Structural diversity using amino acid “Customizable Units”: conversion of hydroxyproline (Hyp) into nitrogen heterocycles

Amino Acids - The ability of amino acid “customizable units” to generate structural diversity is illustrated by the conversion of 4-hydroxyproline (Hyp) units into a variety of nitrogen...     

Partitioning between recoding and termination at a stop codon–selenocysteine insertion sequence

Selenocysteine (Sec) is inserted into proteins by recoding a UGA stop codon followed by a selenocysteine insertion sequence (SECIS). UGA recoding by the Sec machinery is believed to be very inefficient owing to RF2-mediated termination at UGA. Here we show that recoding efficiency in vivo is 30–40% independently of the cell growth rate. Efficient recoding requires sufficient selenium concentrations in the medium. RF2 is an unexpectedly poor competitor of Sec. We recapitulate the major characteristics of SECIS-dependent UGA recoding in vitro using a fragment of fdhF-mRNA encoding a natural bacterial selenoprotein. Only 40% of actively translating ribosomes that reach the UGA codon insert Sec, even in the absence of RF2, suggesting that the capacity to insert Sec into proteins is inherently limited. RF2 does not compete with the Sec incorporation machinery; rather, it terminates translation on those ribosomes that failed to incorporate Sec. The data suggest a model in which early recruitment of Sec-tRNA^Sec–SelB–GTP to the SECIS blocks the access of RF2 to the stop codon, thereby prioritizing recoding over termination at Sec-dedicated stop codons.     

Cells deficient in base-excision repair reveal cancer hallmarks originating from adjustments to genetic instability

Genetic instability, provoked by exogenous mutagens, is well linked to initiation of cancer. However, even in unstressed cells, DNA undergoes a plethora of spontaneous alterations provoked by its inherent chemical instability and the intracellular milieu. Base excision repair (BER) is the major cellular pathway responsible for repair of these lesions, and as deficiency in BER activity results in DNA damage it has been proposed that it may trigger the development of sporadic cancers. Nevertheless, experimental evidence for this model remains inconsistent and elusive. Here, we performed a proteomic analysis of BER deficient human cells using stable isotope labelling with amino acids in cell culture (SILAC), and demonstrate that BER deficiency, which induces genetic instability, results in dramatic changes in gene expression, resembling changes found in many cancers. We observed profound alterations in tissue homeostasis, serine biosynthesis, and one-carbon- and amino acid metabolism, all of which have been identified as cancer cell ‘hallmarks’. For the first time, this study describes gene expression changes characteristic for cells deficient in repair of endogenous DNA lesions by BER. These expression changes resemble those observed in cancer cells, suggesting that genetically unstable BER deficient cells may be a source of pre-cancerous cells.     

Fish assemblage structure in an estuary of the Atlantic Forest biodiversity hotspot (southern Brazil)

We described the fish assemblage in the estuary of the Guaraguaçu River (one of the largest tributaries of the Paranaguá Bay Estuary, located within Brazil’s Atlantic Forest Biosphere Reserve) from June 2005 to May 2006, and assessed the seasonal and spatial effects of abiotic environmental attributes on the fish assemblage structure. Despite some oscillations in salinity, the upper and lower estuaries had year-round persistent oligohaline and polyhaline conditions, respectively. Despite high species richness (55 species), the Guaraguaçu River Estuary fish community contains a few dominant taxa; 11% of the richness accounts for >60% of its density and biomass. The most abundant species (in terms of both biomass and density) was Atherinella brasiliensis. Species whose densities were most strongly associated with the upper estuary were Centropomus parallelus, Ctenogobius schufeldti, Eucinostomus melanopterus, Platanichthys platana, Trinectes paulistanus, and Eugerres brasilianus. Those whose densities were most strongly associated with the lower estuary were A. brasiliensis, Sphoeroides greeleyi, Eucinostomus argenteus, Sphoeroides testudineus, Diapterus rhombeus, and Harengula clupeola. Throughout the year, canonical correspondence analysis identified: (1) the pattern of horizontal stratification of salinity along the river as being the most important variable for explaining most of the fish fauna structure; and (2) a strong relationship between the fish fauna and the salinity gradient along the estuary. Analysis of similarity further confirmed that each estuarine zone supports a year-round persistent and relatively homogeneous fish species assemblage. Total mean density and biomass remained constant over time in each estuarine habitat, but density shifted in the most abundant species, which appears related to recruitment patterns. Such species and abundance persistence likely occurs because seasonal rainfall-induced changes in river discharge are not sufficient to significantly shift runoff and salinity and thus fish assemblage structure (species composition, density and biomass) along the estuary. Such a lack of seasonal fish fauna movement as a response to changes in river flow contrasts with other estuarine systems around the world.     

Creatinyl amino acids—new hybrid compounds with neuroprotective activity

Prolonged oral creatine administration resulted in remarkable neuroprotection in experimental models of brain stroke. However, because of its polar nature creatine has poor ability to penetrate the blood–brain barrier (BBB) without specific creatine transporter (CRT). Thus, synthesis of hydrophobic derivatives capable of crossing the BBB by alternative pathway is of great importance for the treatment of acute and chronic neurological diseases including stroke, traumatic brain injury and hereditary CRT deficiency. Here we describe synthesis of new hybrid compounds—creatinyl amino acids, their neuroprotective activity in vivo and stability to degradation in different media. The title compounds were synthesized by guanidinylation of corresponding sarcosyl peptides or direct creatine attachment using isobutyl chloroformate method. Addition of lipophilic counterion (p‐toluenesulfonate) ensures efficient creatine dissolution in DMF with simultaneous protection of guanidino group towards intramolecular cyclization. It excludes the application of expensive guanidinylating reagents, permits to simplify synthetic procedure and adapt it to large‐scale production. The biological activity of creatinyl amino acids was tested in vivo on ischemic stroke and NaNO₂‐induced hypoxia models. One of the most effective compounds—creatinyl‐glycine ethyl ester increases life span of experimental animals more than two times in hypoxia model and has neuroprotective action in brain stroke model when applied both before and after ischemia. These data evidenced that creatinyl amino acids can represent promising candidates for the development of new drugs useful in stroke treatment. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.