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61.
Many proteins are built from structurally and functionally distinct domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction. 相似文献
62.
Ha JS Ha CE Chao JT Petersen CE Theriault A Bhagavan NV 《Biochimica et biophysica acta》2003,1640(2-3):119-128
In the present study, we used the human EA.hy926 endothelial cell line as the model system to investigate the effect of human serum albumin (HSA) and its structural variants on cholesterol efflux. Initial studies showed that HSA promoted cholesterol efflux in a dose- and time-dependent manner, reaching a plateau at 10 mg/ml at 90 min. As a control, gelatin displayed no significant effect on efflux, while HSA was significantly more efficient than ovalbumin and bovine serum albumin (BSA) in promoting cholesterol efflux. Equal molar concentrations of HSA and apolipoprotein A-I (apoA-I) showed that apoA-I had considerably higher efficiency in efflux. However, the prevailing high plasma concentrations of HSA may compensate for its lower efflux rate compared to apoA-I. To characterize the mechanism of HSA-mediated cholesterol efflux, we studied the effects of cAMP and temperature on efflux using both EA.hy926 endothelial cells and murine RAW 264.7 macrophages. We found that HSA-mediated efflux occurred via a cAMP-independent and relatively temperature-insensitive pathway. We next examined the nature of HSA-cholesterol interaction by comparing the effects of various HSA mutants to wild-type HSA on cholesterol efflux. We found specific interactions between subdomains 2A and 3A and cholesterol, as indicated by the changes in the efflux rate of various HSA mutants. In conclusion, our study provides evidence for the role of HSA in cholesterol efflux, and shows that the substitution of specific amino acid residues in subdomains of 2A and 3A may be important structural determinants in its ability to bind to cholesterol and participate in cholesterol efflux. 相似文献
63.
Valenti L Mathieu J Chancerelle Y Levacher M Chanaud B De Sousa M Strzalko S Dinh-Xuan AT Giroud JP Florentin I 《Cellular immunology》2003,221(1):50-63
We previously showed that an overproduction of nitric oxide (NO) by macrophages was responsible for the collapse of lymphoproliferative responses after burn injury in rats. First, we demonstrate here that 10 days post-burn, the inhibition of splenocyte response to concanavalin-A results from cytostatic, apoptotic, and necrotic effects of NO on activated T cells. This was evidenced by various criteria at the levels of DNA, mitochondria, and plasma membrane. Inhibition of NO synthase by S-methylisothiourea (10 microM) normalized all the parameters. Second, we show that two soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ, restored the proliferative response in a concentration-dependent manner. LY83583 (0.5 microM) rescued T cells from apoptosis. Similar results were obtained with KT5823 (5 microM) a specific inhibitor of protein kinase G (PKG). In contrast, neither LY83583 nor KT5823 inhibited NO-induced necrosis. These results suggest that NO blocked T cells in the G1 phase and induced apoptosis through a sGC-PKG-dependent pathway and necrosis through an independent one. 相似文献
64.
Single-molecule fluorescence resonance energy transfer 总被引:18,自引:0,他引:18
Ha T 《Methods (San Diego, Calif.)》2001,25(1):78-86
Fluorescent resonance energy transfer (FRET) is a powerful technique for studying conformational distribution and dynamics of biological molecules. Some conformational changes are difficult to synchronize or too rare to detect using ensemble FRET. FRET, detected at the single-molecule level, opens up new opportunities to probe the detailed kinetics of structural changes without the need for synchronization. Here, we discuss practical considerations for its implementation including experimental apparatus, fluorescent probe selection, surface immobilization, single-molecule FRET analysis schemes, and interpretation. 相似文献
65.
BNip3 is a member of Bcl-2 family proteins that displays proapoptotic activity. It contains Bcl-2 homology (BH) 3 and single carboxy terminal membrane-anchoring domain (TM), which targets to specific intracellular organelles, especially to mitochondria. Mitochondria play significant roles in apoptosis by releasing apoptogenic factors through large conductance channel known as permeability transition pore (PTP). Although BNip3 associates with mitochondria when overexpressed, apoptotic pathways including mitochondrial cascade and functional domains of BNip3 are still unknown. In this report, we demonstrate that recombinant BNip3 (rBNip3) induces mitochondrial permeability transition (MPT) and cytochrome c release from isolated mitochondria, which are inhibited by the PT inhibitor cyclosporin A (CsA). We further show that carboxy terminal tail of BNip3, but not BH3, is essential for the induction of PT and cytochrome c release on the base of mutational analysis. Moreover, addition of carboxy terminal c-tail to TM substitution mutant, which did not induce the PT and cytochrome c release, restored PT-inducing activity. Taken together, our results suggest that BNip3 exerts proapoptotic activity through PT induction and that carboxy terminal c-tail is crucial for it. 相似文献
66.
Viëtor RJ Newman RH Ha MA Apperley DC Jarvis MC 《The Plant journal : for cell and molecular biology》2002,30(6):721-731
Native cellulose in higher plants forms crystalline fibrils a few nm across, with a substantial fraction of their glucan chains at the surface. The accepted crystal structures feature a flat-ribbon 21 helical chain conformation with every glucose residue locked to the next by hydrogen bonds from O-3' to O-5 and from O-2 to O-6'. Using solid-state NMR spectroscopy we show that the surface chains have a different C-6 conformation so that O-6 is not in the correct position for the hydrogen bond from O-2. We also present evidence consistent with a model in which alternate glucosyl residues are transiently or permanently twisted away from the flat-ribbon conformation of the chain, weakening the O-3' - 0-5 hydrogen bond. Previous molecular modelling and the modelling studies reported here indicate that this 'translational' chain conformation is energetically feasible and does not preclude binding of the surface chains to the interior chains, because the surface chains share the axial repeat distance of the 21 helix. Reduced intramolecular hydrogen bonding allows the surface chains to form more hydrogen bonds to external molecules in textiles, wood, paper and the living plant. 相似文献
67.
Viossat B Daran JC Savouret G Morgant G Greenaway FT Dung NH Pham-Tran VA Sorenson JR 《Journal of inorganic biochemistry》2003,96(2-3):375-385
The purpose of this research was to characterize by X-ray crystallography the ternary dimethylformamide (DMF) Cu(II) complex of acetylsalicylic acid (aspirin), in an effort to compare the structure-activity relationships for the anticonvulsant activity of this and other Cu(II)aspirinate chelates. The ternary DMF Cu(II) complex of aspirin was synthesized and crystals grown from a DMF solution were characterized by single crystal X-ray diffraction. This crystalline material was analyzed for anticonvulsant activity in the Maximal Electroshock (MES) Grand Mal and subcutaneous Metrazol (scMET) Petit Mal models of seizure used to detect anticonvulsant activity. The ternary DMF complex was found to be a monomolecular binuclear complex, tetrakis-mu-(acetylsalicylato)bis(dimethylformamido)dicopper(II) [Cu(II)(2)(aspirinate)(4)(DMF)(2)] with the following parameters: monoclinic, space group P2(1)/n, a=12.259 (1), b=10.228 (1), c=16.987 (1) A, beta=92.07 (1) degrees; V=2128.5 (3) A(3); Z=2. The structure was determined at 180 K from 2903 unique reflections (I>1sigma(I)) to the final values of R=0.030 and wR=0.033 using F. This binuclear complex contains four acetylsalicylate bridging ligands which are related to each other in a two by two symmetry center. The four nearest O atoms around each Cu atom form a closely square planar arrangement with the square pyramidal coordination completed by the dimethylformamide oxygen atom occupying an apical position at a distance of 2.154 (1) A. Each Cu atom is displaced towards the DMF ligand by 0.187 A from the plane of the four O atoms. Electron paramagnetic resonance (EPR) spectra of [Cu(II)(2)(aspirinate)(4)(DMF)(2)] crystals show a strong antiferromagnetic coupling of the copper atoms, similar to that observed with other binuclear copper(II)salicylate compounds. Studies used to detect anticonvulsant activity revealed that [Cu(II)(2)(aspirinate)(4)(DMF)(2)] was an effective anticonvulsant in the MES model of seizure but ineffective against scMET-induced seizures. The monomolecular ternary binuclear [Cu(II)(2)(aspirinate)(4)(DMF)(2)] complex is more effective in inhibiting MES-induced seizures than other binuclear or mononuclear Cu(II) chelates of aspirin including: binuclear polymeric [Cu(II)(2)(aspirinate)(4)], [Cu(II)(2)(aspirinate)(4)(H(2)O)], which is anticipated to be less polymeric, and monomolecular ternary [Cu(II)(2)(aspirinate)(4)(DMSO)(2)] and [Cu(II)(aspirinate)(2)(Pyr)(2)]. These and other chelates appear to be more effective in the scMET model of seizure than [Cu(II)(2)(aspirinate)(4)(DMF)(2)]. These structure-activity relationships support the potential efficacy of Cu chelates of aspirin in treating epilepsies. 相似文献
68.
An unstructured model was developed for batch cultivation of Corynebacterium lactofermentum (ATCC 21799) under controlled dissolved oxygen. The model is capable of predicting batch experiments performed at various initial substrate concentrations. By extending the batch culture model to a fed-batch model and using a heuristic approach to optimize the fed-batch cultivation, it is shown that fed-batch cultivation is superior to batch operation due to increased productivity at high substrate concentrations. 相似文献
69.
Effects of endurance training on three superoxide dismutase isoenzymes in human plasma 总被引:3,自引:0,他引:3
Ookawara T Haga S Ha S Oh-Ishi S Toshinai K Kizaki T Ji LL Suzuki K Ohno H 《Free radical research》2003,37(7):713-719
The effects of endurance training and acute exhaustive exercise on plasma levels of three superoxidedismutase (SOD) isoenzymes and the ability of superoxide generation in neutrophils were studied. Eighteen healthy male students, aged 17-22 years, who volunteered for this study, underwent three months of endurance training in swimming or running. Before and after the training course, they performed acute exercise and blood samples were collected before and after this exercise. The endurance training significantly increased maximal oxygen uptake (VO2max) in all subjects. Neither the endurance training nor the acute exercise affected the plasma CuZn-SOD level. Acute exercise after the training, but not before the training, increased both the plasma Mn-SOD and extracellular SOD (EC-SOD) levels by 33.6 and 33.5%, respectively. The training decreased the EC-SOD level at rest by 22.2%. Acute exercise after the training, but not before the training, increased the plasma lipid peroxide level, suggesting higher oxidative stress in trained subjects during exhaustive exercise. The ability of neutrophils to generate superoxide was increased by the acute exercise, but induction of the superoxide was suppressed after training. These results indicate that EC-SOD levels were changed in a different manner from the CuZn-SOD and Mn-SOD: it was decreased by training but was increased by acute exercise, suggesting that endurance training increases the reserve of EC-SOD in tissues. The results also suggest the possibility of plasma EC-SOD assay as a new index of endurance training. 相似文献
70.
This study examined the effects of shiftwork on the cardiovascular system. The blood pressure (BP) and heart rate variability (HRV) of 134 male workers, who worked 8-hour shifts with rapid rotation of shifts at 3-day intervals, were examined for all the three shifts. In addition, the job stress was measured by Karasek's JCQ 49-item questionnaire and the circadian type was assessed by the morningness-eveningness questionnaire. The smoking and alcohol drinking habits, marital status and past medical history were also obtained. The method of analyzing the measured data based on a mixed model was used to illustrate the association between the shiftwork duration and the BP or HRV. The average age of workers was 29 years (between 25-44). Among them, 77.9% were current smokers, 50% showed the passive type of job strain in Karasek's model. The mean shiftwork duration was 5.21 years (range 5.4 months--10 years). In the circadian type, none of them belonged to a definitely morning type or a definitely evening type. In the multivariate analysis adjusted by age, job strain, shift, circadian rhythm and smoking, the blood pressure showed significantly increasing trends according to shiftwork duration in both the systolic and diastolic BP. The heart rate variability also showed a significantly decreasing trend according to the shiftwork duration in both the parasympathetic and sympathetic functions (p < 0.05). These results suggests that there are negative health effects arising from shiftwork on the cardiovascular system. 相似文献