Proteomic studies of PP2A-B56gamma1 phosphatase complexes reveal phosphorylation-regulated partners in cardiac local signaling

Defects of kinase-phosphatase signaling in cardiac myocytes contribute to human heart disease. The activity of one phosphatase, PP2A, is governed by B targeting subunits, including B56gamma1, expressed in heart cells. As the role of PP2A/B56gamma1 on the heart function remains largely unknown, this study sought to identify protein partners through unbiased, affinity purification-based proteomics combined with the functional validation. The results reveal multiple interactors that are localized in strategic cardiac sites to participate in Ca2+ homeostasis and gene expression, exemplified by the Ca pump, SERCA2a, and the splicing factor ASF/SF2. These results are corroborated by confocal imaging where adenovirally overexpressed B56gamma1 is found in z-line/t-tubule region and nuclear speckles. Importantly, overexpression of B56gamma1 in cultured myocytes dramatically impairs cell contractility. These results provide a global view of B56gamma1-regulated local signaling and heart function.     

Use of dietary diversity score as a proxy indicator of nutrient adequacy of rural elderly people in Sri Lanka

ABSTRACT: BACKGROUND: Macro and micro nutrient deficiencies are public health concerns in most developing countries including Sri Lanka, partly due to monotonous, cereal-based diet that lacks diversity. The objective of the study was to assess validity of food variety score (FVS), dietary diversity score (DDS) and dietary serving score (DSS) as indicators of nutrient adequacy of rural elderly people in Sri Lanka. FINDINGS: A sample of 200 apparently healthy elderly people >60y of age were studied. A single 24 h recall was performed to compute dietary diversity indicators. Pearson's correlation was used to assess the utility of FVS, DDS and DSS as indicators of nutrient adequacy. Sensitivity (Se) and specificity (Spe) analysis were done to determine the most appropriate cut-off points for using FVS and DDS to categorize elderly people with adequate nutrient intake. The average (standard deviation) of the food variety score, dietary diversity score and dietary serving score was 8.4 (2), 4.4 (0.9) and 11.4 (2.5), respectively. Mean adequacy ratio (MAR) of 12 nutrients was 0.39 (39 %). Pearson's correlation coefficients between MAR and FVS was 0.45 (P < 0.01), for DDS it was 0.48 (P < 0.01) and for DSS it was 0.58 (P < 0.01). When maximizing sensitivity and specificity, the best cut-off point for achieving 50 % of MAR was about 9 and 4.5 for FVS and DDS, respectively. CONCLUSION: In conclusion, FVS, DDS and DSS were useful proxy indicators of nutrient adequacy of rural elderly people in Sri Lanka. Indeed, the performance of the indicators is improved when considering the quantities of food consumed.     

Molecular basis for PP2A regulatory subunit B56alpha targeting in cardiomyocytes

Protein phosphatase 2A (PP2A) is a multifunctional protein phosphatase with critical roles in excitable cell signaling. In the heart, PP2A function is linked with modulation of beta-adrenergic signaling and has been suggested to regulate key ion channels and transporters including Na/Ca exchanger, ryanodine receptor, inositol 1,4,5-trisphosphate receptor, and Na/K ATPase. Although many of the functional roles and molecular targets for PP2A in heart are known, little is established regarding the cellular pathways that localize specific PP2A isoform activities to subcellular sites. We report that the PP2A regulatory subunit B56alpha is an in vivo binding partner for ankyrin-B, an adapter protein required for normal subcellular localization of the Na/Ca exchanger, Na/K ATPase, and inositol 1,4,5-trisphosphate receptor. Ankyrin-B and B56alpha are colocalized and coimmunoprecipitate in primary cardiomyocytes. Using multiple strategies, we identified the structural requirements on B56alpha for ankyrin-B association as a 13 residue motif in the B56alpha COOH terminus not present in other B56 family polypeptides. Finally, we report that reduced ankyrin-B expression in primary ankyrin-B(+/-) cardiomyocytes results in disorganized distribution of B56alpha that can be rescued by exogenous expression of ankyrin-B. These new data implicate ankyrin-B as a critical targeting component for PP2A in heart and identify a new class of signaling proteins targeted by ankyrin polypeptides.     

A randomized,double-blind,placebo-controlled,cross-over pilot study to investigate the efficacy of Rest-ZZZ formula in healthy participants with occasional sleeplessness

Sleep and Biological Rhythms - The purpose of this study was&nbsp;to investigate the safety and efficacy of Rest-ZZZ, a natural sleep supplement, in healthy adults without a diagnosed sleep...     

Identification of Lewis x structures of the cell adhesion molecule CEACAM1 from human granulocytes

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed on epithelia, blood vessel endothelia, and leukocytes. A variety of physiological functions have been assigned to CEACAM1. It is involved in the formation of glands and blood vessels, in immune reactions, and in the regulation of tumor growth. As a homophilic and heterophilic adhesion receptor, it signals through different cellular pathways. The existence of special oligosaccharide structures such as Lewis x or sialyl-Lewis x glycans within this highly glycosylated protein has been postulated, but chemical proof is missing so far. Because such structures are known to be essential for different cell-cell recognition and adhesion processes, characterizing the CEACAM1 glycan structure is of pivotal importance in revealing the biological function of CEACAM1. We examine the terminal glycosylation pattern of CEACAM1 from human granulocytes, focusing on Lewis x epitopes. Lewis x-specific antibodies react with immunoaffinity-purified native CEACAM1. Antibody binding was completely abolished by treatment with fucosidase III, confirming a terminal alpha(1-3,4) fucose linkage to the N-acetylglucosamine of lactosamine residues, a key feature of Lewis epitopes. To verify these data, MALDI-TOF MS analysis after stepwise exoglycosidase digestion of the CEACAM1 N-glycan mixture was performed. A complex mixture of CEACAM1-bound oligosaccharides could be characterized with an unusually high amount of fucose. The sequential digestions clearly identified several different Lewis x glycan epitopes, which may modulate the cell adhesive functions of CEACAM1.     

Pro-angiogenic signaling by the endothelial presence of CEACAM1

Here, we demonstrate the expression of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) in angiogenic sprouts but not in large mother blood vessels within tumor tissue. Correspondingly, only human microvascular endothelial cells involved in in vitro tube formation exhibit CEACAM1. CEACAM1-overexpressing versus CEACAM1-silenced human microvascular endothelial cells were used in migration and tube formation assays. CEACAM1-overexpressing microvascular endothelial cells showed prolonged survival and increased tube formation when they were stimulated with vascular endothelial growth factor (VEGF), whereas CEACAM1 silencing via small interfering RNA blocks these effects. Gene array and LightCycler analyses show an up-regulation of angiogenic factors such as VEGF, VEGF receptor 2, angiopoietin-1, angiopoietin-2, tie-2, angiogenin, and interleukin-8 but a down-regulation of collagen XVIII/endostatin and Tie-1 in CEACAM1-overexpressing microvascular endothelial cells. Western blot analyses confirm these results for VEGF and endostatin at the protein level. These results suggest that constitutive expression of CEACAM1 in microvascular endothelial cells switches them to an angiogenic phenotype, whereas CEACAM1 silencing apparently abrogates the VEGF-induced morphogenetic effects during capillary formation. Thus, strategies targeting the endothelial up-regulation of CEACAM1 might be promising for antiangiogenic tumor therapy.     

Role of GlnK in NifL-Mediated Regulation of NifA Activity in Azotobacter vinelandii

In several diazotrophic species of Proteobacteria, P(II) signal transduction proteins have been implicated in the regulation of nitrogen fixation in response to NH(4)(+) by several mechanisms. In Azotobacter vinelandii, expression of nifA, encoding the nif-specific activator, is constitutive, and thus, regulation of NifA activity by the flavoprotein NifL appears to be the primary level of nitrogen control. In vitro and genetic evidence suggests that the nitrogen response involves the P(II)-like GlnK protein and GlnD (uridylyltransferase/uridylyl-removing enzyme), which reversibly uridylylates GlnK in response to nitrogen limitation. Here, the roles of GlnK and GlnK-UMP in A. vinelandii were studied to determine whether the Nif (-) phenotype of glnD strains was due to an inability to modify GlnK, an effort previously hampered because glnK is an essential gene in this organism. A glnKY51F mutation, encoding an unuridylylatable form of the protein, was stable only in a strain in which glutamine synthetase activity is not inhibited by NH(4)(+), suggesting that GlnK-UMP is required to signal adenylyltransferase/adenylyl-removing enzyme-mediated deadenylylation. glnKY51F strains were significantly impaired for diazotrophic growth and expression of a nifH-lacZ fusion. NifL interacted with GlnK and GlnKY51F in a yeast two-hybrid system. Together, these data are consistent with those obtained from in vitro experiments (Little et al., EMBO J., 19:6041-6050, 2000) and support a model for regulation of NifA activity in which unmodified GlnK stimulates NifL inhibition and uridylylation of GlnK in response to nitrogen limitation prevents this function. This model is distinct from one proposed for the related bacterium Klebsiella pneumoniae, in which unmodified GlnK relieves NifL inhibition instead of stimulating it.