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11.
A soluble phosphoinositide-specific phospholipase C (PLC) was purified 58,000-fold from bovine brain. The enzyme, one of six distinct PLC activities detected in brain, accounted for approximately 15% of the soluble phosphatidylinositol-4,5-bisphosphate-phospholipase C (PIP2-PLC) activity in this tissue. The purification scheme included hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on phosphatidylinositol-Sepharose (PI-Sepharose). The enzyme was specifically eluted from the PI-Sepharose with PI, calcium, and detergent. The purified PLC had an estimated molecular weight of 88,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaved as a monomeric protein during sedimentation on glycerol gradients. The enzyme required calcium for activity, exhibited a pH optimum of 6.5, and cleaved only phosphoinositides. The rates of PIP2 and phosphatidyl-4-monophosphate hydrolysis exceeded the rate of PI hydrolysis under all conditions tested. These properties are consistent with a potential role for this PLC in the early events involved in cellular calcium mobilization. 相似文献
12.
Cloning of a lymphocyte homing receptor reveals a lectin domain 总被引:72,自引:0,他引:72
L A Lasky M S Singer T A Yednock D Dowbenko C Fennie H Rodriguez T Nguyen S Stachel S D Rosen 《Cell》1989,56(6):1045-1055
Lymphocytes express cell surface molecules, termed homing receptors, that mediate their selective attachment to specialized high endothelial venules found within secondary lymphoid organs. Previous work has demonstrated that the adhesive interaction between lymphocytes and the endothelium of peripheral lymph nodes appears to involve a lectin-like activity. Moreover, MEL-14, a monoclonal antibody that blocks lymphocyte-peripheral lymph node binding and presumably recognizes the homing receptor mediating this adhesive interaction, appeared to detect the lectin-like receptor. In this paper we describe the cloning of a murine cDNA that encodes the antigen recognized by the MEL-14 antibody. Characterization of the cDNA encoding the putative mouse peripheral lymph node-specific homing receptor shows that it contains a lectin domain that appears to be involved in the binding of lymphocytes to peripheral lymph node endothelium, thus defining a new type of cellular adhesion molecule. This result supports a novel mechanism for the distribution of lymphocyte populations to various lymphoid organs. 相似文献
13.
Expression of human plasminogen cDNA in a baculovirus vector-infected insect cell system 总被引:5,自引:0,他引:5
J Whitefleet-Smith E Rosen J McLinden V A Ploplis M J Fraser J E Tomlinson J W McLean F J Castellino 《Archives of biochemistry and biophysics》1989,271(2):390-399
A cDNA that encodes the human plasminogen (HPg) amino acid sequence has been inserted adjacent to the polyhedrin promoter in the genome of the baculovirus, Autographa californica nuclear polyhedrosis virus, which was then used to infect cultured cells of the farm armyworm, Spodoptera frugiperda. Under the conditions of cell growth employed, recombinant (rec)-HPg was secreted into the medium after 24 h postinfection (p.i.), at which point virtually no rec-HPg antigen remained inside the cells. At 48 h p.i., a maximal level of intact rec-HPg was present in the medium, which underwent substantial proteolytic digestion after that time. The rec-HPg produced by this expression system possessed a molecular weight equivalent to that of plasma [Glu1]-plasminogen. In addition, the rec-HPg adsorbed to Sepharose-lysine, and was eluted with epsilon-aminocaproic acid (EACA). The recombinant protein also interacted with polyclonal antibodies generated to plasma HPg, as well as with a monoclonal antibody directed against a distinct region (kringle 1-3) of the plasma HPg molecule. Finally, the insect-expressed rec-HPg was activatable to plasmin (HPm) by urokinase. The results demonstrate that this expression system produces a full-length functional single-chain rec-HPg, which can be isolated intact from the culture medium, with some consideration for the temporal events that occur in secretion and longer-term degradation of the protein. The fact that this rec-HPg was converted to HPm with a plasminogen activator, and that it interacted with anti-plasma HPg polyclonal and monoclonal antibodies, as well as with the ligand, EACA, indicates that the molecule retains many of its important functional properties and is folded in an integral manner. 相似文献
14.
K Kitagawa B S Rosen B M Spiegelman G E Lienhard L I Tanner 《Biochimica et biophysica acta》1989,1014(1):83-89
The release of adipsin, a serine proteinase with complement factor D activity, from 3T3-L1 adipocytes was measured by quantitative immunoblotting. This protein is secreted constitutively from 3T3-L1 adipocytes, and there is a 2-fold increase in the amount of adipsin released from cells treated with insulin for 1 to 10 min. Longer exposure to insulin had no further effect on the rate of adipsin release. Adipsin does not appear to be anchored by a glycosylphosphatidylinositol moiety, since adipsin which was been released with Triton X-114 from an intracellular membrane fraction partitions into the aqueous phase. Using a previously described procedure for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters (GT vesicles), we show here that these GT vesicles contain an insulin-responsive pool of adipsin. Thus, insulin stimulates the secretion of a soluble protein, adipsin, as well as translocation to the plasma membrane of integral membrane proteins, including the glucose transporter, the transferrin receptors, and the insulin-like growth factor II receptor. 相似文献
15.
Regulated expression of proenkephalin A during ontogenic development of mesenchymal derivative tissues. 总被引:2,自引:0,他引:2 下载免费PDF全文
Proenkephalin A (PEA), a neuropeptide-encoding gene, is widely expressed in the nervous and endocrine systems. Recently, we demonstrated that in addition to its abundance in fetal brain tissue; PEA is markedly expressed in nondifferentiated mesodermal cells of developing fetuses. To evaluate the implication of these findings for the normal development of tissues of mesodermal origin, we examined the expression of PEA in rat mesenchymal tissues during pre- and postnatal development. Using in situ hybridization analysis combined with RNA blots and a Met-enkephalin-specific radioimmunoassay, we showed that (i) PEA mRNA levels in embryonic and newborn mesenchymal derivative tissues were as high as in the developing brain, (ii) PEA mRNA concentrations in these tissues dropped to undetectable levels shortly after birth, and (iii) this mRNA was translated and processed differentially among different mesenchymal tissues, yielding a tissue-specific pattern of PEA-derived peptides. Our results demonstrate multilevel regulation of PEA gene expression during ontogenic development of mesenchymal derivative tissues. The transient expression and the correlation between PEA mRNA and tissue maturation support the notion that peptides encoded by PEA play a significant role in normal development of these tissues. These findings provide a framework for examination of the mechanisms and roles of PEA gene expression during mesenchymal ontogeny. 相似文献
16.
Transcriptional analysis of the mouse beta-casein gene 总被引:1,自引:0,他引:1
17.
18.
Further characterization of the interaction between L-selectin and its endothelial ligands. 总被引:6,自引:0,他引:6
L-Selectin is a lectin-like receptor on lymphocytes which mediates their attachment to high endothelial venules (HEV) within lymph nodes. Previous work has identified HEV-associated endothelial ligands for L-selectin as sialylated, fucosylated and sulphated glycoproteins of approximately 50 kDa and approximately 90 kDa (Sgp50 and Sgp90). The interaction of L-selectin with these ligands is carbohydrate directed, reflecting the involvement of its amino-terminal, calcium-type lectin domain. It has been reported, and we have confirmed, that anti-Ly22 blocks the adhesive function of L-selectin without reducing its binding to a carbohydrate- based ligand PPME (phosphomannan monoester core from Hansenula hostii). The epitope for this monoclonal antibody depends on the epidermal growth factor (EGF) domain of L-selectin. We demonstrate that anti-Ly22 inhibits the interaction of L-selectin with both of the Sgps, thus establishing that the interaction of L-selectin with HEV can be accounted for by the Sgps. Furthermore, the interaction of trypsin fragments of Sgp50 with L-selectin is inhibitable both by an antibody that maps to the lectin domain and by anti-Ly22. These findings raise the possibility that anti-Ly22 is affecting the function of the lectin domain of L-selectin rather than directly antagonizing the EGF domain. Toward a further characterization of L-selectin's carbohydrate specificity, we show that Sgp50 is partially inactivated by the linkage-specific Newcastle Disease virus sialidase (alpha 2,3 linkage). We additionally demonstrate that a sialyl Lewis x-related tetrasaccharide can interact with L-selectin, as has also been demonstrated for E-selectin and P-selectin. 相似文献
19.
20.
Construction of recombinant murine retroviruses that express the human T-cell leukemia virus type II and human T-cell lymphotropic virus type III trans activator genes. 总被引:28,自引:16,他引:12 下载免费PDF全文
Recombinant retroviruses containing the trans activator genes of human T-cell leukemia virus (HTLV) type II and human T-cell lymphotropic virus type III were constructed. The trans activator genes tat II and tat III were inserted into the murine retroviral vector pZIPNEOSV(X)1. Recombinant plasmids were transfected into the psi 2 and psi AM packaging cell lines that produce murine leukemia virions containing no retroviral RNA. Functional tat II and tat III gene products were expressed as demonstrated by trans activation of HTLV type I and II and human T-cell lymphotropic virus type III long terminal repeat-directed gene expression in the respective infected cells. Use of these recombinant vectors permits high-efficiency gene transfer into a wide variety of cells, thereby providing the opportunity to study the biochemical effects associated with tat II and tat III gene expression. 相似文献