587 research outputs found

    Yeast as a model organism for studying the evolution of non-standard genetic codes

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    During the last 30 years, a number of alterations to the standard genetic code have been uncovered both in prokaryotes and eukaryotic nuclear and mitochondrial genomes. But, the study of the evolutionary pathways and molecular mechanisms of codon identity redefinition has been largely ignored due to the assumption that non-standard genetic codes can only evolve through neutral evolutionary mechanisms and that they have no functional significance. The recent discovery of a genetic code change in the genus Candida that evolved through an ambiguous messenger RNA decoding mechanism is bringing that naive assumption to an abrupt end by showing, in a rather dramatic way, that genetic code changes have profound physiological and evolutionary consequences for the species that redefine codon identity. In this paper, the recent data on the evolution of the Candida genetic code are reviewed and an experimental framework based on forced evolution, molecular genetics and comparative and functional genomics methodologies is put forward for the study of non-standard genetic codes and genetic code ambiguity in general. Additionally, the importance of using Saccharomyces cerevisiae as a model organism for elucidating the evolutionary pathway of the Candida and other genetic code changes is emphasised.publishe

    A Knack to Know an Honest Man

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    Alzheimer’s disease (AD) is a multifactorial disorder with several target proteins contributing to its etiology. In search for multifunctional anti-AD drug candidates, taking into account that the acetylcholinesterase (AChE) and beta-amyloid (Aβ) aggregation are particularly important targets for inhibition, the tacrine and benzothiazole (BTA) moieties were conjugated with suitable linkers in a novel series of hybrids. The designed compounds (7a–7e) were synthesized and in vitro as well as in ex vivo evaluated for their capacity for the inhibition of acetylcholinesterase (AChE) and Aβ self-induced aggregation, and also for the protection of neuronal cells death (SHSY-5Y cells, AD and MCI cybrids). All the tacrine–BTA hybrids displayed high in vitro activities, namely with IC50 values in the low micromolar to sub-micromolar concentration range towards the inhibition of AChE, and high percentages of inhibition of the self-induced Aβ aggregation. Among them, compound 7a, with the shortest linker, presented the best inhibitory activity of AChE (IC50 = 0.34 μM), while the highest activity as anti-Aβ42 self-aggregation, was evidenced for compound 7b (61.3%, at 50 μM. The docking studies demonstrated that all compounds are able to interact with both catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. Our results show that compounds 7d and 7e improved cell viability in cells treated with Aβ42 peptide. Overall, these multi-targeted hybrid compounds appear as promising lead compounds for the treatment of Alzheimer’s disease

    Upregulation of tRNA-Ser-AGA-2-1 promotes malignant behavior in normal bronchial cells

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    Serine tRNAs (tRNASer) are frequently overexpressed in tumors and associated with poor prognosis and increased risk of recurrence in breast cancer. Impairment of tRNA biogenesis and abundance also impacts proteome homeostasis, and activates protein quality control systems. Herein, we aimed at testing whether increasing tRNASer abundance could foster tumor establishment through activation of the UPR. In order to do so, firstly we confirmed that the expression of tRNA-Ser-AGA-2-1 [hereafter tRNASer(AGA)] was upregulated by 1.79-fold in Stage I NSCLC tumors when compared to normal adjacent tissue. To study the impact of tRNASer(AGA) in early stage tumorigenesis, we induced its upregulation in a non-tumoral bronchial cell line, BEAS-2B. Upregulation of this tRNA increased cellular proliferation and protein synthesis rate, driven by eIF2α dephosphorylation and ATF4 activation downstream of PERK signaling. Futhermore, tRNASer(AGA) enhanced transformation potential in vitro, and promoted the establishment of slow growing tumors with aggressive features in nude mice. Our work highlights the importance of studying tRNA deregulation on early stage tumorigenesis, as they may be potential malignancy and aggressiveness biomarkers.publishe

    Tacrine-allyl/propargylcysteine-benzothiazole trihybrids as potential anti-Alzheimer's drug candidates

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    The authors acknowledge the Portuguese Fundação para a Ciência e Tecnologia (FCT) for the project UID/QUI/00100/2013, postdoctoral fellowships (RSK and KC). We also thank the doctoral fellowship from Erasmus Namaste program (AH).On continuing our research on new drug candidates for Alzheimer's disease (AD), we have designed, synthesized and evaluated a series of multifunctional trihybrid agents. The design strategy was based on the incorporation of a benzothiazole (BTA) moiety on a series of very recently reported bihybrids, resulting from the conjugation of a tacrine (TAC) with natural based moieties, namely S-allylcysteine (SAC) (garlic constituent) and S-propargylcysteine (SPRC). Thus, in addition to the acetylcholinesterase inhibition (AChEI) and anti-ROS capacity of the bihybrids (TAC-SAC/SPRC), the new trihybrids (TAC-SAC/SPRC-BTA) were endowed with 5-fold capacity for inhibition of the amyloid beta-peptide (Aβ) aggregation. The BTA moiety led also to considerable enhancement of the AChEI on the trihybrids, which molecular modeling suggested to be due to the simultaneous binding to the catalytic active site and peripheral anionic site of AChE. The trihybrids were also assessed for the MAO inhibition, but resulted in lower activity than expected, ascribed to the low accessibility of the propargyl groups to the enzyme active site. Finally, the effects of the compounds on the viability of neuroblastome cells stressedwith Aβ42 and H2O2 showed moderate cell protection. Overall, the performed studies illustrate the importance (and limitations) of enclosing several molecular scaffolds in one molecular entity to allow the modulation of multiple AD targets.PostprintPeer reviewe

    A genetic code alteration generates a proteome of high diversity in the human pathogen Candida albicans

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    Background - Genetic code alterations have been reported in mitochondrial, prokaryotic, and eukaryotic cytoplasmic translation systems, but their evolution and how organisms cope and survive such dramatic genetic events are not understood. Results - Here we used an unusual decoding of leucine CUG codons as serine in the main human fungal pathogen Candida albicans to elucidate the global impact of genetic code alterations on the proteome. We show that C. albicans decodes CUG codons ambiguously and tolerates partial reversion of their identity from serine back to leucine on a genome-wide scale. Conclusion - Such codon ambiguity expands the proteome of this human pathogen exponentially and is used to generate important phenotypic diversity. This study highlights novel features of C. albicans biology and unanticipated roles for codon ambiguity in the evolution of the genetic code.publishe

    Elevated gamma-glutamyl transferase is associated with subclinical inflammation independent of cardiometabolic risk factors in an asymptomatic population: a cross-sectional study

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    BACKGROUND: Serum Gamma-Glutamyl Transferase (GGT), a marker of oxidative stress, has been suggested to be independently associated with cardiovascular disease (CVD) events. We examined the association of serum GGT levels with the burden of subclinical inflammation across a spectrum of metabolic conditions. METHODS: We evaluated 5,446 asymptomatic subjects (43 ± 10 years, 78 % males) who had an employer-sponsored physical between 2008 and 2010. Highly sensitivity C-reactive protein (hsCRP) was measured as a marker of underlying systemic inflammation. A linear regression of GGT quartiles with log transformed hsCRP and a multivariate logistic regression of GGT quartiles with elevated hsCRP (≥3 mg/L) were performed. RESULTS: Median GGT was 31 IU/l (IQR: 22–45 IU/l), 1025 (19 %) had hsCRP ≥ 3 mg/L. The median hsCRP increased with GGT quartiles (Q1: 0.9 mg/L, Q2: 1.1 mg/L, Q3: 1.4 mg/L, Q4: 1.6 mg/L, p < 0.001). Linear regression models showed GGT in the fourth quartile was associated with 0.45 mg/L (95 % CI 0.35, 0.54, p < 0.001) increase in log transformed hsCRP adjusting for risk factors. The Odds Ratio (OR) for an elevated hsCRP (≥3 mg/L) also increased with higher GGT quartiles; GGT Q2 1.44 (95 % CI 1.12, 1.85), GGT Q3 1.89 (95 % CI 1.45, 2.46), GGT Q4 2.22 (95 % CI 1.67, 2.95), compared to GGT Q1. The strength of association increased in the presence of and combination of metabolic conditions. CONCLUSION: In our cohort of asymptomatic individuals a higher serum GGT level was independently associated with increased burden of subclinical inflammation across metabolic states. These findings may explain GGT association with increased CVD risk

    Unveiling the Influence of Carbon Nanotube Diameter and Surface Modification on the Anchorage of L-Asparaginase

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    L-asparaginase (ASNase, EC 3.5.1.1) is an amidohydrolase enzyme known for its anti-cancer properties, with an ever-increasing commercial value. Immobilization has been studied to improve the enzyme’s efficiency, enabling its recovery and reuse, enhancing its stability and half-life time. In this work, the effect of pH, contact time and enzyme concentration during the ASNase physical adsorption onto pristine and functionalized multi-walled carbon nanotubes (MWCNTs and f-MWCNTs, respectively) with different size diameters was investigated by maximizing ASNase relative recovered activity (RRA) and immobilization yield (IY). Immobilized ASNase reusability and kinetic parameters were also evaluated. The ASNase immobilization onto f-MWCNTs offered higher loading capacities, enhanced reusability, and improved enzyme affinity to the substrate, attaining RRA and IY of 100 and 99%, respectively, at the best immobilization conditions (0.4 mg/mL of ASNase, pH 8, 30 min of contact time). In addition, MWCNTs diameter proved to play a critical role in determining the enzyme binding affinity, as evidenced by the best results attained with f-MWCNTs with diameters of 10–20 nm and 20–40 nm. This study provided essential information on the impact of MWCNTs diameter and their surface functionalization on ASNase efficiency, which may be helpful for the development of innovative biomedical devices or food pre-treatment solutionspublishe

    The Yeast PNC1 Longevity Gene Is Up-Regulated by mRNA Mistranslation

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    Translation fidelity is critical for protein synthesis and to ensure correct cell functioning. Mutations in the protein synthesis machinery or environmental factors that increase synthesis of mistranslated proteins result in cell death and degeneration and are associated with neurodegenerative diseases, cancer and with an increasing number of mitochondrial disorders. Remarkably, mRNA mistranslation plays critical roles in the evolution of the genetic code, can be beneficial under stress conditions in yeast and in Escherichia coli and is an important source of peptides for MHC class I complex in dendritic cells. Despite this, its biology has been overlooked over the years due to technical difficulties in its detection and quantification. In order to shed new light on the biological relevance of mistranslation we have generated codon misreading in Saccharomyces cerevisiae using drugs and tRNA engineering methodologies. Surprisingly, such mistranslation up-regulated the longevity gene PNC1. Similar results were also obtained in cells grown in the presence of amino acid analogues that promote protein misfolding. The overall data showed that PNC1 is a biomarker of mRNA mistranslation and protein misfolding and that PNC1-GFP fusions can be used to monitor these two important biological phenomena in vivo in an easy manner, thus opening new avenues to understand their biological relevance

    Evaluation of mutagenicity and antimutagenicity of different fractions of Pterogyne nitens (Leguminosae), using Tradescantia pallida micronuclei assay

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    Pterogyne nitens (Fabaceae-Caesalpinioideae) é uma árvore nativa da América do Sul, onde é empregada na medicina popular para o tratamento da ascaridíase. Recentemente, descrevemos o efeito mutagênico do extrato etanólico das folhas de P. nitens. Dessa forma, o presente estudo teve por objetivo aprofundar a avaliação do potencial mutagênico das frações isoladas das folhas de Pterogyne nitens, acetato de etila (AcOEt), n-butanólica (BuOH) e hidroalcóolica (HA). Quando o efeito mutagênico foi observado somente nas maiores concentrações testadas, o potencial antimutagênico também foi avaliado. Os ensaios mutagênicos e antimutagênicos foram realizados utilizando ensaio de micronúcleo em Trandescantia pallida. Na avaliação de mutagenicidade, observou-se o efeito nas frações AcOEt (0,460 mg/mL), BuOH (0,142, 0,285, 0,570 e 1,14 mg/mL) e HA (0,050, 0,100, 0,200 e 0,400 mg/mL). Considerando que o efeito mutagênico da fração AcOEt foi observado somente na concentração mais elevada (0,460 mg/mL), o potencial antimutagênico da mesma foi avaliado. As concentrações de 0,115 e 0,230 mg/mL da fração AcOEt demonstraram atividade antimutagênica. A partir dos resultados do presente estudo, conclui-se que determinadas frações de P. nitens apresentam mutagenicidade (BuOH e HA), enquanto a fração AcOEt apresentou efeito antimutagênico nas maiores concentrações. Esses resultados tornam o estudo da P. nitens bastante promissor, considerando que esta planta possui distribuição geográfica ampla e tem sido pouco estudada.Pterogyne nitens (Fabaceae-Caesalpinioideae) is a tree native to South American, where it is used in folk treatment of ascaridiasis. Recently, we have been describing the mutagenic effect of the ethanol extract of leaves of P. nitens. Thus, the present study aimed at evaluating the mutagenic potential of the ethyl acetate (EtOAc), n- butanol (BuOH) and hydroalcoholic (HA) fractions. When the mutagenic effect was observed only in the highest tested concentrations, the antimutagenic activity was also evaluated. Both mutagenic and antimutagenic assays were performed using T. pallida micronuclei assay. Mutagenicity was observed between different concentrations of the P nitens fractions, EtOAc (0.460 mg/mL), BuOH (0.142, 0.285, 0.570 and 1.14 mg/mL) and HA (0.050, 0.100, 0.200 and 0.400 mg/mL). Whereas the mutagenic effect of the EtOAc fraction was observed in the highest concentration (0.460 mg/mL), its antimutagenic potential was evaluated. The 0.115 and 0.230 mg/mL concentrations of the EtOAc fraction demonstrated antimutagenic activity. Based on the results of the present study we can conclude that some P. nitens fractions (BuOH and HA) demonstrated mutagenic effects whereas the EtOAc fraction shown low mutagenicity and amtimutagenicity in the two higher concentrations. Those results stimulate the studies with P. nitens, which possess spread geographic distribution and it is still low studied
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