1,720,996 research outputs found
Spectroscopic studies of the super relaxed state of skeletal muscle: a possible target for obesity and type 2 diabetes treatments
Full text linkMyosin and muscles have been studied since the beginning of the last century. The high amount of myosin and its optimized purification protocol started the huge field of the molecular motors around 50 years ago. The importance of myosin is not only due to its physiological role but also for its large contribution to the general biochemistry knowledge. Since myosin is a molecular motor, it represented an important step in the connection between chemistry and physics as fields of studies applied to biology. Myosin, as the fulcrum of biochemical conversion of energy into physical work, is the perfect example of the intimate connection between biochemistry and biophysics.
Several techniques have been used to study molecular muscle functions, and so, myosin function. Some of them are common but very powerful, as fluorescence. Some others are less used in biology, such as electronic paramagnetic resonance (EPR). Back in the 1980s, the first papers about EPR measurements on muscle fibers were published. Muscle fibers, and myosin filaments, are not an ordinary biological sample because of their physiological microscopic and macroscopic order. In such samples, EPR, being sensitive to orientation, becomes really informative. EPR was first used on muscle samples to describe the freedom of the myosin head in all the different physiological states it has to go through to complete a cycle. Using these and other techniques, scientists have been working on myosin, characterizing most aspects of the muscle motor protein. There are several models which try to describe the myosin cycle. They may be really detailed and complex. Generally, they all agree on the steps that the myosin has to go through to perform a power stroke. Recently, a new state of myosin has been identified. This state has been called the “Super Relaxed” (SRX) of myosin because when myosin enters this state the ATPase activity is very low.
In this thesis, we aim to characterize myosin behavior especially by studying the stability and the structural details of the super relaxed state.
First we wanted to explore the super relaxed state by modifying one of its components. We expressed mutants of a subunit associated with myosin and that has an important role in the stability of the super relaxed state. The protein has been exchanged into muscle fibers. The idea is to find a way to sense whether the myosin is in the super relaxed state or not. By collecting data on the effects of these mutants labeled with several probes, we were able to identify the mutant/probe couple able to sense the super relaxed state. We used that fluorimetric assay in a high throughput screening, we were looking for a molecule that was able to destabilize the state. Our belief is that the muscle tissue is a good target for increasing basal metabolism in humans, lowering the stability of the slow ATPase state to improve life quality of people affected by obesity and diabetes. A molecule has been identified and it has been shown to be a good leading compound for further pharmacological studies
Piperine's mitigation of obesity and diabetes can be explained by its up-regulation of the metabolic rate of resting muscle
Full text linkProtocol for measuring force in skinned diaphragm muscle fibers of myopathic SEPN1 knockout mice following chronic tauroursodeoxycholic acid treatment
Full text link: Selenoprotein N1 (SEPN1) is a type II endoplasmic reticulum (ER) glycoprotein. Loss-of-function mutations in the gene encoding for SEPN1 give rise to myopathy. Here, we present a protocol for evaluating the contractility of diaphragmatic muscle fibers of SEPN1 knockout mice following chronic treatment with tauroursodeoxycholic acid (TUDCA). We describe steps for genotyping SEPN1 knockout mice, TUDCA in vivo treatment, diaphragm dissection, and chemical permeabilization. We then detail procedures for single muscle fiber isolation and tension measurement. For complete details on the use and execution of this protocol, please refer to Germani et al.1
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
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
A combined experimental and computational analysis of mantATP turnover in skinned muscle fibers
Full text linkMyosin is the primary motor protein in skeletal muscle, responsible for adenosine triphosphate (ATP) hydrolysis that drives muscle contraction. In addition to force production, resting myosin consumes ATP in futile cycles at two rates, the slower one being associated with the Super Relaxed State (SRX), in contrast to the less inhibited Disordered Relaxed State (DRX). The SRX is typically measured using the mantATP chasing technique, where the decay of a fluorescent ATP analogue is fitted using a multiexponential function. Recently, significant concerns have been raised regarding the use of this technique, particularly when applied to soluble myosin preparations. While skinned fibers offer the advantage of preserving the native thick filament structure and myosin cooperativity, limited diffusion and nonspecific mantATP binding pose challenges. In this study, we combine experimental data and in-silico modeling to dissect the contributions of different components in the mantATP chasing signal. We analyze control skinned fibers and fibers subjected to myosin extraction. Our analysis shows that the nonspecific component partially overlaps with the DRX timescale. In contrast, the slow component linked to myosin SRX nucleotide release is characterized by a time constant that significantly differs from those of the nonspecific signal and DRX, enabling its reliable estimation using this technique. Our findings indicate that evaluating nonspecific mantATP components is necessary to obtain a reliable estimation of both SRX and DRX. We validated our analysis by comparing populations and time constants obtained from chasing with mantATP to mantATPase rates in control conditions and upon piperine-induced SRX destabilization
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
Custom-made 3D-printed boot as a model of disuse-induced atrophy in murine skeletal muscle
Full text linkSkeletal muscle atrophy is characterized by a decrease in muscle mass and strength caused by an imbalance in protein synthesis and degradation. This process naturally occurs upon reduced or absent physical activity, often related to illness, forced bed rest, or unhealthy lifestyles. Currently, no treatment is available for atrophy, and it can only be prevented by overloading exercise, causing severe problems for patients who cannot exercise due to chronic diseases, disabilities, or being bedridden. The two murine models commonly used to induce muscle atrophy are hindlimb suspension and ankle joint immobilization, both of which come with criticalities. The lack of treatments and the relevance of this atrophic process require a unilateral, safe, and robust model to induce muscle atrophy. In this work, we designed and developed a 3D-printed cast to be used for the study of disuse skeletal muscle atrophy. Applying two halves of the cast is non-invasive, producing little to no swelling or skin damage. The application of the cast induces, in 2-weeks immobilized leg, the activation of atrophy-related genes, causing a muscle weight loss up to 25% in the gastrocnemius muscle, and 31% in the soleus muscle of the immobilized leg compared to the control leg. The cross-sectional area of the fibers is decreased by 31% and 34% respectively, with a peculiar effect on fiber types. In the immobilized gastrocnemius, absolute muscle force is reduced by 38%, while normalized force is reduced by 16%. The contralateral leg did not show signs of overload or hypertrophy when compared to free roaming littermates, offering a good internal control over the immobilized limb. Upon removing the cast, the mice effectively recovered mass and force in 3 weeks
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