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151.
Engineering-enhanced protein secretory expression in yeast with application to insulin 总被引:2,自引:0,他引:2
Kjeldsen T Ludvigsen S Diers I Balschmidt P Sorensen AR Kaarsholm NC 《The Journal of biological chemistry》2002,277(21):18245-18248
Adaptation to efficient heterologous expression is a prerequisite for recombinant proteins to fulfill their clinical and biotechnological potential. We describe a rational strategy to optimize the secretion efficiency in yeast of an insulin precursor by structure-based engineering of the folding stability. The yield of a fast-acting insulin analogue (Asp(B28)) expressed in yeast was enhanced 5-fold by engineering a specific interaction between an aromatic amino acid in the connecting peptide and a phenol binding site in the hydrophobic core of the molecule. This insulin precursor is characterized by significantly enhanced folding stability. The improved folding properties enhanced the secretion efficiency of the insulin precursor from 10 to 50%. The precursor remains fully in vitro convertible to mature fast-acting insulin. 相似文献
152.
A novel extinction screen in Arabidopsis thaliana identifies mutant plants defective in early microsporangial development 总被引:3,自引:0,他引:3
Sorensen A Guerineau F Canales-Holzeis C Dickinson HG Scott RJ 《The Plant journal : for cell and molecular biology》2002,29(5):581-594
Few Arabidopsis mutants defective in early male or female germline development have been reported. A novel extinction screen has been devised which permits the identification of mutants deficient in the earliest stages of anther development. Using mutagenized plants carrying GUS reporter constructs driven by tapetal-specific promoters originally derived from Brassica genes, a wide spectrum of mutants have been identified in Arabidopsis, ranging from those defective in archesporial cell differentiation to others expressed later in development. Crosses between these lines and known anther development mutants have enabled the identification of lines carrying mutations in genes expressed during very early anther formation. Initial characterization reveals these early mutants fall into two classes, gne (GUS-negative) 1-like, and gne2-like. Members of the gne1 mutant class initiate all four layers of the anther wall and an appropriate number of sporogenous cells; however, as development proceeds the tapetal and middle-layer cells enlarge, eventually crushing the sporogenous cells. The gne2 class anthers are disrupted at an earlier stage, with the middle and tapetal layers failing to form, and an excess of sporogenous cells developing until the germline aborts late in meiosis II. Analysis of these mutants has already raised questions about the accuracy of current models of angiosperm anther development. 相似文献
153.
Edward J. Beck Roger G. Sorensen Simon J. Slater Manuel Covarrubias 《The Journal of general physiology》1998,112(1):71-84
Protein kinase C inhibits inactivation gating of Kv3.4 K+ channels, and at least two NH2-terminal serines (S15 and S21) appeared involved in this interaction (Covarrubias et al. 1994. Neuron. 13:1403–1412). Here we have investigated the molecular mechanism of this regulatory process. Site-directed mutagenesis (serine → alanine) revealed two additional sites at S8 and S9. The mutation S9A inhibited the action of PKC by ∼85%, whereas S8A, S15A, and S21A exhibited smaller reductions (41, 35, and 50%, respectively). In spite of the relatively large effects of individual S → A mutations, simultaneous mutation of the four sites was necessary to completely abolish inhibition of inactivation by PKC. Accordingly, a peptide corresponding to the inactivation domain of Kv3.4 was phosphorylated by specific PKC isoforms, but the mutant peptide (S[8,9,15,21]A) was not. Substitutions of negatively charged aspartate (D) for serine at positions 8, 9, 15, and 21 closely mimicked the effect of phosphorylation on channel inactivation. S → D mutations slowed the rate of inactivation and accelerated the rate of recovery from inactivation. Thus, the negative charge of the phosphoserines is an important incentive to inhibit inactivation. Consistent with this interpretation, the effects of S8D and S8E (E = Glu) were very similar, yet S8N (N = Asn) had little effect on the onset of inactivation but accelerated the recovery from inactivation. Interestingly, the effects of single S → D mutations were unequal and the effects of combined mutations were greater than expected assuming a simple additive effect of the free energies that the single mutations contribute to impair inactivation. These observations demonstrate that the inactivation particle of Kv3.4 does not behave as a point charge and suggest that the NH2-terminal phosphoserines interact in a cooperative manner to disrupt inactivation. Inspection of the tertiary structure of the inactivation domain of Kv3.4 revealed the topography of the phosphorylation sites and possible interactions that can explain the action of PKC on inactivation gating. 相似文献
154.
3-(3-Carboxyphenyl)alanine, (3-carboxyphenyl)glycine, 3-(3-carboxy-4-hydroxyphenyl)alanine and (3-carboxy-4-hydroxyphenyl)glycine occur in all parts of Reseda luteola. The concentrations of the two diastereoisomers of 2(S)-4-hydroxy-4-methylglutamic acid undergo seasonal variation, the highest concentrations occurring in the first part of the summer. Highest concentrations are found in the inflorescences. The two diastereoisomers of 2(S)-4-hydroxy-2-aminopimelic acid occur in appreciable amounts in all parts of the plant. They are easily transformed into two structurally different lactones, one of which is very unstable. The structures of these amino acids have been confirmed by synthesis. Green parts of R. luteola also contain substantial quantities of γ-glutamylglutamic acid and glutathione. 相似文献
155.
Lactation markedly increases nutrient requirements in both rodents and ruminants. This is met mostly by increased food intake, but there are also adaptations to increase metabolic efficiency. Despite such changes, lactating animals usually experience periods of negative energy balance. This is not due to a physical constraint on food intake, at least in the rat. Leptin, a hormone secreted by adipocytes, plays an important role in the regulation of appetite and energy balance. During lactation, serum leptin concentration is decreased in both rodents and ruminants, and the nocturnal rise in concentration is lost in rats. Hypoleptinaemia in lactation is primarily a result of negative energy balance. There is also increased clearance of serum leptin, and the attenuation of the nocturnal rise in leptin in rats is at least partly due to the suckling stimulus. Hypoleptinaemia is not the major factor driving hyperphagia in lactating rats, but it probably facilitates the increased food intake. Leptin may play a more important role in this respect in lactating ruminants. Leptin is probably involved in other adaptations that increase metabolic efficiency during lactation. The ability of hypothalamic neuropeptides to respond to leptin does not appear to be altered by lactation in either rodents or ruminants. The reason why lactating animals do not respond to hypoleptinaemia with a further increase in appetite, thereby achieving energy balance, appears to be due to a failure to respond to changes in neuropeptides which mediate the effects of leptin. 相似文献
156.
C. S. Wang D. Gianola D. A. Sorensen J. Jensen A. Christensen J. J. Rutledge 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1994,88(2):220-230
A replicated selection experiment aimed at increasing litter size (total number of pigs born per litter) in Danish Landrace pigs was conducted from 1984 to 1991. The experiment included two selection and two control lines. In each generation, 30 and 14 first litters were produced in selection and control lines, respectively, and dams produced two litters. Each replicate, consisting of one selection and one control line, was founded from 60 families chosen randomly from the population at large. Family selection was practiced, and the criterion was the predicted breeding value for litter size computed using a repeatability animal model, and taking into account all available information. The data consisted of 947 records from 523 dams (424 dams had two litters) representing five cycles of selection of increased litter size. Data were analyzed from a Bayesian perspective, based on marginal posterior distributions of genetic parameters of interest. Marginalization was achieved using Gibbs sampling, with a single chain length of 1 205 000. After discarding the first 5 000 iterations, a sample was drawn every ten iterations, so 120 000 samples in total were saved. Densities were estimated and plotted, and summary statistics were computed from the estimated densities. The posterior means (± standard error) of heritability and repeatability were 0.22 ± 0.06 and 0.32 ± 0.05, respectively. These point estimates of genetic parameters were within the range of literature values, although on the high side. The posterior mean (± standard error) of genetic response to selection, defined as the difference between the mean breeding values of the selected lines and that of the base population, was 1.37 ± 0.43 pigs after five cycles of selection. The regression (through the origin) of breeding values in the selected lines on generation was 0.25 ± 0.08 pigs. Several informative priors constructed from information obtained with field data in this population were used to examine their influence on inferences. The priors were influential because of the relatively small scale of the experiment. An analysis excluding data from one of the control lines gave smaller genetic variance and heritability, and a smaller response to selection. However, it appears that selection for litter size is effective, but that the true rate of response is probably smaller than data from this experiment suggest. 相似文献
157.
Ann Marie Sorensen Douglas Bowman Daniel T. Baran 《Journal of cellular biochemistry》1993,52(2):237-242
1α,25-Dihydroxyvitamin D3 increases intracellular calcium in rat osteoblast-like cells that possess the classic receptor (ROS 17/2.8) as well as those that lack the classic receptor (ROS 24/1), indicating that a separate signalling system mediates this rapid nongenomic action. To determine the intracellular sites of this calcium increase, cytosolic and nuclear fluorescence (340 nm/380 nm ratio) were measured in Fura 2AM loaded ROS 17/2.8 cells using digital microscopy. Within 5 min, cytosolic fluorescence increased by 29% (P < 0.05) and nuclear fluorescence by 30% (P < 0.01) after exposure to 1α,25-dihydroxyvitamin D3 (20 nM). This effect was blocked by the inactive epimer 1β,25-dihydroxyvitamin D3. In an individual cell, cytosolic and nuclear fluorescence increased gradually after 1, 3, and 5 min exposure to vitamin D. Nuclei were then isolated from ROS 17/2.8 cells to directly measure the hormone's effect on nuclear calcium. The calcium content of Fura 2AM loaded nuclei was not affected by increasing the calcium concentration in the incubation buffer from 50 nM to 200 nM. After 5 min, 1α,25-dihydroxyvitamin D3, 20 nM, increased the calcium of isolated nuclei in medium containing 50 nM calcium and 200 nM calcium. 1β,25-dihydroxyvitamin D3, 20 nM, had no effect on nuclear calcium but blocked the 1α,25-dihydroxyvitamin D3 induced rise in the isolated nuclei. The results indicate that the nuclear membrane of the ROS 17/2.8 cells contain calcium permeability barriers and transport systems that are sensitive to and specific for 1α,25-dihydroxyvitamin D3. 1α,25-Dihydroxyvitamin D3 rapidly increases nuclear calcium levels in both intact cells and isolated nuclei suggesting that rapid nongenomic activation of nuclear calcium may play a functional role in osteoblastic activity. 相似文献
158.
Elsie M. B. Sorensen Nancy K. R. Smith Cindy S. Boecker Daniel Acosta 《In vitro cellular & developmental biology. Plant》1984,20(10):771-779
Summary Parenchymal hepatocytes from neonatal rats were isolated, cultured about 24 h, exposed to cadmium with or without calcium,
and processed for scanning electron microscopy. To assess the severity of cadmium-induced changes, exposed hepatocytes were
categorized based upon the extent of morphological damage. Differences in surface blebbing, alterations in microvilli, variations
in the degree of swelling, and changes in cell shape were used to categorize the severity of cell damage. A double-blind morphometric
analysis (a geometricostatistical processing of two-dimensional data for the collection of three-dimensional information)
of cellular changes was conducted for each exposure time and for each concentration of cadmium in the presence or absence
of calcium. Significant decreases occurred in the percent relative volume of normal, flattened cells present in cultures exposed
for 30 min to 50 or 100 μM cadmium in the absence of calcium. In contrast, the percent relative volume of severely damaged spherical cells was significantly
increased after exposure to solutions containing 50 or 100 μM cadmium and lacking calcium. Percent relative volume of intermediate cells (which were slightly swollen and showed changes
in microvillar number) was significantly increased following a 30 min exposure to all cadmium concentrations in the absence
of calcium. The examination of hepatocytes exposed for 60 min showed that the degree of cadmium-induced cytotoxicity was more
severe in the absence of calcium than was the case for the hepatocyte cultures exposed for 30 min: approximately 30% more
spherical cells and 30% fewer flattened cells were present if cultures were exposed in the absence of calcium for 60 min compared
to those exposed for 30 min. The degree of blebbing was significantly greater at all cadmium concentrations in the absence
of calcium. The presence of calcium, therefore, reduced cadmium-induced cytotoxicity in primary cultures of rat hepatocytes
subjected to morphometric analysis after scanning electron microscopy. 相似文献
159.
Bovine intestinal 5'-nucleotidase has been partially purified and characterized for comparison with two other phosphohydrolases from the same tissue, alkaline phosphatase and 5'-nucleotide phosphodiesterase, which are closely related structurally and mechanistically. Kinetic studies with a variety of nucleotides and phosphonate analogs show that, although 5'-nucleotidase is a monoesterase like alkaline phosphatase, it more closely resembles 5'-nucleotide phosphodiesterase in its high affinity and specificity for nucleotide binding. 5'-Nucleotidase is bound very strongly by an affinity column containing a bound phosphonate analog of ADP but is not bound by an affinity column containing a non nucleotide phosphonate which selectively binds alkaline phosphatase. 5'-Nucleotidase is strongly bound by immobilized antibodies prepared against 5'-nucleotide phosphodiesterase, and is less strongly bound by immobilized antibodies prepared against alkaline phosphatase. We conclude that 5'-nucleotidase is structurally more similar to 5'-nucleotide phosphodiesterase than to another monoesterase, alkaline phosphatase. 相似文献
160.
N Alberding R H Austin S S Chan L Eisenstein H Frauenfelder D Good K Kaufmann M Marden T M Nordlund L Reinisch A H Reynolds L B Sorensen G C Wagner K T Yue 《Biophysical journal》1978,24(1):319-334
Using fast flash photolysis, we have measured the binding of CO to carboxymethylated cytochrome c and to heme c octapeptide as a function of temperature (5 degrees-350 degreesK) over an extended time range (100 ns(-1) ks). Experiments used a microsecond dye laser (lambda = 540 nm), and a mode-locked frequency-doubled Nd-glass laser (lambda = 530 nm). At low temperatures (5 degrees-120 degreesK) the rebinding exhibits two components. The slower component (I) is nonexponential in time and has an optical spectrum corresponding to rebiding from an S = 2, CO-free deoxy state. The fast component (I*) is exponential in time with a lifetime shorter than 10 mus and an optical spectrum different from the slow component. In myoglobin and the separated alpha and beta chains of hemoglobin, only process I is visible. The optical absorption spectrum of I* and its time dependence suggest that it may correspond to recombination from an excited state in which the iron has not yet moved out of the heme plane. The temperature dependences of both processes have been measured. Both occur via quantum mechanical tunneling at the lowest temperatures and via over-the-barrier motion at higher temperatures. 相似文献