Browsing by Author "Jonathan Allen, Committee Member"

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Antimicrobial Susceptibility Profiles and Clonal Population of Multidrug Resistant Campylobacter coli Isolates from Commercially Grown Turkeys.
(2011-02-04) Ahmad, Harun; Sophia Kathariou, Committee Chair; Jonathan Allen, Committee Member; Donna Carver, Committee Member; Deborah Threadgill, Committee Member; Sophia Kathariou, Committee Member
The Biology and Regulation of Activating Transcription Factor 3 (ATF3) by Nonsteroidal Anti-inflammatory Drugs (NSAIDs) and Dietary Compounds with Chemopreventive Activity.
(2005-04-19) Bottone, Frank Gerard Jr.; Thomas E. Eling, Committee Co-Chair; Jack Odle, Committee Member; Jonathan Allen, Committee Member; Brenda Alston-Mills, Committee Co-Chair
Nonsteroidal anti-inflammatory drugs (NSAIDs) are effective chemopreventive agents in various tissue types such as colon and breast. Until recently their mode of action was thought to be solely through inhibition of cyclooxygenase-2 (Cox-2), which along with its products such as prostaglandin E2, are upregulated in tumors. However, gene regulation may, in part, explain the alterations in invasion, apoptosis, and/or cell proliferation seen with NSAIDs. In this report, we utilized microarray analysis of colorectal cancer cells treated with low, non-toxic concentrations of sulindac sulfide to show that the active metabolite of this NSAID and potent cancer chemopreventive drug regulates the expression of a variety of genes. Several genes related to cell growth and apoptosis, while others were transcription factors, which are important regulators of gene expression. NSAIDs such as the Cox-1 specific inhibitor SC-560 and the Cox-2 specific inhibitor SC-58125 modulated the expression of these genes in HCT-116 colorectal cancer cells, which correlated with the biological activity but not Cox-2 inhibitory activity of these compounds. Activating Transcription Factor 3 (ATF3) was one gene identified as induced by a variety of NSAIDs and confirmed in a variety of cell lines in our laboratory. ATF3 was induced by a variety of other compounds with cancer chemopreventive activity such as the PPAR gamma ligand troglitazone (TGZ) and the dietary compounds diallyl disulfide (DADS), resveratrol, and genistein. To ascertain the biological significance of the induction of ATF3, we overexpressed ATF3 in the sense and antisense orientation. Overexpression of ATF3 in the sense orientation reduced the size of mouse tumor xenografts by 54 percent in vivo. One explanation for the biological activity of ATF3 is down-stream gene modulation. Using microarray analysis, overexpression of ATF3 in the sense orientation regulated several genes related to invasion and metastasis. ATF3 overexpression decreased focus formation and inhibited invasion potential in vitro to a similar degree as sulindac sulfide treatment. Conversely, antisense ATF3 overexpression increased invasion potential and focus formation. Therefore, the biological activity of these compounds may be linked to the gene regulator role of ATF3. Lastly, we demonstrated that ATF3 is modulated by the transcription factor Early Growth Response Gene-1 (Egr-1). The induction of ATF3 by sulindac sulfide and TGZ at the mRNA, protein, and promoter level required Egr-1 and the Extracellular regulated kinase-1/2 (Erk-1/2) MAPK pathway. In conclusion, NSAIDs and other chemopreventive compounds alter the expression of a number of genes, in particular transcription factors, which may be linked to the biological activity of these compounds. Lastly, we dispute the dogma that ATF3 is solely a stress response gene and provide evidence that ATF3 has anti-cancer activity warranting further investigation.
Comparative Compositional and Biological Properties of Muscadine and Cabernet franc Grape Skins
(2009-07-20) Liu, Alison Ann; Jonathan Allen, Committee Member; Lisa Dean, Committee Member; Leon C. Boyd, Committee Chair
Numerous studies have documented the health benefits of moderate consumption of wines, and especially red wines. Much of this activity has been attributed to the extraction of phytochemicals from the skin and seeds during on-skin fermentation procedures. This study focused on the extraction and biological properties of phytochemicals from freeze dried skins of muscadine and Cabernet franc. The Cabernet franc grapes are a good source of resveratrol whereas muscadine grapes are an excellent source of ellagic acid with the resveratrol content varying considerably from batch to batch. Both grapes contain a complex mixture of phytochemicals, many of which have been shown to have potential health benefits in vitro. The skins of two muscadine cultivars, Carlos and Noble, and the skins of Cabernet franc grapes obtained during two harvests were freeze-dried, extracted with 80:20 methanol-water, and analyzed for antioxidant capacity followed by high pressure liquid chromatography (HPLC) and HPLC coupled with time of flight mass spectrometry (HPLC-TOF-MS) analyses for phenolic compounds. The anticarcinogenic activity of extracts was determined against LNCaP prostate carcinoma cells with the XTT proliferation assay and the caspase-3 assay used to determine apoptosis. The 2006 Cabernet franc extract contained 70.3 ÃŽÂ¼g/g resveratrol and no ellagic acid, whereas muscadine extracts contained 4.2-12.2 ÃŽÂ¼g resveratrol and 117-269 ÃŽÂ¼g ellagic acid per g dried sample. Neither resveratrol nor ellagic acid was detected in the 2007 Cabernet franc extract. The antioxidant capacity was measured using the oxygen radical absorbance capacity (ORAC) assay. ORAC values of grape skin extracts ranged from 110-354 ÃŽÂ¼mol Trolox equivalents per g dried sample with Carlos being the lowest, followed by Cabernet franc, and Noble being the highest. At concentrations of 5 mg/mL, all skin extracts induced apoptosis in LNCaP cells as the increases in caspase-3 and other closely related proteases were significantly different from the controls. LNCaP proliferation was also inhibited at doses of 5 mg/mL. This study shows that even though the phytochemical composition of muscadine and Cabernet franc differ considerably, as by products of the winemaking industry, they both may have economic and potential health benefits as dietary supplements.
Estrogen Response Element and the Promoter Context of the Human and Mouse Lactoferrin Genes Influence Estrogen Receptor alpha-Mediated Transactivation Activity in Mammary Gland Cells
(2004-12-01) Stokes, Kenya; William L. Miller, Committee Member; Mark Conkling, Committee Member; Jonathan Allen, Committee Member; Christina T. Teng, Committee Co-Chair; Brenda Alston-Mills, Committee Co-Chair
The purpose of this research has been to determine whether an extended estrogen response element half-site (ERRE) contributes to the differential estrogen responses of the human and mouse lactoferrin estrogen response element (ERE) in the context of their natural promoters. This research utilized molecular biology techniques to evaluate gene activation. Transfections of MCF-7 cells showed that liganded ER-alpha activates transcription of the human lactoferrin ERE 4-fold higher than the mouse lactoferrin ERE in the context of their natural promoters. Since the ERRE of the human lactoferrin gene naturally occurs 18 bp upstream from the ERE and is absent in the mouse lactoferrin gene promoter, we created a chimeric mouse lactoferrin CAT reporter, which now encodes the ERRE in the identical location as in the human lactoferrin gene. The addition of the ERRE in the mouse lactoferrin gene rendered this reporter extremely responsive to estrogen stimulation. We also demonstrated that the conformation of the estrogen receptor bound to the ERE alone or in the presence of ERRE differed and that the ERRE influenced the selectivity of coactivators in liganded ER-alpha-mediated transcriptional activity. Like the lactoferrin gene ERE, most known natural estrogen response elements are imperfect palindromes that differ from the consensus by at least a 1 base pair change and confer different levels of ER transcriptional activation compared to the consensus ERE. In contrast to our transient transfection data showing a lower estrogen response of the mouse lactoferrin ERE compared to the human lactoferrin ERE in the context of their natural promoters, in vivo data showed that the gene is robustly transcribed in response of estrogen in both species. Therefore, this research model can be applied to studies of genes that have different hormone responses in vivo versus in vitro in an attempt to identify non-typical estrogen response elements that influence ER-alpha-mediated transactivation.
Investigation of Molecular Forces Involved in Gelation of Commercially Prepared Soy Protein Isolates
(2002-09-26) McKlem, Lacey Kay; Jonathan Allen, Committee Member; Christopher R. Daubert, Committee Member; Prachaub Kwanyuen, Committee Co-Chair; Tyre C. Lanier, Committee Co-Chair
Gelling behavior of soy protein isolates (SPI) commercially prepared from two soybean cultivars, Prolina and Brim, was studied. SPI (prepared at 12% protein w/w) were predenatured by commercial processing and gelled immediately upon hydration, prior to heat treatment. This research first investigated the effects of temperature, holding time, and reheating on thermal reversibility and reformability of these commercial SPI gels. Secondly, chemical reagents were added to determine the molecular forces contributing to their gelation. Small strain rheology was used to determine the effect of temperature (40 – 90 °C), holding time at elevated temperature (0 or 30 min), and reheating on thermal reversibility of Prolina and Brim gels. Final gel rigidity (measured by storage modulus G') increased with increasing endpoint temperature, and holding at each endpoint temperature further increased the final gel rigidity. During heating, all gels exhibited a decrease in G', which was largely reversible upon cooling. Prolina and Brim gels exhibited increases in G' during holding at temperatures ranging from 50 – 90 °C. Reheating did not enhance or diminish the gel rigidity. Vane fracture testing was utilized to investigate temperature effects and reformability of cooked gels. Fracture stress of heated and cooled gels was significantly higher than initial gels, while fracture deformation was significantly lower. Cooked gels were rechopped and gels reformed at 10 °C, again strengthening upon subsequent heating and cooling. Again, additional thermal treatment did not enhance or diminish gel properties. To investigate contribution of molecular forces, the chemical reagents urea (weakens hydrogen bonding and hydrophobic interactions) and dithiothreitol (DTT) (reduces disulfide bonds) were added to Prolina and Brim SPI and resulting gelling behavior was monitored during small strain rheology. Control (deionized water) and urea-added samples gelled initially and G' decreased upon heating to 80 °C, whereas DTT-added samples did not gel initially. Gel rigidity of both control and DTT-added samples increased during holding at 80 °C. Final gel rigidity of urea- and DTT-added samples was lower than control samples; the sum of the final G' values for the urea- and DTT-added samples neared the final G' value of the control sample for both Prolina and Brim gels. Urea had a more detrimental effect on gel rigidity of Brim gels, whereas DTT had a similar effect on Prolina and Brim. Prolina and Brim gels (unheated and heated) were solubilized in various chemical reagents. Addition of urea + DTT completely solubilized all samples, while solubilization in either reagent alone was greater than solubilization with the control treatment. More protein was solubilized from gels that exhibited lower gel rigidity. Results from this investigation indicate that hydrogen bonding, hydrophobic interactions, and disulfide bonding all play roles in commercial SPI gelation. Disulfide bonding is important in the initial gel network as indicated by a high degree of solubilization in DTT and formation of gel prior to heating by pastes containing urea. Disulfide bonds are not essential for gel formation, but their presence adds rigidity to the gel network. Hydrogen bonding and hydrophobic interactions, however, are the primary forces contributing to gelation of these commercially prepared SPI. This is evidenced by the extensive reformability of the gels as well as the similar effects of heating and holding on Prolina and Brim gels. More specifically, increases in G' during holding and cooling were attributed to hydrophobic interactions and hydrogen bonding, respectively.
Statistical Topics in Disease Gene Mapping
(2003-04-14) Meng, Zhaoling; Bruce S. Weir, Committee Chair; Margaret G. Ehm, Committee Co-Chair; Zhao-Bang Zeng, Committee Member; Russ Wolfinger, Committee Member; Greg Gibson, Committee Member; Jonathan Allen, Committee Member
Efforts in disease gene mapping have achieved a great deal of success in mendelain diseases, but made slower progress in common disease studies because of their complexity. The rapid development of genetics and molecular technologies provides an immense amount of DNA data; developing powerful and efficient statistical methodologies is under high demand. This dissertation explored some aspects of the problem. The power of two genome-wide disease gene mapping strategies is investigated. One applies linkage analysis and then linkage disequilibrium (LD) tests to markers within linked regions. The other looks for LD with disease using all markers. The results showed that the genome-wide association based tests are much more likely to identify genes. Genotyping closely spaced Single Nucleotide Polymorphisms (SNPs) frequently yields highly correlated data due to extensive LD, and gives association studies unnecessary and unaffordable burden when these markers don't yield significantly different information. Two procedures are developed to select an optimum subset of SNPs that could be efficiently genotyped on larger numbers of samples while retaining most of the information based on genotypes of a large initial set of SNPs on a small number of samples. One utilizes a spectral decomposition method based on matrices of pair-wise LD, and the other extends David Clayton's htSNP selection method. Properties of the procedures are studied; minimum sample sizes needed for achieving consistent results are recommended; the procedures are evaluated using experimental data. Studying gene-treatment interaction is a long desired problem. When the genetic variation that is being tested is not specific functional sites but randomly selected polymorphisms, a source of randomness is introduced. A mixed effect model is developed to fit fixed treatment effects, random haplotypic effects, and random gene-treatment interactions in this scenario; likelihood ratio tests are applied for testing the random effects. Our simulation results showed that the mixed effect model is valid and generally behaves better than the fixed haplotypic effects model in the exploratory phase of a study.