Mice and cattle with genetic deficiencies in myostatin exhibit dramatic increases in skeletal muscle mass, suggesting that myostatin normally suppresses muscle growth. Whether this increased muscling results from prenatal or postnatal lack of myostatin activity is unknown. Here we show that myostatin circulates in the blood of adult mice in a latent form that can be activated by acid treatment. Systemic overexpression of myostatin in adult mice was found to induce profound muscle and fat loss analogous to that seen in human cachexia syndromes. These data indicate that myostatin acts systemically in adult animals and may be a useful pharmacologic target in clinical settings such as cachexia, where muscle growth is desired.

In this review we examine techniques, software, and statistical analyses used in label-free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labelling techniques, and forward-looking applications for label-free quantitative data are presented. We conclude that label-free quantitative proteomics is a reliable, versatile, and cost-effective alternative to labelled quantitation.

The majority of disulfide-linked cytosolic proteins are thought to be enzymes that transiently form disulfide bonds while catalyzing oxidation-reduction (redox) processes. Recent evidence indicates that reactive oxygen species can act as signaling molecules by promoting the formation of disulfide bonds within or between select redox-sensitive proteins. However, few studies have attempted to examine global changes in disulfide bond formation following reactive oxygen species exposure. Here we isolate and identify disulfide-bonded proteins (DSBP) in a mammalian neuronal cell line (HT22) exposed to various oxidative insults by sequential nonreducing/reducing two-dimensional SDS-PAGE combined with mass spectrometry. By using this strategy, several known cytosolic DSBP, such as peroxiredoxins, thioredoxin reductase, nucleoside-diphosphate kinase, and ribonucleotide-diphosphate reductase, were identified. Unexpectedly, a large number of previously unknown DSBP were also found, including those involved in molecular chaperoning, translation, glycolysis, cytoskeletal structure, cell growth, and signal transduction. Treatment of cells with a wide range of hydrogen peroxide concentrations either promoted or inhibited disulfide bonding of select DSBP in a concentration-dependent manner. Decreasing the ratio of reduced to oxidized glutathione also promoted select disulfide bond formation within proteins from cytoplasmic extracts. In addition, an epitope-tagged version of the molecular chaperone HSP70 forms mixed disulfides with both ␤4-spectrin and adenomatous polyposis coli protein in the cytosol. Our findings indicate that disulfide bond formation within families of cytoplasmic proteins is dependent on the nature of the oxidative insult and may provide a common mechanism used to control multiple physiological processes.Oxidative stress occurs when the rate of reactive oxygen species (ROS) 1 generation exceeds the detoxification abilities of the cell, and it has been implicated in many degenerative diseases. It is frequently argued that ROS cause relatively nonspecific damage to vital cellular components such as lipids, DNA, and proteins. However, emerging evidence indicates that ROS can cause specific protein modifications that may lead to a change in the activity or function of the oxidized protein (1, 2). Several major forms of oxidative modifications can occur on amino acid residue side chains including carbonylation, nitrosylation, and oxidation of methionine to methionine sulfoxide (3). Protein sulfhydryls can be oxidized to protein disulfides and sulfenic acids as well as more highly oxidized states such as the sulfinic and sulfonic acid forms of protein cysteines (4). Under non-stressed conditions, disulfide bond formation occurs primarily in the oxidizing environment of the endoplasmic reticulum (ER) in eukaryotic cells (5). The sulfhydryl groups in the vast majority of protein cysteine residues (Cys-SH) have a pK a Ͼ8.0 and, in the reducing environment of the cytoplasm, remain protonated at physiological pH. Thus,...

Abstract-Bovine aortic smooth muscle cell (BASMC) cultures undergo mineralization on addition of the organic phosphate donor, ␤-glycerophosphate (␤GP). Mineralization is characterized by apatite deposition on collagen fibrils and the presence of matrix vesicles, as has been described in calcified vascular lesions in vivo as well as in bone and teeth. In the present study, we used this model to investigate the molecular mechanisms driving vascular calcification. We found that BASMCs lost their lineage markers, SM22␣ and smooth muscle ␣-actin, within 10 days of being placed under calcifying conditions. Conversely, the cells gained an osteogenic phenotype as indicated by an increase in expression and DNA-binding activity of the transcription factor, core binding factor ␣1 (Cbfa1). Moreover, genes containing the Cbfa1 binding site, OSE2, including osteopontin, osteocalcin, and alkaline phosphatase were elevated. The relevance of these in vitro findings to vascular calcification in vivo was further studied in matrix GLA protein null (MGP Ϫ/Ϫ ) mice whose arteries spontaneously calcify. We found that arterial calcification was associated with a similar loss in smooth muscle markers and a gain of osteopontin and Cbfa1 expression. These data demonstrate a novel association of vascular calcification with smooth muscle cell phenotypic transition, in which several osteogenic proteins including osteopontin, osteocalcin, and the bone determining factor Cbfa1 are gained. The findings suggest a positive role for SMCs in promoting vascular calcification.