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136.
Recombinant plasmid pGC20 containing (GC)9-insert into SmaI site of pUC19 has been used to study the inhibition of cleavage by six restriction endonucleases; KpnI, SacI, EcoRI and also BamHI, XbaI and SalI, due to Z-DNA formation in negatively supercoiled plasmid. The recognition sites of these enzymes were located at different distances on both sides of the (CG)10-sequence. It was shown that the inhibition of the cleavage by KpnI, SacI and EcoRI was decreased in this series as fast as the distance between recognition site and B-Z junction was increased, and no inhibition of cleavage by EcoRI was found. However, such a correlation was not found in the series of BamHI, XbaI and SalI. In contrast with EcoRI the cleavage by SalI was inhibited completely. These results indicate the difference for "sensitivity" of restriction endonucleases to the structural perturbations of DNA associated with B-Z junctions. It seems to depend on features of the enzyme-substrate interaction mechanisms and also on recognition and flanking sequences of DNA. Consequently, experiments with the inhibition of the cleavage by any enzyme can not help to determine the dimension of the region of DNA with altered structure. 相似文献
137.
Expression of N-linked sialyl Le(x) determinants and O-glycans in the carbohydrate moiety of human amniotic fluid transferrin during pregnancy 总被引:3,自引:0,他引:3
Transferrin, a glycoprotein involved in iron transport in body fluids, was
isolated from amniotic fluid of a hydramniospatient by sequential
anion-exchange chromatography and gel filtration. The N-glycans of human
amniotic fluid transferrin (hAFT) were enzymatically liberated by PNGase-F
digestion, isolated by gel filtration and fractionated by (high-pH)
anion-exchange chromatography. After alkaline borohydride treatment of
native hAFT, the released O-glycans were isolated by gel filtration and
fractionated by anion-exchange chroma-tography. Structure elucidation of 14
N- and 2 O-glycans was performed by 500 or 600 MHz1H-NMR spectroscopy.
Besides conventional N-glycans established earlier for human serum
transferrin (hST), new (alpha1-3)-fucosylated N- glycans were found,
representing sialyl Le(x) elements. Furthermore, as compared to hST, a
higher degree of (alpha1-6)-fucosylation and an increase in branching from
di- to triantennary compounds has been detected. The presence of O-glycans
is demonstrated for the first time in transferrin.
相似文献
138.
The events leading to the completion of cytokinesis after the formation of the midbody and intercellular bridge in D-98S cells were studied with light and electron microscopy. Pairs of daughter cells corresponding to different stages of cytokineses, as determined previously form time lapse films, were selected from embedded monolayers for serial sectioning. Separation of daughter cells is preceded by the reduction in diameter of the intercellular bridge from 1-1.5 μm to approx. 0.2 μm. Two processes contribute to this reduction: (a) The intercellular bridge becomes gradually thinner after telophase; a progressive breakdown of midbody structures accompanies this change; and (b) the more significant contribution to reduction in bridge diameter occurs through the localized constriction of a segment of the intercellular bridge.. The microtubules within the constricted portion of the bridge are forced closer together, and some microtubules disappear as this narrowing progresses. The plasma membrane over the narrowed segments is thrown into a series of wavelike ripples. Separation of daughter cells is achieved through movements of the cells which stretch and break the diameter-reduced bridge. The midbody is discarded after separation and begins to deteriorate. Occasional pairs of daughter cells were found in which incomplete karyokineses resulted in their nuclei being connected by a strand of nuclear material traversing the bridge and midbody. Such cells do not complete cytokinesis but merge together several hours after telophase. This merging of daughter cells coincides with the nearly complete breakdown of the midbody. 相似文献
139.
Secretion and degradation of parathormone as a function of intracellular maturation of hormone pools 下载免费PDF全文
The biosynthesis, processing, and secretion of parthormone and the effect of calcium on these processes were measured in dispersed porcine parthyroid cells incubated with [(35)S]methionine. Proparathormone was detected at 10 min, the earliest time measured, and was rapidly and apparently quantitatively converted to parathormone. The half-life of the prohomormone pool was 15 min. Secretion of parathormone was detected by 20 min. In pulse-chase experiments there was a period between 20 and 40 min during which the wave of newly-synthesized parathormone was secreted. After 40 min during little additional radioactive hormone was secreted, but dibutyryl cyclic AMP, an agent that can mobilize stored parathormone, when added to the incubation mixtures enhanced radioactive parathormone secretion but only after 60 min, although it increased net hormone secretion as determined by radioimmunoassay to the same extent at all times studied. When the ionized calcium concentration of the medium was lowered, more radioactive hormone was secreted at all times but the effect was greatest on that hormone that was synthesized less than 60 min previously ; however, net hormone secretion in contrast to radioactive hormone was enhanced equally at all intervals. These data could mean that the refractoriness to secretion of parathormone 40-60 min of age was related to maturation of secretory container preparatory to storage. Low calcium (0.5 mM) stimulated hormone secretion up to fivefold compared to high calcium (3.0 mM) but did not affect synthesis of parathormone or proparathormne or conversion of the latter to hormone. During processing at least 70 percent of the intracellular parathormone was lost, presumably through proteolysis and this degradation was greater at high calcium. These data have been interpreted in light of the concept that two secretable pools of parathormone exist within the parathyroid. 相似文献
140.
A kinetic study of 1H leads to 3H exchange in C(8) H-groups of purinic residues in DNA. 总被引:1,自引:1,他引:0 下载免费PDF全文
The kinetic study of 1H leads to 3H exchange in C(8) H-groups of purinic residues of DNAs with different G-C content as well as in corresponding dNMP mixtures have been carried out. The present results show that 1H--3H exchange in DNA is retarded (as compared to the exchange in dNMP mixtures) to a lesser extent (Kret =2.4-2.8) than in RNA (Kret=6-8). The degree of retardation in these polymers is practically independent of their nucleotide composition. Assuming the ylide mechanism of exchange reaction it is suggested that the lower rate of 1H leads to 3H exchange in C(8) H-groups of purinic residues in polynucleotides of A-form (RNA and other polyribonucleotides) as compared to those of B-form (DNA and other polydeoxyribonucleotides) might be accounted for by decreased availability of C(8) H-groups for OH-ions of the solvent due to a different microenviroment of these groups in A- and B-type helixes. 相似文献