Cleavage of pyrene-stabilized RNA bulge loops by trans-(±)-cyclohexane-1,2-diamine

Chemical agents that cleave HIV genome can be potentially used for anti-HIV therapy. In this report, the cleavage of the upper stem-loop region of HIV-1 TAR RNA was studied in a variety of buffers containing organic catalysts. trans-(±)-Cyclohexane-1,2-diamine was found to cleave the RNA with the highest activity (31%, 37°C, 18 h). Cleavage of the RNA in trans-(±)-cyclohexane-1,2-diamine buffer was also studied when the RNA was hybridized with complementary DNAs. A pyrene-modified C3 spacer was incorporated to the DNA strand to facilitate the formation of a RNA bulge loop in the RNA/DNA duplex. In contrast, unmodified DNAs cannot efficiently generate RNA bulge loops, regardless of the DNA sequences. The results showed that the pyrene-stablized RNA bulge loops were efficiently and site-specifically cleaved by trans-(±)-cyclohexane-1,2-diamine

The Leucine Zipper Domains of the Transcription Factors GCN4 and c-Jun Have Ribonuclease Activity

Basic-region leucine zipper (bZIP) proteins are one of the largest transcription factor families that regulate a wide range of cellular functions. Owing to the stability of their coiled coil structure leucine zipper (LZ) domains of bZIP factors are widely employed as dimerization motifs in protein engineering studies. In the course of one such study, the X-ray structure of the retro-version of the LZ moiety of yeast transcriptional activator GCN4 suggested that this retro-LZ may have ribonuclease activity. Here we show that not only the retro-LZ but also the authentic LZ of GCN4 has weak but distinct ribonuclease activity. The observed cleavage of RNA is unspecific, it is not suppressed by the ribonuclease A inhibitor RNasin and involves the breakage of 3′,5′-phosphodiester bonds with formation of 2′,3′-cyclic phosphates as the final products as demonstrated by HPLC/electrospray ionization mass spectrometry. Several mutants of the GCN4 leucine zipper are catalytically inactive, providing important negative controls and unequivocally associating the enzymatic activity with the peptide under study. The leucine zipper moiety of the human factor c-Jun as well as the entire c-Jun protein are also shown to catalyze degradation of RNA. The presented data, which was obtained in the test-tube experiments, adds GCN4 and c-Jun to the pool of proteins with multiple functions (also known as moonlighting proteins). If expressed in vivo, the endoribonuclease activity of these bZIP-containing factors may represent a direct coupling between transcription activation and controlled RNA turnover. As an additional result of this work, the retro-leucine zipper of GCN4 can be added to the list of functional retro-peptides

Fluorescence-Quenched Substrates for Live Cell Imaging of Human Glucocerebrosidase Activity

Deficiency of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) leads to abnormal accumulation of glucosyl ceramide in lysosomes and the development of the lysosomal storage disease known as Gaucher’s disease. More recently, mutations in the GBA1 gene that encodes GCase have been uncovered as a major genetic risk factor for Parkinson’s disease (PD). Current therapeutic strategies to increase GCase activity in lysosomes involve enzyme replacement therapy (ERT) and molecular chaperone therapy. One challenge associated with developing and optimizing these therapies is the difficulty in determining levels of GCase activity present within the lysosomes of live cells. Indeed, visualizing the activity of endogenous levels of any glycoside hydrolases, including GCase, has proven problematic within live mammalian cells. Here we describe the successful modular design and synthesis of fluorescence-quenched substrates for GCase. The selection of a suitable fluorophore and quencher pair permits the generation of substrates that allow convenient time-dependent monitoring of endogenous GCase activity within cells as well as localization of activity within lysosomes. These efficiently quenched (∼99.9%) fluorescent substrates also permit assessment of GCase inhibition in live cells by either confocal microscopy or high content imaging. Such substrates should enable improved understanding of GCase in situ as well the optimization of small-molecule chaperones for this enzyme. These findings also suggest routes to generate fluorescence-quenched substrates for other mammalian glycoside hydrolases for use in live cell imaging

O Brasil e a construção da ordem econômica internacional contemporânea Brazil and the making of the modern global economic order

Entre o Congresso de Viena, no qual estiveram representados apenas oito Estados "cristãos", as Conferências de Paz da Haia e o Tratado de Versalhes, que envolveram pouco mais de duas dezenas de países, e o atual sistema onusiano, praticamente universal, a sociedade internacional conheceu uma profunda democratização nos últimos dois séculos, mesmo que os fundamentos do poder político e econômico não tenham conhecido modificação substancial. Esse fenômeno de ampliação da antiga "democracia censitária" é particularmente visível na elaboração de normas e instituições para o relacionamento econômico internacional, em que as organizações multilaterais de cooperação técnica e econômica, dentre as quais se destacam o Fundo Monetário Internacional, o Banco Mundial e a Organização Mundial do Comércio, desempenham relevante papel na construção da interdependência global. Este ensaio histórico segue, na longa duração, a evolução do multilateralismo, fundamentalmente em sua vertente econômica, e examina a inserção internacional do Brasil, um dos poucos países da periferia a ter participado ativamente da construção da ordem econômica internacional em várias épocas, por meio de sua presença nas mais diversas conferências multilaterais que presidiram ao nascimento dessas organizações intergovernamentais de cooperação.<br>Starting from the Congress of Vienna, in which only eight "Christian" states assisted, through the Hague Peace conferences and the Versailles treaty, mobilizing no more than two dozens countries, to the current UNO system, virtually universal, international society has undergone a deep democratization in the last two centuries, even if the sources of political and economic power and its distribution among countries have been substantially maintained. This process of enlargement of the old "restrictive democracy" is mostly evident in the institutional rule making for the international economic relations, where multilateral organizations for technical and economic cooperation - among them the IMF, the World Bank and the WTO - have a significant role in reinforcing the global interdependence among states. This historical essay follows the evolution of multilateralism, in the longue durée, with particular attention to its economic features, and examines Brazil's international insertion in the world economy, as one of the few "peripheric" countries which took an active part in the making of "international economic order". Indeed, Brazil was present at the creation of most, if not all, intergovernmental organizations and took part in various multilateral conferences from the 19 th century to our times