1,720,988 research outputs found
Mechanism of ribosomal recruitment of initiator tRNA in bacteria
No full textInitiation of mRNA translation in prokaryotes requires the small ribosomal subunit (30S), three initiation factors, IF1, IF2 and IF3, initiator tRNA (fMet-tRNAfMet) and the large ribosomal subunit (50S). During initiation, the 30S ribosomal subunit interacts with the three factors and binds in a presumably random manner fMet-tRNAfMet and mRNA. This initially formed 30S pre-initiation complex is a kinetic intermediate of the stable 30S initiation complex which is formed upon base pairing of the anticodon of fMet-tRNAfMet and the intiation codon of the mRNA in the P-site. In a subsequent step the 50S subunit binds to the 30S initiation complex giving rise to a 70S initiation complex. During this step, a molecule of GTP bound to IF2 is hydrolyzed, the initiation factors are released from the ribosome and fMet-tRNAfMet is stabilized in the P site ready to enter in the elongation phase of translation. In turn, each of these major steps involves numerous elemental steps, whose sequence and timing are largely unknown. How the fMettRNAfMet is bound to the ribosome during the assembly of the initiation complex is one of the many questions still remaining open. According to the classical model, IF2 acts as a carrier of fMet-tRNAfMet to the ribosome. An alternative model predicts that the IF2, prebound to the 30S subunit awaits the arrival of fMet-tRNAfMet with which it interacts only at the ribosomal surface. This work presents results of a kinetic analysis of binding to the 30S subunit of various ribosomal ligands (Initiation Factors, fMet-tRNAfMet, mRNA), performed with fast kinetic techniques (rapid filter binding, fluorescence stopped flow). The data obtained strongly support the model in which IF2 is not a fMettRNAfMet carrier. The elemental rate constants determined here give us a more detailed description of the process of 30S initiation complex assembly, of the interplay of the various ribosomal ligands and of the functional significance of different structural modules of IF2
Emerging therapeutic strategies for sarcoglycanopathy
No full textIntroduction: Sarcoglycanopathy is the name shared by four rare autosomal recessive muscular dystrophies (LGMD2 C-F) that are usually characterized by early onset and rapid progression and an accompanying loss of independent walking since adolescence. Respiratory problems are frequent, and dilated cardiomyopathy may occur, although milder forms have also been described. However, sarcoglycanopathy is currently incurable, and we herein aim to describe the state of the art in the field of treatments for this disease.
Areas covered: We summarize the pathogenesis of sarcoglycanopathy, with particular emphasis on the molecular mechanism(s) underlying the disease. We describe the very few published cases of symptomatic treatment with steroids and the gene therapy approaches that have entered phase I/II clinical trials. We then present emerging novel therapeutic strategies explored at the preclinical stage that are expected to replace the defective gene (cell therapy), address general effects of the disease, or address the primary events of the pathogenic mechanism (small molecule-based therapy).
Expert opinion: Anti-inflammatory strategies, which are at present empirically applied, warrant further exploration. Although promising and currently being evaluated in clinical trials, gene therapy remains associated with concerns and requires additional confirmation. Thus, novel strategies targeting different aspects of the disease pathogenic mechanism are highly anticipated
Targeting of PFKFB3 with miR-206 but not mir-26b inhibits ovarian cancer cell proliferation and migration involving FAK downregulation
Full text link: Few studies explored the role of microRNAs (miRNAs) in the post-transcriptional regulation of glycolytic proteins and downstream effectors in ovarian cancer cells. We recently showed that the functional activation of the cytoskeletal regulator FAK in endothelial cells is fostered by the glycolytic enhancer 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). We tested the hypothesis that miR-206 and mir-26b, emerging onco-suppressors targeting PFKFB3 in estrogen-dependent tumors, would regulate proliferation and migration of serous epithelial ovarian cancer (EOC) cells via common glycolytic proteins, i.e., GLUT1 and PFKFB3, and downstream FAK. PFKFB3 was overexpressed in SKOV3, and its pharmacological inhibition with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) significantly reduced cell proliferation and motility. Both miR-206 and miR-26b directly targeted PFKFB3 as evaluated by a luciferase reporter assay. However, endogenous levels of miR-26b were higher than those of miR-206, which was barely detectable in SKOV3 as well as OVCAR5 and CAOV3 cells. Accordingly, only the anti-miR-26b inhibitor concentration-dependently increased PFKFB3 levels. While miR-206 overexpression impaired proliferation and migration by downregulating PFKFB3 levels, the decreased PFKFB3 protein levels related to miR-26 overexpression had no functional consequences in all EOC cell lines. Finally, consistent with the migration outcome, exogenous miR-206 and miR-26b induced opposite effects on the levels of total FAK and of its phosphorylated form at Tyr576/577. 3PO did not prevent miR-26b-induced SKOV3 migration. Overall, these results support the inverse relation between endogenous miRNA levels and their tumor-suppressive effects and suggest that restoring miR-206 expression represents a potential dual anti-PFKFB3/FAK strategy to control ovarian cancer progression
Synthesis and Evaluation of Bithiazole Derivatives As Potential α-Sarcoglycan Correctors
Full text link4′-Methyl-4,5′-bithiazoles were previously identified as cystic fibrosis transmembrane regulator (CFTR) correctors, thus being able to correct folding defective mutants of the channel regulating chloride transport through the membrane. Additionally, bithiazole derivative C17 was reported to recover α-sarcoglycan in vitro and in vivo. We report here the synthesis of two new derivatives of C17, in which the two sides of the bithiazole scaffold were modified. The synthesized compounds and the corresponding precursors were tested in myogenic cells to evaluate the expression of α-sarcoglycan. The results highlighted that both substitutions of
the bithiazole scaffold are important to achieve the maximum recovery of the α-sarcoglycan mutant. Nonetheless, partial preservation of the activity was observed. Accordingly, this paves the way to further derivatizations/optimization and target fishing studies, which were preliminarily performed in this study as a proof of concept, allowing the investigation of the molecular mechanisms leading to the α-sarcoglycan correction
Human Tyrosinase Produced in Insect Cells: A Landmark for the Screening of New Drugs Addressing its Activity
No full textHuman tyrosinase is the first enzyme of the multistep process of melanogenesis. It catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine and the following oxidation of o-diphenol to the corre- sponding quinone, L-dopaquinone. In spite of its biomedi- cal relevance, its reactivity is far from being fully understood, mostly because of the lack of a suitable expression system. Indeed, until now, studies on substrates and inhibitors of tyrosinases have been performed in vitro almost exclusively using mushroom or bacterial enzymes. We report on the production of a recombinant human tyrosinase in insect cells (Sf9 line). Engineering the pro- tein, improving cell culture conditions, and setting a suit- able purification protocol optimized product yield. The obtained active enzyme was truthfully characterized with a number of substrate and inhibitor molecules. These results were compared to those gained from a parallel analysis of the bacterial (Streptomyces antibioticus) enzyme and those acquired from the literature for mushroom tyrosinase, showing that the reactivity of the human enzyme appears unique and pointing out the great bias introduced when using non-human tyrosinases to measure the inhibitory efficacy of new molecules. The described enzyme is therefore an indispensable paradigm in testing pharma- ceutical or cosmetic agents addressing tyrosinase activity
Differential Analysis of Gly211Val and Gly286Val Mutations Affecting Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA1) in Congenital Pseudomyotonia Romagnola Cattle
Full text linkCongenital pseudomyotonia in cattle (PMT) is a rare skeletal muscle disorder, clinically characterized by stiffness and by delayed muscle relaxation after exercise. Muscle relaxation impairment is due to defective content of the Sarco(endo)plasmic Reticulum Ca2+ ATPase isoform 1 (SERCA1) protein, caused by missense mutations in the ATP2A1 gene. PMT represents the only mammalian model of human Brody myopathy. In the Romagnola breed, two missense variants occurring in the same allele were described, leading to Gly211Val and Gly286Val (G211V/G286V) substitutions. In this study, we analyzed the consequences of G211V and G286V mutations. Results support that the reduced amount of SERCA1 is a consequence of the G211V mutation, the G286V mutation almost being benign and the ubiquitin–proteasome system (UPS) being involved. After blocking the proteasome using a proteasome inhibitor, we found that the G211V mutant accumulates in cells at levels comparable to those of WT SERCA1. Our conclusion is that G211/286V mutations presumably originate in a folding-defective SERCA1 protein, recognized and diverted to degradation by UPS, although still catalytically functional, and that the main role is played by G211V mutation. Rescue of mutated SERCA1 to the sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca2+ concentration and prevent the appearance of pathological signs, paving the way for a possible therapeutic approach against Brody disease
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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