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51.
Isoelectric focusing of glutathione S-transferases from rat liver and kidney 总被引:2,自引:2,他引:0
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The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently. 相似文献
52.
Georgirene D. Vladutiu Richard M. Fike Valerie T. Amigone 《In vitro cellular & developmental biology. Plant》1981,17(7):588-592
Summary Fibroblasts derived from patients with I-cell disease have been shown to accumulate many natural substrates including a three
to fourfold increase in sialic acid content compared to that found in normal fibroblasts. This diverse accumulation of storage
material is due to a massive deficiency of multiple lysosomal hydrolases as they are preferentially excreted into the culture
fluid. There is evidence that the I-cell plasma membrane itself is abnormal with respect to certain transferase activities
and in its sensitivity to freezing and Triton X-100. In this study, we have shown that a neuraminidase-sensitive substrate,
and perhaps others in I-cell fibroblasts, contribute to an increased electronegativity of the I-cell fibroblast surface and
to the cells' sensitivity to freezing. We also found that neuraminidase treatment of I-cell fibroblasts before preservative
freezing in liquid nitrogen enables the cells to adapt more easily to subculture upon thawing.
This project was supported in part by National Institutes of Health (NIH) BRSG Grant RR-05493, NIH Grant 1-R01-HD-11453-01-A1,
National Science Foundation Grant PCM 77-05733, and Maternal and Child Health Service Project 417. Georgirene D. Vladutiu
is the recipient of Research Career Development Award 1K04 HD 00312-01A1 from the National Institutes of Health. 相似文献
53.
Seed maturation of Pisum sativum cv. Progress No. 9 proceeds more slowly in winter than in summer even when the parent plants are grown in greenhouse conditions with light-and heat-supplementation. For parent plants grown under summer and winter conditions the metabolism of [3H]GA9 in cultured seeds is qualitatively different in seeds of equivalent age and qualitatively the same in seeds of equivalent weight. 13-Hydroxylation of [3H]GA9[3H]GA20 is restricted to early stages of seed development. 2-Hydroxylation of [3H]GA92-OH-[3H]GA9 has only been observed at a stage of development after endogenous GA9 has accumulated. 2-OH-GA9 has been shown to be endogenous to pea and is named GA51. H2-GA31 and its conjugate have not been shown to be present in pea and may be induced metabolites of [3H]GA9. The metabolism of GA20GA29 is used to illustrate a technique of feeding [2H][3H]GAs in order to distinguish a metabolite from the same endogenous compound. The in vitro conversion of [3H]GA20[3H]GA29, and the virtual non-metabolism of [3H]GA29 have been confirmed for seeds in intact fruits. These results are discussed in relation to the apparent absence of conjugated GAs in mature pea seeds.Abbreviations GAn
gibberellin An
- GC
gas chromatography
- GC-MS
combined gas chromatography-mass spectrometry
- GC-RC
combined gas chromatography-radio counting
- Me
methyl ester
- RT
etention time
- SICM
selected ion current monitoring
- TLC
thin layer chromatography
- TMS
trimethyl silyl ether
The author is née Frydman 相似文献
54.
David L. Brautigam Balwant S. Khatra Thomas R. Soderling Edmond H. Fischer 《Archives of biochemistry and biophysics》1982,219(1):228-235
An Mn2+-activated phosphoprotein phosphatase of Mr = 80,000 from rabbit muscle catalyzes the dephosphorylation of skeletal muscle proteins that are phosphorylated by either phosphorylase kinase or cAMP-dependent protein kinase. Phosphorylase or glycogen synthase labeled by phosphorylase kinase at seryl residues 14 or 7, respectively, are both dephosphorylated by the phosphatase. Phosphorylase a and glycogen synthase compete with one another for the phosphatase. The phosphatase discriminates between different sites labeled by the cAMP-dependent protein kinase: glycogen synthase phosphorylated either to 1.0 or 1.8 mol phosphate/mol, or phosphorylase kinase phosphorylated on its β-subunit serve as substrates for the phosphatase, but the phosphorylase kinase α-subunit, the phosphorylated phosphatase inhibitor 1, or casein do not. Histone fraction IIA, phosphorylated by the catalytic subunit, was a poor substrate even at a concentration of 100 μm. Phosphorylation of the α-subunit of phosphorylase kinase had no influence on the kinetics of dephosphorylation of the β-subunit. Thus, the Mr = 80,000 phosphatase meets the functional definition of a protein phosphatase 1 [Cohen, P. (1978) Curr. Top. Cell. Regul.14, 117–196]. Furthermore, from a comparison of the known phosphorylated sites of these proteins, it appears that the phosphatase discriminates between different sites present in the phosphoproteins tested on the basis of the Km values for the reactions. It displays a preferential activity toward proteins with a primary structure wherein basic residues are two positions amino-terminal from the phosphoserine, AgrLysX-YSer(P) or LysArgX-YSer(P), rather and one residue away, ArgArgX-Ser(P). 相似文献
55.
In Smittia and other chironomid embryos, both anterior and posterior egg halves can give rise to either anterior or posterior segments. Upon various types of experimental interference, eggs may develop one of four basic body patterns: normal embryos, double cephalons, double abdomens, or inverted embryos. From a previous model of anteroposterior determination, we derive four sets of predictions for the results of combined ultraviolet irradiation and centrifugation experiments. While most of the actual results are in agreement with the predictions, some are not. Most of the discrepancies are resolved in a modified version of the model. According to the new model, anterior and posterior egg halves contain both anterior and posterior cytoplasmic determinants. These are thought to be mutually repressive, and to control an overall determination for either anterior or posterior development. Centrifugation and ultraviolet irradiation appear to affect the relative strength of anterior determinants in one or both of the egg halves, thus modifying the probabilities for the four basic body patterns to develop. Different frequencies of these patterns, which have been obtained after similar experimental treatment of different chironomid species, can be ascribed to species-specific variation in the ultraviolet sensitivity of anterior and posterior determinants. 相似文献
56.
The patterns of termination of DNA replication in human embryonic MRC-5 fibroblasts at four passage levels have been examined by autoradiography. Only chromosome 9 showed statistically significant differences in the time of replication among cultures of different ages. This chromosome terminated replication earlier at later passages than at earlier passages, primarily because of differences in the time of replication of the centromere region. Because very few differences were observed at different passage levels, we conclude that changes in the order of chromosome replication are unlikely to contribute to the phenomenon of in vitro senescence. 相似文献
57.
Nancy Auestad Rose A. Korsak Jack W. Morrow John Edmond 《Journal of neurochemistry》1991,56(4):1376-1386
The oxidation of the fatty acids octanoate and palmitate to CO2 and the ketone bodies acetoacetate and D-(-)-3-hydroxybutyrate was examined in astrocytes that were prepared from cortex of 2-day-old rat brain and grown in primary culture to confluence. Accumulation of acetoacetate (by mass) in the culture medium of astrocytes incubated with octanoate (0.3-0.5 mM) was 50-90 nmol C2 units h-1 mg of protein-1. A similar rate was obtained using radiolabeled tracer methodology with [1-14C]octanoate as labeled substrate. The results from the radiolabeled tracer studies using [1-14C]- and [7-14C]octanoate and [1-14C]-, [13-14C]-, and [15-14C]palmitate indicated that a substantial proportion of the omega-terminal four-carbon unit of these fatty acids bypassed the beta-ketothiolase step of the beta-oxidation pathway and the 3-hydroxy-3-methylglutaryl (HMG)-CoA cycle of the classic ketogenic pathway. The [14C]acetoacetate formed from the 1-14C-labeled fatty acids, obligated to pass through the acetyl-CoA pool, contained 50% of the label at carbon 3 and 50% at carbon 1. By contrast, the [14C]acetoacetate formed from (omega-1)-labeled fatty acids contained 90% of the label at carbon 3 and 10% at carbon 1, whereas that formed from the (omega-3)-labeled fatty acid contained 20% of the label at carbon 3 and 80% at carbon 1. These results indicate that acetoacetate is primarily formed either by the action of 3-oxo-acid-CoA transferase (EC 2.8.3.5) or acetoacetyl-CoA deacylase (EC 3.1.2.11) or both on acetoacetyl-CoA and not by the action of the mitochondrial HMG-CoA cycle involving HMG-CoA lyase (EC 4.1.3.4), which was readily detected, and HMG-CoA synthase (EC 4.1.3.5), which was barely measurable. 相似文献
58.
Leslie M. Loew Lawrence B. Cohen James Dix Eric N. Fluhler Valerie Montana Guy Salama Wu Jian-young 《The Journal of membrane biology》1992,130(1):1-10
The fast potentiometric indicator di-4-ANEPPS is examined in four different preparations: lipid vesicles, red blood cells, squid giant axon, and guinea pig heart. The dye gives consistent potentiometric responses in each of these systems, although some of the detailed behavior varies. In lipid vesicles, the dye displays an increase in fluorescence combined with a red shift of the excitation spectrum upon hyperpolarization. Similar behavior is found in red cells where a dual wavelength radiometric measurement is also demonstrated. The signal-to-noise ratio of the potentiometric fluorescence response is among the best ever recorded on the voltage-clamped squid axon. The dye is shown to be a faithful and persistent monitor of cardiac action potentials with no appreciable loss of signal or deterioration of cardiac activity for periods as long as 2 hr with intermittent illumination every 10 min. These results, together with previously published applications of the dye to a spherical lipid bilayer model and to cells in culture, demonstrate the versatility of di-4-ANEPPS as a fast indicator of membrane potential. 相似文献
59.
Methane oxidation rates were measured in boreal forest soils using seven techniques that provide a range of information on soil CH4 oxidation. These include: (a) short-term static chamber experiments with a free-air (1.7 ppm CH4) headspace, (b) estimating CH4 oxidation rates from soil CH4 distributions and (c)222Rn-calibrated flux measurements, (d) day-long static chamber experiments with free-air and amended (+20 to 2000 PPM CH4) headspaces, (e) jar experiments on soil core sections using free-air and (f) amended (+500 ppm CH4) headspaces, and (g) jar experiments on core sections involving tracer additions of14CH4. Short-term unamended chamber measurements,222Rn-calibrated flux measurements, and soil CH4 distributions show independently that the soils are capable of oxidizing atmospheric CH4 at rates ranging to < 2 mg m–2 d–1. Jar experiments with free-air headspaces and soil CH4 profiles show that CH4 oxidation occurs to a soil depth of 60 cm and is maximum in the 10 to 20 cm zone. Jar experiments and chamber measurements with free-air headspaces show that CH4 oxidation occurs at low (< 0.9 ppm) thresholds. The14CH4-amended jar experiments show the distribution of end products of CH4 oxidation; 60% is transformed to CO2 and the remainder is incorporated in biomass. Chamber and jar experiments under amended atmospheres show that these soils have a high capacity for CH4 oxidation and indicate potential CH4 oxidation rates as high as 867 mg m–2 d–1. Methane oxidation in moist soils modulates CH4 emission and can serve as a negative feedback on atmospheric CH4 increases. 相似文献
60.
Charles M. Paden Bruce S. McEwen Jack Fishman Lenore Snyder Valerie DeGroff 《Journal of neurochemistry》1982,39(2):512-520
Abstract: We have examined the ability of various steroids to compete for high-affinity binding of 3H-labeled ligands to catecholamine receptors in membranes prepared from rat cerebral cortex, striatum, and anterior pituitary. Ligands employed were: [3H]WB4101, [3H]prazosin, [3H]yohimbine, and [3H]clonidine (alpha-noradrenergic); [3H]dihydroalprenolol (beta-noradrenergic); [3H]spiperone and [3H]ADTN (dopaminergic). Only the 17β estrogens were effective and only binding of [3H]spiperone and [3H]ADTN in striatum and [3H]WB4101 and [3H]prazosin in cerebral cortex was reduced. Thus putative dopaminergic and alpha1-noradrenergic sites alone appear to recognize estrogens. A slight competitive effect on [3H]spiperone binding to anterior pituitary membranes was also observed. Among the 17β estrogens tested, the most effective in all cases was the catechol estrogen 2-hydroxyestradiol (2-OHE2). The ability of 2-OHE2 (IC50= 20–30 μM) to inhibit ligand binding to alpha1 receptors was comparable to that of norepinephrine (IC50= 10–20 μM), whereas for dopamine receptors in striatum and pituitary 2-OHE2 was an order of magnitude less effective than dopamine (IC30= 12 μM) in reducing binding of 3H ligands. Estradiol-17β and 2-hydroxyestrone were also able to inhibit binding, but the order of steroid potency was different for alpha1 and dopaminergic receptors. Progesterone, testosterone, and corticosterone were without effect in all cases. These results show that there is specificity of steroid interactions with catecholamine receptors in the brain, both in terms of steroid structure and receptor type. The possible relevance of these interactions to neuroendocrine function is discussed. 相似文献