.0 showed a marked growth inhibition of tooth germ. Moreover, no developmental arrest was found in the cultured tooth germ at E15.0 treated with nucleolin AS S-ODN. Real time PCR was performed to examine the mRNA expression of nucleolin-related genes, and a significant reduction in the midkine mRNA expression was thus observed in the mouse mandible after being treated with nucleolin AS S-ODN. This inhibition assay indicated that nucleolin could thus be involved in the early stage of tooth germ initiation and morphogenesis, possibly by regulating the midkine expression. 相似文献
86.
Contreras L Gomez-Puertas P Iijima M Kobayashi K Saheki T Satrústegui J 《The Journal of biological chemistry》2007,282(10):7098-7106
Ca(2+) regulation of the Ca(2+) binding mitochondrial carriers for aspartate/glutamate (AGCs) is provided by their N-terminal extensions, which face the intermembrane space. The two mammalian AGCs, aralar and citrin, are members of the malate-aspartate NADH shuttle. We report that their N-terminal extensions contain up to four pairs of EF-hand motifs plus a single vestigial EF-hand, and have no known homolog. Aralar and citrin contain one fully canonical EF-hand pair and aralar two additional half-pairs, in which a single EF-hand is predicted to bind Ca(2+). Shuttle activity in brain or skeletal muscle mitochondria, which contain aralar as the major AGC, is activated by Ca(2+) with S(0.5) values of 280-350 nm; higher than those obtained in liver mitochondria (100-150 nm) that contain citrin as the major AGC. We have used aralar- and citrin-deficient mice to study the role of the two isoforms in heart, which expresses both AGCs. The S(0.5) for Ca(2+) activation of the shuttle in heart mitochondria is about 300 nm, and it remains essentially unchanged in citrin-deficient mice, although it undergoes a drastic reduction to about 100 nm in aralar-deficient mice. Therefore, aralar and citrin, when expressed as single isoforms in heart, confer differences in Ca(2+) activation of shuttle activity, probably associated with their structural differences. In addition, the results reveal that the two AGCs fully account for shuttle activity in mouse heart mitochondria and that no other glutamate transporter can replace the AGCs in this pathway. 相似文献
87.
Sakai T Sakaue H Nakamura T Okada M Matsuki Y Watanabe E Hiramatsu R Nakayama K Nakayama KI Kasuga M 《The Journal of biological chemistry》2007,282(3):2038-2046
The increase in the mass of adipose tissue during the development of obesity can arise through an increase in cell size, an increase in cell number, or both. Here we show that long term maintenance of C57BL/6 mice on a high fat diet (for approximately 25 weeks) induces an initial increase in adipocyte size followed by an increase in adipocyte number in white adipose tissue. The latter effect was found to be accompanied by up-regulation of expression of the gene for the F-box protein Skp2 as well as by downregulation of the cyclin-dependent kinase inhibitor p27(Kip1), a principal target of the SCF(Skp2) ubiquitin ligase, in white adipose tissue. Ablation of Skp2 protected mice from the development of obesity induced either by a high fat diet or by the lethal yellow agouti (A(y)) mutation, and this protective action was due to inhibition of the increase in adipocyte number without an effect on adipocyte hypertrophy. The reduction in the number of adipocyte caused by Skp2 ablation also inhibited the development of obesity-related insulin resistance in the A(y) mutant mice, although the reduced number of beta cells and reduced level of insulin secretion in Skp2-deficient mice resulted in glucose intolerance. Our observations thus indicate that Skp2 controls adipocyte proliferation during the development of obesity. 相似文献
88.
Cooke PS Holsberger DR Cimafranca MA Meling DD Beals CM Nakayama K Nakayama KI Kiyokawa H 《Obesity (Silver Spring, Md.)》2007,15(6):1400-1408
Objective: The etiology of some obesity may involve adipocyte hyperplasia. However, the role of adipocyte number in establishing adipose mass is unclear. Cyclin‐dependent kinase inhibitor p27 regulates activity of cyclin/cyclin‐dependent kinase complexes responsible for cell cycle progression. This protein is critical for establishing adult adipocyte number, and p27 knockout increases adult adipocyte number. The SCF (for Skp1‐Cullin‐F‐box protein) complex targets proteins such as p27 for ubiquitin‐proteosome degradation; the F box protein S phase kinase‐associated protein 2 (Skp2), a component of the SCF complex, specifically recognizes p27 for degradation. We used Skp2 knockout (Skp2?/?) mice to test whether Skp2 loss decreased adipose mass and adipocyte number. Research Methods and Procedures: We measured body weight, adipose mass, adipocyte diameter and number, and glucose tolerance in wild‐type (WT), Skp2?/?, and p27?/?Skp2?/? mice. Mouse embryo fibroblasts (MEFs) from WT and Skp2?/? fetuses were differentiated to determine whether Skp2 directly affected adipogenesis. Results: Skp2?/? mice had a 50% decrease in both subcutaneous and visceral fat pad mass and adipocyte number; these decreases exceeded those in body weight, kidney, or muscle. To test the hypothesis that Skp2 effects on adipocyte number involved p27 accumulation, we used p27?/?Skp2?/? double knockout mice. The Skp2?/? decrements in adipocyte number and fat pad mass were totally reversed in p27?/?Skp2?/? mice. Adipogenesis was inhibited in MEFs from Skp2?/? vs. WT mice, and this inhibition was absent in MEFs from p27?/?Skp2?/? mice. Discussion: Our results indicate that Skp2 regulates adipogenesis and ultimate adipocyte number in vivo; thus, Skp2 may contribute to obesity involving adipocyte hyperplasia. 相似文献
89.
90.
Actin-rich spherical extrusion induced in okadaic acid-treated K562 cells by crosslinking of membrane microdomains. 总被引:1,自引:0,他引:1
Takeshi Baba Keiko Udaka Nobuo Terada Hideho Ueda Yasuhisa Fujii Shinichi Ohno Satoshi B Sato 《The journal of histochemistry and cytochemistry》2003,51(2):245-252
Interconnection between surface microdomains and the actin cytoskeleton is vital to various cellular activities. We studied the responses of okadaic acid (OKA)-treated K562 leukemia cells to crosslinking of membrane microdomains. Although OKA alone induced clustering of surface-bound F-actin, addition of a biotinylated poly(ethylene glycol) derivative of cholesterol (bPEG-Chol) and subsequent binding of streptavidin (SA) further induced accumulation of the clusters, resulting in the formation of a spherical cell extrusion. This extrusion was also induced by direct crosslinking of a raft marker, CD59, and ganglioside GM1. In addition, we found that knockout of the gene encoding Fyn kinase inhibited formation of the spherical extrusion in murine T-cells. In bPEG-Chol/SA-treated cells, CD59, ganglioside GM1, and clathrin/AP-2 were all accumulated on the surface of the actin-rich extrusion, whereas dynamin and transferrin receptors were unaffected. Intermediate filaments, mitochondria, and other vesicles also accumulated. These results suggest that crosslinking of membrane domains exaggerates the linkage between actin and a defined set of membrane proteins in OKA-treated cells. 相似文献