A strategy for the identification of proteins targeted by thioredoxin

Thioredoxins are 12-kDa proteins functional in the regulation of cellular processes throughout the animal, plant, and microbial kingdoms. Growing evidence with seeds suggests that an h-type of thioredoxin, reduced by NADPH via NADP-thioredoxin reductase, reduces disulfide bonds of target proteins and thereby acts as a wakeup call in germination. A better understanding of the role of thioredoxin in seeds as well as other systems could be achieved if more were known about the target proteins. To this end, we have devised a strategy for the comprehensive identification of proteins targeted by thioredoxin. Tissue extracts incubated with reduced thioredoxin are treated with a fluorescent probe (monobromobimane) to label sulfhydryl groups. The newly labeled proteins are isolated by conventional two-dimensional electrophoresis: (i) nonreducing/reducing or (ii) isoelectric focusing/reducing SDS/PAGE. The isolated proteins are identified by amino acid sequencing. Each electrophoresis system offers an advantage: the first method reveals the specificity of thioredoxin in the reduction of intramolecular vs. intermolecular disulfide bonds, whereas the second method improves the separation of the labeled proteins. By application of both methods to peanut seed extracts, we isolated at least 20 thioredoxin targets and identified 5—three allergens (Ara h2, Ara h3, and Ara h6) and two proteins not known to occur in peanut (desiccation-related and seed maturation protein). These findings open the door to the identification of proteins targeted by thioredoxin in a wide range of systems, thereby enhancing our understanding of its function and extending its technological and medical applications.

The structure of bovine F1-ATPase complexed with the peptide antibiotic efrapeptin.

In the previously determined structure of mitochondrial F1-ATPase determined with crystals grown in the presence of adenylyl-imidodiphosphate (AMP-PNP) and ADP, the three catalytic beta-subunits have different conformations and nucleotide occupancies. AMP-PNP and ADP are bound to subunits beta TP and beta DP, respectively, and the third beta-subunit (beta E) has no bound nucleotide. The efrapeptins are a closely related family of modified linear peptides containing 15 amino acids that inhibit both ATP synthesis and hydrolysis by binding to the F1 catalytic domain of F1F0-ATP synthase. In crystals of F1-ATPase grown in the presence of both nucleotides and inhibitor, efrapeptin is bound to a unique site in the central cavity of the enzyme. Its binding is associated with small structural changes in side chains of F1-ATPase around the binding pocket. Efrapeptin makes hydrophobic contacts with the alpha-helical structure in the gamma-subunit, which traverses the cavity, and with subunit beta E and the two adjacent alpha-subunits. Two intermolecular hydrogen bonds could also form. Intramolecular hydrogen bonds probably help to stabilize efrapeptin's two domains (residues 1-6 and 9-15, respectively), which are connected by a flexible region (beta Ala-7 and Gly-8). Efrapeptin appears to inhibit F1-ATPase by blocking the conversion of subunit beta E to a nucleotide binding conformation, as would be required by an enzyme mechanism involving cyclic interconversion of catalytic sites.

Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium.

We describe a protease, named "thiocalsin," that is activated by calcium but only after reductive activation by thioredoxin, a small protein with a redox-active disulfide group that functions widely in regulation. Thiocalsin appeared to be a 14-kDa serine protease that functions independently of calmodulin. The enzyme, purified from germinating wheat grain, specifically cleaved the major indigenous storage proteins, gliadins and glutenins, after they too had been reduced, preferentially by thioredoxin. The disulfide groups of the enzyme, as well as its protein substrates, were reduced by thioredoxin via NADPH and the associated enzyme, NADP-thioredoxin reductase. The results broaden the roles of thioredoxin and calcium and suggest a joint function in activating thiocalsin, thereby providing amino acids for germination and seedling development.

Discovery of a brain promoter from the human transferrin gene and its utilization for development of transgenic mice that express human apolipoprotein E alleles.

Transgenic mice carrying heterologous genes directed by a 670-bp segment of the regulatory sequence from the human transferrin (TF) gene demonstrated high expression in brain. Mice carrying the chimeric 0.67kbTF-CAT gene expressed TF-CAT in neurons and glial cells of the nucleus basalis, the cerebrum, corpus callosum, cerebellum, and hippocampus. In brains from two independent TF-CAT transgenic founder lines, copy number of TF-CAT mRNA exceeded the number of mRNA transcripts encoding either mouse endogenous transferrin or mouse endogenous amyloid precursor protein. In two transgenic founder lines, the chloramphenicol acetyltransferase (CAT) protein synthesized from the TF-CAT mRNA was estimated to be 0.10-0.15% of the total soluble proteins of the brain. High expression observed in brain indicates that the 0.67kbTF promoter is a promising director of brain expression of heterologous genes. Therefore, the promoter has been used to express the three common human apolipoprotein E (apoE) alleles in transgenic mouse brains. The apoE alleles have been implicated in the expression of Alzheimer disease, and the human apoE isoforms are reported to interact with different affinities to the brain beta-amyloid and tau protein in vitro. Results of this study demonstrate high expression and production of human apoE proteins in transgenic mouse brains. The model may be used to characterize the interaction of human apoE isoforms with other brain proteins and provide information helpful in designing therapeutic strategies for Alzheimer disease.

Tabaquismo y motivación para dejar de fumar en el área de Salud Mental del Centro San Juan de Dios de Ciempozuelos

La prevalencia de consumo de tabaco en personas con trastornos mentales oscila entre un 42% y un 78% dependiendo del tipo de trastorno que padezcan (Solty, Crockford, White y Currie, 2009; Stockings et al., 2013), encontrándose las mayores tasas de consumo de tabaco y de dependencia a la nicotina en las personas con trastorno mental grave ingresadas en centros psiquiátricos (Lineberry, Allen, Nash, y Galardy, 2009). Es poco habitual que a los fumadores con psicopatología se les ofrezca tratamiento para disminuir o abandonar el consumo de tabaco, basándose en parte esta decisión en la creencia de que no están motivados para dejar de fumar. Sin embargo, se ha demostrado que muchos de estos fumadores muestran deseos de dejar de fumar, han realizado algún intento en el último año para conseguirlo y refieren que ya no disfrutan fumando, siendo una de sus principales motivaciones la preocupación por el propio estado de salud (Etter, Mohr, Garin y Etter, 2004; Mann-Wrobel, Bennett, Weiner, Buchanan y Ball, 2011)...

Rotten Symbol Mongering: Scapegoating in Post-9/11 American War Literature

A rhetorical approach to the fiction of war offers an appropriate vehicle by which one may encounter and interrogate such literature and the cultural metanarratives that exist therein. My project is a critical analysis—one that relies heavily upon Kenneth Burke’s dramatistic method and his concepts of scapegoating, the comic corrective, and hierarchical psychosis—of three war novels published in 2012 (The Yellow Birds by Kevin Powers, FOBBIT by David Abrams, and Billy Lynn’s Long Halftime Walk by Ben Fountain). This analysis assumes a rhetorical screen in order to subvert and redirect the grand narratives the United States perpetuates in art form whenever it goes to war. Kenneth Burke’s concept of ad bellum purificandum (the purification of war) sought to bridge the gap between war experience and the discourse that it creates in both art and criticism. My work extends that project. I examine the symbolic incongruity of convenient symbols that migrate from war to war (“Geronimo” was used as code for Osama bin Laden’s death during the S.E.A.L team raid; “Indian Country” stands for any dangerous land in Iraq; hajji is this generation’s epithet for the enemy other). Such an examination can weaken our cultural “symbol mongering,” to borrow a phrase from Walker Percy. These three books, examined according to Burke’s methodology, exhibit a wide range of approaches to the soldier’s tale. Notably, however, whether they refigure the grand narratives of modern culture or recast the common redemptive war narrative into more complex representations, this examination shows how one can grasp, contend, and transcend the metanarrative of the typical, redemptive war story.

Is Regulatory Convergence Efficient? CES Germany & Europe Working Papers, No. 02.3, May 1992

One of the important themes in the new institutionalism is the convergence of market regulations in a world with three powerful clusters of countries (Western Europe, North America, and East Asia) on a small number of regimes, like disorganized capitalism, free market capitalism, and coordinated market capitalism. This paper examines the political-economic theory of regulatory convergence. It reconstructs and compares three welfarist approaches: the optimal regulatory regime (Tinbergen), the rule of constitutional law (Buchanan), and regulatory rivalry (Hayek). The paper concludes that most plausible results of convergence theory are completely opposite to the expressed political intentions of the theorists. Tinbergen's theory predicts neoliberalism, not social democracy. The theories of Buchanan and Hayek predict respectively a consensual or spontaneous formation of corporatist regulations, not the return of classical constitutionalism or liberalism. The paper summons new institutionalists to repair the weak scientific elements of convergence theory and to make a distinction between the ideological origins of this theory and its unintended ideological consequences.

A critical introduction to the Old Testament /

Includes index.

The legal status of women in the United States of America /

Some numbers have multiple revisions.

Luther's reply to King Henry VIII, now first Englished after the lapse of four centuries,

Mode of access: Internet.

Scrap tires as artificial reefs /

"An environmental protection publication (SW-119)"--T.p. verso.

Cases decided in the Supreme Court of the Cape of Good Hope, during the years [1868-1879] /

Mode of access: Internet.

1921 annotations to Corpus juris : 1-22 C.J. ; and Workmen's compensation acts third volume permanent cyc annotations : contains annotations from all cases decided since the original treatises were written ; also errata corrected, new principles announced, etc. /

On cover: Annotations, Corpus juris-Cyc service. Permanent volume. 1921. C.J., A-Ev ; Cyc, Ex-Z.