AN INVESTIGATION INTO THE MOLECULAR MECHANISMS OF ANDROGEN ACTION IN THE SERTOLI CELL

Steroid hormones regulate target cell function via quantitative and qualitative changes in RNA and protein synthesis. In the testis, androgens are known to play an important role in the regulation of spermatogenesis. The Sertoli cell (SC), whose function is thought to be supportive to the developing germ cell, has been implicated as an androgen target cell. Although cytoplasmic androgen receptors and chromatin acceptor sites for androgen-receptor complexes have been found in SC, effects on RNA synthesis have not previously been demonstrated. In this study, SC RNA synthetic activity was characterized and the effect of testosterone on SC nuclear transcriptional activity in vitro assessed. SC exhibited two fold increases in RNA and ribonucleotide pool concentrations during sexual maturation. These changes appeared to correlate with a previously observed increase in protein concentration per cell over an age span of 15-60 days. Following incubation with ('3)H-uridine, SC from older animals incorporated more label into RNA than SC from younger animals. Since the relative concentration of cytidine nucleotides was higher in SC from older rats, the age-related increase in tritium incorporation may reflect an associated increase in incorporation of ('3)H-CMP into RNA. Alternatively, the enhanced labeling may be the result of either a change in the base composition of the RNA resulting in a higher proportion of CMP and UMP in the RNA, or compartmentalization of the nucleotide pools. The physiologic consequences of these maturational alterations of nucleotide pools remains to be elucidated. RNA polymerase activities were characterized in intact nuclei obtained from cultured rat SC. (alpha)-Amanitin resistant RNA polymerase I+III activity was identical when measured in low or high ionic strength (0.05 M or 0.25 M ammonium sulfate (AS)) in the presence of MnCl(,2) or MgCl(,2), with a divalent cation optimum of 1.6 mM. RNA polymerase II was most active in 0.25 M AS and 1.6 mM MnCl(,2). The apparent Km of RNA polymerase II for UTP was 0.016 mM in 0.05 M AS and 0.037 mM in 0.25 M AS. The apparent Km values for total polymerase activity was 0.008 mM and 0.036 mM at low and high ionic strenghts, respectively. These data indicate that Sertoli cell RNA polymerase activities have catalytic properties characteristic of eukaryotic polymerase activities in general. In the presence of 21 (mu)M testosterone, RNA polymerase II activity increased two fold at 15 minutes, then declined but was still elevated over control values six hours after androgen addition. Polymerase I+III activity was not greatly affected by testosterone. The stimulation of polymerase II measured at 15 minutes was dose-dependent, with a maximum at 0.53 nM and no further stimulation up to 10('-5) M (ED(,50) = 0.25 nM testosterone), and was androgen specific. The results of preliminary RNA isolation and characterization experiments suggested that the synthesis of several species of RNA was enhanced by testosterone administration. These findings have great potential importance since they represent the first demonstration of a direct effect of androgens on the transcriptional process in the Sertoli cell. Furthermore, the results of these studies constitute further evidence that the Sertoli cell is a target for androgen action in the testis. ^

Molecular consequences of the loss of 3'→5' exonuclease activity of DNA polymerases delta and epsilon

The DNA replication polymerases δ and ϵ have an inherent proofreading mechanism in the form of a 3'→5' exonuclease. Upon recognition of errant deoxynucleotide incorporation into DNA, the nascent primer terminus is partitioned to the exonuclease active site where the incorrectly paired nucleotide is excised before resumption of polymerization. The goal of this project was to identify the cellular and molecular consequences of an exonuclease deficiency. The proofreading capability of model system MEFs with EXOII mutations was abolished without altering polymerase function.^ It was hypothesized that 3'→5' exonucleases of polymerases δ and ϵ are critical for prevention of replication stress and important for sensitization to nucleoside analogs. To test this hypothesis, two aims were formulated: Determine the effect of the exonuclease active site mutation on replication related molecular signaling and identify the molecular consequences of an exonuclease deficiency when replication is challenged with nucleoside analogs.^ Via cell cycle studies it was determined that larger populations of exonuclease deficient cells are in the S-phase. There was an increase in levels of replication proteins, cell population growth and DNA synthesis capacity without alteration in cell cycle progression. These findings led to studies of proteins involved in checkpoint activation and DNA damage sensing. Finally, collective modifications at the level of DNA replication likely affect the strand integrity of DNA at the chromosomal level.^ Gemcitabine, a DNA directed nucleoside analog is a substrate of polymerases δ and ϵ and exploits replication to become incorporated into DNA. Though accumulation of gemcitabine triphosphate was similar in all cell types, incorporation into DNA and rates of DNA synthesis were increased in exonuclease defective cells and were not consistent with clonogenic survival. This led to molecular signaling investigations which demonstrated an increase in S-phase cells and activation of a DNA damage response upon gemcitabine treatment.^ Collectively, these data indicate that the loss of exonuclease results in a replication stress response that is likely required to employ other repair mechanisms to remove unexcised mismatches introduced into DNA during replication. When challenged with nucleoside analogs, this ongoing stress response coupled with repair serves as a resistance mechanism to cell death.^

Investigating the effects of the 2007 Texas tobacco tax increase on the smoking behavior of adults

Smoking is major cause of premature mortality and morbidity in the United States. The health consequences of tobacco usage are increasingly concentrated in minority and lower socioeconomic groups. One of the most effective means of deterring tobacco consumption and generating revenue to fund prevention activities is the levying of excise taxes. In 2007 the state of Texas increased the excise tax on cigarettes by $1.00 per pack. This study sought to determine if there was a significant effect on smoking prevalence in the state by examining Behavioral Risk Factor Surveillance System (BRFSS) data for two years leading up to the tax increase-2005 and 2006- and two years post tax increase -2007 and 2008. Results were compared against a chi square distribution and three multiple logistic regression models were created to adjust for race/ethnicity, age, education and income. Results from this study show that there was not a significant decrease in smoking prevalence for most of the groups stratified by age, income and ethnicity. There was not a significant decrease in the younger adults aged 18-34 by income, ethnicity, or education. Smoking prevalence increased for some groups, e.g., Hispanic females. In the regression models, the tax effect was not significant. While overall prevalence decreased by 9%, there were not significant reductions among non-White or Hispanic survey participants. Taxed sales dropped by approximately 17% according to the Texas Comptroller. Without BRFSS data measuring daily cigarette consumption among current smokers, now not assessed, it is impossible to determine whether the discrepancy in reported prevalence and taxes sales is attributable to consumption of fewer cigarettes among smokers or tax avoidance.^

Exploring the association of children's engagement in nature and outdoor activity with physical activity: A systematic review

The natural environment and green spaces are settings that may facilitate physical activity and, as a result, combat childhood obesity and benefit children's physical health. A systematic review was conducted to assess the effect of children's engagement in outdoor activity on children's physical activity levels. A total of 169 articles were initially identified, of which 11 were eligible for inclusion in the systematic review. Studies were heterogeneous: cross-sectional, RCT, cohort, and direct observation. Study participants were between the ages of 3-15 years, and physical activity was measured by accelerometers, pedometers, direct observation or surveys. A majority of the studies (9/11) found a positive association between time spent outdoors and physical activity in children and adolescents. Of these 9 studies, 5 found this association specifically between time spent outdoors in greenspace and physical activity. Despite limitations, the findings of this review support the positive association between time spent outdoors and physical activity in children and adolescents, and the notion that children and adolescents who spend more time outdoors are more physically active. This demonstrates the need to use outdoor environments as settings for children's and adolescents' physical activity.^