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171.
Domain interchange analyses and site-directed mutagenesis indicate that the His107 residue of the human subunit hGSTM1 has a pronounced influence on catalysis of nucleophilic aromatic substitution reactions, and a H107S substitution accounts for the marked differences in the properties of the homologous hGSTM1-1 (His107) and hGSTM4-4 (Ser107) glutathione S-transferases. Reciprocal replacement of His107 and Ser107 in chimeric enzymes results in reciprocal conversion of catalytic properties. With 1-chloro-2, 4-dinitrobenzene as a substrate, the His107 residue primarily influences the pH dependence of catalysis by lowering the apparent pKa of kcat/Km from 7.8 for the Ser107-containing enzymes to 6.3 for the His107-containing enzymes. There is a parallel shift in the pKa for thiolate anion formation of enzyme-bound GSH. Y6F mutations have no effect on the pKa for these enzymes. Crystal structures of hGSTM1a-1a indicate that the imidazole ring of His107 is oriented toward the substrate binding cleft approximately 6 A from the GSH thiol group. Thus, His107 has the potential to act as a general base in proton transfer mediated through an active site water molecule or directly following a modest conformational change, to promote thiolate anion formation. All wild-type enzymes and H107S chimera have nearly identical equilibrium constants for formation of enzyme-GSH complexes (Kd values of 1-2 x 10(-)6 M); however, KmGSH and Ki values for S-methylglutathione inhibition determined by steady-state kinetics are nearly 100-fold higher. The functions of His107 of hGSTM1a-1a are unexpected in view of a substantial body of previous evidence that excluded participation of histidine residues in the catalytic mechanisms of other glutathione S-transferases. Consequences of His107 involvement in catalysis are also substrate-dependent; in contrast to 1-chloro-2,4-dinitrobenzene, for the nucleophilic addition reaction of GSH to ethacrynic acid, the H107S substitution has no effect on catalysis presumably because product release is rate-limiting. 相似文献
172.
MN Isupov AA Antson EJ Dodson GG Dodson IS Dementieva LN Zakomirdina KS Wilson Z Dauter AA Lebedev EH Harutyunyan 《Canadian Metallurgical Quarterly》1998,276(3):603-623
The X-ray structure of tryptophanase (Tnase) reveals the interactions responsible for binding of the pyridoxal 5'-phosphate (PLP) and atomic details of the K+ binding site essential for catalysis. The structure of holo Tnase from Proteus vulgaris (space group P2(1)2(1)2(1) with a = 115.0 A, b = 118.2 A, c = 153.7 A) has been determined at 2.1 A resolution by molecular replacement using tyrosine phenol-lyase (TPL) coordinates. The final model of Tnase, refined to an R-factor of 18.7%, (Rfree = 22.8%) suggests that the PLP-enzyme from observed in the structure is a ketoenamine. PLP is bound in a cleft formed by both the small and large domains of one subunit and the large domain of the adjacent subunit in the so-called "catalytic" dimer. The K+ cations are located on the interface of the subunits in the dimer. The structure of the catalytic dimer and mode of PLP binding in Tnase resemble those found in aspartate amino-transferase, TPL, omega-amino acid pyruvate aminotransferase, dialkylglycine decarboxylase (DGD), cystathionine beta-lyase and ornithine decarboxylase. No structural similarity has been detected between Tnase and the beta 2 dimer of tryptophan synthase which catalyses the same beta-replacement reaction. The single monovalent cation binding site of Tnase is similar to that of TPL, but differs from either of those in DGD. 相似文献
173.
It is commonly assumed that essentially all of the water in cells has the same ideal motional and colligative properties as does water in bulk liquid state. This assumption is used in studies of volume regulation, transmembrane movement of solutes and electrical potentials, solute and solution motion, solute solubility and other phenomena. To get at the extent and the source of non-ideally behaved water (an operational term dependent on the measurement method), we studied the motional and colligative properties of water in cells, in solutions of amino acids and glycine peptides whose surface characteristics are known, and in solution of bovine serum albumin, hemoglobin and some synthetic polypeptides. Solutions of individual amino acids with progressively larger hydrophobic side chains showed one perturbed water molecule (structured-slowed in motion) per nine square angstroms of hydrophobic surface area. Water molecules adjacent to hydrophobic surfaces form pentagonal structural arrays, as shown by X-ray diffraction studies, that are reported to be disrupted by heat, electric field, hydrostatic pressure and phosphorylation state. Hydrophilic amino acids demonstrated water destructuring (increased motion) that was attributed to dielectric realignment of dipolar water molecules in the electric field between charge groups. In solutions of proteins, several methods indicate the equivalent of 2-8 layers of structured water molecules extending beyond the protein surface, and we have recently demonstrated that induced protein conformational change modifies the extent of non-ideally behaved water. Water self-diffusion rate as measured in three different cell types was about half that of bulk water, indicating that most of the water in these cells was slower in motion than bulk water. In different cell types the extent of osmotically perturbed water ranged from less that half to almost all of the intracellular water. The assumption that essentially all intracellular water has ideal osmotic and motional behavior is not supported by the experimental findings. The non-ideally of cell water is an operational term. Therefore, the amount of non-ideally behaving water is dependent on the characteristics of water targeted, i.e. the measurement method, and a large fraction of it is explainable in mechanistic terms at a molecular level based on solute-solvent interactions. 相似文献
174.
AP Zis LN Yatham RW Lam CM Clark M Srisurapanont K McGarvey 《Canadian Metallurgical Quarterly》1996,15(3):263-270
The corneal wound-healing properties of fibronectin (FN) and a fibronectin hydrolysate (FNH) have been evaluated in comparison with commonly used drugs. Nonpenetrating bilateral surgical keratectomy was performed in male albino rabbits. The left eye was treated with the active product, whereas the right eye served as a control (vehicle). The healing area was measured by planimetry after fluorescein staining at 0, 24, 48, and 72 h after keratectomy. Histological and immunohistochemical analysis of the healing process was also performed. Results were as follows: (a) Nandrolone (p < 0.005) and asiaticoside (p < 0.001), both at 10 mg/ml, in eyedrops delayed the healing process. (b) An ointment containing vitamin A and amino acids also delayed the process but at the limit of statistical significance (p = 0.055). (c) FNH (20-80 mg prot/ml) significantly improved the quality and shortened the time of the healing process at 60 mg prot/ml and above. (d) Human FN (100-800 micrograms/ml) did not affect the healing process. Immunohistochemical analysis demonstrated that FNH accelerated the appearance of endogenous FN in the damaged cornea earlier than in the control eyes. It is concluded that FNH may be useful in the management of corneal wounds, whereas the effectiveness of FN is doubtful. 相似文献
175.
VV Frol'kis SM Novikova LN Bohats'ka RI Potapenko TH Mozzhukhina OK Kul'chyts'ky? MK Burchyns'ka HV Kopylova 《Canadian Metallurgical Quarterly》1997,43(3-4):3-10
Development and regression of experimental atherosclerosis in rabbits is an expressed age-related phenomenon, showing the increase in occurrence and pronouncement of atherosclerotic process with age. A new approach to the disease treatment by means of olivomycin is described. It has shown that while influencing considerably the protein biosynthesis of experimental animal, one can accelerate the regression of atherosclerosis by means of olivomycin and reduce protein and lipid metabolism indicators characterizing the disease development. 相似文献
176.
Coupling of the c-Cbl protooncogene product to ErbB-1/EGF-receptor but not to other ErbB proteins 总被引:1,自引:0,他引:1
G Levkowitz LN Klapper E Tzahar A Freywald M Sela Y Yarden 《Canadian Metallurgical Quarterly》1996,12(5):1117-1125
The ErbB family of transmembrane tyrosine kinases includes the receptor for EGF (ErbB-1), two receptors for NDF/heregulin (ErbB-3 and ErbB-4) and an orphan receptor (ErbB-2). In order to examine the possibility that distinct signal transduction pathways are coupled to each ErbB protein, we examined the interaction of individual ligand-stimulated receptors with the c-Cbl protein, a protooncogene-encoded signaling molecule previously identified in hematopoietic cells. We report that c-Cbl undergoes rapid and sustained phosphorylation on tyrosine residues upon stimulation of fibroblast and epithelial cell lines with ligands of ErbB-1. By contrast, activation of either ErbB-3 or ErbB-4 by NDF did not affect tyrosine phosphorylation of c-Cbl. Likewise, activation of a chimeric ligand-stimulatable ErbB-2 by a heterologous ligand was ineffective. Despite rapidity of the EGF effect, we observed no association of c-Cbl with activated ErbB-1, implying that the interaction is indirect. Our in vitro experiments suggest that a candidate mediator of the interaction is the Grb-2/Ash adaptor protein, which is constitutively bound to c-Cbl. These results indicate that different ErbB proteins can couple to distinct signaling pathways, and therefore their physiological functions are probably non-redundant. 相似文献
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We discuss some points on which we agree, and others on which we disagree, with Cheeseman's target article. Particular agreements include the need for an eclectic approach; disagreements include the misleading distinction between probabilistic and logical reasoning regarding the notion of truth, and also some matters of nonmonotonicity. 相似文献