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871.
Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor 总被引:62,自引:0,他引:62
K Miyazawa H Tsubouchi D Naka K Takahashi M Okigaki N Arakaki H Nakayama S Hirono O Sakiyama K Takahashi 《Biochemical and biophysical research communications》1989,163(2):967-973
Amino acid sequences of four peptide fragments of human hepatocyte growth factor purified from the plasma of patients with fulminant hepatic failure were determined. Based on the amino acid sequence of one of the fragments, two oligodeoxyribonucleotide mixtures were synthesized and used to screen a human placenta cDNA library. On the screening, two overlapping cDNA clones for human hepatocyte growth factor were isolated and the nucleotide sequence of the cDNA was determined. The entire primary structure of the protein was deduced from the sequence. The protein consists of 728 amino acid residues, including a possible signal peptide at the N-terminus. The sequence revealed that the heavy and light chains which comprise the protein are encoded by the same mRNA and are produced from a common translation product by proteolytic processing. 相似文献
872.
The effect of ATP on the formation, spectral properties, and reactions of [beta-(2-furyl)acryloyl]glyceraldehyde-3-phosphate dehydrogenase (FA-GPDH) has been investigated. The chromophoric FA-GPDH has the advantage of providing spectrophotometric signals of the interaction of acyl enzyme with nucleotides and dinucleotides. The results are consistent with the exclusive existence of two acyl-enzyme conformations previously inferred from the interaction of the acyl enzyme with NAD+ and NADH. ATP interaction stabilizes a conformation different from that stabilized by NAD+. The inhibitory effects of ATP on these reactions are consistent with the reported inhibitory effect of ATP on the steady-state reaction with the true substrate. The physiological significance of these results to the regulation of glycolysis, via the ligand-dependent fate of 3-phosphoglycerol-GPDH, is discussed. 相似文献
873.
Isolation and characterization of biologically active murine interleukin-1 alpha derived from expression of a synthetic gene in Escherichia coli 总被引:3,自引:0,他引:3
G O Daumy J M Merenda A S McColl G C Andrews A E Franke K F Geoghegan I G Otterness 《Biochimica et biophysica acta》1989,998(1):32-42
A murine interleukin-1 alpha (mIL-1 alpha) gene coding for amino acids 115 to 270 of the precursor protein (Lomedico, P.T., Gubler, U., Hellmann, C.P., Dukovich, M., Giri, J.G., Pan, Y.E., Collier, K., Semionow, R., Chua, A.O. and Mizel, S.B. (1984) Nature 312, 458-462) was chemically synthesized and expressed in Escherichia coli. mIL-1 alpha, in the form of insoluble inclusion bodies, accounted for approx. 30% of total cellular protein produced by the recombinant strain. A simple isolation protocol was developed in which inclusion body material was first solubilized in 3 M guanidine hydrochloride, and the mIL-1 alpha was then simultaneously purified and allowed to fold to its active conformation by dialysis against distilled water. This procedure yielded pure, biologically active mIL-1 alpha with 41% recovery of the mIL-1 alpha present in the guanidine hydrochloride extract. The purified preparation had the expected amino acid composition, a molar absorptivity of 28,200 M-1.cm-1 and a pI of 5.2. No methionyl-mIL-1 alpha was detected by N-terminal sequence analysis, and the endotoxin level was less than 10 pg per micrograms of mIL-1 alpha. The specific biological activity was 3.10(7) units/mg in a co-mitogenic thymocyte proliferation assay. In addition to full-length mIL-1 alpha, the preparation contained N-terminally truncated mIL-1 alpha species (mainly des-4 and des-6 amino acid forms). The truncated species were isolated and found to have the same biological activity as the complete polypeptide. Thus, the active fragment of mIL-1 alpha appears to consist of a proteinase-sensitive N-terminal region which is not essential for activity, and a proteinase-resistant core which harbors the essential determinants of its cytokine function. 相似文献
874.
Novel human proalbumin variant with intact dibasic sequence facilitates identification of its converting enzyme 总被引:1,自引:0,他引:1
We describe here the identification of a new genetic variant of human proalbumin with an N-terminal sequence of Arg-Gly-Val-Phe-Arg-Arg-Val-Ala-His-Lys-. Proalbumin Blenheim (10%) and mature albumin Blenheim (38%) with an initial sequence of Val-Ala-His-Lys-make up nearly half the serum albumin in affected individuals. Despite retaining an intact dibasic processing site, proalbumin Blenheim (1 Asp----Val) enters the circulation unprocessed. The observed ratio of proalbumin to albumin can be accounted for by proteolysis in the periphery. Employed as a potential substrate, proalbumin Blenheim provides a unique means of identifying the physiologically relevant proalbumin convertase. In vitro studies showed that the variant is readily cleaved by trypsin. However, it is not cleaved by the proposed proalbumin convertase, a membrane-bound Ca2+-dependent proteinase prepared from rat liver Golgi vesicles, which gives authentic cleavage of normal human proalbumin. 相似文献
875.
The partial collapse of dipolar and chemical shift tensors for peptide NH and for the amide NH at cross-link sites in cell wall peptidoglycan, of intact lyophilized cells of Aerococcus viridans, indicates NH vector root-mean-square fluctuations of 23 degrees. This result is consistent with the local mobility calculated in typical picosecond regime computer simulations of protein dynamics in the solid state. The experimental root-mean-square angular fluctuations for both types of NH vectors increase to 37 degrees for viable wet cells at 10 degrees C. The similarity in mobilities for both general protein and cell wall peptidoglycan suggests that one additional motion in wet cells involves cooperative fluctuations of segments of cell walls, attached proteins, and associated cytoplasmic proteins. 相似文献
876.
Fluid-phase interactions between hematologic cells and those of the vessel wall were studied in order to define a role for lipoxygenase products as cell signals in the control of vascular cholesterol metabolism. A functional parameter for hydroxy acids in this system has not been previously demonstrated. We report herein for the first time a biochemical effect of lipoxygenase-derived eicosanoids in the modulation of cholesterol metabolism in smooth muscle cells. Products of platelet-neutrophil interactions served as cell signals in vitro to modulate cholesterol metabolism. We demonstrate that 12-HETE, 12,20-DiHETE, and 12-HETE-1,20-dioic acid activate both lysosomal and cytoplasmic cholesteryl ester (CE) hydrolytic activities, although no effect was observed on CE synthetic (ACAT) activity. The platelet lipoxygenase product, 12-HETE, was the most effective stimulator of CE hydrolysis in the smooth muscle cell, and its conversion to 12,20-DiHETE and the dioic acid derivative by the neutrophils was not necessary for the activation of CE hydrolase. A 2-fold enhancement on CE hydrolysis occurred and was independent of any "cross-activation" by hydroxy acids on production of cyclooxygenase or other lipoxygenase products. The activation of cytoplasmic CE hydrolysis had a lesser cofactor dependence on bile salts in the presence of 12-HETE. This suggested a reduced requirement for surface-active agents in an enzyme-substrate interaction where enzymes are hydrolyzing insoluble lipid substrates. Moreover, 12-HETE induced an additive effect with several lipolytic hormones in the activation of CE catabolism.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
877.
E O Dillingham A Lasslo G Carter-Burks S E Bond R Gollamudi 《Biochimica et biophysica acta》1989,990(2):128-132
The inhibitory potencies of carbamoylpiperidinoalkane and N-alkylnipecotoylpiperazine derivatives on ADP-stimulated human blood platelet aggregation, serotonin (5-HT) release and platelet factor 4 (PF-4) release were evaluated. The procedure was designed to allow concurrent determination of all three sets of values. Most compounds were more than twice as potent in blocking PF-4 (X = 91 +/- 1 (S.E., n = 7)%) compared to their inhibition of 5-HT (X = 38 +/- 1(S.E., n = 6)%) release; the one compound which failed to meet these criteria was still decidedly more powerful in impeding PF-4 than 5-HT release. Since the compounds' platelet aggregation-inhibitory values were within the same range as their 5-HT release-blocking potencies, but had a strikingly greater impact in arresting PF-4 release, it is suggested that the platelet plasma membrane and the lining enveloping the dense bodies may share certain commonalities, while the sheathing encasing the alpha-granules may differ from both in a tangible manner. 相似文献
878.
879.
Direct observation of the enzyme-intermediate complex of 5-enolpyruvylshikimate-3-phosphate synthase by 13C NMR spectroscopy 总被引:2,自引:0,他引:2
The interaction of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, 4,5-dideoxyshikimate 3-phosphate (ddS3P), and [2-13C]-and [3-13C]phosphoenolpyruvate (PEP) has been examined by 13C NMR spectroscopy. Although no resonances due to a dead-end intermediate complex could be detected, an enzyme active site specific formation of pyruvate was observed. The interaction of EPSP synthase with shikimate 3-phosphate (S3P) and [2-13C]- or [3-13C]PEP has been examined by 13C NMR spectroscopy. With [2-13C]PEP, in addition to the resonances due to [2-13C]PEP and [8-13C]EPSP, new resonances appeared at 164.8, 110.9, and 107.2 ppm. The resonance at 164.8 ppm has been assigned to enzyme-bound EPSP. The resonance at 110.9 ppm has been assigned to C-8 of an enzyme-free tetrahedral intermediate of the sort originally proposed by Levin and Sprinson [Levin, J. G., & Sprinson, D. B. (1964) J. Biol. Chem. 239, 1142-1150] and recently independently observed by Anderson et al. [Anderson, K. S., Sikorski, J. A., Benesi, A. J., & Johnson, K. A. (1988) J. Am. Chem. Soc. 110, 6577-6579]. The resonance at 107.2 ppm has been assigned to an enzyme-bound intermediate whose structure is closely related to that of the tetrahedral intermediate. With [3-13C]PEP, new resonances appeared at 88.9, 26.2, 25.5, and 24.5 ppm. The resonance at 88.9 ppm has been assigned to enzyme-bound EPSP. The resonance at 26.2 ppm, which was found to correlate with 1.48 ppm by isotope-edited multiple quantum coherence 1H NMR spectroscopy, has been assigned to the methyl group 4-hydroxy-4-methylketoglutarate.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
880.
Construction of an artificial bifunctional enzyme, beta-galactosidase/galactose dehydrogenase, exhibiting efficient galactose channeling 总被引:5,自引:0,他引:5
The in-frame fusion between two oligomeric enzymes, beta-galactosidase and galactose dehydrogenase, is described. The lacZ gene was fused to the 3' end of the galdh gene with a linker encoding only three amino acids. The purified artificial bifunctional enzyme displayed the enzymic activity of both gene products. The hybrid protein was found in two major forms, consisting of four and six subunits, but other forms could also be identified. The molecular weight of each subunit was determined to be 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bifunctional enzyme shows kinetic advantages over the identical native system in conversion of lactose to galactonolactone. A higher steady-state rate and a reduction of the transient time are observed. This phenomenon is especially pronounced at low initial substrate concentrations and when the pH is adjusted to a level at which the galactose dehydrogenase activity is much higher than that of the beta-galactosidase. 相似文献