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441.
Kelly RM Leemhuis H Gätjen L Dijkhuizen L 《The Journal of biological chemistry》2008,283(16):10727-10734
Small molecule inhibitors play an essential role in the selective inhibition of enzymes associated with human infection and metabolic disorders. Targeted enzymes may evolve toward inhibitor resistance through selective incorporation of mutations. Acquisition of insensitivity may, however, result in profound devolution of native enzyme function and stability. We therefore investigated the consequential effects on native function and stability by evolving a cyclodextrin glucanotransferase (CGTase) enzyme toward insensitivity to the small molecule inhibitor of the protein, acarbose. Error-prone PCR mutagenesis was applied to search the sequence space of CGTase for acarbose-insensitive variants. Our results show that all selected mutations were localized around the active site of the enzyme, and in particular, at the acceptor substrate binding sites, highlighting the regions importance in acarbose inhibition. Single mutations conferring increased resistance, K232E, F283L, and A230V, raised IC(50) values for acarbose between 3,500- and 6,700-fold when compared with wild-type CGTase but at a significant cost to catalytic efficiency. In addition, the thermostability of these variants was significantly lowered. These results reveal not only the relative ease by which resistance may be acquired to small molecule inhibitors but also the considerable cost incurred to native enzyme function and stability, highlighting the subsequent constraints in the further evolutionary potential of inhibitor-resistant variants. 相似文献
442.
A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing 总被引:1,自引:0,他引:1
Green RE Malaspinas AS Krause J Briggs AW Johnson PL Uhler C Meyer M Good JM Maricic T Stenzel U Prüfer K Siebauer M Burbano HA Ronan M Rothberg JM Egholm M Rudan P Brajković D Kućan Z Gusić I Wikström M Laakkonen L Kelso J Slatkin M Pääbo S 《Cell》2008,134(3):416-426
A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000 year-old Neandertal individual with 8341 mtDNA sequences identified among 4.8 Gb of DNA generated from approximately 0.3 g of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs, and allows an estimate of the divergence date between the two mtDNA lineages of 660,000 +/- 140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared with other primate lineages, suggesting that the effective population size of Neandertals was small. 相似文献
443.
Human Urotensin-II (hU-II) is a cyclic 11-amino acid peptide that plays a role in cardiovascular homeostasis. Its receptor is a member of the class A of G-protein-coupled receptors, called GPR14. In recent years, several nonpeptide ligands have been reported in the literature. Most were identified by high-throughput screening and optimized by medicinal chemistry methods. Other nonpeptide ligands were discovered starting from the 3D structure of hU-II or other ligands. They were identified by a virtual screening approach based on a 3D pharmacophore or by structural similarity with others cyclic peptides. In this review, nonpeptide agonists and antagonists are presented in relation to structure-activity relationships. 相似文献
444.
Forbes EK Sander C Ronan EO McShane H Hill AV Beverley PC Tchilian EZ 《Journal of immunology (Baltimore, Md. : 1950)》2008,181(7):4955-4964
Boosting bacillus Calmette-Guérin (BCG)-primed mice with a recombinant adenovirus expressing Mycobacterium tuberculosis Ag 85A by different administration routes has very different effects on protection against aerosol challenge with M. tuberculosis. Mice boosted intradermally make very strong splenic CD4 and CD8 Th1 cytokine responses to Ag 85A, but show no change in lung mycobacterial burden over BCG primed animals. In contrast, intranasally boosted mice show greatly reduced mycobacterial burden and make a much weaker splenic response but a very strong lung CD4 and CD8 response to Ag 85A and an increased response to purified protein derivative. This effect is associated with the presence in the lung of multifunctional T cells, with high median fluorescence intensity and integrated median fluorescence intensity for IFN-gamma, IL-2, and TNF. In contrast, mice immunized with BCG alone have few Ag-specific cells in the lung and a low proportion of multifunctional cells, although individual cells have high median fluorescence intensity. Successful immunization regimes appear to induce Ag-specific cells with abundant intracellular cytokine staining. 相似文献
445.
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447.
Allogeneic bone marrow transplant is a life-saving procedure for adults and children that have high-risk or relapsed hematological malignancies. Incremental advances in the procedure, as well as expanded sources of donor hematopoietic cell grafts have significantly improved overall rates of success. Yet, the outcomes for patients for whom suitable donors cannot be found remain a significant limitation. These patients may benefit from a hematopoietic cell transplant wherein a relative donor is fully haplotype mismatched. Previously this procedure was limited by graft rejection, lethal graft-versus-host disease, and increased treatmentrelated toxicity. Recent approaches in haplo-identical transplantation have demonstrated significantly improved outcomes. Based on years of incremental preclinical research into this unique form of bone marrow transplant, a range of approaches have now been studied in patients in relatively large phase Ⅱ trials that will be summarized in this review. 相似文献
448.
Lescot E Sopkova-de Oliveira Santos J Colloc'h N Rodrigo J Milazzo-Segalas I Bureau R Rault S 《Proteins》2008,73(1):173-184
Human urotensin-II (hU-II) is a cyclic peptide that plays a central role in cardiovascular homeostasis and is considered to be the most potent mammalian vasoconstrictor identified to date. It is a natural ligand of the human urotensin-II (hUT-II) receptor, a member of the family of rhodopsin-like G-protein-coupled receptors. To understand the molecular interactions of hU-II and certain antagonists with the hUT-II receptor, a model of the hUT-II receptor in an active conformation with all its connecting loops was constructed by homology modeling. The initial model was placed in a pre-equilibrated lipid bilayer and re-equilibrated by several procedures of energy minimization and molecular dynamics simulations. Docking studies were performed for hU-II and for a series of nonpeptide hUT-II receptor antagonists in the active site of the modeled receptor structure. Results of the hU-II docking study are in agreement with our previous work and with experimental data showing the contribution of the extracellular loops II and III to ligand recognition. The docking of hU-II nonpeptide antagonists allows identification of key molecular interactions and confirms a previously reported hU-II antagonist pharmacophore model. The results of the present studies will be used in structure-based drug design for developing novel antagonists for the hUT-II receptor. 相似文献
449.
Binder BM O'malley RC Wang W Moore JM Parks BM Spalding EP Bleecker AB 《Plant physiology》2004,136(2):2913-2920
Responses to the plant hormone ethylene are mediated by a family of five receptors in Arabidopsis that act in the absence of ethylene as negative regulators of response pathways. In this study, we examined the rapid kinetics of growth inhibition by ethylene and growth recovery after ethylene withdrawal in hypocotyls of etiolated seedlings of wild-type and ethylene receptor-deficient Arabidopsis lines. This analysis revealed that there are two phases to growth inhibition by ethylene in wild type: a rapid phase followed by a prolonged, slower phase. Full recovery of growth occurs approximately 90 min after ethylene removal. None of the receptor null mutations tested had a measurable effect on the two phases of growth inhibition. However, loss-of-function mutations in ETR1, ETR2, and EIN4 significantly prolonged the time for recovery of growth rate after ethylene was removed. Plants with an etr1-6;etr2-3;ein4-4 triple loss-of-function mutation took longer to recover than any of the single mutants, while the ers1;ers2 double mutant had no effect on recovery rate, suggesting that receiver domains play a role in recovery. Transformation of the ers1-2;etr1-7 double mutant with wild-type genomic ETR1 rescued the slow recovery phenotype, while a His kinase-inactivated ETR1 construct did not. To account for the rapid recovery from growth inhibition, a model in which clustered receptors act cooperatively is proposed. 相似文献
450.
Effect of yeast inoculation rate on the metabolism of contaminating lactobacilli during fermentation of corn mash 总被引:1,自引:0,他引:1
Two separate 4 (bacterial concentrations)×6 (yeast concentrations) full factorial experiments were conducted in an attempt to identify a novel approach to minimize the effects caused by bacterial contamination during industrial production of ethanol from corn. Lactobacillus plantarum and Lactobacillus paracasei, commonly occurring bacterial contaminants in ethanol plants, were used in separate fermentation experiments conducted in duplicate using an industrial strain of Saccharomyces cerevisiae, Allyeast Superstart. Bacterial concentrations were 0, 1×106, 1×107 and 1×108 cells/ml mash. Yeast concentrations were 0, 1×106, 1×107, 2×107, 3×107, and 4×107 cells/ml mash. An increased yeast inoculation rate of 3×107 cells/ml resulted in a greater than 80% decrease (P<0.001) and a greater than 55% decrease (P<0.001) in lactic acid production by L. plantarum and L. paracasei, respectively, when mash was infected with 1×108 lactobacilli/ml. No differences (P>0.25) were observed in the final ethanol concentration produced by yeast at any of the inoculation rates studied, in the absence of lactobacilli. However, when the mash was infected with 1×107 or 1×108 lactobacilli/ml, a reduction of 0.7–0.9% v/v (P<0.005) and a reduction of 0.4–0.6% v/v (P<0.005) in the final ethanol produced was observed in mashes inoculated with 1×106 and 1×107 yeast cells/ml, respectively. At higher yeast inoculation rates of 3×107 or 4×107 cells/ml, no differences (P>0.35) were observed in the final ethanol produced even when the mash was infected with 1×108 lactobacilli/ml. The increase in ethanol corresponded to the reduction in lactic acid production by lactobacilli. This suggests that using an inoculation rate of 3×107 yeast cells/ml reduces the growth and metabolism of contaminating lactic bacteria significantly, which results in reduced lactic acid production and a concomitant increase in ethanol production by yeast. 相似文献