Changes in Achilles Tendon Thickness Following a 10 – Week Heavy Load Eccentric Exercise Program

Achilles tendinopathy (AT) is a common overuse injury in running or jumping activities where the tendon is unable to properly respond to the load. AT increases tendon thickness while decreasing stiffness and function (1). Studies have shown that tendon properties can be positively influenced by load (5,6). Therefore, AT has typically been treated conservatively through physical therapy, with eccentric calf strengthening exercises as the focus of the treatment (2)

The geometry and efficacy of cation–π interactions in a diagonal position of a designed β-hairpin

Cation–π interactions are common in proteins, but their contribution to the stability and specificity of protein structure has not been well established. In this study, we examined the impact of cation–π interactions in a diagonal position of a β-hairpin peptide through comparison of the interaction of Phe or Trp with Lys or Arg. The diagonal interactions ranged from −0.20 to −0.48 kcal/mole. Our experimental values for the diagonal cation–π interactions are similar to those found in α-helical studies. Upfield shifting of the Lys and Arg side chains indicates that the geometries of cation–π interactions adopted in the 12-residue β-hairpin are comparable to those found in protein structures. The Lys was found to interact through the polarized Cɛ, and the Arg is stacked against the aromatic ring of Phe or Trp. Folding of these peptides was found to be enthalpically favorable (ΔH° ∼ −3 kcal/mole) and entropically unfavorable (ΔS°∼ −8 cal mole−1 K−1)

Injection of wild-type cytoplasm and poly(A)+ RNA provokes phenotype rescue in spätzle mutant Drosophila embryos

spätzle (spz), a maternal effect gene of Drosophila, is involved in the establishment of the dorso-ventral axis during embryogenesis. Eggs from females lacking the spz gene product develop into completely dorsalized embryos, i.e. the ventral and lateral pattern elements fail to develop. Upon injection of either cytoplasm or poly(A)+ RNA from early wild-type embryos, spz embryos develop lateral pattern elements represented by Filzkörper and in the case of injected cytoplasm additional ventral pattern elements represented by ventral setae. Wild-type cytoplasm retains the rescuing activity longer than the poly(A)+ RNA fraction does, and cytoplasm is always more effective in provoking the rescue than poly(A)+ RNA. Mosaic females containing spz germ cells surrounded by spz + tissues were generated by pole cell transplantations; a mutant genotype in the germ cells is sufficient to produce all aspects of the spz mutant phenotype, suggesting that the maternal source of spz gene product is the germ line

Identification of a p53-based portable degron based on the MDM2-p53 binding region

In recent years the ubiquitin proteasome system (UPS) has garnered increasing interest as a target for chemotherapeutics. Due to the success of the proteasome inhibitors Bortezomib and Carfilzomib in the treatment of multiple myeloma, several new compounds have been developed to target E3 ubiquitin ligases and the proteasome in numerous human cancers. This has increased the need for new analytical methods to precisely measure intracellular enzyme activity in cells. A key component of a desired analytical method is a substrate that is capable of rapid intracellular ubiquitination yet easily incorporated into the next generation of more sophisticated UPS reporters. Portable degradation sequences, or degrons, have the ability to bind to E3 ligases and promote substrate ubiquitination when the sequence is presented in isolation or appended to other entities such as fluorescent peptide-based reporters. Previous work identified an E3 ligase (MDM2)-binding element at p53 amino acids 92-112, which was later demonstrated to be rapidly ubiquitinated in cytosolic lysates effectively functioning as a transportable degron. In this work, a shortened p53 sequence within amino acids 92-112 that displayed rapid ubiquitination kinetics was identified. A nine-member peptide library was synthesized using sequence elements of various sizes and lengths, all based on the initial 22 amino acid long sequence, containing a single ubiquitination site lysine. The ubiquitination kinetics were determined using a combination of gel electrophoresis and analytical high performance liquid chromatography (HPLC) to rank the members of the library and identify the optimal ubiquitination sequence. This analysis identified the five amino acid sequence, KGSYG, corresponding to residues 105-108 with an added N-terminal lysine, as a portable degron since this sequence demonstrated the most rapid ubiquitination kinetics

A Comparative Analysis of the Ubiquitination Kinetics of Multiple Degrons to Identify an Ideal Targeting Sequence for a Proteasome Reporter

<div><p>The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins. The conjugation of a polyubiquitin chain, or polyubiquitination, to a target protein requires an increasingly diverse cascade of enzymes culminating with the E3 ubiquitin ligases. Protein recognition by an E3 ligase occurs through a specific sequence of amino acids, termed a degradation sequence or degron. Recently, degrons have been incorporated into novel reporters to monitor proteasome activity; however only a limited few degrons have successfully been incorporated into such reporters. The goal of this work was to evaluate the ubiquitination kinetics of a small library of portable degrons that could eventually be incorporated into novel single cell reporters to assess proteasome activity. After an intensive literary search, eight degrons were identified from proteins recognized by a variety of E3 ubiquitin ligases and incorporated into a four component degron-based substrate to comparatively calculate ubiquitination kinetics. The mechanism of placement of multiple ubiquitins on the different degron-based substrates was assessed by comparing the data to computational models incorporating first order reaction kinetics using either multi-monoubiquitination or polyubiquitination of the degron-based substrates. A subset of three degrons was further characterized to determine the importance of the location and proximity of the ubiquitination site lysine with respect to the degron. Ultimately, this work identified three candidate portable degrons that exhibit a higher rate of ubiquitination compared to peptidase-dependent degradation, a desired trait for a proteasomal targeting motif.</p></div