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1.
The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.  相似文献   
2.
J. E. Devitt  G. E. Catton 《CMAJ》1966,94(18):929-932
Four postmenopausal women are described in whom breast cancer responded both to bilateral adrenalectomy and bilateral oophorectomy, and subsequently, after relapse, to estrogen therapy. This paradoxical finding demonstrates the complexity of the response of breast carcinoma to hormone manipulations. Simple estrogen dependence and estrogen suppression of pituitary mammotrophins are seen to be inadequate explanations of this phenomenon. Seven fundamental observations are listed that have to be accounted for by hypotheses concerning the endocrinology of breast cancer. It is suggested that in the past we have perhaps overlooked (1) the difficulty of extrapolating observations on experimental animal tumours to spontaneous human neoplasms, (2) the fact that there may be more than one type of breast cancer, and (3) the important role that must be played by the different tissues bearing the metastases.  相似文献   
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A few philosophers of biology have recently explicitly rejected Essential Membership, the doctrine that if an individual organism belongs to a taxon, particularly a species, it does so essentially. But philosophers of biology have not addressed the broader issue, much discussed by metaphysicians on the basis of modal intuitions, of what is essential to the organism. In this paper, I address that issue from a biological basis, arguing for the Kripkean view that an organism has a partly intrinsic, partly historical, essence. The arguments appeal to the demands of biological explanation and are analogous to arguments that I have given elsewhere that a taxon has a partly intrinsic, partly historical, essence. These conclusions about the essences of individuals and taxa yield an argument for Essential Membership. Finally, I cast doubt on LaPorte’s objection to that doctrine arising from the view that a species cannot survive having a daughter.  相似文献   
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Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere–protein linkage was stable for ≥90 h at 37 °C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake.Many proteomic techniques can be used to build a picture of a protein with unknown function, but eventually the individual protein''s activity must be studied. Traditional transfection of encoding DNA permits intracellular expression, but often at uncontrolled, nonphysiological levels. Moreover, DNA transfection can neither deliver protein–inhibitor complexes nor readily deliver multiple proteins in a single experiment and thus exploit knowledge from proteomic protein–protein interaction analyses. In contrast, a truly generic protein transduction reagent could theoretically address all possibilities. We believe that polymeric microspheres could fulfill this role, and we have recently synthesized and characterized dual-functionalized, bio-compatible microspheres that permit intracellular tracking (1). Herein, we now report the development of those microspheres into a protein transduction reagent that can carry protein stably, deliver it efficiently to cells, release the protein in the cytoplasm, and concurrently permit fluorescent imaging of transduced cells.Phagocytosis of microspheres was first observed over 30 years ago (2). Perhaps more unexpectedly, uptake of polystyrene microspheres has recently been reported in many other, nonphagocytic cell types, some of which are traditionally considered to be resistant to DNA transfection and/or protein transduction. For example, microspheres are taken up readily by primary immune cells (3), embryonic stem cells (4), human neural stem cells (5), differentiating mouse neural stem cells (5), and several nonphagocytic cell lines (3, 6, 7). In all instances, the reported efficiency of cellular uptake is high, with “beadfection” of up to 90% of cells being typical (4, 5, 8). No additional reagents aside from the microspheres themselves are required in order to promote cellular uptake, and critically, no toxicity has been observed in any of the cell types beadfected, including HEK293T and L929 cells 2 days after beadfection (8), E14g2a embryonic stem cells 3 days after beadfection (4), and mouse and human neural stem cells 30 days after beadfection (5). In the latter case, the microspheres did not have any deleterious effect on the differentiation of human neural stem cells 30 days after beadfection (5).The mechanism of microsphere entry is also nontoxic, and compelling evidence has been published recently that polystyrene-based microspheres (from 0.2 μm to as large as 2 μm) enter cells via a non-endocytosis, energy-independent mechanism (8). Although unusual, such a mechanism is consistent with claims for the commercial reagent Chariot™ (9). Interestingly, a non-endocytic, energy-independent mechanism has also been reported for the entry of rhenium cluster/polymer hybrid particles into HeLa cells (10). Failure of the microspheres to be endocytosed, at least via a clathrin-dependent mechanism, is perhaps to be predicted, as their diameter considerably exceeds that of clathrin-coated vesicles (typically 100 nm). Bradley and co-workers (8) propose that the entry mechanism for polystyrene-based microspheres is one of passive diffusion in which the microsphere interacts with the membrane, anchors, and, after membrane reorganization, enters the cell, resulting in direct cytoplasmic localization.For functional analysis following transduction, the avoidance of endocytosis or phagocytosis is particularly relevant, as endocytosed particles are destined for endosomes and then, normally, for the lysosomes. The lowered pH of the endosome and, more seriously, the acidic and hydrolytic environment of the lysosome risk disruption of the protein structure and/or function. In contrast, for vaccine delivery (where liposomes can be employed), such exposure is advantageous because protein breakdown forms an essential part of antigen presentation. The potential for protein breakdown in endosomes is also irrelevant for the delivery of protein/peptide drugs such as insulin (for which microencapsulation has proven effective for long-term controlled drug release (11, 12)), as these drugs typically function in the extracellular environment, often exerting their effects by binding to membrane-bound receptors. Thus, although vehicles such as liposomes and nanoparticles are employed both extensively and successfully as drug and vaccine delivery vectors in vivo (1316), they are far from ideal for studying the biological effect of a delivered protein in vitro. Colloidal particles are also endocytosed (17), and therefore these delivery vehicles may present similar disadvantages.Traditionally, protein transduction domains such as HIV TAT (1820) or other cell-penetrating peptides (2123) are used to deliver proteins to cells. Whereas positively charged peptides such as TAT are thought to enter the cells via macropinocytosis (reviewed in Ref. 24), a recent publication suggests that at least some cell-penetrating peptide/bio-cargo complexes (siRNA) are endocytosed (25). Here, although the cargoes avoid the lysosomes, acidification of the endosome is required for endosomal escape of the delivered cargo, and indeed, acidification appears to be a recurring requirement for endosomal escape of biomolecular cargoes using cell-penetrating peptides (reviewed in Ref. 24). Consequently, cell-penetrating peptides are unlikely to become generic tools for functional protein delivery.In contrast, the recent demonstrations that polystyrene microspheres can carry a variety of molecular cargoes with them into the cytoplasm (4, 5, 7, 26, 27) make them particularly exciting as potential vectors for delivering functional proteins and/or protein complexes. β-Galactosidase retains its activity when delivered via this route (7), confirming the potential of microspheres to act as generic protein-delivery vehicles. However, delivered proteins have to date remained tethered to the microspheres, and thus existing studies are limited to proteins that are active in the cytoplasm and, critically, retain their activity when immobilized on polystyrene. For the broad-based study of protein function, the subsequent release of the delivered protein within the cell is desirable.An ideal technology would deliver any protein to any cell type and release that protein in the cell, where it could undertake its normal activity. Here we report the first example of such a microsphere-based approach. Protein is delivered on microspheres and then released in the cell by the reducing cytoplasmic environment. This release is mediated by a linker that attaches the protein stably and covalently to the microspheres in vitro but intracellularly is cleaved over a period of hours. It has already been shown that microspheres are taken up with high efficiency by a range of cell types and can carry a variety of cargoes. Because the chemistry of the linker described herein is amenable to linkage with any molecule containing a free amine moiety, the technology provides a new generic platform for in vitro, cell-based delivery of individual proteins, protein complexes, protein mixtures, or other amino-functionalized molecules.  相似文献   
6.
A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within the complex sequential processes that result in phagocytosis and degradation of apoptotic cells. Intercellular adhesion molecule 3 (ICAM-3, also known as CD50), a human leukocyte-restricted immunoglobulin super-family (IgSF) member, has previously been implicated in apoptotic cell clearance, although its precise role in the clearance process is ill defined. The main objective of this work is to further characterise the function of ICAM-3 in the removal of apoptotic cells. Using a range of novel anti-ICAM-3 monoclonal antibodies (mAbs), including one (MA4) that blocks apoptotic cell clearance by macrophages, alongside apoptotic human leukocytes that are normal or deficient for ICAM-3, we demonstrate that ICAM-3 promotes a domain 1-2-dependent tethering interaction with phagocytes. Furthermore, we demonstrate an apoptosis-associated reduction in ICAM-3 that results from release of ICAM-3 within microparticles that potently attract macrophages to apoptotic cells. Taken together, these data suggest that apoptotic cell-derived microparticles bearing ICAM-3 promote macrophage chemoattraction to sites of leukocyte cell death and that ICAM-3 mediates subsequent cell corpse tethering to macrophages. The defined function of ICAM-3 in these processes and profound defect in chemotaxis noted to ICAM-3-deficient microparticles suggest that ICAM-3 may be an important adhesion molecule involved in chemotaxis to apoptotic human leukocytes.  相似文献   
7.
  总被引:4,自引:4,他引:4  
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9.
Presence of the dihydrouridine (D) stem in the mitochondrial cysteine tRNA is unusually variable among lepidosaurian reptiles. Phylogenetic and comparative analyses of cysteine tRNA gene sequences identify eight parallel losses of the D-stem, resulting in D-arm replacement loops. Sampling within the monophyletic Acrodonta provides no evidence for reversal. Slipped-strand mispairing of noncontiguous repeated sequences during replication or direct replication slippage can explain repeats observed within cysteine tRNAs that contain a D-arm replacement loop. These two mechanisms involving replication slippage can account for the loss of the cysteine tRNA D-stem in several lepidosaurian lineages, and may represent general mechanisms by which the secondary structures of mitochondrial tRNAs are altered.   相似文献   
10.
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