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2.
One-step gene disruption by cotransformation to isolate double auxotrophs in Candida albicans 总被引:11,自引:0,他引:11
Summary The Candida albicans LEU2 gene was disrupted by substituting lambda DNA for a small deletion within the LEU2 gene. Cotransformation with a selectable URA3 ARS vector was used to introduce a linear fragment containing the disruption into the genome of a C. albicans ura3 deletion mutant. Cotransformants containing the lambda DNA were identified by colony hybridization and the URA3 plasmid was subsequently cured. Leu2 disrupted heterozygotes were detected by Southern hybridization and one disruptant was subsequently treated with UV irradiation. Only one leu2 ura3 mutant (SGY-484) was isolated out of 11,000 mutagenized cells. SGY-484 was transformed to Leu+ with either the C. albicans or Saccharomyces cerevisiae LEU2 gene. Southern hybridization analysis revealed that the mutant is not homozygous for the disruption; the leu2 mutation reverts and is most likely a point mutation. Unexpectedly, an ade2 ura3 mutant was isolated from the same mutagenesis. 相似文献
3.
Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor
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M Ewert B D Shivers H Lüddens H M?hler P H Seeburg 《The Journal of cell biology》1990,110(6):2043-2048
mAbs bd 17, bd 24, and bd 28 raised against bovine cerebral gamma-aminobutyric acid (GABAA)/benzodiazepine receptors were analyzed for their ability to detect each of 12 GABAA receptor subunits expressed in cultured mammalian cells. Results showed that mAb bd 17 recognizes epitopes on both beta 2 and beta 3 subunits while mAb bd 24 is selective for the alpha 1 subunit of human and bovine, but not of rat origin. The latter antibody reacts with the rat alpha 1 subunit carrying an engineered Leu at position four, documenting the first epitope mapping of a GABAA receptor subunit-specific mAb. In contrast to mAbs bd 17 and bd 24, mAb bd 28 reacts with all GABAA receptor subunits tested but not with a glycine receptor subunit, suggesting the presence of shared epitopes on subunits of GABA-gated chloride channels. 相似文献
4.
Elsa A. Schmauder-Chock Stephen P. Chock Myra L. Patchen 《The Histochemical journal》1994,26(2):142-151
Summary The application of an antibody against tumour necrosis factor-alpha (TNF) to thin sections of plastic-embedded mouse tissue has identified sites of TNF activity in normal and endotoxin-treated C3N/HeN mice. Prior to endotoxin treatment, TNF was observed in the secretory granules of the antibacterial Paneth cell and one type of crypt endocrine cell. Four hours after endotoxin treatment, these two types of intestinal cell were found to have degranulated. In addition, endotoxin treatment resulted in the appearance of TNF in the secretory granules of all eosinophils, neutrophils and monocytes in the bone marrow, spleen, lung and the proximal intestine. TNF was also observed in the internal elastic lamina (IEL) of arterioles. These results suggest that the process of TNF induction specifically targets the immune system and the vasculature. An invasive stimulus, such as circulating endotoxin, can provoke the immune cells to be armed with TNF. That same stimulus may cause arteriole smooth muscle cells to secrete TNF. TNF secretion in the presence of arteriole smooth muscle cells may play a role in the adjustment of arteriole tone. In the venules, TNF may be responsible for platelet and neutrophil accumulation which leads to embolism formation. 相似文献
5.
The R1 abdominal retractor muscles of the insect Tenebrio molitor change position during the course of metamorphosis. These muscles detach from the epidermal tendon cells at their anterior ends, and migrate in a posterior direction, parallel to the body axis, to form completely new attachments shortly before adult emergence. Movement is preceded by the loss of sarcomere structure, and the muscles migrate in a partially dedifferentiated condition, closely accompanied by satellite cells and haemocytes. Movement appears to result from the extension of muscle processes towards the epidermis posterior to the larval attachment sites, which contact reciprocal processes extended from the epidermis. Contacts at the new posterior sites are then reinforced, and relinquished at the anterior. This cycle is subsequently repeated. It is envisaged that migration ceases when the muscles encounter a contour in the epidermal gradient known to specify the position of the adult muscle attachment sites. This positional information may be encoded in the epidermal basal lamina. The muscles then redifferentiate, with concurrent differentiation of new epidermal tendon cells. Development of adult muscle attachments appears to require reciprocal morphogenetic interactions between muscle and epidermis. 相似文献
6.
Richard R. Shivers 《Tissue & cell》1977,9(1):43-56
Hemocytes, which contain large cytoplasmic granules, invade the multilamellate glial sheath of ventral ganglion nerve roots of the crayfish following surgical interruption of these nerves. Electron microscopic examination of sections of plasticembedded tissues and replicas of freeze-cleaved ganglion roots reveals numerous slender cytoplasmic extensions of the hemocytes present in damaged nerve sheaths. Many of these microvillous extensions contact glial cells and filamentous extracellular masses. At sites of contact, the microvilli are flattened and occasionally electron-dense material is present in the hemocyte cytoplasm subjacent to the plasma membrane that is closely apposed to a glial cell or connective tissue. Intramembranous surfaces of hemocyte plasmalemmae exposed by freeze-fracture, exhibit particle aggregates 700–2500 Å in diameter. Individual particles are 95–105 Å in diameter. Since the particle aggregates correspond in overall dimension and position in the cell to the sites of contact of hemocyte processes with other sheath components, it is assumed that the two structures are equivalent and represent a junctional complex very similar in structure to some hemidesmosomes. Results from this study strongly suggest that granulated crustacean hemocytes, in response to surgical injury of nerves, invade the damaged nerve sheath and identify damaged glial cells and connective tissue by forming slender cytoplasmic processes which contact elements of the sheath. Tissue components contacted by the hemocytes may subsequently be phagocytosed by them. This is the first report of an invertebrate hemocyte-mediated response to tissue damage in which evidence is presented that the hemocyte may identify necrotic cells and extracellular matrix by forming junctional complexes with them. Crustacean hemocytes, therefore, are likely much more complex functionally than has been previously estimated. 相似文献
7.
Primary culture of capillary endothelium from rat brain 总被引:11,自引:0,他引:11
P D Bowman A L Betz D Ar J S Wolinsky J B Penney R R Shivers G W Goldstein 《In vitro》1981,17(4):353-362
To provide an in vitro system for studies of brain capillary function we developed a method for culture of brain capillary endothelial cells. Capillaries were isolated from rat brain and enzymatically treated to remove the basement membrane and contaminating pericytes. Subsequent Percoll gradient centrifugation resulted in a homogeneous population of capillary endothelial cells that attached to a collagen substrate and incorporated [3H]thymidine. Evidence for the endothelial nature of these cells was provided by the presence of Factor VIII antigen and angiotensin converting enzyme activity and by the failure of platelets to adhere to the cell surface. In addition, the cells were joined together by tight junctions. Thus, primary cultures of these cells retained both endothelial and blood-brain barrier features. 相似文献
8.
Martin Poe Myra N. Williams Norma J. Greenfield Karst Hoogsteen 《Biochemical and biophysical research communications》1975,67(1):240-247
Proton magnetic resonance studies of 1:1 complexes of E. coli dihydrofolate reductase with folate and methotrexate were performed. A resonance at 1850 Hz in 1:1 enzyme-folate was assigned as the C-7 proton of bound folate by comparison with the spectra of enzyme complexed with folate specifically deuterated at C-7. The first order rate constant for folate dissociation was calculated to be less than 110 sec?1. Four of the five histidine residues exhibited the same pK's and chemical shifts in the two complexes with pK values of 8.0, 7.3, 6.5 and ~5. However, one histidine increased its pK by 0.7 units (6.25→6.95) and its C-2 proton resonance shifted upfield 50 Hz when folate was substituted for methotrexate. Comparison of these results with those of chemical modification and ultraviolet difference spectroscopy experiments suggests that this histidine may be in the folate binding site — possibly near the pteridine portion of that site. 相似文献
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10.
Ingrid Elise Amlie Hegertun Kristin Marie Sulheim Gundersen Elisabeth Kleppa Siphosenkosi Gift Zulu Svein Gunnar Gundersen Myra Taylor Jane D. Kvalsvig Eyrun Floerecke Kjetland 《PLoS neglected tropical diseases》2013,7(3)