miRNAs as serum biomarkers for Duchenne muscular dystrophy

Dystrophin absence in Duchenne muscular dystrophy (DMD) causes severe muscle degeneration. We describe that, as consequence of fibre damage, specific muscle-miRNAs are released in to the bloodstream of DMD patients and their levels correlate with the severity of the disease. The same miRNAs are abundant also in the blood of mdx mice and recover to wild-type levels in animals ‘cured’ through exon skipping. Even though creatine kinase (CK) blood levels have been utilized as diagnostic markers of several neuromuscular diseases, including DMD, we demonstrate that they correlate less well with the disease severity. Although the analysis of a larger number of patients should allow to obtain more refined correlations with the different stages of disease progression, we propose that miR-1, miR-133, and miR-206 are new and valuable biomarkers for the diagnosis of DMD and possibly also for monitoring the outcomes of therapeutic interventions in humans. Despite many different DMD therapeutic approaches are now entering clinical trials, a unifying method for assessing the benefit of different treatments is still lacking

Anions and the contraction of glycerol-extracted muscle fibers

Glycerinated rabbit psoas fibers 5-19 days old were immersed in contracting solutions with and without varying concentrations of different sodium salts. Raising the pH of the solution in small steps identified a threshold pH above which the fibers rapidly developed maximum tension. In solutions with added chloride or acetate (0-0.15 ), threshold pH and maximum tension changed only slightly. In solutions with added nitrate, bromide, or iodide, fibers developed much less tension, according to the series: CH3COO- > Cl- > NO3- > Br- > I-. Addition of excess calcium to the last solutions produced no further tension. After 15 min in NO3-, Br-, or I- solution, fibers partially regained tension when reimmersed in acetate but not chloride solution. Fibers that had developed tension in chloride solution lost some of it when reimmersed in nitrate, bromide, or iodide solution, according to the same anion series, but they retained more tension than they could develop initially in the latter solution.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32830/1/0000205.pd

Tropomyosin-mediated Regulation of Cytoplasmic Myosins

The ability of the actin-based cytoskeleton to rapidly reorganize is critical for maintaining cell organization and viability. The plethora of activities in which actin polymers participate require different biophysical properties, which can vary significantly between the different events that often occur simultaneously at separate cellular locations. In order to modify the biophysical properties of an actin polymer for a particular function, the cell contains diverse actin-binding proteins that modulate the growth, regulation and molecular interactions of actin-based structures according to functional requirements. In metazoan and yeast cells, tropomyosin is a key regulator of actin-based structures. Cells have the capacity to produce multiple tropomyosin isoforms, each capable of specifically associating as copolymers with actin at distinct cellular locations to fine-tune the functional properties of discrete actin structures. Here, we present a unifying theory in which tropomyosin isoforms critically define the surface landscape of copolymers with cytoplasmic ?- or ?-actin. Decoration of filamentous actin with different tropomyosin isoforms determines the identity and modulates the activity of the interacting myosin motor proteins. Conversely, changes in the nucleotide state of actin and posttranslational modifications affect the composition, morphology, subcellular localization and allosteric coupling of the associated actin-based superstructures

Temperature-pressure studies on magnesium-activated adenosinetriphosphatase from skeletal muscle

The effects of pressure, temperature, and pH on the activity of purified rabbit skeletal muscle magnesium-activated ATPase were measured. The enzyme is rapidly and irreversibly inactivated by both temperature and pressure and shows no evidence of reversible denaturation. The extent of irreversible inactivation depends on the amount of pressure applied and is largest at the optimum pH of the enzyme.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32346/1/0000416.pd

Analysis of biophysical and functional consequences of Tropomyosin - fluorescent protein fusions.

The dynamic nature of actin polymers is modulated to facilitate a diverse range of cellular processes. These dynamic properties are modulated by different isoforms of Tropomyosin, which are recruited to distinct subpopulations of actin polymers to differentially modulate their functional properties. This makes them an attractive target for labelling discrete actin populations. We have assessed the effect of different fluorescent labelling strategies for this protein. Although tropomyosin fluorescent fusions decorate actin in vivo, they are either non-functional or perturb regulation of actin nucleation and cell cycle timings. Thus conclusions and physiological relevance should be carefully evaluated when using tropomyosin fusions

Regulation of phosphorylase kinase by low concentrations of Ca ions upon muscle contraction: the connection between metabolism and muscle contraction and the connection between muscle physiology and Ca-dependent signal transduction

It had long been one of the crucial questions in muscle physiology how glycogenolysis is regulated in connection with muscle contraction, when we found the answer to this question in the last half of the 1960s. By that time, the two principal currents of muscle physiology, namely, the metabolic flow starting from glycogen and the mechanisms of muscle contraction, had already been clarified at the molecular level thanks to our senior researchers. Thus, the final question we had to answer was how to connect these two currents. We found that low concentrations of Ca ions (10−7–10−4 M) released from the sarcoplasmic reticulum for the regulation of muscle contraction simultaneously reversibly activate phosphorylase kinase, the enzyme regulating glycogenolysis. Moreover, we found that adenosine 3′,5′-monophosphate (cyclic AMP), which is already known to activate muscle phosphorylase kinase, is not effective in the absence of such concentrations of Ca ions. Thus, cyclic AMP is not effective by itself alone and only modifies the activation process in the presence of Ca ions (at that time, cyclic AMP-dependent protein kinase had not yet been identified). After a while, it turned out that our works have not only provided the solution to the above problem on muscle physiology, but have also been considered as the first report of Ca-dependent protein phosphorylation, which is one of the central problems in current cell biology. Phosphorylase kinase is the first protein kinase to phosphorylate a protein resulting in the change in the function of the phosphorylated protein, as shown by Krebs and Fischer. Our works further showed that this protein kinase is regulated in a Ca-dependent manner. Accordingly, our works introduced the concept of low concentrations of Ca ions, which were first identified as the regulatory substance of muscle contraction, to the vast field of Ca biology including signal transduction