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11.
Oxidative decarboxylation of [1-14C]pyruvate was studied in primary cultures of neurons and of astrocytes. The rate of this process, which is a measure of carbon flow into the tricarboxylic acid (TCA) cycle and which is inhibited by its end product, acetyl CoA, was determined under conditions which would either elevate or reduce the components of the malate-aspartate shuttle (MAS). Addition of aspartate (1 mM) was found to stimulate pyruvate decarboxylation in astrocytes whereas addition of glutamate (or glutamine) had no effect. Since aspartate is a precursor for extramitochondrial malate, and thus intramitochondrial oxaloacetate, whereas glutamate and glutamine are not, this suggests that an increase in oxaloacetate level stimulates TCA cycle activity. Conversely, a reduction of the glutamate content by 3 mM ammonia, which might reduce exchange between glutamate and aspartate across the mitochondrial membrane, suppressed pyruvate decarboxylation. This effect was abolished by addition of glutamate or glutamine or exposure to methionine sulfoximine (MSO). These findings suggest that impairment of MAS activity by removal of MAS constituents decreases TCA cycle activity whereas replenishment of these compounds restores the activity of the TCA cycle. No corresponding effects were observed in neurons. 相似文献
12.
The effect of intraperitoneal administration of L-methionine-DL-sulphoximine (MSI) was studied on branched-chain amino acid transaminases (BCAA-T) in different regions of rat brain and in liver. Administration of an acute dose of MSI (300 mg/kg body weight) resulted in a significant decrease in leucine aminotransferase activity in cerebral cortex, cerebellum, and brain-stem, while the activity of isoleucine aminotransferase was enhanced in hippocampus, corpus striatum, brain stem, and midbrain. Activities of both these enzymes changed marginally or remained unaltered in other regions of the brain. Valine aminotransferase showed a significant decrease in all the regions of the brain except in cerebellum. Following the administration of a sub-acute dose of MSI (150 mg/kg body wt.), the activities of the three BCAA aminotransferases were found to be enhanced in all regions of the brain. The results are discussed in relation to the utilization of BCAA for the production of glutamate and glutamine in hyperammonemia. 相似文献
13.
K. Madhava Madyastha N. S. R. Krishna Murthy 《Applied microbiology and biotechnology》1988,28(4-5):324-329
Summary Incubation of acetates of geraniol, citronellol and linalool with Aspergillus niger resulted in their hydrolysis to corresponding alcohols which were further hydroxylated to their respective 8-hydroxy derivatives. In the case of linalyl acetate, besides linalool and 8-hydroxylinalool, small amounts of geraniol and -terpineol were also formed. Microsomes (105 000xg sediment) prepared from induced cells of A. niger were found to convert (1-3H)citronellol to 8-hydroxy citronellol in the presence of NADPH and O2. The pH optimum for the hydroxylase was found to be 7.6. 相似文献
14.
Y Ma B I Wilson S Bijvoet H E Henderson E Cramb G Roederer M R Ven Murthy P Julien H D Bakker J J Kastelein 《Genomics》1992,13(3):649-653
We have previously reported two common lipoprotein lipase (LPL) gene mutations underlying LPL deficiency in the majority of 37 French Canadians (Monsalve et al., 1990. J. Clin. Invest. 86: 728-734; Ma et al., 1991. N. Engl. J. Med. 324: 1761-1766). By examining the 10 coding exons of the LPL gene in another French Canadian patient, we have identified a third missense mutation that is found in two of the three remaining patients for whom mutations are undefined. This is a G to A transition in exon 6 that results in a substitution of asparagine for aspartic acid at residue 250. Using in vitro site-directed mutagenesis, we have confirmed that this mutation causes a catalytically defective LPL protein. In addition, the Asp250----Asn mutation was also found on the same haplotype in an LPL-deficient patient of Dutch ancestry, suggesting a common origin. This mutation alters a TaqI restriction site in exon 6 and will allow for rapid screening in patients with LPL deficiency. 相似文献
15.
Hydrolysis of p-nitrophenyl-beta-D-glucoside by the beta-glucosidase of a thermophilic and cellulolytic fungus, Humicola insolens was stimulated by two-fold in the presence of high concentrations of beta-mercaptoethanol. This enzyme did not have any free sulfhydryl groups and high concentrations of beta-mercaptoethanol (5% v/v) reduced all of the three disulfide bonds present in the enzyme. In contrast, the hydrolysis of cellobiose and cellulose polymers was inhibited by 50% under the same conditions. Sodium dodecyl sulfate (1% w/v) even in combination with beta-mercaptoethanol did not show any significant effects on this enzyme. These unusual properties suggest that this enzyme may be of significant importance for understanding the structure of the enzyme. 相似文献
16.
Biosynthesis and turnover of a 34-kDa protein growth factor in human cytotrophoblasts 总被引:1,自引:0,他引:1
S Roy-Choudhury A Sen-Majumdar U Murthy V S Mishra H J Kliman J E Nestler J F Strauss M Das 《European journal of biochemistry》1988,172(3):777-783
Recently we isolated a new protein growth factor of 34 kDa from synctial membranes of human placenta. In its polypeptide molecular mass, antigenic structure, receptor binding specificity and partial amino acid sequence, it is unlike several known growth factors, hormones and other proteins. Here we report studies on its biosynthesis and turnover in cultured cytotrophoblasts from term human placenta. Expression of the 34-kDa protein in these cells was studied by immunoprecipitation and Western blot analyses using a highly specific antibody. The experiments have produced the following results. a) Immunostaining and Western blot analyses have demonstrated the presence of immunoreactive 34-kDa protein in isolated cytotrophoblasts. The protein is present in both freshly isolated cells and in cells that have fused in culture to form multinuclear syncytiotrophoblasts. b) Trophoblastic biosynthesis of the protein has been demonstrated by in vitro translation of cellular mRNA and by metabolic labelling experiments with intact cells. c) Pulse-chase experiments show that biosynthesis of the protein does not involve any detectable precursors of higher or lower molecular mass. d) Studies on turnover indicate that the synthesized protein is unusually stable with a half-life of 50-70 h. 相似文献
17.
A non-kallikrein arginine esterase (esterase I) has been purified from dog urine and characterized. The enzyme was purified by a three-step procedure, including ion exchange chromatography on DEAE-Sephacel, affinity chromatography on p-aminobenzamidine-Sepharose, and final gel filtration on Ultrogel AcA-54. The purified preparation gave three protein bands on polyacrylamide gel electrophoresis, all of which had esterolytic activity. The enzyme has a specific activity of 601 esterase units/mg protein. It has negligible kininogenase activity. Esterase I gave two closely migrating protein bands on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of 34,000 and 33,300. Esterase I is a glycoprotein with a pH optimum of 9.5 and a pI of 4.62. The enzyme is strongly inhibited by a host of inhibitors including aprotinin, leupeptin, antipain, soybean trypsin inhibitor, lima bean trypsin inhibitor, and DPhe-Phe-Arg-chloromethyl ketone (I50 in the 10(-9)-10(-8) M range). However, p-aminobenzamidine, N alpha-p-tosyl-lysyl chloromethyl ketone and phenylmethylsulfonyl fluoride were weak inhibitors, with I50 values in the 10(-5)-10(-7) M range. The enzyme preferentially hydrolyzes Pro-Arg bonds. Among fluorogenic substrates used in this study, butyloxycarbonyl-Val-Pro-Arg-methylcoumarinamide (alpha-thrombin substrate) was found to be the best, with a Km of 1.7 microM and a kcat/Km of 6.3 s.microM-1. However, esterase I does not convert fibrinogen to fibrin nor activate plasminogen to plasmin. Esterase I is immunologically distinct from dog urinary kallikrein, having no cross-reactivity with antibodies against dog kallikrein. 相似文献
18.
The genomic sequences of several RNA plant viruses including cucumber mosaic virus, brome mosaic virus, alfalfa mosaic virus
and tobacco mosaic virus have become available recently. The former two viruses are icosahedral while the latter two are bullet
and rod shaped, respectively in particle morphology. The non-structural 3a proteins of cucumber mosaic virus and brome mosaic
virus have an amino acid sequence homology of 35% and hence are evolutionarily related. In contrast, the coat proteins exhibit
little homology, although the circular dichroism spectrum of these viruses are similar. The non-coding regions of the genome
also exhibit variable but extensive homology. Comparison of the brome mosaic virus and alfalfa mosaic virus sequences reveals
that they are probably related although with a much larger evolutionary distance. The polypeptide folds of the coat protein
of three biologically distinct isometric plant viruses, tomato Bushy stunt virus, southern bean mosaic virus and satellite
tobacco necrosis virus have been shown to display a striking resemblance. All of them consist of a topologically similar 8-standard
β-Barrel. The implications of these studies to the understanding of the evolution of plant viruses will be discussed. 相似文献
19.
The Scatchard plot in a radioreceptor assay depends upon the definition of specific binding and the quality of the iodinated
hormone used. Iodination of protein hormones may alter it so that it no longer binds to the receptor and methods are available
to measure the extent of this inactivation. When appropriate corrections are made for specific binding and the amount of inactive
iodinated hormone in an assay, both qualitative and quantitative differences were observed in estimates of binding capacity
and affinity in some well characterised hormone receptor systems.
Theoretical predictions derived from Scatchard analysis of irreversible unimolecular hormone-receptor interactions were applicable,
both qualitatively and quantitatively to two irreversible hormone-receptor systems. A method described permits a more accurate
estimate of capacity from radioreceptor assay data. 相似文献
20.
Kinetic studies of the binding and dissociation of [125I]-human growth hormone to rabbit liver and mammary gland membrane receptors have showed that the binding of [125I]-human growth hormone was largely irreversible to liver membrane receptors and completely to the solubilised mammary gland
receptor. As Scatchard analysis assumes complete reversibility of the hormone-receptor interaction the validity of estimates
of affinity and capacity of receptors derived by this analysis may be questionable.
Theoretical considerations show that in unimolecular irreversible interactions of hormone and receptor, a nonlinear (concave)
or a linear Scatchard plot can be obtained. In linear Scatchard plots the capacity of the receptor obtained by extrapolation
represents an overestimation of true capacity. This overestimation correlates with the value of the intercept in the Scatchard
plot. 相似文献