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31.
The studies reported here demonstrate that ATP may be used in lieu of EDTA to inhibit nuclease digestion of DNA and chromatin. Because ATP is a milder chelator than EDTA and is a biochemical common to the cellular microenvironment in vivo, critical studies of cellular processes that require native structure to be maintained are more feasible without the presence of strong chelators. During the digestion of chromatin into its components by nuclease treatment, ATP assures the retention of nucleoprotein compaction, particularly for large to intermediate-sized oligosomes (2400bp–1000bp in length). ATP used at a concentration of 3.3 mM appears to be somewhat better than EDTA, 1.0 mM, for minimizing degradation of nuclease-treated chromatin. However, termination of nuclease digestion of chromatin and minimization of further degradation by the addition of ATP to a concentration of 1.0 mM was almost equivalent to the addition of EDTA to a concentration of 1.0 mM. Slightly more degradation was observed for the latter condition. In addition, ATP can be used to inhibit endogenous nuclease activity when specific restriction enzymes are needed. Standard low ionic strength DNP, deoxyribonucleoprotein, and DNA electrophoresis of proteinized and deproteinized chromatin oligomers, respectively, indicated that ATP effectively inhibits staphylococcal nuclease. Low ionic strength nucleoprotein electrophoresis to resolve staphylococcal nuclease-digested chromatin indicates that as little as 10–4 M EDTA can promote structural unfolding resulting in changes in apparent mobilities for chromatin oligomers 250 and 600 by in length. Comparative digestion of chromatin with staphlococcal nuclease followed by reaction termination by ATP or EDTA showed that this observation was not merely the result of degradation due to inefficiency of ATP enzyme inhibition.  相似文献   
32.
An experiment was designed to answer two questions as they apply to random skin-flap survival: Is there a therapy that can improve random skin-flap survival when given postoperatively? And if so, when does one start such a therapy? Fluosol-DA 20% (Fluosol) has increased random skin-flap survival when given preoperatively in our laboratory. An experiment was devised to see if it could rescue failing flaps. One-hundred Sprague-Dawley rats were divided into a control (N = 25) and five experimental groups (N = 15). All had 10 X 13 cm reverse McFarlane random flaps raised and reinset. The experimental groups underwent hemodilution with either Ringer's lactate or Fluosol at 4, 8, and 12 hours after flap elevation. All were kept in 50% oxygen for 72 hours postoperatively. The flaps and their corresponding necrotic areas were measured on day 7. As to when to institute a therapy, we simultaneously evaluated the use of a microfluorometer as a monitor of flap survival. Analysis of flap survival showed little difference between control and experimental Ringer's lactate or Fluosol groups. Analysis of the microfluorometric data led to the following points. First, as a monitor of flap viability, it is limited by a lack of specificity and sensitivity. Second, comparison of the data from portions of the flap destined to live with those destined to die suggests that it may not be failure of circulatory inflow that leads to flap death.  相似文献   
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Granule cells were dissociated from rat cerebella with a procedure that yields a 98% pure cell population. Potassium currents in these cells were studied using the patch-clamp technique. Depolarizing pulses of 10 mV step and 100 ms duration from a holding potential of –80 mV elicited two different potassium outward currents: a transient, low-voltage activated component and a long lasting, high-voltage activated component. At +30 mV, the total current reached an amplitude of 2 nA (mean value of 15 experiments). The reversal potential of the transient current, estimated by measuring tail currents, was –77 mV, close to that predicted by the Nernst equation. The transient current was half inactivated with a holding potential of –78 mV and completely inactivated with –50 mV or more positive holding potentials. Finally, the current decay could be fitted by the sum of two exponentials with time constants of about 20 and 250 ms.  相似文献   
35.
We have characterized further the molecular basis of human inherited propionyl CoA carboxylase deficiency by measuring steady state levels of the mRNAs coding for the enzyme's two protein subunits (alpha and beta) and by estimating initial synthesis and steady state levels of the protein subunits in skin fibroblasts from controls and affected patients. We studied cell lines from both major complementation groups (pccA and pccBC) corresponding, respectively, to defects in the carboxylase's alpha and beta subunits. Analysis of pccA lines revealed the absence of alpha chain mRNA in three and an abnormally small alpha-mRNA in a fourth. Despite the presence of normal beta-mRNA in each of these pccA lines, there was complete absence of both alpha and beta protein subunits under steady state conditions, even though new synthesis and mitochondrial import of beta precursors was normal. Results in nine pccBC lines revealed normal alpha mRNA in each, while the amounts of beta-mRNA were distinctly reduced in every case. Correspondingly, alpha protein subunits were present in normal amounts at steady-state, but beta subunits were uniformly decreased. In addition, in six of the nine beta deficient cell lines, partially degraded beta-subunits were observed. To help interpret these results, synthesis and stability of carboxylase subunits were studied in intact HeLa cells using a pulse-chase protocol. Whereas alpha chains were stable over the four hour interval studied, beta chains--initially synthesized in large excess over alpha chains--were degraded rapidly reaching equivalence with alpha chains after two hours.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   
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Summary The hematological features of cold-adapted, red-blooded Antarctic teleosts has prompted this study on the relationship between hemoglobin molecular structure and oxygen-binding properties. The hemolysates from 21 species of 5 families contained one component (Hb 1), often accompanied by an additional, minor one (Hb 2, 5%–10% of total). On the other hand, 3 species of Zoarcidae, a non-endemic family, had 4–5 components. All purified hemoglobins from the former group, but only 1–2 of the 4–5 hemoglobins of Zoarcidae, showed a strong Root effect (pH regulation of oxygen binding). Globins from each hemoglobin have been purified and characterised with respect to molecular structure in several species. The similarity between the complete amino acid sequence of one -chain and those of non-Antarctic -chains is lower than that among the latter sequences, suggesting independent pathways of evolution.Presented at the 5th SCAR Symposium on Antarctic Biology, Hobart, Australia (August 29th-September 3rd, 1988)  相似文献   
38.
We surveyed primary care physicians in Canada to determine their current practices regarding the detection and treatment of hyperlipidemia in asymptomatic adults 20 years of age or more and to determine the role of selected patient characteristics (age, sex and the presence of coronary heart disease [CHD] risk factors) in their management decisions. The self-administered questionnaire was completed by 428 of 804 family physicians and general practitioners. The proportion of physicians who reported having tested at least 50% of their adult patients varied from 29% to 85% and was related to the number of CHD risk factors present and the patient''s age. The proportion of respondents who reported starting dietary or drug therapy among patients with a cholesterol level of 6.2 mmol/L or less increased as the number of CHD risk factors increased and was not related to patient age or sex. According to the factors examined our results suggest that primary care physicians in Canada select patients for screening and treatment mainly on the basis of CHD risk factors present and that their approach is more conservative than that recommended by the Canadian and US consensus conferences.  相似文献   
39.
The human type I keratins K16 and K14 are coexpressed in a number of epithelial tissues, including esophagus, tongue, and hair follicles. We determined that two genes encoding K16 and three genes encoding K14 were clustered in two distinct segments of chromosome 17. The genes within each cluster were tightly linked, and large parts of the genome containing these genes have been recently duplicated. The sequences of the two K16 genes showed striking homology not only within the coding sequences, but also within the intron positions and sequences and extending at least 400 base pairs 5' upstream and 850 base pairs 3' downstream from these genes. Despite the strong homologies between these two genes, only one of the genes encoded a protein which assembled into keratin filaments when introduced into simple epithelial cells. While there were no obvious abnormalities in the sequence of the other gene, its promoter seemed to be significantly weaker, and even a hybrid gene with the other gene's promoter gave rise to a much reduced mRNA level after gene transfection. To demonstrate that the functional K16 gene that we identified was in fact responsible for the K16 expressed in human tissues, we made a polyclonal antiserum which recognized our functional K16 gene product in both denatured and filamentous form and which was specific for bona fide human K16.  相似文献   
40.
We have measured the rates of isotope exchange at the nitrogen of the indole ring of Trp-63 of lysozyme and of L-tryptophan as a function of solution viscosity. We have used two cosolvents, glycerol and ethylene glycol, to modify the relative viscosity. We have derived the appropriate kinetic equations for the alternative possibilities that the exchange takes place either in solution or in the intact protein matrix. Because we chose to study the proton-catalyzed exchange reaction, the rate of it is not expected to be diffusion-limited. We confirmed this by measuring the exchange from tryptophan. These results and the known effects of glycerol and ethylene glycol on the solvation of indole allow us to predict that if the exchange reaction takes place in a protein matrix the effects of the two cosolvents when compared under isoviscous conditions should be identical. This is what we find for Trp-63 in lysozyme at 15, 20 and 26 degrees C. The slope of the linear plot of log k vs. log relative viscosity is 0.6. This strongly supports a model for conformational fluctuations where transient solvation takes place without major changes in protein folding. The most interesting feature of our findings is the fact that a slow reaction admittedly not diffusion-limited shows, when taking place in a protein matrix, a linear dependence on solution viscosity. We suggest that what we observe is the effect of damping of movement of the side chain expressed as a change in the friction along the reaction coordinate in the corresponding phase space. The presence of such effects stresses the validity and usefulness of Kramers model of rate processes for reactions taking place in a protein matrix. Such behavior is predicted by several of the recently proposed general mechanisms of enzyme catalysis.  相似文献   
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