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81.
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83.
The in vivo rat hepatocyte autoradiographic assay for unscheduled DNA synthesis (UDS) described by Mirsalis et al, and its in vitro counterpart described earlier by Williams have been employed by us for 4 years. Our experience is that the in vivo assay performs as described in the literature. We have therefore concentrated in this initial paper on the key practical factors we have found to govern the assay sensitivity and reproducibility. This has been achieved by a discussion of the assay performance with two potent rat hepatocarcinogens [the novel azo compound 6-dimethylaminophenylazobenzthiazole (6BT) and the reference agent 2-acetylaminofluorene (2AAF)] and a non-carcinogen of similar structure to 6BT [5-dimethylaminophenylazoindazole (51)]. Assay responses were compared with the effect of these chemicals in the Salmonella mutation assay. We conclude that the in vivo liver UDS assay has a critical role to play as a complement to rodent bone marrow cytogenic assays when conducting assessment studies on agents defined as genotoxic in vitro. However, the in vivo assay is resource-consuming and false results could consequently arise due to incomplete evaluations. Methods to counteract this danger are discussed and criteria for assessing weak UDS responses are suggested. 相似文献
84.
Fox JT Renter DG Sanderson MW Thomson DU Lechtenberg KF Nagaraja TG 《Applied and environmental microbiology》2007,73(16):5253-5260
Our objective was to evaluate methods for identifying cattle with high concentrations of Escherichia coli O157 in their feces. In two experiments, feces were collected from cattle orally inoculated with nalidixic acid (Nal)-resistant E. coli O157, and direct plating of diluted feces on sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) containing Nal was considered the gold standard (GS) method. In experiment 1, methods evaluated were preenrichment direct streak, immunomagnetic separation with most probable number (MPN), and postenrichment direct streak with MPN, all using CT-SMAC. The mean concentration of Nal-resistant E. coli O157 in samples (n = 59) by use of the GS was 3.6 log10 CFU/g. The preenrichment streak detected >3.0 log10 CFU/g samples with a 74.4% sensitivity and 68.8% specificity. Postenrichment direct streak-MPN and immunomagnetic separation-MPN concentrations were correlated significantly with GS concentrations (r = 0.53 and r = 0.39, respectively). In experiment 2 (480 samples), pre- and postenrichment direct streaking performed in triplicate and spiral plating on CT-SMAC were evaluated. For preenrichment streaks, sensitivity was 79.7% and specificity was 96.7% for detecting >3.0 log10 CFU/g when the criterion was positive cultures on at least two plates. For spiral plating at that concentration, sensitivity and specificity were 83.9% and 56.3%, respectively. Postenrichment streaking performed relatively poorly. Triplicate preenrichment streaks of 1:10-diluted feces on CT-SMAC may be useful for identifying cattle shedding high concentrations of E. coli O157. Estimates of sensitivity and specificity enable appropriate application of methods and interpretation of results and may enhance applied research, surveillance, and risk assessments. 相似文献
85.
Stellingwerff T Boon H Jonkers RA Senden JM Spriet LL Koopman R van Loon LJ 《American journal of physiology. Endocrinology and metabolism》2007,292(6):E1715-E1723
Intramyocellular triacylglycerol (IMTG) has been suggested to represent an important substrate source during exercise. In the present study, IMTG utilization during exercise is assessed through the use of various methodologies. In addition, we identified differences in the use of intramyocellular lipids deposited in the immediate subsarcolemmal (SS) area and those stored in the more central region of the fiber. Contemporary stable isotope technology was applied in combination with muscle tissue sampling before and immediately after 3 h of moderate-intensity cycling exercise (62 +/- 2% Vo(2 max)) in eight well-trained male cyclists. Continuous infusions with [U-13C]palmitate and [6,6-(2)H2]glucose were applied to quantify plasma free fatty acid (FFA) and glucose oxidation rates and to estimate whole body IMTG and glycogen use. Both immunohistochemical analyses of oil red O (ORO)-stained muscle cross sections and biochemical triacylglycerol (TG) extraction were performed to assess muscle lipid content. During exercise, plasma FFA, muscle (and/or lipoprotein)-derived TG, plasma glucose, and muscle glycogen oxidation contributed 24 +/- 2, 22 +/- 3, 11 +/- 1, and 43 +/- 3% to total energy expenditure, respectively. In accordance, a significant net decline in muscle lipid content was observed following exercise as assessed by ORO staining (67 +/- 8%) and biochemical TG extraction (49 +/- 8%), and a positive correlation was observed between methods (r = 0.56; P < 0.05). Lipid depots located in the SS area were utilized to a greater extent than the more centrally located depots. This is the first study to show significant use of IMTG as a substrate source during exercise in healthy males via the concurrent implementation of three major methodologies. In addition, this study shows differences in resting subcellular intramyocellular lipid deposit distribution and in the subsequent net use of these deposits during exercise. 相似文献
86.
Brooks NL Trent CM Raetzsch CF Flurkey K Boysen G Perfetti MT Jeong YC Klebanov S Patel KB Khodush VR Kupper LL Carling D Swenberg JA Harrison DE Combs TP 《The Journal of biological chemistry》2007,282(48):35069-35077
Glucose metabolism is altered in long-lived people and mice. Although it is clear that there is an association between altered glucose metabolism and longevity, it is not known whether this link is causal or not. Our current hypothesis is that decreased fasting glucose utilization may increase longevity by reducing oxygen radical production, a potential cause of aging. We observed that whole body fasting glucose utilization was lower in the Snell dwarf, a long-lived mutant mouse. Whole body fasting glucose utilization may be reduced by a decrease in the production of circulating glucose. Our isotope labeling analysis indicated both gluconeogenesis and glycogenolysis were suppressed in Snell dwarfs. Elevated circulating adiponectin may contribute to the reduction of glucose production in Snell dwarfs. Adiponectin lowered the appearance of glucose in the media over hepatoma cells by suppressing gluconeogenesis and glycogenolysis. The suppression of glucose production by adiponectin in vitro depended on AMP-activated protein kinase, a cell mediator of fatty acid oxidation. Elevated fatty acid oxidation was indicated in Snell dwarfs by increased utilization of circulating oleic acid, reduced intracellular triglyceride content, and increased phosphorylation of acetyl-CoA carboxylase. Finally, protein carbonyl content, a marker of oxygen radical damage, was decreased in Snell dwarfs. The correlation between high glucose utilization and elevated oxygen radical production was also observed in vitro by altering the concentrations of glucose and fatty acids in the media or pharmacologic inhibition of glucose and fatty acid oxidation with 4-hydroxycyanocinnamic acid and etomoxir, respectively. 相似文献
87.
Wei WH de Koning DJ Penman JC Finlayson HA Archibald AL Haley CS 《Animal genetics》2007,38(3):222-226
Ear size and erectness are important conformation measurements in pigs. An F(2) population established by crossing European Large White (small, erect ears) with Chinese Meishan (large, flop ears) was used to study the genetic influence of the two ear traits for the first time. A linkage map incorporating 152 markers on 18 autosomal chromosomes was utilised in a genome scan for QTL. Significant QTL were found on SSC1, 5, 7, 9 and 12 for the two traits. The QTL on SSC5 and SSC7 had major effects and were significant at the genome-wide level (P < 0.01). The QTL on SSC1 for ear erectness also had a major effect and was genome-wide significant (P < 0.01). The 95% confidence interval (CI) of the ear size QTL on SSC5 spanned only 4 cM. The QTL on SSC7 for the two ear traits each had a CI of <20 cM, and their positions overlapped with those of the major QTL affecting subcutaneous fat depths on the same chromosome. This study provides insights on the complex genetic influences underlying pig ear traits and will facilitate positional candidate gene analysis to identify causative DNA variants. 相似文献
88.
Vincent J. Tepedino Diane Gail Alston Brosi A. Bradley Trent R. Toler Terry L. Griswold 《Biodiversity and Conservation》2007,16(11):3083-3094
Capitol Reef National Park in central Utah, USA surrounds 22 managed fruit orchards started over a century ago by Mormon pioneers.
Honey bees are imported for pollination, although the area in which the Park is embedded has over 700 species of native bees,
many of which are potential orchard pollinators. We studied the visitation of native bees to apple, pear, apricot, and sweet
cherry over 2 years. Thirty species of bees visited the flowers but, except for pear flowers, most were uncommon compared
to honey bees. Evidence that honey bees prevented native bees from foraging on orchard crop flowers was equivocal: generally,
honey bee and native bee visitation rates to the flowers were not negatively correlated, nor were native bee visitation rates
positively correlated with distance of orchards from honey bee hives. Conversely, competition was tentatively suggested by
much larger numbers of honey bees than natives on the flowers of apples, apricots and cherry; and by the large increase of
native bees on pears, where honey bee numbers were low. At least one-third of the native bee species visiting the flowers
are potential pollinators, including cavity-nesting species such as Osmia lignaria propinqua, currently managed for small orchard pollination in the US, plus several fossorial species, including one rosaceous flower
specialist (Andrena milwaukiensis). We suggest that gradual withdrawal of honey bees from the Park would help conserve native bee populations without decreasing
orchard crop productivity, and would serve as a demonstration of the commercial value of native pollinators. 相似文献
89.
90.
Chemical shift assignment is the first step in all established protocols for structure determination of uniformly labeled
proteins by NMR. The explosive growth in recent years of magic-angle spinning (MAS) solid-state NMR (SSNMR) applications is
largely attributable to improved methods for backbone and side-chain chemical shift correlation spectroscopy. However, the
techniques developed so far have been applied primarily to proteins in the size range of 5–10 kDa, despite the fact that SSNMR
has no inherent molecular weight limits. Rather, the degeneracy inherent to many 2D and 3D SSNMR spectra of larger proteins
has prevented complete unambiguous chemical shift assignment. Here we demonstrate the implementation of 4D backbone chemical
shift correlation experiments for assignment of solid proteins. The experiments greatly reduce spectral degeneracy at a modest
cost in sensitivity, which is accurately described by theory. We consider several possible implementations and investigate
the CANCOCX pulse sequence in detail. This experiment involves three cross polarization steps, from H to CA[i], CA[i] to N[i],
and N[i] to C′[i−1], followed by a final homonuclear mixing period. With short homonuclear mixing times (<20 ms), backbone
correlations are observed with high sensitivity; with longer mixing times (>200 ms), long-range correlations are revealed.
For example, a single 4D experiment with 225 ms homonuclear mixing time reveals ∼200 uniquely resolved medium and long-range
correlations in the 56-residue protein GB1. In addition to experimental demonstrations in the 56-residue protein GB1, we present
a theoretical analysis of anticipated improvements in resolution for much larger proteins and compare these results in detail
with the experiments, finding good agreement between experiment and theory under conditions of stable instrumental performance. 相似文献