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61.
Degradation of focal adhesion proteins during nocodazole-induced apoptosis in rat-1 cells 总被引:4,自引:0,他引:4
Kook S Shim SR Kim JI Ahnn JH Jung YK Paik SG Song WK 《Cell biochemistry and function》2000,18(1):1-7
Nocodazole, a microtubule-disrupting agent, induced apoptosis in Rat-1 cells, as indicated by changes in cell morphology, DNA fragmentation, and eventual cell death. During nocodazole-induced apoptosis, normally flat cells became rounded in shape and detached from the extracellular matrix. These morphological changes appeared to be closely associated with degradation of focal adhesion proteins, including p130cas, p125(FAK) and paxillin. p130cas was also degraded in cells treated with staurosporine or etoposide, suggesting that degradation of focal adhesion proteins is a characteristic feature of apoptosis. Nocodazole-induced apoptosis was antagonized by Bcl-2: degradation of focal adhesion proteins was suppressed and cell viability was enhanced in bcl-2 over-expressing cells, even in the presence of nocodazole. Further study of the molecular mechanism of Bcl-2 activation should provide an understanding of the apoptosis induced by disruption of the microtubule network. 相似文献
62.
Park HS Huh SH Kim Y Shim J Lee SH Park IS Jung YK Kim IY Choi EJ 《The Journal of biological chemistry》2000,275(12):8487-8491
Selenium, an essential biological trace element, exerts its modulatory effects in a variety of cellular events including cell survival and death. In our study we observed that selenite protects HEK293 cells from cell death induced by ultraviolet B radiation (UVB). Exposure of HEK293 cells to UVB radiation resulted in the activation of caspase-3-like protease activity, and pretreatment of the cells with z-DEVD-fmk (N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), a caspase-3 inhibitor, prevented UVB-induced cell death. Interestingly, enzymatic activity of caspase-3-like protease in cell lysates of UVB-exposed cells was repressed in vitro by the presence of selenite. Selenite also inhibited the in vitro activity of purified recombinant caspase-3 in cleaving Ac-DEVD-pNA (N-acetyl-Asp-Glu-Asp-p-nitroanilide) or ICAD(L) (inhibitor of a caspase-activated deoxyribonuclease) and in the induction of DNA fragmentation. The inhibitory action of selenite on a recombinant active caspase-3 could be reversed by sulfhydryl reducing agents, such as dithiothreitol and beta-mercaptoethanol. Furthermore, pretreatment of cells with selenite suppressed the stimulation of the caspase-3-like protease activity in UVB-exposed cells, whereas dithiothreitol and beta-mercaptoethanol reversed this suppression of the enzymatic activity. Taken together, our data suggest that selenite inhibits caspase-3-like protease activity through a redox mechanism and that inhibition of caspase-3-like protease activity may be the mechanism by which selenite exerts its protective effect against UVB-induced cell death. 相似文献
63.
Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils 总被引:15,自引:0,他引:15
Takeyama K Dabbagh K Jeong Shim J Dao-Pick T Ueki IF Nadel JA 《Journal of immunology (Baltimore, Md. : 1950)》2000,164(3):1546-1552
Oxidative stress has been implicated in the pathogenesis of inflammatory diseases of airways. Here we show that oxidative stress causes ligand-independent activation of epidermal growth factor receptors (EGFR) and subsequent activation of mitogen-activated protein kinase kinase (MEK)-p44/42 mitogen-activated protein kinase (p44/42mapk), resulting in mucin synthesis in NCI-H292 cells. Exogenous hydrogen peroxide and neutrophils activated by IL-8, FMLP, or TNF-alpha increased EGFR tyrosine phosphorylation and subsequent activation of p44/42mapk and up-regulated the expression of MUC5AC at both mRNA and protein levels in NCI-H292 cells. These effects were blocked by selective EGFR tyrosine kinase inhibitors (AG1478, BIBX1522) and by a selective MEK inhibitor (PD98059), whereas a selective platelet-derived growth factor receptor tyrosine kinase inhibitor (AG1295), a selective p38 MAPK inhibitor (SB203580), and a negative compound of tyrosine kinase inhibitors (A1) were without effect. Neutrophil supernatant-induced EGFR tyrosine phosphorylation, activation of p44/42mapk, and MUC5AC synthesis were inhibited by antioxidants (N-acetyl-cysteine, DMSO, dimethyl thiourea, or superoxide dismutase); neutralizing Abs to EGFR ligands (EGF and TGF-alpha) were without effect, and no TGF-alpha protein was found in the neutrophil supernatant. In contrast, the EGFR ligand, TGF-alpha, increased EGFR tyrosine phosphorylation, activation of p44/42mapk, and subsequent MUC5AC synthesis, but these effects were not inhibited by antioxidants. These results implicate oxidative stress in stimulating mucin synthesis in airways and provide new therapeutic approaches in airway hypersecretory diseases. 相似文献
64.
Distinct and redundant functions of mu1 medium chains of the AP-1 clathrin-associated protein complex in the nematode Caenorhabditis elegans 下载免费PDF全文
In the nematode Caenorhabditis elegans, there exist two micro1 medium chains of the AP-1 clathrin-associated protein complex. Mutations of unc-101, the gene that encodes one of the micro1 chains, cause pleiotropic effects (). In this report, we identified and analyzed the second mu1 chain gene, apm-1. Unlike the mammalian homologs, the two medium chains are expressed ubiquitously throughout development. RNA interference (RNAi) experiments with apm-1 showed that apm-1 and unc-101 were redundant in embryogenesis and in vulval development. Consistent with this, a hybrid protein containing APM-1, when overexpressed, rescued the phenotype of an unc-101 mutant. However, single disruptions of apm-1 or unc-101 have distinct phenotypes, indicating that the two medium chains may have distinct functions. RNAi of any one of the small or large chains of AP-1 complex (sigma1, beta1, or gamma) showed a phenotype identical to that caused by the simultaneous disruption of unc-101 and apm-1, but not that by single disruption of either gene. This suggests that the two medium chains may share large and small chains in the AP-1 complexes. Thus, apm-1 and unc-101 encode two highly related micro1 chains that share redundant and distinct functions within AP-1 clathrin-associated protein complexes of the same tissue. 相似文献
65.
Caspase-mediated cleavage of p130cas in etoposide-induced apoptotic Rat-1 cells 总被引:5,自引:0,他引:5 下载免费PDF全文
Kook S Shim SR Choi SJ Ahnn J Kim JI Eom SH Jung YK Paik SG Song WK 《Molecular biology of the cell》2000,11(3):929-939
Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell-cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD(416)G and DSPD(748)G) in vitro, and point mutations substituting the Asp of DVPD(416)G and DSPD(748)G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD(416)G generated a 74-kDa fragment, which was in turn cleaved at DSPD(748)G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas-paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix. 相似文献
66.
Transforming growth factor-beta (TGF-beta) has been known as a potent immunosuppressive cytokine that can induce apoptosis in lymphoid cells. We established an IL-2-independent cell line, CTLL-2A, from murine T cell line CTLL-2. CTLL-2A expressed higher levels of CD95, CD69, and CD18 molecules than CTLL-2 did, suggesting a more activated state in CTLL-2A than in the CTLL-2 by phenotype. Exposing both CTLL-2 and CTLL-2A to TGF-beta results in differential apoptosis patterns defined by DNA fragmentation and plasma membrane alteration. Among the bcl-2 family members, bcl-2, bcl-w, and bcl-x(L) were also differently expressed in these two cell lines. In CTLL-2A, bcl-x(L) was amplified as a major anti-apoptotic molecule, and TGF-beta-induced cell death was more enhanced than in the original cell line. Caspase 1-like protease was activated by TGF-beta treatment and consequently it cleaved bcl-x(L) in CTLL-2A. TGF-beta-induced DNA fragmentation and cleavage of bcl-x(L) were inhibited by pretreatment with tetra peptide caspase 1 inhibitor, YVAD.cmk. These findings suggest that TGF-beta induces cell death in activated murine T cells through cleavage of bcl-x(L) via activated caspase 1-like protease, which may act as an important executor in that process. 相似文献
67.
The secondary, tertiary, and quaternary structures of the Synechocystis Cph1 phytochrome were investigated by absorption and circular dichroism spectroscopy, size exclusion chromatography, and limited proteolysis. The Cph1 protein was coexpressed with a bacterial thioredoxin in Escherichia coli, reconstituted in vitro with tetrapyrrole chromophores, and purified by chitin affinity chromatography. The resultant Cph1 holoproteins were essentially pure and had the specific absorbance ratio (SAR) of 0.8-0.9. Circular dichroism spectroscopy and limited proteolysis showed that the chromophore binding induced marked conformational changes in the Cph1 protein. The alpha-helical content increased to 42-44% in the holoproteins from 37% in the apoprotein. However, no significant difference in the secondary structure was detected between the Pr and Pfr forms. The tertiary structure of the Cph1 apoprotein appeared to be relatively flexible but became more compact and resistant to tryptic digestion upon chromophore binding. Interestingly, a small chromopeptide of about 30 kDa was still predominant even after longer tryptic digestion. The N-terminal location of this chromopeptide was confirmed by expression in E. coli and in vitro reconstitution with chromophores of the 32.5 kDa N-terminal fragment of the Cph1 protein. This chromopeptide was fully photoreversible with the spectral characteristic similar to that of the full-size Cph1 protein. The Cph1 protein forms dimers through the C-terminal region. These results suggest that the prokaryotic Cph1 phytochrome shares the structural and conformational characteristics of plant phytochromes, such as the two-domain structure consisting of the relatively compact N-terminal and the relatively flexible C-terminal regions, in addition to the chromophore-induced conformational changes. 相似文献
68.
69.
Mycelial protoplast isolation and regeneration of Lentinus lepideus 总被引:14,自引:0,他引:14
Generation of fungal protoplast is essential for fusion and transformation systems. Protoplast fusion offers great potential for the improvement of industrially important microorganisms. To establish conditions for the protoplast isolation and regeneration of the mycelia of Lentinus lepideus, various enzymes and osmotic stabilizers were examined. To investigate suitable medium for the culture of L. lepideus, the mycelia were grown in ten different media at 28 degrees C for 10 days. Among them potato dextrose agar (PDA) medium was found to be the best for colony growth. When Novozym 234, cellulase and beta-glucuronidase were added to the mycelia in combination or alone, Novozym 234 alone at the concentration of 10 mg/ml was the most effective for the protoplast yield. Purified spherical protoplasts of the mycelia were osmotically hypersensitive and further incubation of the mycelia with the lytic enzyme resulted in the older parts of the hyphae swollen. When we applied various osmotic stabilizers at the fixed concentration of 0.6 M on the protoplasts, the yields of protoplasts were increased until 4-hr incubation. However application of sucrose or MgSO4 led to further protection of protoplasts after that time and reached a plateau on 5- and 7-hr incubations, respectively. The suitable incubation time and optimal pH with the lytic enzyme for the maximum release of protoplasts were 6 hrs of incubation and pH 5, respectively. When we examined various osmotic stabilizers for the regeneration of the protoplast, the complete medium containing 0.6 M sucrose induced highest hyphal growth with regeneration frequency of 3.28%. 相似文献
70.
Substituted indoles (5-bromo-, 5-fluoro-, 5-chloro-, 5-hydroxy-, 4-methyl-, 5-methyl-, 6-methyl-, and 7-methylindole) were fed at 1 mM for 10 to 21 days into the tissue cultures of Polygonum tinctorium including cell suspension, root and shoot cultures. They were converted into the corresponding indirubin and indigo derivatives with the exception of 5-hydroxyindole. The yield ranged between 0.7–1.1 mg/g dried weight for indigos and 0.007–0.024 mg/g dried weight for indirubins. Presence of the corresponding indican derivatives in the shoot culture suggested that the pigment production proceeded through the indican formation. © Rapid Science Ltd. 1998 相似文献