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71.
Plasmid DNA of six Escherichia fergusonii colicinogenic strains (three producers of colicin E1, two of Ib and one of Ia) was isolated and the colicin-encoding regions of the corresponding Col plasmids were sequenced. Two new variants of colicin E1, one of colicin Ib, and one of colicin Ia were identified as well as new variants of the colicin E1 and colicin Ib immunity proteins and the colicin E1 lysis polypeptide. The recombinant Escherichia coli producer harboring pColE1 from E. fergusonii strain EF36 (pColE1-EF36) was found to be only partially immune to E1 colicins produced by two other E. fergusonii strains suggesting that pColE1-EF36 may represent an ancestor ColE1 plasmid. 相似文献
72.
In the past few years considerable progress regarding the knowledge of the human genome map has been achieved. As a result, attempts to use gene therapy in patients’ management are more and more often undertaken. The aim of gene therapy is to replace defective genes in vivo and/or to promote the long-term endogenous synthesis of deficient protein. In vitro studies improve the production of human recombinant proteins, such as insulin (INS), growth hormone (GH), insulin-like growth factor-1 (IGF-1) and erythropoietin (EPO), which could have therapeutic application. Unfortunately, genetic methods developed for therapeutic purposes are increasingly being used in competitive sports. Some new substances (e.g., antibodies against myostatin or myostatin blockers) might be used in gene doping in athletes. The use of these substances may cause an increase of body weight and muscle mass and a significant improvement of muscle strength. Although it is proven that uncontrolled manipulation of genetic material and/or the introduction of recombinant proteins may be associated with health risks, athletes are increasingly turning to banned gene doping. At the same time, anti-doping research is undertaken in many laboratories around the world to try to develop and refine ever newer techniques for gene doping detection in sport. Thanks to the World Anti-Doping Agency (WADA) and other sports organizations there is a hope for real protection of athletes from adverse health effects of gene doping, which at the same time gives a chance to sustain the idea of fair play in sport. 相似文献
73.
Many protein-protein interactions (PPIs) are compelling targets for drug discovery, and in a number of cases can be disrupted by small molecules. The main goal of this study is to examine the mechanism of binding site formation in the interface region of proteins that are PPI targets by comparing ligand-free and ligand-bound structures. To avoid any potential bias, we focus on ensembles of ligand-free protein conformations obtained by nuclear magnetic resonance (NMR) techniques and deposited in the Protein Data Bank, rather than on ensembles specifically generated for this study. The measures used for structure comparison are based on detecting binding hot spots, i.e., protein regions that are major contributors to the binding free energy. The main tool of the analysis is computational solvent mapping, which explores the surface of proteins by docking a large number of small “probe” molecules. Although we consider conformational ensembles obtained by NMR techniques, the analysis is independent of the method used for generating the structures. Finding the energetically most important regions, mapping can identify binding site residues using ligand-free models based on NMR data. In addition, the method selects conformations that are similar to some peptide-bound or ligand-bound structure in terms of the properties of the binding site. This agrees with the conformational selection model of molecular recognition, which assumes such pre-existing conformations. The analysis also shows the maximum level of similarity between unbound and bound states that is achieved without any influence from a ligand. Further shift toward the bound structure assumes protein-peptide or protein-ligand interactions, either selecting higher energy conformations that are not part of the NMR ensemble, or leading to induced fit. Thus, forming the sites in protein-protein interfaces that bind peptides and can be targeted by small ligands always includes conformational selection, although other recognition mechanisms may also be involved. 相似文献
74.
75.
Remnant trees, spared from cutting when tropical forests are cleared for agriculture or grazing, act as nuclei of forest regeneration following field abandonment. Previous studies on remnant trees were primarily conducted in active pasture or old fields abandoned in the previous 2–3 years, and focused on structure and species richness of regenerating forest, but not species composition. Our study is among the first to investigate the effects of remnant trees on neighborhood forest structure, biodiversity, and species composition 20 years post-abandonment. We compared the woody vegetation around individual remnant trees to nearby plots without remnant trees in the same second-growth forests (“control plots”). Forest structure beneath remnant trees did not differ significantly from control plots. Species richness and species diversity were significantly higher around remnant trees. The species composition around remnant trees differed significantly from control plots and more closely resembled the species composition of nearby old-growth forest. The proportion of old-growth specialists and generalists around remnant trees was significantly greater than in control plots. Although previous studies show that remnant trees may initially accelerate secondary forest growth, we found no evidence that they locally affect stem density, basal area, and seedling density at later stages of regrowth. Remnant trees do, however, have a clear effect on the species diversity, composition, and ecological groups of the surrounding woody vegetation, even after 20 years of forest regeneration. To accelerate the return of diversity and old-growth forest species into regrowing forest on abandoned land, landowners should be encouraged to retain remnant trees in agricultural or pastoral fields. 相似文献
76.
Livan Alonso-Sarduy Paolo De Los Rios Fabrizio Benedetti Dusan Vobornik Giovanni Dietler Sandor Kasas Giovanni Longo 《PloS one》2014,9(7)
Proteins can switch between different conformations in response to stimuli, such as pH or temperature variations, or to the binding of ligands. Such plasticity and its kinetics can have a crucial functional role, and their characterization has taken center stage in protein research. As an example, Topoisomerases are particularly interesting enzymes capable of managing tangled and supercoiled double-stranded DNA, thus facilitating many physiological processes. In this work, we describe the use of a cantilever-based nanomotion sensor to characterize the dynamics of human topoisomerase II (Topo II) enzymes and their response to different kinds of ligands, such as ATP, which enhance the conformational dynamics. The sensitivity and time resolution of this sensor allow determining quantitatively the correlation between the ATP concentration and the rate of Topo II conformational changes. Furthermore, we show how to rationalize the experimental results in a comprehensive model that takes into account both the physics of the cantilever and the dynamics of the ATPase cycle of the enzyme, shedding light on the kinetics of the process. Finally, we study the effect of aclarubicin, an anticancer drug, demonstrating that it affects directly the Topo II molecule inhibiting its conformational changes. These results pave the way to a new way of studying the intrinsic dynamics of proteins and of protein complexes allowing new applications ranging from fundamental proteomics to drug discovery and development and possibly to clinical practice. 相似文献
77.
Ingrid M. Bonilla Victor P. Long III Pedro Vargas-Pinto Patrick Wright Andriy Belevych Qing Lou Kent Mowrey Jae Yoo Philip F. Binkley Vadim V. Fedorov Sandor Gy?rke Paulus M. L. Janssen Ahmet Kilic Peter J. Mohler Cynthia A. Carnes 《PloS one》2014,9(10)
The role of IKCa in cardiac repolarization remains controversial and varies across species. The relevance of the current as a therapeutic target is therefore undefined. We examined the cellular electrophysiologic effects of IKCa blockade in controls, chronic heart failure (HF) and HF with sustained atrial fibrillation. We used perforated patch action potential recordings to maintain intrinsic calcium cycling. The IKCa blocker (apamin 100 nM) was used to examine the role of the current in atrial and ventricular myocytes. A canine tachypacing induced model of HF (1 and 4 months, n = 5 per group) was used, and compared to a group of 4 month HF with 6 weeks of superimposed atrial fibrillation (n = 7). A group of age-matched canine controls were used (n = 8). Human atrial and ventricular myocytes were isolated from explanted end-stage failing hearts which were obtained from transplant recipients, and studied in parallel. Atrial myocyte action potentials were unchanged by IKCa blockade in all of the groups studied. IKCa blockade did not affect ventricular myocyte repolarization in controls. HF caused prolongation of ventricular myocyte action potential repolarization. IKCa blockade caused further prolongation of ventricular repolarization in HF and also caused repolarization instability and early afterdepolarizations. SK2 and SK3 expression in the atria and SK3 in the ventricle were increased in canine heart failure. We conclude that during HF, IKCa blockade in ventricular myocytes results in cellular arrhythmias. Furthermore, our data suggest an important role for IKCa in the maintenance of ventricular repolarization stability during chronic heart failure. Our findings suggest that novel antiarrhythmic therapies should have safety and efficacy evaluated in both atria and ventricles. 相似文献
78.
Jay Rasmussen Brandon H Gilroyed Tim Reuter Sandor Dudas Norman F Neumann Aru Balachandran 《朊病毒》2014,8(1):136-142
Prions, the causative agent of chronic wasting disease (CWD) enter the environment through shedding of bodily fluids and carcass decay, posing a disease risk as a result of their environmental persistence. Plants have the ability to take up large organic particles, including whole proteins, and microbes. This study used wheat (Triticum aestivum L.) to investigate the uptake of infectious CWD prions into roots and their transport into aerial tissues. The roots of intact wheat plants were exposed to infectious prions (PrPTSE) for 24 h in three replicate studies with PrPTSE in protein extracts being detected by western blot, IDEXX and Bio-Rad diagnostic tests. Recombinant prion protein (PrPC) bound to roots, but was not detected in the stem or leaves. Protease-digested CWD prions (PrPTSE) in elk brain homogenate interacted with root tissue, but were not detected in the stem. This suggests wheat was unable to transport sufficient PrPTSE from the roots to the stem to be detectable by the methods employed. Undigested PrPTSE did not associate with roots. The present study suggests that if prions are transported from the roots to the stems it is at levels that are below those that are detectable by western blot, IDEXX or Bio-Rad diagnostic kits. 相似文献
79.
Andriy E. Belevych Hsiang-Ting Ho Radmila Terentyeva Ingrid M. Bonilla Dmitry Terentyev Cynthia A. Carnes Sandor Gyorke George E. Billman 《PloS one》2013,8(10)
It has been proposed that dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) can reduce the risk of ventricular arrhythmias in post-MI patients. Abnormal Ca2+ handling has been implicated in the genesis of post-MI ventricular arrhythmias. Therefore, we tested the hypothesis that dietary n-3 PUFAs alter the vulnerability of ventricular myocytes to cellular arrhythmia by stabilizing intracellular Ca2+ cycling. To test this hypothesis, we used a canine model of post-MI ventricular fibrillation (VF) and assigned the animals to either placebo (1 g/day corn oil) or n-3 PUFAs (1-4 g/day) groups. Using Ca2+ imaging techniques, we examined the intracellular Ca2+ handling in myocytes isolated from post-MI hearts resistant (VF-) and susceptible (VF+) to VF. Frequency of occurrence of diastolic Ca2+ waves (DCWs) in VF+ myocytes from placebo group was significantly higher than in placebo-treated VF- myocytes. n-3 PUFA treatment did not decrease frequency of DCWs in VF+ myocytes. In contrast, VF- myocytes from the n-3 PUFA group had a significantly higher frequency of DCWs than myocytes from the placebo group. In addition, n-3 PUFA treatment increased beat-to-beat alterations in the amplitude of Ca2+ transients (Ca2+ alternans) in VF- myocytes. These n-3 PUFAs effects in VF- myocytes were associated with an increased Ca2+ spark frequency and reduced sarcoplasmic reticulum Ca2+ content, indicative of increased activity of ryanodine receptors. Thus, dietary n-3 PUFAs do not alleviate intracellular Ca2+ cycling remodeling in myocytes isolated from post-MI VF+ hearts. Furthermore, dietary n-3 PUFAs increase vulnerability of ventricular myocytes to cellular arrhythmia in post-MI VF- hearts by destabilizing intracellular Ca2+ handling. 相似文献
80.
Large anionic multilamellar liposomes containing 71% membrane cholesterol (MLV) caused complement (C) activation in human serum in vitro, as reflected in significant rises in S protein-bound terminal complex (SC5b-9) and C3a-desarg levels. Increasing the albumin content in serum by 1-4 g/100 ml led to 50-100% further increase in MLV-induced C activation, while higher amounts of exogenous human serum albumin (HSA) gradually lost the capability to potentiate liposomal C activation. HSA alone had no influence on SC5b-9 formation at any level below 12%. Complement activation by liposomes and the potentiating effect of supplemental HSA were greatly reduced or eliminated in the absence of C1q or in the presence of 10 mM EGTA/2.5 mM Mg2+, pointing to the involvement of the classical pathway. Potentiation of C activation by supplemental HSA was not unique to MLV-induced activation, as deposition of HSA on the membrane of ‘Centricon’ ultrafiltration units also potentiated the C-activating effect of the polycarbonate membrane. Fatty acid (FA) or non-monomeric protein contamination in HSA were unlikely to be playing a role in the described effects, as 96% pure, FA-rich (Buminate) and 99% pure, FA-free HSA had identical effects on liposomal C activation. While highlighting a new modulatory mechanism on liposomal C activation, the above data raise the possibility that deposition of extravasated HSA at sites of tissue injury may serve a hitherto unrecognized proinflammatory function. 相似文献