Coordinated changes in mRNA turnover, translation, and RNA processing bodies in bronchial epithelial cells following inflammatory stimulation.

Bronchial epithelial cells play a pivotal role in airway inflammation, but little is known about posttranscriptional regulation of mediator gene expression during the inflammatory response in these cells. Here, we show that activation of human bronchial epithelial BEAS-2B cells by proinflammatory cytokines interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) leads to an increase in the mRNA stability of the key chemokines monocyte chemotactic protein 1 and IL-8, an elevation of the global translation rate, an increase in the levels of several proteins critical for translation, and a reduction of microRNA-mediated translational repression. Moreover, using the BEAS-2B cell system and a mouse model, we found that RNA processing bodies (P bodies), cytoplasmic domains linked to storage and/or degradation of translationally silenced mRNAs, are significantly reduced in activated bronchial epithelial cells, suggesting a physiological role for P bodies in airway inflammation. Our study reveals an orchestrated change among posttranscriptional mechanisms, which help sustain high levels of inflammatory mediator production in bronchial epithelium during the pathogenesis of inflammatory airway diseases.

Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells.

Development of transcriptional pulsing approaches using the c-fos and Tet-off promoter systems greatly facilitated studies of mRNA turnover in mammalian cells. However, optimal protocols for these approaches vary for different cell types and/or physiological conditions, limiting their widespread application. In this study, we have further optimized transcriptional pulsing systems for different cell lines and developed new protocols to facilitate investigation of various aspects of mRNA turnover. We apply the Tet-off transcriptional pulsing strategy to investigate ARE-mediated mRNA decay in human erythroleukemic K562 cells arrested at various phases of the cell cycle by pharmacological inhibitors. This application facilitates studies of the role of mRNA stability in control of cell-cycle dependent gene expression. To advance the investigation of factors involved in mRNA turnover and its regulation, we have also incorporated recently developed transfection and siRNA reagents into the transcriptional pulsing approach. Using these protocols, siRNA and DNA plasmids can be effectively cotransfected into mouse NIH3T3 cells to obtain high knockdown efficiency. Moreover, we have established a tTA-harboring stable line using human bronchial epithelial BEAS-2B cells and applied the transcriptional pulsing approach to monitor mRNA deadenylation and decay kinetics in this cell system. This broadens the application of the transcriptional pulsing system to investigate the regulation of mRNA turnover related to allergic inflammation. Critical factors that need to be considered when employing these approaches are characterized and discussed.

Validity and reliability of a measure of Latina mother's nutritional self-efficacy

Background. Not only has obesity played a role in Texas adults but it is also becoming a large issue among low-income Latino children. In Latino children between 2-5 years of age, the Pediatric Nutrition Surveillance data in 1997 found the prevalence of obesity was 12 percent, highest among all ethnic groups. Children learn what and how to eat from their environment. Despite many mothers being working mothers they are still the principal caregivers and source of influence on their toddler's diet. Self-efficacy, a concept created by Albert Bandura, one's belief that one is capable of performing a behavior needed to reach an intended goal, is increasingly becoming important in nutrition and health education. This study is important to understand the degree of impact that a mother's self-efficacy will have on a child's diet. This is useful knowing if influencing a mother's self-efficacy could improve a child's diet to prevent certain public health issues such as obesity and diabetes. The purpose of this study was to examine nutrition self-efficacy of Latina mothers, focusing on sweets and beverage and if their self-efficacy impacted their child's diet. Methods. The data was collected during July-September 2008. Mothers were recruited from two federally qualified San Antonio health centers. In order to qualify, participants had to be Hispanic with children of toddler age. Mothers were informed of incentives available upon completion. The interview consisted of demographic info, a set of five self-efficacy questions repeated at completion, testing reliability and a 24-hour food recall diary asked of the participant's child's diet. Results. There were 225 mothers who participated between both clinics. The Crohnbach alpha scores for the two different times the self-efficacy questions were asked were .44 corresponding to the first time and .49 for the second time. The three most common beverages reported were milk, juice, and water. The mothers who met or gave their child more milk than recommended by the scientific community, 800mg of calcium/3 cups (24oz) set, had a higher self-efficacy score than those who did not meet the standard at all. Mothers who gave their children more juice than the standard recommends, 4-6oz for children 1-6 years of age, had slightly higher self-efficacy scores than mother's who simply met the standard. In general, the lower the mother's self-efficacy, the more sweets they gave their child and vice versa. Conclusion. This study's Kappa values were adequate and this research showed that Latina mothers did in fact have high self-efficacy. In general some of the children's diets did not reflect the current scientific nutrition recommendations. In order to improve self-efficacy and have an impact on children's diets, the scientific community has a responsibility to make recommendations that are easily understood and can be put into practice. The public health community needs to ensure that we encourage those we serve to be more active in their health and educate them about what constitutes good health and nutrition for both themselves and their children.^

Ligand-stimulated internalization of a Dictyostelium discoideum G protein-coupled cAMP receptor

The social amoeba, Dictyostelium discoideum, undergoes a remarkable starvation-induced program of development that transforms a population of unicellular amoebae into a fruiting body composed of resistant spores suspended on a stalk. During this development, secreted cAMP drives chemotaxis of the amoebae, leading to their aggregation, and subsequent differentiation and morphogenesis. Four sequentially expressed G protein-coupled receptors (GPCRs) for cAMP play critical roles in this process. The first of these, cAR1, is essential for aggregation as it mediates chemotaxis as well as the propagation of secreted cAMP waves throughout aggregating populations. Ligand-induced internalization has been shown to regulate a variety of GPCRs. However, little was known at the outset of this study about the role of internalization in the regulation of cAR1 function or, for that matter, in developmental systems in general. For this study, cAMP-induced cAR1 internalization was assessed by measuring (1) the reduction of cell surface binding sites for [ 3H]cAMP and (2) the redistribution of YFP-tagged receptors to the cell's interior, cAMP was found to induce little or no loss of ligand binding (LLB) in vegetative cells. However, the ability to induce LLB increased progressively over the initial 6 hrs of development, reaching ∼70% in cells undergoing aggregation. Despite these reductions in surface binding, detectable cAR1-YFP redistribution could be induced by cAMP only after the cells reached the mound stage (10 hrs) and was found to occur naturally by the ensuing slug stage (18 hrs). Site-directed substitution of a cluster of 5 serines in the receptor's cytoplasmic tail that was previously shown to be the principal site of cAMP-induced cAR1 phosphorylation impaired both LLB and receptor redistribution and furthermore resulted in mound-stage developmental arrest, suggesting that phosphorylation of cAR1 is a prerequisite for its internalization and that cAR1 internalization is required for post-aggregative development. To assess the involvement of clathrin mediated endocytosis, Dictyostelium cells lacking the clathrin light chain gene (clc-) or either of two dynamin genes were examined and found to be defective in LLB and, in the case of clc- cells, also cAR1 redistribution and turnover. Furthermore, cAR1 overexpression in clc- cells (like the serine mutant in wild-type cells) promoted developmental arrest in mounds. The mound-arrest phenotype was also recapitulated in a wild-type background by the specific expression of cAR1 in prestalk cells (but not prespore cells), suggesting that development depends critically on internalization and clearance of cAR1 from these cells. Persistent cAR1 expression following aggregation was found to be associated with aberrant expression of prestalk and prespore genes, which may adversely affect development in the prestalk cell lineage. The PI3 kinase-TORC2 signal transduction pathway, known to be important for Dictyostelium chemotaxis and internalization of yeast pheromone receptors, was examined using chemical inhibitors and null cells and found to be necessary for cAR1 internalization. In conclusion, cAR1 was shown to be similar to other GPCRs in that its internalization depends on phosphorylation of cytoplasmic domain serines, utilizes clathrin and dynamin, and involves the TORC2 complex. In addition, the findings presented here that cAR1 internalization is both developmentally regulated and required for normal development represent a novel regulatory paradigm that might pertain to other GPCRs known to play important roles in the development of humans and other metazoans. ^

Growth and development of body fat distribution from skinfold measurements: Longitudinal principal components

Longitudinal principal components analyses on a combination of four subcutaneous skinfolds (biceps, triceps, subscapular and suprailiac) were performed using data from the London Longitudinal Growth Study. The main objectives were to discover at what age during growth sex differences in body fat distribution occur and to see if there is continuity in body fatness and body fat distribution from childhood into the adult status (18 years). The analyses were done for four age sectors (3mon-3yrs, 3yrs-8yrs, 8yrs-18yrs and 3yrs-18yrs). Longitudinal principal component one (LPC1) for each age interval in both sexes represents the population mean fat curve. Component two (LPC2) is a velocity of fatness component. Component three (LPC3) in the 3mon-3yrs age sector represents infant fat wave in both sexes. In the next two age sectors component three in males represents peaks and shifts in fat growth (change in velocity), while in females it represents body fat distribution. Component four (LPC4) in the same two age sectors is a reversal in the sexes of the patterns seen for component three, i.e., in males it is body fat distribution and in females velocity shifts. Components five and above represent more complicated patterns of change (multiple increases and decreases across the age interval). In both sexes there is strong tracking in fatness from middle childhood to adolescence. In males only there is also a low to moderate tracking of infant fat with middle to late childhood fat. These data are strongly supported in the literature. Several factors are known to predict adult fatness among the most important being previous levels of fatness (at earlier ages) and the age at rebound. In addition we found that the velocity of fat change in middle childhood was highly predictive of later fatness (r $\approx -$0.7), even more so than age at rebound (r $\approx -$0.5). In contrast to fatness (LPC1), body fat distribution (LPC3-LPC4) did not track well even though significant components of body fat distribution occur at each age. Tracking of body fat distribution was higher in females than males. Sex differences in body fat distribution are non existent. Some sex differences are evident with the peripheral-to-central ratios after age 14 years. ^

The application of principal component analysis on human development indicators: Temporal approach from 1999 to 2010

Background and Objective. Ever since the human development index was published in 1990 by the United Nations Development Programme (UNDP), many researchers started searching and corporative studying for more effective methods to measure the human development. Published in 1999, Lai’s “Temporal analysis of human development indicators: principal component approach” provided a valuable statistical way on human developmental analysis. This study presented in the thesis is the extension of Lai’s 1999 research. ^ Methods. I used the weighted principal component method on the human development indicators to measure and analyze the progress of human development in about 180 countries around the world from the year 1999 to 2010. The association of the main principal component obtained from the study and the human development index reported by the UNDP was estimated by the Spearman’s rank correlation coefficient. The main principal component was then further applied to quantify the temporal changes of the human development of selected countries by the proposed Z-test. ^ Results. The weighted means of all three human development indicators, health, knowledge, and standard of living, were increased from 1999 to 2010. The weighted standard deviation for GDP per capita was also increased across years indicated the rising inequality of standard of living among countries. The ranking of low development countries by the main principal component (MPC) is very similar to that by the human development index (HDI). Considerable discrepancy between MPC and HDI ranking was found among high development countries with high GDP per capita shifted to higher ranks. The Spearman’s rank correlation coefficient between the main principal component and the human development index were all around 0.99. All the above results were very close to outcomes in Lai’s 1999 report. The Z test result on temporal analysis of main principal components from 1999 to 2010 on Qatar was statistically significant, but not on other selected countries, such as Brazil, Russia, India, China, and U.S.A.^ Conclusion. To synthesize the multi-dimensional measurement of human development into a single index, the weighted principal component method provides a good model by using the statistical tool on a comprehensive ranking and measurement. Since the weighted main principle component index is more objective because of using population of nations as weight, more effective when the analysis is across time and space, and more flexible when the countries reported to the system has been changed year after year. Thus, in conclusion, the index generated by using weighted main principle component has some advantage over the human development index created in UNDP reports.^

TURNOVER RATE OF THE NEURONAL CONNEXIN CX36 IN HELA CELLS

Electrical synapses formed of the gap junction protein Cx36 show a great deal of functional plasticity, much dependent on changes in phosphorylation state of the connexin. However, gap junction turnover may also be important for regulating cell-cell communication, and turnover rates of Cx36 have not been studied. Connexins have relatively fast turnover rates, with short half-lives measured to be 1.5 to 3.5 hours in pulse-chase analyses of connexins (Cx26 and Cx43) in tissue culture cells and whole organs. We utilized HaloTag technology to study the turnover rate of Cx36 in transiently transfected HeLa cells. The HaloTag protein forms irreversible covalent bonds with chloroalkane ligands, allowing pulse-chase experiments to be performed very specifically. The HaloTag open reading frame was inserted into an internal site in the C-terminus of Cx36 designed not to disrupt the regulatory phosphorylation sites and not to block the C-terminal PDZ interaction motif. Functional properties of Cx36-Halo were assessed by Neurobiotin tracer coupling, live cell imaging, and immunostaining. For the pulse-chase study, transiently transfected HeLa cells were pulse labeled with Oregon Green (OG) HaloTag ligand and chase labeled at various times with tetramethylrhodamine (TMR) HaloTag ligand. Cx36-Halo formed large junctional plaques at sites of contact between transfected HeLa cells and was also contained in a large number of intracellular vesicles. The Cx36-Halo transfected HeLa cells supported Neurobiotin tracer coupling that was regulated by activation and inhibition of PKA in the same manner as wild-type Cx36 transfected cells. In the pulse-chase study, junctional protein labeled with the pulse ligand (OG) was gradually replaced by newly synthesized Cx36 labeled with the chase ligand (TMR). The half-life for turnover of protein in junctional plaques was 2.8 hours. Treatment of the pulse-labeled cells with Brefeldin A (BFA) prevented the addition of new connexins to junctional plaques, suggesting that the assembly of Cx36 into gap junctions involves the traditional ER-Golgi-TGN-plasma membrane pathway. In conclusion, Cx36-Halo is functional and has a turnover rate in HeLa cells similar to that of other connexins that have been studied. This turnover rate is likely too slow to contribute substantially to short-term changes in coupling of neurons driven by transmitters such as dopamine, which take minutes to achieve. However, turnover may contribute to longer-term changes in coupling.

Regulation of phosphatidylinositide turnover in the myometrium by cAMP: Implications for the control of uterine contraction

Regulation of uterine quiescence involves the integration of the signaling pathways regulating uterine contraction and relaxation. Uterine contractants increase intracellular calcium through receptor/GαqPLC coupling, resulting in contraction of the myometrium. Elevation of cAMP concentration has been correlated with relaxation of the myometrium. However, the mechanism of cAMP action in the uterus is unclear. ^ Both endogenous and exogenous increases in cAMP inhibited oxytocin-stimulated phosphatidylinositide turnover in an immortalized pregnant human myometrial cell line (PHM1-41). This inhibition was reversed by cAMP-dependent protein kinase (PKA) inhibitors, suggesting the involvement of PKA. cAMP inhibited phosphatidyinositide turnover stimulated by different agonists in different cell lines. These data suggest that the cAMP inhibitory mechanism is neither cell nor receptor dependent, and inhibits Gαq/PLCβ1 and PLCβ3 coupling. ^ The subcellular localization of PKA occurs via PKA binding to A-Kinase-Anchoring-Proteins (AKAP), and peptides that inhibit this association have been developed (S-Ht31). S-Ht31 blocked cAMP-stimulated PKA activity and decreased PKA concentration in PHM1-41 cell plasma membranes. S-Ht31 reversed the ability of CPT-cAMP, forskolin and relaxin to inhibit phosphatidylinositide turnover in PHM1-41 cells. Overlay analysis of both PHM1-41 cell and nonpregnant rat myometrium found an AKAPs of 86 kDa and 150 kDa associated with the plasma membrane, respectively. These data suggest that PKA anchored to the plasma membrane via AKAP150/PKA anchoring is involved in the cAMP inhibitory mechanism. ^ CPT-cAMP and isoproterenol inhibited phosphatidylinositide turnover in rat myometrium from days 12 through 20 of gestation. In contrast, neither agent was effective in the 21 day pregnant rat myometrium. The decrease in the cAMP inhibitory mechanism was correlated with a decrease in PKA and an increase in protein phosphatase 2B (PP2B) concentration in rat myometrial plasma membranes on day 21 of gestation. In myometrial total cell homogenates, both PKA and PP2B concentration increased on day 21. S-Ht31 inhibited cAMP inhibition of phosphatidylinositide turnover in day 19 pregnant rat myometrium. Both PKA and PP2B coimmunoprecipitated with an AKAP150 in a gestational dependent manner, suggesting this AKAP localizes PKA and PP2B to the plasma membrane. ^ These data presented demonstrate the importance of the cAMP inhibitory mechanism in regulating uterine contractility. ^

Mechanism of translationally coupled mRNA turnover mediated by c-fos protein-coding-region determinant

Proto-oncogene c-fos is a member of the class of early-response genes whose transient expression plays a crucial role in cell proliferation, differentiation, and apoptosis. Degradation of c- fos mRNA is an important mechanism for controlling c-fos expression. Rapid mRNA turnover mediated by the protein-coding-region determinant (mCRD) of the c-fos transcript illustrates a functional interplay between mRNA turnover and translation that coordinately influences the fate of cytoplasmic mRNA. It is suggested that mCRD communicates with the 3′ poly(A) tail via an mRNP complex comprising mCRD-associated proteins, which prevents deadenylation in the absence of translation. Ribosome transit as a result of translation is required to alter the conformation of the mRNP complex, thereby eliciting accelerated deadenylation and mRNA decay. To gain further insight into the mechanism of mCRD-mediated mRNA turnover, Unr was identified as an mCRD-binding protein, and its binding site within mCRD was characterized. Moreover, the functional role for Unr in mRNA decay was demonstrated. The result showed that elevation of Unr protein level in the cytoplasm led to inhibition of mRNA destabilization by mCRD. In addition, GST pull-down assay and immuno-precipitation analysis revealed that Unr interacted with PABP in an RNA-independent manner, which identified Unr as a novel PABP-interacting protein. Furthermore, the Unr interacting domain in PABP was characterized. In vivo mRNA decay experiments demonstrated a role for Unr-PABP interaction in mCRD-mediated mRNA decay. In conclusion, the findings of this study provide the first evidence that Unr plays a key role in mCRD-mediated mRNA decay. It is proposed that Unr is recruited by mCRD to initiate the formation of a dynamic mRNP complex for communicating with poly(A) tail through PABP. This unique mRNP complex may couple translation to mRNA decay, and perhaps to recruit the responsible nuclease for deadenylation. ^