Proteomic analysis of rat soleus muscle undergoing hindlimb suspension-induced atrophy and reweighting hypertrophy

A proteomic analysis was performed comparing normal rat soleus muscle to soleus muscle that had undergone either 0.5, 1, 2, 4, 7, 10 and 14 days of hindlimb suspension-induced atrophy or hindlimb suspension-induced atrophied soleus muscle that had undergone 1 hour, 8 hour, 1 day, 2 day, 4 day and 7 days of reweighting-induced hypertrophy. Muscle mass measurements demonstrated continual loss of soleus mass occurred throughout the 21 days of hindlimb suspension; following reweighting, atrophied soleus muscle mass increased dramatically between 8 hours and 1 day post reweighting. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 29 soleus proteins. Reweighting following atrophy demonstrated statistically significant changes in the relative levels of 15 soleus proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both atrophied and hypertrophied soleus muscle. Five differentially regulated proteins from the hindlimb suspended atrophied soleus muscle were identified while five proteins were identified in the reweighting-induced hypertrophied soleus muscles. The identified proteins could be generally grouped together as metabolic proteins, chaperone proteins and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the skeletal muscle proteome occur during disuse-induced soleus muscle atrophy and reweighting hypertrophy.     

Erythroid expression of the human alpha-spectrin gene promoter is mediated by GATA-1- and NF-E2-binding proteins

alpha-Spectrin is a highly expressed membrane protein critical for the flexibility and stability of the erythrocyte. Qualitative and quantitative defects of alpha-spectrin are present in the erythrocytes of many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. We wished to determine the regulatory elements that determine the erythroid-specific expression of the alpha-spectrin gene. We mapped the 5' end of the alpha-spectrin erythroid cDNA and cloned the 5' flanking genomic DNA containing the putative alpha-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, an alpha-spectrin gene erythroid promoter with binding sites for GATA-1- and NF-E2-related proteins was identified. Both binding sites were required for full promoter activity. In transgenic mice, a reporter gene directed by the alpha-spectrin promoter was expressed in yolk sac, fetal liver, and erythroid cells of bone marrow but not adult reticulocytes. No expression of the reporter gene was detected in nonerythroid tissues. We conclude that this alpha-spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells.     

Control of Ser2448 phosphorylation in the mammalian target of rapamycin by insulin and skeletal muscle load

We have investigated the effects of insulin, amino acids, and the degree of muscle loading on the phosphorylation of Ser(2448), a site in the mammalian target of rapamycin (mTOR) phosphorylated by protein kinase B (PKB) in vitro. Phosphorylation was assessed by immunoblotting with a phosphospecific antibody (anti-Ser(P)(2448)) and with mTAb1, an activating antibody whose binding is inhibited by phosphorylation in the region of mTOR that contains Ser(2448). Incubating rat diaphragm muscles with insulin increased Ser(2448) phosphorylation but did not change the total amount of mTOR. Insulin, but not amino acids, activated PKB, as evidenced by increased phosphorylation of both Ser(308) and Thr(473) in the kinase. Ser(2448) phosphorylation was also modulated by muscle-loading. Overloading the rat plantaris muscle by synergist muscle ablation, which promotes hypertrophy of the plantaris muscle, increased Ser(2448) phosphorylation. In contrast, unloading the gastrocnemius muscle by hindlimb suspension, which promotes atrophy of the muscle, decreased Ser(2448) phosphorylation, an effect that was fully reversible. Neither overloading nor hindlimb suspension significantly changed the total amount of mTOR. In summary, our results demonstrate that atrophy and hypertrophy of skeletal muscle are associated with decreases and increases in Ser(2448) phosphorylation, suggesting that modulation of this site may have an important role in the control of protein synthesis.     

Elasmobranch immune cells as a source of novel tumor cell inhibitors: Implications for public health

Reports that elasmobranchs (sharks, skates, and rays) may havea low incidence of disease have stimulated interest in understandingthe role of their immune system in this apparent resistance.Although research in this area may potentially translate intoapplications for human health, a basic understanding of theelasmobranch immune system components and how they functionis essential. As in higher vertebrates, elasmobranch fishespossess thymus and spleen, but in the absence of bone marrowand lymph nodes, these fish have evolved unique lymphomyeloidtissues, namely epigonal and Leydig organs. As conditions forshort-term culture of elasmobranch immune cells have becomebetter understood, the opportunity to examine functional activityof cytokine-like factors derived from conditioned culture mediumhas resulted in the identification of growth inhibitory activityagainst a variety of tumor cell lines. Specifically, the mediumenriched by short term culture of bonnethead shark (Sphyrnatiburo) epigonal cells (epigonal conditioned medium, ECM) hasbeen shown to inhibit the growth of mammalian tumor cell lines,including fibrosarcoma (WEHI-164), melanoma (A375.S2), B-celllymphoma (Daudi), T-cell leukemia (Jurkat), pancreatic cancer(PANC-1), ovarian cancer (NIH:OVCAR-3), and three breast carcinomacell lines (MCF7, HCC38, Hs578T). Of the cell lines tested,WEHI-164, A375.S2, Daudi, and Jurkat cells were among the mostsensitive to growth inhibitory activity of ECM whereas PANC-1and NIH:OVCAR-3 cells were among the least sensitive. In addition,ECM demonstrated preferential growth inhibition of malignantcells in assays against two different malignant/non-malignantcell line pairs (HCC38/HCC38 BL and Hs 578T/Hs 578Bst). Separationof protein components of ECM using SDS-PAGE resulted in a veryreproducible pattern of three major bands corresponding to molecularsizes of approximately 40–42 kD, 24 kD, and 17 kD. Activityis lost after heating at 75°C for 30 min, and can be diminishedby treatment with proteinase K and protease. Activity is notaffected by treating with trypsin, DNase I or RNase A.     

The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice

Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults.     

Evidence that pH induced activation of the rat hepatic glucocorticoid-receptor complex is irreversible

The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15 degrees C, 30 min), and then returning it to pH 6.5 for a second incubation (15 degrees C, 30 min). Activation was quantitated by measuring the binding of [3H]triamcinolone acetonide [( 3H]TA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [3H]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [3H]TA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and DEAE-cellulose chromatography have been used to study pH induced activation of the glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible.