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1.
A tumor-promoting phorbol ester, [3H]phorbol-12,13-dibutyrate, may bind to a homogeneous preparation of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) in the simultaneous presence of Ca2+ and phospholipid. This tumor promoter does not bind simply to phospholipid nor to the enzyme per se irrespective of the presence and absence of Ca2+. All four components mentioned above appear to be bound together, and the quaternary complex thus produced is enzymatically fully active for protein phosphorylation. Phosphatidylserine is most effective. Various other phorbol derivatives which are active in tumor promotion compete with [3H]phorbol-12,13-dibutyrate for the binding, and an apparent dissociation binding constant of the tumor promoter is 8 nM. This value is identical with the activation constant for protein kinase C and remarkably similar to the dissociation binding constant that is described for intact cell surface receptors. The binding of the phorbol ester is prevented specifically by the addition of diacylglycerol, which serves as activator of protein kinase C under physiological conditions. Scatchard analysis suggests that one molecule of the tumor promoter may bind to every molecule of protein kinase C in the presence of Ca2+ and excess phospholipid. It is suggestive that protein kinase C is a phorbol ester-receptive protein, and the results presented seem to provide clues for clarifying the mechanism of tumor promotion.  相似文献
2.
Two oligonucleotides 14-bases long were synthesized, one complementary to rabbit beta-globin DNA (R beta G14A) and the other with the same sequence except for a single base change (T for C) (R beta G14B). Hybridization conditions were established such that R beta G14A would hybridize to globin DNA while R beta G14B would not. We also synthesized a mixture of 13-base long oligonucleotides (R beta G13Mix), representing eight of the possible coding sequences for amino acids 15-19 of rabbit beta-globin. One of the eight is complementary to globin DNA. R beta G13Mix was found to hybridize specifically to globin DNA under conditions where oligonucleotides forming single base pair mismatches do not. Furthermore, R beta G13Mix was shown to hybridize specifically to colonies containing a plasmid with a globin DNA insert. These results are discussed with respect to a general procedure for screening recombinant clones for those containing DNA coding for a protein of known amino acid sequence.  相似文献
3.
Two new mAbs (M/K-1 and M/K-2) define an adhesion molecule expressed on stromal cell clones derived from murine bone marrow. The protein is similar in size to a human endothelial cell adhesion molecule known as VCAM-1 or INCAM110. VCAM-1 is expressed on endothelial cells in inflammatory sites and recognized by the integrin VLA-4 expressed on lymphocytes and monocytes. The new stromal cell molecule is a candidate ligand for the VLA-4 expressed on immature B lineage lymphocytes and a possible homologue of human VCAM-1. We now report additional similarities in the distribution, structure, and function of these proteins. The M/K antibodies detected large cells in normal bone marrow, as well as rare cells in other tissues. The antigen was constitutively expressed and functioned as a cell adhesion molecule on cultured murine endothelial cells. It correlated with the presence of mRNA which hybridized to a human VCAM-1 cDNA probe. Partial NH2 terminal amino acid sequencing of the murine protein revealed similarities to VCAM-1 and attachment of human lymphoma cells to murine endothelial cell lines was inhibited by the M/K antibodies. All of these observations suggest that the murine and human cell adhesion proteins may be related. The antibodies selectively interfered with B lymphocyte formation when included in long term bone marrow cultures. Moreover, they caused rapid detachment of lymphocytes from the adherent layer when added to preestablished cultures. The VCAM-like cell adhesion molecule on stromal cells and VLA-4 on lymphocyte precursors may both be important for B lymphocyte formation.  相似文献
4.
Purification of human erythropoietin.   总被引:43,自引:0,他引:43  
Human erythropoietin, derived from urine of patients with aplastic anemia, has been purified to apparent homogeneity. The seven-step procedure, which included ion exchange chromatography, ethanol precipitation, gel filtration, and adsorption chromatography, yielded a preparation with a potency of 70,400 units/mg of protein in 21% yield. This represents a purification factor of 930. The purified hormone has a single electrophoretic component in polyacrylamide gels at pH 9, in the presence of sodium dodecylsulfate at pH 7, and in the presence of Triton X-100 at pH 6. Two fractions of the same potency and molecular size, by sodium dodecyl sulfate gel electrophoresis, but differing slightly in mobility at pH 9, were obtained at the last step of fractionation. The nature of the difference between these two components is not yet understood.  相似文献
5.
Monocytes/macrophages exposed to LPS show reduced responses to second stimulation with LPS, which is termed LPS tolerance. In this study, we investigated molecular mechanism of LPS tolerance in macrophages. Mouse peritoneal macrophages pre-exposed to LPS exhibited reduced production of inflammatory cytokines in a time- and dose-dependent manner. Activation of neither IL-1 receptor-associated kinase nor NF-kappaB was observed in macrophages that became tolerant by LPS pretreatment, indicating that the proximal event in Toll-like receptor 4 (TLR4)-MyD88-dependent signaling is affected in tolerant macrophages. Although TLR4 mRNA expression significantly decreased within a few hours of LPS pretreatment and returned to the original level at 24 h, the surface TLR4 expression began to decrease within 1 h, with a gradual decrease after that, and remained suppressed over 24 h. A decrease in inflammatory cytokine production in tolerant macrophages well correlates with down-regulation of the surface TLR4 expression, which may explain one of the mechanisms for LPS tolerance.  相似文献
6.
As a model of the reperfusion injury found in stroke, we have exposed neurons to hypoxia followed by reoxygenation. Neurons treated with hypoxia/reoxygenation (H/R) respond by activating nuclear factor-kappaB (NFkappaB), releasing cytochrome c from their mitochondria, and ultimately dying. Further supporting an apoptotic mechanism, expression of the antiapoptotic Bcl-2 and Bcl-x proteins was increased following H/R. In this model, adenoviral-mediated transduction of lkappaB expression inhibited NFkappaB activation and significantly accelerated cytochrome c release and caspase-dependent neuronal death. At the same time, expression of mutated lkappaB prevented the increased expression of endogenous Bcl-2 and Bcl-x. In the presence of mutated lkappaB, singular overexpression of only Bcl-2 by adenoviral-mediated transduction significantly inhibited cytochrome c release, caspase-3-like activation, and cell death in response to H/R. These findings suggest a pathway where NFkappaB activation induces overexpression of Bcl-2 and Bcl-x, which function to prevent apoptotic cell death following H/R treatments.  相似文献
7.
The human MD-2 molecule is associated with the extracellular domain of human Toll-like receptor 4 (TLR4) and greatly enhances its LPS signaling. The human TLR4-MD-2 complex thus signals the presence of LPS. Little is known, however, about cell surface expression and LPS signaling of the TLR4-MD-2 complex in vivo. We cloned mouse MD-2 molecularly and established a unique mAb MTS510, which reacted selectively with mouse TLR4-MD-2 but not with TLR4 alone in flow cytometry. Mouse MD-2 expression in TLR4-expressing cells enhanced LPS-induced NF-kappaB activation, which was clearly inhibited by MTS510. Thioglycolate-elicited peritoneal macrophages expressed TLR4-MD-2, which was rapidly down-regulated in the presence of LPS. Moreover, LPS-induced TNF-alpha production by peritoneal macrophages was inhibited by MTS510. Collectively, the TLR4-MD-2 complex is expressed on macrophages in vivo and senses and signals the presence of LPS.  相似文献
8.
Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.  相似文献
9.
The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) in order to develop functional assays for the half-transporter, MXR/BCRP/ABCP1. A measure of FTC-inhibitable efflux was generated for each compound in a series of MXR-overexpressing drug-selected cell lines and in ten unselected cell lines which were used to determine if the four fluorescent compounds were sensitive enough to detect the low MXR levels found in drug-sensitive cell lines. FTC-inhibitable efflux of mitoxantrone and prazosin was found in four of the ten cell lines, SF295, KM12, NCI-H460, and A549, and low but detectable levels of MXR mRNA were also observed by Northern analysis in these cells. FTC-inhibitable mitoxantrone and prazosin efflux in both selected and unselected cell lines was found to correlate well with MXR levels as determined by Northern blotting, r(2)=0.89 and r(2)=0.70 respectively. In contrast, rhodamine and LysoTracker were not able to reliably detect MXR. Cytotoxicity assays performed on two of the four unselected cell lines confirmed increased sensitivity to mitoxantrone in the presence of FTC. FTC was found to be a specific inhibitor of MXR, with half-maximal inhibition of MXR-associated ATPase activity at 1 microM FTC. Short term selections of the SF295, KM12, NCI-H460 and A549 cell lines in mitoxantrone resulted in a small but measurable increase in MXR by both Northern blot and functional assay. These studies show that flow cytometric measurement of FTC-inhibitable mitoxantrone or prazosin efflux is a sensitive and specific method for measuring the function of the MXR half-transporter in both selected and unselected cell lines.  相似文献
10.
The significance of drinking green tea in prevention of two of the main lifestyle-related diseases, cancer and cardiovascular disease, was demonstrated in terms of a prospective cohort study on a total of 8,552 general residents in Saitama Prefecture, Japan. On the basis of the follow-up study, we revealed decreased relative risk of cancer incidence for those consuming over 10 cups a day, compared with those consuming below 3 cups: 0.54 (95% confidence interval, 0.22-1.34) for men, 0.57 (0.34-0.98) for women, and 0.59 (0.35-0.98) for both sexes. Furthermore, a significant delay in cancer onset was associated with increased consumption of green tea. Next, decreased relative risk of death from cardiovascular disease was 0.58 (0.34-0.99) for men, 0.82 (0.49-1.38) for women, and 0.72 (0.60-1.04) for members of both sexes consuming over 10 cups a day. Finally, we evaluated the life-prolonging effects of drinking green tea on cumulative survival, using the life table.  相似文献
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