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91.
Comparison of Phosphoenolpyruvate-Carboxykinase from Autotrophically and Heterotrophically Grown Euglena and Its Role during Dark Anaerobiosis 下载免费PDF全文
Euglena gracilis (1224-5/9) contains phosphoenolpyruvate carboxykinase when grown autotrophic with CO2 in the light. Its yield is higher when an additional carbon source like glucose has been added. The enzyme is lacking in cells provided with CO2 alone and kept in the dark, whereas highest yields result if both glucose and CO2 are provided together in the dark. The enzyme was purified by ammonium sulfate precipitation, gel filtration on Sephacryl S-300 and affinity chromatography on GMP-Sepharose. The latter step was most effective to protect the enzyme from inactivation. Its homogeneity was tested electrophoretically and immunologically. Enzymes from autotrophic and heterotrophically grown cells have identical pH optima and similar isoelectric points. The molecular weight was different: 761,000 for the enzyme from autotrophic and 550,000 for that from heterotrophic cells as determined by gel filtration. The subunit molecular weight of both enzymes is nearly the same. The kinetic data of the enzymes are slightly different. Glycolytic and tricarboxylic acid cycle intermediates are of limited influence on enzyme activity and inhibitory in unphysiological high concentrations. From Ouchterlony double immunodiffusion and enzyme-linked immunosorbent assay, it is evident that the enzyme is localized in the cytosol. With the latter quantification test the phosphoenolpyruvate carboxykinase protein content was found 10 times higher in heterotrophically grown cells than when cultivated under autotrophic conditions. 相似文献
92.
Role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex 总被引:1,自引:0,他引:1
The role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex has been investigated by site-directed mutagenesis. The residues Phe56, Pro60, and Tyr63 of hirudin were replaced by a number of different amino acids, and the kinetics of the inhibition of thrombin by the mutant proteins were determined. Phe56 could be replaced by aromatic amino acids without significant loss in binding energy. While substitution of Phe56 by alanine decreased the binding energy (delta G degrees b by only 1.9 kJ mol-1, replacement of this residue by amino acids with branched side chains caused larger decreases in delta G degrees b. For example, the mutant Phe56----Val displayed a decrease in delta G degrees b of 10.5 kJ mol-1. Substitution of Pro60 by alanine or glycine resulted in a decrease in delta G degrees b of about 6 kJ mol-1. Tyr63 could be replaced by phenylalanine without any loss in binding energy, and replacement of this residue by alanine caused a decrease of 2.2 kJ mol-1 in delta G degrees b. Substitution of Tyr63 by residues with branched side chains resulted in smaller decreases in delta G degrees b than those seen with the corresponding substitutions of Phe56; for example, the mutant Tyr63----Val showed a decrease in binding energy of 5.1 kJ mol-1. The effects of the mutations are discussed in terms of the crystal structure of the thrombin-hirudin complex. 相似文献
93.
The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein that occurs in developmentally regulated isoforms in the vertebrate central nervous system. Monoclonal antibodies (mAbs) against the GlyR distinguish neonatal and adult GlyR proteins by identifying distinct alpha subunit variants within these receptor isoforms. Here, bacterially expressed fusion proteins of the rat GlyR alpha 1 subunit were used to localize the major antigenic epitopes of this protein within its N-terminal 105 amino acids. Synthetic peptides allowed further fine mapping of two mAb binding domains. MAb 2b, specific for the adult alpha 1 subunit, bound to a peptide corresponding to amino acids 1-10, whereas mAb 4a, which recognizes both neonatal and adult GlyR isoforms, reacted with a peptide representing residues 96-105 of the alpha 1 polypeptide. These data define unique and common antigenic epitopes on GlyR alpha subunit variants. 相似文献
94.
The inhibitory glycine receptor (GlyR) of rat spinal cord contains an intrinsic transmembrane channel mediating agonist-gated anion flux. Here, synthetic peptides modelled after the predicted transmembrane domains M2 and M4 of its ligand-binding subunit were incorporated into lipid vesicle membranes and black lipid bilayers to analyze their channel forming capabilities. Both types of peptides prohibited the establishment of, or dissipated, preexisting transmembrane potentials in the vesicle system. Incorporation of peptide M2 into the black lipid bilayer elicited randomly gated single channel events with various conductance states and life-times. Peptide M4 increased the conductance of the bilayer without producing single channels. Exchange of the terminal arginine residues of peptide M2 by glutamate resulted in a significant shift towards cation selectivity of the respective channels as compared to peptide M2. In conclusion, the peptide channels observed differed significantly from native GlyR in both conductivity and ion-selectivity indicating that individual synthetic transmembrane segments are not sufficient to mimic a channel protein composed of subunits with multiple transmembrane segments. 相似文献
95.
Significance of nucleotide sequence alignments: a method for random sequence permutation that preserves dinucleotide and codon usage 总被引:10,自引:0,他引:10
The similarity of two nucleotide sequences is often expressed in terms of
evolutionary distance, a measure of the amount of change needed to
transform one sequence into the other. Given two sequences with a small
distance between them, can their similarity be explained by their base
composition alone? The nucleotide order of these sequences contributes to
their similarity if the distance is much smaller than their average
permutation distance, which is obtained by calculating the distances for
many random permutations of these sequences. To determine whether their
similarity can be explained by their dinucleotide and codon usage, random
sequences must be chosen from the set of permuted sequences that preserve
dinucleotide and codon usage. The problem of choosing random dinucleotide
and codon-preserving permutations can be expressed in the language of graph
theory as the problem of generating random Eulerian walks on a directed
multigraph. An efficient algorithm for generating such walks is described.
This algorithm can be used to choose random sequence permutations that
preserve (1) dinucleotide usage, (2) dinucleotide and trinucleotide usage,
or (3) dinucleotide and codon usage. For example, the similarity of two
60-nucleotide DNA segments from the human beta-1 interferon gene
(nucleotides 196-255 and 499-558) is not just the result of their nonrandom
dinucleotide and codon usage.
相似文献
96.
Modulation of a Recombinant Glycine Transporter (GLYT1b) by Activation of Protein Kinase C 总被引:6,自引:3,他引:3
Abstract: Treatment of human embryonic kidney cells (HEK 293 cells) expressing the mouse glycine transporter 1 (GLYT1b) with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) decreased specific [3 H]glycine uptake. This down-regulation resulted from a reduction of the maximal transport rate and was blocked by the PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine. The inhibitory effect of PMA treatment was also observed after removing all five predicted phosphorylation sites for PKC in GLYT1b by site-directed mutagenesis. These data indicate that glycine transport by GLYT1b is modulated by PKC activation; however, this regulation may involve indirect phosphorylation mechanisms. 相似文献
97.
Temperature-Sensitive Expression of Drosophila Neuronal Nicotinic Acetylcholine Receptors 总被引:4,自引:2,他引:2
Stuart J. Lansdell Bertram Schmitt Heinrich Betz †David B. Sattelle Neil S. Millar 《Journal of neurochemistry》1997,68(5):1812-1819
Abstract: Heterologous expression of cloned Drosophila nicotinic acetylcholine receptor (nAChR) subunits indicates that these proteins misfold when expressed in mammalian cell lines at 37°C. This misfolding can, however, be overcome either by growing transfected mammalian cells at lower temperatures or by the expression of Drosophila nAChR subunits in a Drosophila cell line. Whereas the Drosophila nAChR β subunit (SBD) cDNA, reported previously, lacked part of the SBD coding sequence, here we report the construction and expression of a full-length SBD cDNA. We have examined whether problems in expressing functional Drosophila nAChRs in either Xenopus oocytes or mammalian cell lines can be attributed to an inability of these expression systems to assemble correctly Drosophila nAChRs. Despite expression in what might be considered a more native cellular environment, we have been unable to detect functional nAChRs in a Drosophila cell line unless Drosophila nAChR subunit cDNAs are coexpressed with vertebrate nAChR subunits. Our results indicate that the folding of Drosophila nAChR subunits is temperature-sensitive and strongly suggest that the inability of these Drosophila nAChR subunits to generate functional channels in the absence of vertebrate subunits is due to a requirement for coassembly with as yet unidentified Drosophila nAChR subunits. 相似文献
98.
Primary cultures of mouse spinal cord express the neonatal isoform of the inhibitory glycine receptor 总被引:14,自引:0,他引:14
Expression of the inhibitory glycine receptor complex was investigated in primary cultures of fetal mouse spinal cord using sensitive immunomethods. In these cells, glycine receptor is predominantly of the neonatal isoform characterized by a low affinity for the antagonist strychnine. It contains a ligand binding subunit that differs from that of the adult receptor in antigenic epitopes and apparent molecular weight. Whereas in vivo the neonatal receptor isoform is completely replaced by the adult isoform within 3 weeks after birth, this exchange of subtypes is not seen in culture. The increased expression of the cytoplasmic glycine receptor-associated polypeptide of 93 kd occurring after birth is also seen under culture conditions. Purification of glycine receptor from cultures yielded polypeptides of 49 kd and 93 kd, suggesting that the membrane-spanning core of the neonatal receptor may be a homooligomer composed of 49 kd subunits. About half of the 49 kd subunit is cleaved by trypsinization of the cultures, indicating a predominant cell surface localization of the receptor. Pulse-labeling experiments revealed the 49 kd subunit to be a metabolically stable glycoprotein (half-life approximately 2 days). After its synthesis, a transition time of 30-45 min is required for acquisition of a strychnine binding conformation. 相似文献
99.
The effect of metyrapone on the activity of the steroid 17alpha-hydroxylase from rat testis was evaluated. A competitive pattern of inhibition was observed after analysis of data using a least mean squares computer analysis. The substrate for the hydroxylase induced a Type I difference spectrum in an active suspension of Triton treated microsomes. The magnitude of this spectral change was dependent on steroid concentration and was diminished by metyrapone. The effect of metyrapone was abolished at infinite steroid concentration. These results confirm the participation of cytochrome P-450 as a reactant in the 17alpha-hydroxylase reaction. 相似文献
100.
Summary
D-Glucose and D-xylose addition to not-growing Rhodotorula gracilis cells brings about alterations in pyruvate kinase and phosphoenolpyruvate carboxykinase activities characteristic for glycolysis and gluconeogenesis, respectively.Abbreviations used PK
Pyruvate kinase (EC 2.7.1.40)
- PEPCK
Phosphoenolpyruvate carboxykinase (EC 4.1.1.32)
- PFK
Phosphofructokinase (EC 2.7.1.11) 相似文献