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41.
42.
Polychlorinated biphenyl isomers and congeners as inducers of both 3-methylcholanthrene- and phenobarbitone-type microsomal enzyme activity 总被引:1,自引:0,他引:1
Highly purified synthetic polychlorinated biphenyls substituted in the meta and para positions of both phenyl rings and at one ortho position were administered to male Wistar rats and the effects of these compounds on the microsomal drug-metabolising enzymes were evaluated. The in vivo effects of these compounds were determined by measuring the microsomal benzo[a]pyrene hydroxylase, dimethylaminoantipyrine N-demethylase and NADPH-cytochrome c reductase enzyme activities, the cytochrome b5 content and the relative peak intensities and spectral shifts of the reduced microsomal cytochrome P-450 : CO and ethylisocyanide binding difference spectra. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC), 2,2',4,4'-tetrachlorobiphenyl (TCBP-II) (a PB-type inducer), 3,3',4,4'-tetrachlorobiphenyl (TCBP-I) (an MC-type inducer), PB plus MC (coadministered) and TCBP-II + TCBP-I (coadministered) to the test animals. At dosage levels of 30 and 150 mumol . kg-1, pretreatment with 2,3,3',4,4'-pentachlorobiphenyl (PCBP-II), 2,3',4,4',5-pentachlorobiphenyl (PCBP-I), 2,3,3',4,4',5-hexachlorobiphenyl (HCBP-II) and 2,3,3',4,4',5-hexachlorobiphenyl (HCBP-III) gave hepatic microsomes with enzymic and spectral properties consistent with a mixed pattern of induction. These polychlorinated biphenyl (PCB) isomers and congeners have been identified as either major or minor components of the commercial PCB mixtures and must contribute to their activity as MC-type inducers. The only PCB isomer in this series which was not a mixed type inducer was 2,3',4,4',5,5'-hexachlorobiphenyl (HCBP-I) which appeared to be a PB-type inducer. This contrasted to the mixed-type activity observed for 2,3',4,4',5,5'-hexabromobiphenyl which was isolated from a commercial polybrominated biphenyl (PBB) mixture. 相似文献
43.
Summary. The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration
of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms
of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as “hydrophilic” and “hydrophobic” (H. J. Prochaska and P. Talalay,
Journal of Biological Chemistry 261: 1372–1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic
ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured
their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance
the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic
acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H2O2-Fe2+. Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards
ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing
the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol
was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol
may be one of the physiologic functions of this enzyme.
Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003
RID="*"
ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio
Severo Ochoa, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain. 相似文献
44.
Silvia C. Locatelli-Hoops Inna Gorshkova Klaus Gawrisch Alexei A. Yeliseev 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(10):2045-2056
Human peripheral cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural and functional studies on CB2 may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB2 and immobilization at solid interfaces. Expression of CB2 as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB2 was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB2 was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB2 from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB2 and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB2. 相似文献
45.
Oliver Beckstein Elizabeth J. Denning Juan R. Perilla 《Journal of molecular biology》2009,394(1):160-1449
Adenylate kinase (AdK), a phosphotransferase enzyme, plays an important role in cellular energy homeostasis. It undergoes a large conformational change between an open and a closed state, even in the absence of substrate. We investigate the apo-AdK transition at the atomic level both with free-energy calculations and with our new dynamic importance sampling (DIMS) molecular dynamics method. DIMS is shown to sample biologically relevant conformations as verified by comparing an ensemble of hundreds of DIMS transitions to AdK crystal structure intermediates. The simulations reveal in atomic detail how hinge regions partially and intermittently unfold during the transition. Conserved salt bridges are seen to have important structural and dynamic roles; in particular, four ionic bonds that open in a sequential, zipper-like fashion and, thus, dominate the free-energy landscape of the transition are identified. Transitions between the closed and open conformations only have to overcome moderate free-energy barriers. Unexpectedly, the closed state and the open state encompass broad free-energy basins that contain conformations differing in domain hinge motions by up to 40°. The significance of these extended states is discussed in relation to recent experimental Förster resonance energy transfer measurements. Taken together, these results demonstrate how a small number of cooperative key interactions can shape the overall dynamics of an enzyme and suggest an “all-or-nothing” mechanism for the opening and closing of AdK. Our efficient DIMS molecular dynamics computer simulation approach can provide a detailed picture of a functionally important macromolecular transition and thus help to interpret and suggest experiments to probe the conformational landscape of dynamic proteins such as AdK. 相似文献
46.
Background
The deposition of aggregated β-amyloid peptide senile plaques and the accumulation of arginine within the astrocytes in the brain of an Alzheimer's patient are classic observations in the neuropathology of the disease. It would be logical, in the aetiology and pathogenesis, to investigate arginine-metabolising enzymes and their intimate association with amyloid peptides.Methods
Neuronal nitric oxide synthase (nNOS) was isolated, purified and shown, through fluorescence quenching spectroscopy and fluorescence resonance energy transfer (FRET), to interact with structural fragments of Aβ1–40 and be catalytic towards amyloid fibril formation.Results
Only one binding site on the enzyme was available for binding. Two amyloid peptide fragments of Aβ1–40 (Aβ17–28 and Aβ25–35) had Stern–Volmer values (KSV) of 0.111 μM−1 and 0.135 μM−1 indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. The polarity of this active site precludes binding of the predominantly hydrophobic amyloid peptide fragments contained within Aβ17–28 and within two glycine zipper motifs [G-X-X-X-G-X-X-X-G] [Aβ29–37] and bind to the enzyme at a site remote to the active region.Conclusions
The interaction and binding of Aβ17–28 and Aβ25–35 to nNOS causes the movement of two critical tryptophan residues of 0.77 nm and 0.57 nm respectively towards the surface of the enzyme.General significance
The binding of Aβ-peptide fragments with nNOS has been studied by spectrofluorimetry. The information and data presented should contribute towards understanding the mechanism for deposition of aggregated Aβ-peptides and fibrillogenesis in senile plaques in an AD brain. 相似文献47.
Traxlmayr MW Wozniak-Knopp G Antes B Stadlmayr G Rüker F Obinger C 《Journal of biotechnology》2011,155(2):193-202
Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL - a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops. 相似文献
48.
Haiyan Luo Sanghun Sin Joseph M. McDonough Carmen R. Isasi Raanan Arens David M. Wootton 《Journal of biomechanics》2014
Background
Improvements in obstructive sleep apnea syndrome (OSAS) severity may be associated with improved pharyngeal fluid mechanics following adenotonsillectomy (AT). The study objective is to use image-based computational fluid dynamics (CFD) to model changes in pharyngeal pressures after AT, in obese children with OSAS and adenotonsillar hypertrophy.Methods
Three-dimensional models of the upper airway from nares to trachea, before and after AT, were derived from magnetic resonance images obtained during wakefulness, in a cohort of 10 obese children with OSAS. Velocity, pressure, and turbulence fields during peak tidal inspiratory flow were computed using commercial software. CFD endpoints were correlated with polysomnography endpoints before and after AT using Spearman?s rank correlation (rs).Results
Apnea hypopnea index (AHI) decreases after AT was strongly correlated with reduction in maximum pressure drop (dPTAmax) in the region where tonsils and adenoid constrict the pharynx (rs=0.78, P=0.011), and with decrease of the ratio of dPTAmax to flow rate (rs=0.82, P=0.006). Correlations of AHI decrease to anatomy, negative pressure in the overlap region (including nasal flow resistance), or pressure drop through the entire pharynx, were not significant. In a subgroup of subjects with more than 10% improvement in AHI, correlations between flow variables and AHI decrease were stronger than in all subjects.Conclusions
The correlation between change in dPTAmax and improved AHI suggests that dPTAmax may be a useful index for internal airway loading due to anatomical narrowing, and may be better correlated with AHI than direct airway anatomic measurements. 相似文献49.
Agonist stimulation of G protein-coupled receptors causes receptor activation, phosphorylation, beta-arrestin binding and receptor internalization. Angiotensin II (AngII) causes rapid internalization of the AT1 receptors, whereas AngII-bound AT2 receptors do not internalize. Although the activation of the rat AT1A receptor with AngII causes translocation of beta-arrestin2 to the receptor, no association of this molecule with the AT2 receptor can be detected after AngII treatment with confocal microscopy or bioluminescence resonance energy transfer. These data demonstrate that the two subtypes of angiotensin receptors have different mechanisms of regulation. 相似文献
50.
Estella A. Newcombe Kiersten M. Ruff Ashish Sethi Angelique R. Ormsby Yasmin M. Ramdzan Archa Fox Anthony W. Purcell Paul R. Gooley Rohit V. Pappu Danny M. Hatters 《Journal of molecular biology》2018,430(10):1442-1458
Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington's disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild-type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen–deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity. 相似文献