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Gopinath Chattopadhyay Raghavan Varadarajan 《Protein science : a publication of the Protein Society》2019,28(6):1127-1134
With advancements in high‐throughput generation of phenotypic data on mutant proteins, it has become important to individually characterize different proteins or their variants rapidly and with minimal sample consumption. We have made use of a nano differential scanning fluorimetric device, from NanoTemper technologies, to rapidly carry out isothermal chemical denaturation and measure folding/unfolding kinetics of proteins and compared these to corresponding data obtained from conventional spectrofluorimetry. We show that using sample volumes 10‐50‐fold lower than with conventional fluorimetric techniques, one can rapidly and accurately measure thermodynamic and kinetic stability, as well as folding/unfolding kinetics. This method also facilitates characterization of proteins that are difficult to express and purify. 相似文献
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Germination and seedling growth of rice was studied in NaCl and PEG 6000 solutions having osmotic potentials −0.2, −0.4, −0.6
and −0.8 MPa.
At isoosmotic concentrations, the NaCl proved more harmful to germination, seedling growth, per cent moisture content of seedling
organs as well as mobilization of food matter from seed to the growing seedlings. This fact suggested that in rice, at least
in the early stage, a specific ion effect rather than osmotic effect is the prime cause of salt injury. Compared to susceptible
cultivar, the tolerant one was less inhibited by salinity. 相似文献
36.
Cholesterol organization in membranes at low concentrations: effects of curvature stress and membrane thickness 下载免费PDF全文
Cholesterol is often found distributed nonrandomly in domains in biological and model membranes and has been reported to be distributed heterogeneously among various intracellular membranes. Although a large body of literature exists on the organization of cholesterol in plasma membranes or membranes with high cholesterol content, very little is known about organization of cholesterol in membranes containing low amounts of cholesterol. Using a fluorescent cholesterol analog (25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol, or NBD-cholesterol), we have previously shown that cholesterol may exhibit local organization even at very low concentrations in membranes, which could possibly be attributable to transbilayer tail-to-tail dimers. This is supported by similar observations reported by other groups using cholesterol or dehydroergosterol, a naturally occurring fluorescent cholesterol analog which closely mimics cholesterol. In this paper, we have tested the basic features of cholesterol organization in membranes at low concentrations using spectral features of dehydroergosterol. More importantly, we have investigated the role of membrane surface curvature and thickness on transbilayer dimer arrangement of cholesterol using NBD-cholesterol. We find that dimerization is not favored in membranes with high curvature. However, cholesterol dimers are observed again if the curvature stress is relieved. Further, we have monitored the effect of membrane thickness on the dimerization process. Our results show that the dimerization process is stringently controlled by a narrow window of membrane thickness. Interestingly, this type of local organization of NBD-cholesterol at low concentrations is also observed in sphingomyelin-containing membranes. These results could be significant in membranes that have very low cholesterol content, such as the endoplasmic reticulum and the inner mitochondrial membrane, and in trafficking and sorting of cellular cholesterol. 相似文献
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Chattopadhyay MK Murakami Y Matsufuji S 《The Journal of biological chemistry》2001,276(24):21235-21241
The mechanism of the regulatory degradation of ornithine decarboxylase (ODC) by polyamines was studied in fission yeast, Schizosaccharomyces pombe. To regulate cellular spermidine experimentally, we cloned and disrupted S-adenosylmethionine decarboxylase gene (spe2) in S. pombe. The null mutant of spe2 was devoid of spermidine and spermine, accumulated putrescine, and contained a high level of ODC. Addition of spermidine to the culture medium resulted in rapid decrease in the ODC activity caused by the acceleration of ODC degradation, which was dependent on de novo protein synthesis. A fraction of ODC forming an inactive complex concomitantly increased. The accelerated ODC degradation was prevented either by knockout of antizyme gene or by selective inhibitors of proteasome. Thus, unlike budding yeast, mammalian type antizyme-mediated ODC degradation by proteasome is operating in S. pombe. 相似文献
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1. We have examined the interaction of tertiary amine local anesthetics with the bovine hippocampal serotonin1A (5-HT1A) receptor, an important member of the G-protein-coupled receptor superfamily. 2. The local anesthetics inhibit specific agonist and antagonist binding to the 5-HT1A receptor at a clinically relevant concentration range of the anesthetics. This is accompanied by a concomitant reduction in the binding affinity of the 5-HT1A receptor to the agonist. Interestingly, the extent of G-protein coupling of the receptor is reduced in the presence of the local anesthetics. 3. Fluorescence polarization measurements using depth-dependent fluorescent probes show that procaine and lidocaine do not show any significant change in membrane fluidity. On the other hand, tetracaine and dibucaine were found to alter fluidity of the membrane as indicated by a fluorescent probe which monitors the headgroup region of the membrane. 4. The local anesthetics showed inhibition of agonist binding to the 5-HT1A receptor in membranes depleted of cholesterol more or less to the same extent as that of control membranes in all cases. This suggests that the inhibition in ligand binding to the 5-HT1A receptor brought about by local anesthetics is independent of the membrane cholesterol content. 5. Our results on the effects of the local anesthetics on the ligand binding and G-protein coupling of the 5-HT1A receptor support the possibility that G-protein-coupled receptors could be involved in the action of local anesthetics. 相似文献
39.
In Population Genetics, two populations are distinguished from each other on the basis of the differences in the distributions
of the alleles at the locus or loci under consideration. These differences are measured by a “genetic distance” between the
two populations (not to be confused with genetic distance between two loci, which is based on recombination fractions) and
they play a major role in inferences at the population level. Several measures of genetic distance have been proposed by different
authors (Sanghvi 1953; Cavalli-Sforza and Edwards 1967; Jukes and Cantor 1969; Nei 1972; Kimura 1980; Reynoldset al 1983; reviews in Felsenstein 1991; Nei and Kumar 2000). Most of these measures are actually dissimilarity measures and not
mathematically true distance measures (B-Rao and Majumdar 1999). Independently, and much before the geneticists, statisticians
too were concerned with the idea of distinguishing between two (statistical) populations. In order to discriminate between
two populations on the basis of one or more characters, divergence measures like “Mahalanobis’D
2 statistic” or “Mahalanobis’ generalized distance” (1936) and “Bhattacharyya’s distance” (1943, 1946), Kullback-Leibler’s
divergence measure (1951) etc. have been proposed by statisticians. Mukherjee and Chattopadhyaya (1986) have mentioned measures
based on distances, association between two attributes and discrimination function. There are similarities between the distance
measures defined by applied scientists and by theoreticians. Felsenstein (1985) shows that three of the allele frequency-based
genetic distance measures were anticipated by Bhattacharyya (1946). Nei and Takezaki (1994) have also studied the effectiveness
of several genetic distance measures in the context of phylogenetic analysis, including Bhattacharyya’s distance measure. 相似文献
40.