scholarly journals Assessment of Colorimetric Reporter Enzymes in the PURE System

Author(s):  
Caitlin E. Sharpes ◽  
John B. McManus ◽  
Steven M. Blum ◽  
Glory E. Mgboji ◽  
Matthew W. Lux
Keyword(s):  
1981 ◽  
Vol 46 (04) ◽  
pp. 749-751 ◽  
Author(s):  
E Cofrancesco ◽  
A Vigo ◽  
E M Pogliani

SummaryThe ability of heparin and related glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, factor Xa and plasmin in plasma and in a purified system containing antithrombin III (At III) was studied using chromogenic peptide substrate assaysThere was a good correlation between the charge density of the mucopolysaccharides and the activities investigated. While the difference between potentiation of the antithrombin activity by GAGs in plasma and in the purified system was slight, the inhibition of factor Xa in plasma was more pronounced than in the presence of purified At III, indicating the mechanisms for GAGs-potentiated inhibition of thrombin and factor Xa are not identical.For the antiplasmin activity, there was a good correlation between the chemical structure and biological activity only in the pure system, confirming that the antithrombin-GAG complex plays a very limited role in the inactivation of plasmin in plasma.


2021 ◽  
Vol 534 ◽  
pp. 519-525 ◽  
Author(s):  
Keita Tsukamoto ◽  
Takehiro Ando ◽  
Daisuke Fuji ◽  
Takumi Yokoyama ◽  
Yukio Takamori ◽  
...  

2006 ◽  
Vol 80 (12) ◽  
pp. 5992-6002 ◽  
Author(s):  
Yasumasa Iwatani ◽  
Hiroaki Takeuchi ◽  
Klaus Strebel ◽  
Judith G. Levin

ABSTRACT APOBEC3G (APO3G), a cytidine deaminase with two zinc finger domains, inhibits human immunodeficiency virus type 1 replication in the absence of Vif. Here, we provide a comprehensive molecular analysis of the deaminase and nucleic acid binding activities of human APO3G using a pure system containing only one protein component, i.e., highly purified, catalytically active enzyme expressed in a baculovirus system. We demonstrate that APO3G deaminates cytosines in single-stranded DNA (ssDNA) only, whereas it binds efficiently to ssDNA and ssRNA, about half as well to a DNA/RNA hybrid, and poorly to double-stranded DNA and RNA. In addition, the base specificities for deamination and binding of ssDNA are not correlated. The minimum length required for detection of APO3G binding to an ssDNA oligonucleotide in an electrophoretic mobility shift assay is 16 nucleotides. Interestingly, if nucleocapsid protein and APO3G are present in the same reaction, we find that they do not interfere with each other's binding to RNA and a complex containing the RNA and both proteins is formed. Finally, we also identify the functional activities of each zinc finger domain. Thus, although both zinc finger domains have the ability to bind nucleic acids, the first zinc finger contributes more to binding and APO3G encapsidation into virions than finger two. In contrast, deamination is associated exclusively with the second zinc finger. Moreover, zinc finger two is more important than finger one for the antiviral effect, demonstrating a correlation between deaminase and antiviral activities.


Author(s):  
Charles Manga Fombad

One reason why dictatorships flourished in Africa until the 1990s was that constitutions concentrated excessive powers in presidents. The democratic revival of the 1990s led to the introduction of new or substantially revised constitutions in a number of countries that for the first time sought to promote constitutionalism, good governance, and respect for the rule of law. A key innovation was the introduction of provisions providing for separation of powers. However, in many cases the reintroduction of multipartyism did not lead to thorough constitutional reform, setting the scene for a subsequent struggle between opposition parties, civil society, and the government, over the rule of law. This reflects the complex politics of constitutionalism in Africa over the last 60 years. In this context, it is important to note that most of the constitutions introduced at independence had provided for some degree of separation of powers, but the provisions relating to this were often vaguely worded and quickly undermined. Despite this, the doctrine of separation of powers has a long history, and the abundant literature on it shows that there is no general agreement on what it means or what its contemporary relevance is. Of the three main models of separation of powers, the American one, which comes closest to a “pure” system of separation of powers, and the British, which involves an extensive fusion of powers, have influenced developments in anglophone Africa. The French model, which combines elements of the British and American models but in which the executive predominates over the other two branches, has influenced developments in all civilian jurisdictions in Africa, particularly those in francophone Africa. The common denominator among the models is the desire to prevent tyrannical and arbitrary government by separating powers but doing so in a manner that allows for limited interference through checks and balances on the principle that le pouvoir arrête le pouvoir. The combined Anglo-American (common law) and French (civil law) models received during the colonial period remain applicable today, but despite its adoption in the 1990s, the effectiveness of the doctrine of separation of powers in limiting governmental abuse has been curtailed by the excessive powers African presidents still enjoy and the control they exercise over dominant parties in legislatures. South Africa in its 1996 Constitution, followed by Kenya in 2010 and Zimbabwe in 2013, entrenched a number of hybrid institutions of accountability that have the potential not only to complement the checks and balances provided by the traditional triad but also to act where it is unable or unwilling to do so. The advent of these institutions has given the doctrine of separation of powers renewed potency and relevance in advancing Africa’s faltering constitutionalism project.


2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Takehiro Nishikawa ◽  
Takeshi Sunami ◽  
Tomoaki Matsuura ◽  
Tetsuya Yomo

Directed evolution of proteins is a technique used to modify protein functions through “Darwinian selection.”In vitrocompartmentalization (IVC) is anin vitrogene screening system for directed evolution of proteins. IVC establishes the link between genetic information (genotype) and the protein translated from the information (phenotype), which is essential for all directed evolution methods, by encapsulating both in a nonliving microcompartment. Herein, we introduce a new liposome-based IVC system consisting of a liposome, the protein synthesis using recombinant elements (PURE) system and a fluorescence-activated cell sorter (FACS) used as a microcompartment,in vitroprotein synthesis system, and high-throughput screen, respectively. Liposome-based IVC is characterized byin vitroprotein synthesis from a single copy of a gene in a cell-sized unilamellar liposome and quantitative functional evaluation of the synthesized proteins. Examples of liposome-based IVC for screening proteins such as GFP andβ-glucuronidase are described. We discuss the future directions for this method and its applications.


1999 ◽  
Vol 10 (08) ◽  
pp. 1391-1397 ◽  
Author(s):  
A. P. YOUNG

This paper discusses how slow dynamics can occur in the vicinity of quantum phase transitions. In addition to critical slowing down, there are two additional sources of slow dynamics. Firstly, even for a pure system, there is a phase coherence time which diverges as the temperature tends to zero. This is hard to see from numerical studies in imaginary time and real time dynamics are needed. Secondly, in random systems, Griffiths–McCoy singularities occur at low temperature because of rare regions which are "locally in the wrong phase" due to of statistical fluctuations in the random interactions. These are particularly strong for systems with discrete, e.g., Ising, symmetry. The form of Griffiths–McCoy singularities in real time is not understood.


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