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61.
Lipid A from the nitrogen-fixing bacterium Rhizobium leguminosarum displays many structural differences compared with lipid A of Escherichia coli. R. leguminosarum lipid A lacks the usual 1- and 4'-phosphate groups but is derivatized with a galacturonic acid substituent at position 4'. R. leguminosarum lipid A often contains an aminogluconic acid moiety in place of the proximal glucosamine 1-phosphate unit. Striking differences also exist in the secondary acyl chains attached to E. coli versus R. leguminosarum lipid A, specifically the presence of 27-hydroxyoctacosanoate and the absence of laurate and myristate in R. leguminosarum. Recently, we have found that lipid A isolated by pH 4.5 hydrolysis of R. leguminosarum cells is more heterogeneous than previously reported (Que, N. L. S., Basu, S. S., White, K. A., and Raetz, C. R. H. (1998) FASEB J. 12, A1284 (abstr.)). Lipid A species lacking the 3-O-linked beta-hydroxymyristoyl residue on the proximal unit contribute to this heterogeneity. We now describe a membrane-bound deacylase from R. leguminosarum that removes a single ester-linked beta-hydroxymyristoyl moiety from some lipid A precursors, including lipid X, lipid IVA, and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA. The enzyme does not cleave E. coli lipid A or lipid A precursors containing an acyloxyacyl moiety on the distal glucosamine unit. The enzyme is not present in extracts of E. coli or Rhizobium meliloti, but it is readily demonstrable in membranes of Pseudomonas aeruginosa, which also contains a significant proportion of 3-O-deacylated lipid A species. Optimal reaction rates are seen between pH 5.5 and 6.5. The enzyme requires a nonionic detergent and divalent metal ions for activity. It cleaves the monosaccharide lipid X at about 5% the rate of lipid IVA and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA. 1H NMR spectroscopy of the deacylase reaction product, generated with lipid IVA as the substrate, confirms unequivocally that the enzyme cleaves only the ester-linked beta-hydroxymyristoyl residue at the 3-position of the glucosamine disaccharide. 相似文献
62.
Que-Gewirth NL Ribeiro AA Kalb SR Cotter RJ Bulach DM Adler B Girons IS Werts C Raetz CR 《The Journal of biological chemistry》2004,279(24):25420-25429
Leptospira interrogans differs from other spirochetes in that it contains homologs of all the Escherichia coli lpx genes required for the biosynthesis of the lipid A anchor of lipopolysaccharide (LPS). LPS from L. interrogans cells is unusual in that it activates TLR2 rather than TLR4. The structure of L. interrogans lipid A has now been determined by a combination of matrix-assisted laser desorption ionization time-of-flight mass spectrometry, NMR spectroscopy, and biochemical studies. Lipid A was released from LPS of L. interrogans serovar Pomona by 100 degrees C hydrolysis at pH 4.5 in the presence of SDS. Following purification by anion exchange and thin layer chromatography, the major component was shown to have a molecular weight of 1727. Mild hydrolysis with dilute NaOH reduced this to 1338, consistent with the presence of four N-linked and two O-linked acyl chains. The lipid A molecules of both the virulent and nonvirulent forms of L. interrogans serovar Icterohaemorrhagiae (strain Verdun) were identical to those of L. interrogans Pomona by the above criteria. Given the selectivity of L. interrogans LpxA for 3-hydroxylaurate, we propose that L. interrogans lipid A is acylated with R-3-hydroxylaurate at positions 3 and 3' and with R-3-hydroxypalmitate at positions 2 and 2'. The hydroxyacyl chain composition was validated by gas chromatography and mass spectrometry of fatty acid methyl esters. Intact hexa-acylated lipid A of L. interrogans Pomona was also analyzed by NMR, confirming the presence a beta-1',6-linked disaccharide of 2,3-diamino-2,3-dideoxy-d-glucopyranose units. Two secondary unsaturated acyl chains are attached to the distal residue. The 1-position of the disaccharide is derivatized with an axial phosphate moiety, but the 4'-OH is unsubstituted. (1)H and (31)P NMR analyses revealed that the 1-phosphate group is methylated. Purified L. interrogans lipid A is inactive against human THP-1 cells but does stimulate tumor necrosis factor production by mouse RAW264.7 cells. 相似文献
63.
Que-Gewirth NL Lin S Cotter RJ Raetz CR 《The Journal of biological chemistry》2003,278(14):12109-12119
The structures of Rhizobium leguminosarum and Rhizobium etli lipid A are distinct from those found in other Gram-negative bacteria. Whereas the more typical Escherichia coli lipid A is a hexa-acylated disaccharide of glucosamine that is phosphorylated at positions 1 and 4', R. etli and R. leguminosarum lipid A consists of a mixture of structurally related species (designated A-E) that lack phosphate. A conserved distal unit, comprised of a diacylated glucosamine moiety with galacturonic acid residue at position 4' and a secondary 27-hydroxyoctacosanoyl (27-OH-C28) as part of a 2' acyloxyacyl moiety, is present in all five components. The proximal end is heterogeneous, differing in the number and lengths of acyl chains and in the identity of the sugar itself. A proximal glucosamine unit is present in B and C, but an unusual 2-amino-2-deoxy-gluconate moiety is found in D-1 and E. We now demonstrate that membranes of R. leguminosarum and R. etli can convert B to D-1 in a reaction that requires added detergent and is inhibited by EDTA. Membranes of Sinorhizobium meliloti and E. coli lack this activity. Mass spectrometry demonstrates that B is oxidized in vitro to a substance that is 16 atomic mass units larger, consistent with the formation of D-1. The oxidation of the lipid A proximal unit is also demonstrated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in the positive and negative modes using the model substrate, 1-dephospho-lipid IV(A). With this material, an additional intermediate (or by product) is detected that is tentatively identified as a lactone derivative of 1-dephospho-lipid IV(A). The enzyme, presumed to be an oxidase, is located exclusively in the outer membrane of R. leguminosarum as judged by sucrose gradient analysis. To our knowledge, an oxidase associated with the outer membranes of Gram-negative bacteria has not been reported previously. 相似文献
64.
An unusual feature of lipid A from plant endosymbionts of the Rhizobiaceae family is the presence of a 27-hydroxyoctacosanoic acid (C28) moiety. An enzyme that incorporates this acyl chain is present in extracts of Rhizobium leguminosarum, Rhizobium etli, and Sinorhizobium meliloti but not Escherichia coli. The enzyme transfers 27-hydroxyoctacosanate from a specialized acyl carrier protein (AcpXL) to the precursor Kdo2 ((3-deoxy-d-manno-octulosonic acid)2)-lipid IV(A). We now report the identification of five hybrid cosmids that direct the overexpression of this activity by screening approximately 4000 lysates of individual colonies of an R. leguminosarum 3841 genomic DNA library in the host strain S. meliloti 1021. In these heterologous constructs, both the C28 acyltransferase and C28-AcpXL are overproduced. Sequencing of a 9-kb insert from cosmid pSSB-1, which is also present in the other cosmids, shows that acpXL and the lipid A acyltransferase gene (lpxXL) are close to each other but not contiguous. Nine other open reading frames around lpxXL were also sequenced. Four of them encode orthologues of fatty acid and/or polyketide biosynthetic enzymes. AcpXL purified from S. meliloti expressing pSSB-1 is fully acylated, mainly with 27-hydroxyoctacosanoate. Expression of lpxXL in E. coli behind a T7 promoter results in overproduction in vitro of the expected R. leguminosarum acyltransferase, which is C28-AcpXL-dependent and utilizes (3-deoxy-d-manno-octulosonic acid)2-lipid IV(A) as the acceptor. These findings confirm that lpxXL is the structural gene for the C28 acyltransferase. LpxXL is distantly related to the lauroyltransferase (LpxL) of E. coli lipid A biosynthesis, but highly significant LpxXL orthologues are present in Agrobacterium tumefaciens, Brucella melitensis, and all sequenced strains of Rhizobium, consistent with the occurrence of long secondary acyl chains in the lipid A molecules of these organisms. 相似文献
65.
66.
Endotoxin biosynthesis in Pseudomonas aeruginosa: enzymatic incorporation of laurate before 3-deoxy-D-manno-octulosonate. 总被引:1,自引:0,他引:1 下载免费PDF全文
Unlike Escherichia coli, living cells of Pseudomonas aeruginosa can complete the fatty acylation of lipid A when the biosynthesis of 3-deoxy-D-manno-octulosonate (Kdo) is inhibited (R. C. Goldman, C. C. Doran, S. K. Kadam, and J. O. Capobianco, J. Biol. Chem. 263:5217-5233, 1988). In this study, we demonstrate the presence of a novel enzyme in extracts of P. aeruginosa that can transfer lauroyl-acyl carrier protein (ACP) to a tetraacyl disaccharide-1,4'-bis-phosphate precursor of lipid A (termed lipid IVA) that accumulates in Kdo-deficient mutants of E. coli. Comparable E. coli extracts cannot transfer laurate from lauroyl-ACP to lipid IVA, only to (Kdo)2-lipid IVA (K. A. Brozek, and C. R. H. Raetz, J. Biol. Chem. 265:15410-15417, 1990). P. aeruginosa extracts do not utilize myristoyl- or R-3-hydroxymyristoyl-ACP instead of lauroyl-ACP to acylate lipid IVA. Laurate incorporation in P. aeruginosa extracts is dependent upon time, protein concentration, and the presence of Triton X-100 but is inhibited by lauroyl-coenzyme A. P. aeruginosa extracts transfer only one laurate to lipid IVA, whereas E. coli extracts can transfer two laurates to (Kdo)2-lipid IVA. These results demonstrate that incorporation of laurate into lipid A does not require prior attachment of Kdo in all gram-negative bacteria. 相似文献
67.
Mutants of Escherichia coli K12, defective in phosphatidylserine synthetase (pss), can be isolated as temperature-sensitive, conditional lethals. When cultivated at intermediate temperatures (30 degrees), such mutants contain approximately 3 times more phosphatidylglycerol plus cardiolipin (and less phosphatidylethanolamine) than normal. We now wish to report that, under these conditions, the pss-8 mutant is hypersensitive to certain antibiotics, especially to streptomycin, kanamycin, and gentamicin, although also to ampicillin and novobiocin. At 30 degrees, the membrane protein and fatty acid composition of pss-8 is nearly normal, i.e. identical with an isogenic pss+ organism. Radiochemical labeling and bacteriophage growth studies show that lipopolysaccharide is also unaltered. Therefore, the antibiotic hypersensitivity of pss-8 differs from previously reported hypersensitivities, associated with lipopolysaccharide defects. These results suggest that the polar phospholipid headgroups may play an important role in maintaining the barrier function of the outer gramnegative membrane and that putative inhibitors of the phosphatidylserine synthetase might potentiate the action of numerous antibiotics currently in clinical use. 相似文献
68.
Partial purification and characterization of cytidine 5''-diphosphate-diglyceride hydrolase from membranes of Escherichia coli. 总被引:10,自引:5,他引:5 下载免费PDF全文
Cytidine 5'-diphosphate (CDP)-diglyceride is hydrolyzed to phosphatidic acid and cytidine 5'-monophosphate by a specific membrane-bound enzyme in cell-free extracts of Escherichia coli. The hydrolase can be extracted from the particulate fraction with Triton X-100 and purified 1,000-fold in the presence of this detergent. Several nucleoside disphosphate diglycerides were synthesized to determine the substrate specificity of the hydrolase. CDP-diglyceride was hydrolyzed preferentially, although uridine 5'-diphosphate-diglyceride, guanosine 5'-diphosphate-diglyceride, and adenosine 5'-diphosphate (ADP)-diglyceride were also slowly hydrolyzed. Surprisingly, the purified enzyme did not catalyze detectable cleavage of deoxy-CDP (dCDP)-diglyceride. The liponucleotide pool of E. coli contains dCDP-diglyceride and CDP-diglyceride in approximately equal amounts (Raetz and Kennedy, 1973). Water-soluble nucleoside pyrophosphates, such as CDP-choline, nicotinamide adenine dinucleotide, or adenosine 5'-triphosphate are not attacked by this specific hydrolase. Hydrolysis of CDP-diglyceride is strongly inhibited by adenosine 5'-monophosphate and by ADP-diglyceride. 相似文献
69.
The kinetics of glucose liberation from lactose by means of the beta-glactosidase from Aspergillus niger has been studied in a wide range of the main variables. The analysis shows that the kinetic models proposed so far are not adequate. The main finding is that the reaction rate is not linearly correlated to the enzyme concentration-it increase more than proportionally. This nonlinear relationship results because this lactase can distinguish between alpha-and beta-galactose alpha-Galactose acts as competitive and anticompetitive inhibitor while beta-galactose is a competitive one. The competitive inhibition of the alpha-anomer is approximately 12 times more sever than that of the beta-anomer. The kinetics, including a simplified model for the mutarotation of galactose is given for a temperature of 50 degrees C at a pH of 3.5-the most likely conditions for the application of this lactase in acid whey treatment. 相似文献
70.
UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the reversible transfer of an R-3-hydroxyacyl chain from R-3-hydroxyacyl-acyl carrier protein to the glucosamine 3-OH of UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A is required for the growth and virulence of most Gram-negative bacteria, making its biosynthetic enzymes intriguing targets for the development of new antibacterial agents. LpxA is a member of a large family of left-handed beta-helical proteins, many of which are acyl- or acetyltransferases. We now demonstrate that histidine-, lysine-, and arginine-specific reagents effectively inhibit LpxA of Escherichia coli, whereas serine- and cysteine-specific reagents do not. Using this information in conjunction with multiple sequence alignments, we constructed site-directed alanine substitution mutations of conserved histidine, lysine, and arginine residues. Many of these mutant LpxA enzymes show severely decreased specific activities under standard assay conditions. The decrease in activity corresponds to decreased k(cat)/K(m,UDP-GlcNAc) values for all the mutants. With the exception of H125A, in which no activity is seen under any assay condition, the decrease in k(cat)/K(m,UDP-GlcNAc) mainly reflects an increased K(m,UDP-GlcNAc). His(125) of E. coli LpxA may therefore function as a catalytic residue, possibly as a general base. LpxA does not catalyze measurable UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc hydrolysis or UDP-GlcNAc/UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc exchange, arguing against a ping-pong mechanism with an acyl-enzyme intermediate. 相似文献