Identification of the Acetylation and Ubiquitin-Modified Proteome during the Progression of Skeletal Muscle Atrophy

Skeletal muscle atrophy is a consequence of several physiological and pathophysiological conditions including muscle disuse, aging and diseases such as cancer and heart failure. In each of these conditions, the predominant mechanism contributing to the loss of skeletal muscle mass is increased protein turnover. Two important mechanisms which regulate protein stability and degradation are lysine acetylation and ubiquitination, respectively. However our understanding of the skeletal muscle proteins regulated through acetylation and ubiquitination during muscle atrophy is limited. Therefore, the purpose of the current study was to conduct an unbiased assessment of the acetylation and ubiquitin-modified proteome in skeletal muscle during a physiological condition of muscle atrophy. To induce progressive, physiologically relevant, muscle atrophy, rats were cast immobilized for 0, 2, 4 or 6 days and muscles harvested. Acetylated and ubiquitinated peptides were identified via a peptide IP proteomic approach using an anti-acetyl lysine antibody or a ubiquitin remnant motif antibody followed by mass spectrometry. In control skeletal muscle we identified and mapped the acetylation of 1,326 lysine residues to 425 different proteins and the ubiquitination of 4,948 lysine residues to 1,131 different proteins. Of these proteins 43, 47 and 50 proteins were differentially acetylated and 183, 227 and 172 were differentially ubiquitinated following 2, 4 and 6 days of disuse, respectively. Bioinformatics analysis identified contractile proteins as being enriched among proteins decreased in acetylation and increased in ubiquitination, whereas histone proteins were enriched among proteins increased in acetylation and decreased in ubiquitination. These findings provide the first proteome-wide identification of skeletal muscle proteins exhibiting changes in lysine acetylation and ubiquitination during any atrophy condition, and provide a basis for future mechanistic studies into how the acetylation and ubiquitination status of these identified proteins regulates the muscle atrophy phenotype.     

PTMScan direct: identification and quantification of peptides from critical signaling proteins by immunoaffinity enrichment coupled with LC-MS/MS

Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in signaling in a wide array of experimental systems, combining the specificity of traditional biochemical methods with the high number of data points and dynamic range of proteomic methods.     

Sexual segregation in ungulates: individual behaviour and the missing link

Previous "explanations" of sexual segregation in ungulates establish no more than a prerequisite for habitat segregation because they do not include a model of competitive habitat selection. Here we provide one based on the ideal free distributions of mutually competing, optimally foraging, individual deer. We parameterised our model using field data collected from a population of fallow deer (Dama dama) in a Mediterranean forest. The predictions of the inter-sex competition model were in full agreement with observational data, but those of single sex distributions (conventional theory) were not. The "conventional" hypothesis, that segregation arises simply from sex differences, predicted no more than moderate (20–40%) levels of segregation, even in optimal conditions. By introducing inter-sex resource competition, the predicted segregation can generally more than double and full segregation becomes possible in some circumstances. The modelling showed segregation to be density-dependent, varying in complicated ways with season and animal density. Sensitivity analysis showed the vulnerability of the "conventional" understanding of environmental variation and uncertainty. Using our competition model we show that as diet difference increases, direct competition between the sexes declines, so that as males increasingly differ from females, segregation declines and the two sexes are more likely to be found mixed (as long as the chosen food is available to both in the same area). Conversely, small differences among male and female deer are amplified by both food depletion and inter-sex competition to give substantial levels of segregation. The theoretical framework on which our model is built strongly suggests that sexual dimorphism in the context of scramble competition may be the fundamental cause of sexual habitat-segregation among ungulates.     

Cimiracemates A-D,phenylpropanoid esters from the rhizomes of Cimicifuga racemosa

Four phenylpropanoid esters, cimiracemates A-D (1-4), along with three known compounds, isoferulic acid, ferulic acid and methyl caffeate were isolated from the EtOAc fraction of the rhizome of Cimicifuga racemosa. The structures of the esters were elucidated by means of spectral data, including 2D NMR spectroscopy.     

Constituents of the stems of Macrococculus pomiferus and their inhibitory activities against cyclooxygenases-1 and -2

As part of our program directed towards the discovery of new cancer chemopreventive agents from plants, the EtOAc-soluble extract of the stems of M. pomiferus was found to inhibit the enzyme cyclooxygenase-2 (COX-2). Bioassay-directed fractionation of this extract led to the isolation of two dibenzylbutyrolactone lignans, (8R,8'R)-3'-O-demethyl-5-hydroxymatairesinol (1) and (8R,8'R)-3'-O-demethyl-5-methoxymatairesinol (2), as well as seven known compounds, (-)-5'-methoxyyatein (3), blumenol A, (-)-deoxypodophyllotoxin (anthricin), (-)-deoxypodorhizone, 2,6-dimethoxyhydroquinone, 4-hydroxybenzaldehyde, and beta-sitosterol glucoside. The structures of compounds 1 and 2 were determined using spectroscopic data (1D and 2D NMR, and HREIMS), and the 8R and 8'R absolute stereochemistry was established for both 1 and 2 on the basis of their CD spectra. All isolates obtained in the present study were evaluated for their inhibitory effects with both COX-1 and -2. Of these, only 5'-methoxyyatein (3) showed weak activity against COX-2, while all other compounds isolated were inactive. The COX-2 inhibitory activity of the EtOAc extract was also traced to the presence of several common fatty acids by LC-MS.     

Activity-guided isolation of constituents of Cerbera manghas with antiproliferative and antiestrogenic activities

Two new cardenolides, (-)-14-hydroxy-3beta-(3-O-methyl-6-deoxy-alpha-L-rhamnosyl)-11a lpha, 12alpha-epoxy-(5beta,14beta,17betaH)-card-20 (22)-enolide (1), (-)-14-hydroxy-3beta-(3-O-methyl-6-deoxy-alpha-L-glucopyranosyl)-11al pha,12alpha-epoxy-(5beta,14beta,17betaH)-card -20(22)-enolide (2), and a known cardenolide, (-)-17beta-neriifolin (3), were isolated from the roots of Cerbera manghas as antiproliferative and antiestrogenic principles when evaluated against a human colon cancer cell line (Col2) and the Ishikawa cell line, respectively. Two known lignans, (-)-olivil (4) and (-)-cycloolivil (5), were also isolated but were inactive in the assay systems used.     

Inhibition of insect juvenile hormone synthesis by phorbol 12-myristate 13-acetate

The synthesis of insect juvenile hormone III (JH III) by isolated corpora allata of the cockroach Diploptera punctata incubated in vitro is inhibited by phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate and 1-oleyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate and diolein are inactive. The inhibitory effect of phorbol 12-myristate 13-acetate is fully reversed by 2E,6E-farnesol or by 2E,6E-farnesoic acid. It is highest in corpora allata that are past their peak in secretory activity or that have been inhibited by injections of 20-hydroxyecdysone. This effect of phorbol esters implicates protein kinase C in the regulation of insect corpus allatum activity.     

Seedling survivorship,growth, and response to disturbance in Belizean mangal

Species zonation patterns across tidal gradients in mangrove forests are formed by successful seedling establishment and maintained by replacement of adults by conspecific seedlings. These two processes rarely have been examined experimentally in neotropical mangal. We studied survivorship and growth of seedlings of two species of mangrove, Rhizophora mangle L. and Avicennia germinans (L.) Steam, across a tidal gradient in Belize, Central America. Propagules of each species were planted in common gardens at tidal elevations corresponding to lowest low water (LLW), mean water (MW), and highest high water (HHW). Sixty-nine percent of Rhizophora seedlings planted at MW and 56% of those planted at LLW survived 1 year. Forty-seven percent of MW Avicennia seedlings also survived 1 year. No individuals of either species survived at HHW, and neither did any LLW Avicennia seedlings. Among the surviving Rhizophora seedlings, LLW seedlings grew more rapidly in terms of height, diameter, leaf production, and biomass than did MW seedlings. Insect herbivory was twice as high on MW seedlings as on LLW Rhizophora seedlings. We also examined the response of established Rhizophora seedlings to experimental removal of the adult Rhizophora canopy. Seedlings in canopy removal areas had higher survivorship, grew twice as fast, produced more leaves, and had less than half the herbivory of seedlings growing beneath an intact canopy. These results provide insights into underlying causes and maintenance of zonation in Caribbean mangrove forests.     

Sun-shade adaptability of the red mangrove,Rhizophora mangle (Rhizophoraceae): Changes through ontogeny at several levels of biological organization

Rhizophora mangle L., the predominant neotropical mangrove species, occupies a gradient from low intertidal swamp margins with high insolation, to shaded sites at highest high water. Across a light gradient, R. mangle shows properties of both “light-demanding” and “shade-tolerant” species, and defies designation according to existing successional paradigms for rain forest trees. The mode and magnitude of its adaptability to light also change through ontogeny as it grows into the canopy. We characterized and compared phenotypic flexibility of R. mangle seedlings, saplings, and tree modules across changing light environments, from the level of leaf anatomy and photosynthesis, through stem and whole-plant architecture. We also examined growth and mortality differences among sun and shade populations of seedlings over 3 yr. Sun and shade seedling populations diverged in terms of four of six leaf anatomy traits (relative thickness of tissue layers and stomatal density), as well as leaf size and shape, specific leaf area (SLA), leaf internode distances, disparity in blade–petiole angles, canopy spread: height ratios, standing leaf numbers, summer (July) photosynthetic light curve shapes, and growth rates. Saplings showed significant sun/shade differences in fewer characters: leaf thickness, SLA, leaf overlap, disparity in bladepetiole angles, standing leaf numbers, stem volume and branching angle (first-order branches only), and summer photosynthesis. In trees, leaf anatomy was insensitive to light environment, but leaf length, width, and SLA, disparities in bladepetiole angles, and summer maximal photosynthetic rates varied among sun and shade leaf populations. Seedling and sapling photosynthetic rates were significantly depressed in winter (December), while photosynthetic rates in tree leaves did not differ in winter and summer. Seasonal and ontogenetic changes in response to light environment are apparent at several levels of biological organization in R. mangle, within constraints of its architectural baiiplan. Such variation has implications for models of stand carbon gain, and suggest that response flexibility may change with plant age.