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1.
2.
Purified rat liver UDP-GlcNAc:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II (Bendiak, B., and Schachter, H. (1987) J. Biol. Chem. 262, 5775-5783) has been characterized kinetically, and its substrate specificity and inhibition characteristics have been determined. Kinetic data indicate an ordered, or largely ordered sequential mechanism, with UDP-GlcNAc binding prior to the acceptor. The minimal acceptor structure required for full activity is: (Formula: see text) The acceptor molecule must have a terminal Man alpha 1-6 residue, and a terminal GlcNAc beta 1-2Man alpha 1-3 branch to display any activity, but does not require the reducing GlcNAc residue, as the enzyme was about 50% as active after reduction of this residue to N-acetylglucosaminitol. Additional residues (Gal beta 1-4 on the GlcNAc beta 1-2Man alpha 1-3 arm, or a bisecting GlcNAc beta 1-4 on the beta-Man residue) abolish catalytic activity. These results suggest a rigid order in the biosynthesis of all N-linked complex oligosaccharides (bisected and nonbisected bi-, tri-, and tetraantennary), since the enzyme must act to completion prior to the action of either UDP-Gal:GlcNAc beta 1-4 galactosyltransferase or N-acetylglucosaminyltransferase III to make such structures. Inhibition studies with nucleotides, sugars, nucleotide-sugars, and their respective analogues revealed that analogues of UDP and UTP, in which the hydrogen at the 5 position of the uracil was substituted with -CH3, bromine, or mercury (as the mercaptide) were good reversible inhibitors of the enzyme, whereas substitution at other sites lessened the inhibitory potency, usually to a large degree. 相似文献
3.
Summary Membrane-impermeant and -permeant maleimides were applied to characterize the location and function of the sulfhydryl (SH) groups essential for the facilitated diffusion mediated by the human erythrocyte glucose transport protein. Three such classes have been identified. Type I SH is accessible to membrane-impermeant reagents at the outer (exofacial) surface of the intact erythrocyte. Alkylation of this class inhibits glucose transport; D-glucose and cytochalasin B protect against the alkylation. Type II SH is located at the inner (endofacial) surface of the membrane and is accessible to the membrane-impermeant reagent glutathione maleimide only after lysis of the erythrocyte. D-glucose enhances, while cytochalasin B reduces, the alkylation of Type II SH by maleimides. Reaction of Types I and II SH with an impermeant maleimide increases the half-saturation concentration for binding of D-glucose to erythrocyte membranes. By contrast, inactivation of Type III SH markedly decreases the half-saturation concentration for the binding of D-glucose and other transported sugars. Type III SH is inactivated by the relatively lipid-soluble reagents N-ethylmaleimide (NEM) and dipyridyl disulfide, but not by the impermeant glutathione maleimide. Type III SH is thus located in a hydrophobic membrane domain. A kinetic model constructed to explain these observations indicates that Type III SH is required for the translocation event in a hydrophobic membrane domain which leads to the dissociation of glucose bound to transport sites at the membrane surfaces. 相似文献
4.
Inka Brockhausen Arthur A Grey Henrianna Pang Harry Schachter Jeremy P Carver 《Glycoconjugate journal》1988,5(4):419-448
Sixteen asparagine-linked oligosaccharides ranging in size from (Man)2(GlcNAc)2 (Fuc)1 to (GlcNAc)6(Man)3(GlcNAc)2 were obtained from human 1-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis
bisecting GlcNAc
- DMSO
dimethylsulfoxide
- FAB
fast atom bombardment
- Fuc
l-fucose
- Gal
d-galactose
- GLC
gas-liquid chromatography
- GlcNAc or Gn
N-acetyl-d-glucosamine
- HPLC
high performance liquid chromatography
- Man or M
d-mannose
- MES
2-(N-morpholino)ethanesulfonate
- MS
mass spectrometry
- NMR
nuclear magnetic resonance
- PIPES
piperazine-N,N-bis(2-ethane sulfonic acid)
the nomenclature of the oligosaccharides is shown in Table 1. 相似文献
5.
The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells. 相似文献
6.
Scott Pownall Christine A. Kozak Keith Schappert Mohan Sarkar Eric Hull Harry Schachter Jamey D. Marth 《Genomics》1992,12(4)
The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:α-3-
-mannoside β-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells. 相似文献
7.
Calcium alters the acyl chain composition and lipid fluidity of rat hepatocyte plasma membranes in vitro 总被引:1,自引:0,他引:1
Calcium ion decreases the lipid fluidity of isolated rat hepatocyte plasma membranes by modulating the activity of membrane enzymes which alter the lipid composition. To explore the mechanism of the effect of the cation, eight fluorophores were used to assess lipid fluidity via estimations of either steady-state fluorescence polarization or excimer fluorescence intensity. The results demonstrate that the reduction in fluidity occurs in the hydrophobic interior of the bilayer and that both the dynamic and static (lipid order) components of fluidity are affected by treatment with calcium. Analysis of the membrane lipids demonstrates that calcium treatment decreases the arachidonic acid content of the polar lipid fraction and, thereby, reduces the double-bond index of the fatty acids. This change in composition, which is expected to reduce the lipid fluidity, may result from activation by calcium of the endogenous hepatocyte plasma membrane phospholipase A2. 相似文献
8.
Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides 总被引:20,自引:0,他引:20
H Schachter 《Biochimie et biologie cellulaire》1986,64(3):163-181
Detailed studies on the enzyme machinery responsible for the biosynthesis of protein-bound oligosaccharides of the Asn-GlcNAc and Ser(Thr)-GalNAc linkage types have allowed the formulation of some general rules which explain, at least in part, the branching patterns and microheterogeneity of these structures. These rules are discussed under the following headings: competition of two or more enzymes for a common substrate; controls at the level of enzyme substrate specificity (e.g., critical sugar residues which turn enzyme activity on or off, branch specificity, and the role of the polypeptide in the glycoprotein substrate); substrate availability. 相似文献
9.
Influence of cholesterol content on red cell membrane viscoelasticity and fluidity. 总被引:4,自引:0,他引:4
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The purpose of this investigation was to correlate the viscoelastic properties and lipid fluidity of the red blood cell membrane to its lipid composition. The viscoelastic properties of human red cells that had been enriched or depleted in cholesterol were determined by the micropipette technique. The lipid fluidity of the outer and inner leaflets of the erythrocyte membrane was concurrently assessed by steady state fluorescence depolarization. The elastic modulus and the viscosity moduli of the erythrocyte membrane showed no significant differences between the cholesterol-modified and the control cells. Cholesterol enrichment decreased the lipid fluidity of the outer membrane leaflet alone, and cholesterol depletion increased the fluidity mainly of the inner leaflet. 相似文献
10.
Chlamydia trachomatis was recovered from the cervices of 9.8 percent (268/2,729) of women attending seven family planning clinics. The infection rate varied from 5.5 percent to 22.5 percent in different clinics. Chlamydial infection could be associated with younger age, nulliparity, being black and use of oral contraceptives. Most (70 percent) of the chlamydial infections were inapparent and presumptive indicators for antichlamydial therapy that are useful for symptomatic women will not make a major impact on this reservoir. It is concluded that chlamydial cultures are needed to deal with the high prevalence of these infections. 相似文献