Browsing by Author "Jack Odle, Committee Member"

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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.
DFM/Probiotic effects on gastrointestinal tract development and immune function of broiler chicken
(2009-11-11) Qiu, Rongsheng; Jim Croom, Committee Co-Chair; Jerry Spears, Committee Member; Jack Odle, Committee Member; Matt Koci, Committee Chair
Dietary and housing effects on growth performance, gut health and Salmonella colonization of Salmonella-challenged broilers
(2007-11-26) Bohorquez, Diego V.; Peter R. Ferket, Committee Chair; Edgar Oviedo, Committee Member; Matt Koci, Committee Member; Jack Odle, Committee Member
The Effect of Early Post-Hatch Dietary Amino Acid Levels on Satellite Cell Dynamics in Turkeys.
(2007-07-05) Nierobisz, Lidia Sylwia; Jack Odle, Committee Member; Vernon Felts, Committee Member; Paul Mozdziak, Committee Chair
Satellite cells are defined as myonuclear myogenic stem cells residing between sarcolemma and basal lamina of the myofiber. Myofiber number is established during embryonic development. Post-hatch and post-natal muscle growth occurs exclusively through an increase in myofiber size. The increase of myofiber size in early post-hatch turkeys is predominantly dependent on the contribution of new myonuclei to pre-existing myofibers by the mitotically active satellite cell population. Preliminary data in broilers has revealed that supplementation with amino acid deficient diet immediately post-hatch results in an increase satellite cell mitotic activity in Pectoralis thoracicus muscle in 3-day-old chicks as compared to birds fed adequate and above requirement amino acid levels. Additionally, chicks denied feed for first three days post-hatch exhibited significantly lower satellite cell mitotic activity in Pectoralis thoracicus muscle as compared to remaining treatment groups. The hypothesis tested in the current study was that turkeys supplemented with crude protein and amino acid deficient diet early post-hatch would produce higher meat yield at 140 days. Since very little is known about the impact of early nutrition on the satellite cell activity in commercially raised turkeys, the objective of the present study was to measure the effect of different levels of dietary crude protein and amino acids (0.88 NRC, 1.00 NRC, and 1.12 NRC) as well as feed deprivation on the satellite cell mitotic activity, Pectoralis thoracicus muscle weight, and body weight of commercially raised turkey males. At 3, 4, 5, 8, and 11 days of age, birds from each treatment group were injected with 5-bromo-2'-deoxyuridine (BrdU) to label mitotically active cells. The right Pectoralis thoracicus muscle was harvested one hour after BrdU injection followed by BrdU and PI immunohistochemistry and measurement of myofiber diameters. On the third day post-hatch satellite cell mitotic activity was the highest (P<0.05) in 0.88 NRC treatment group and the lowest (P<0.05) in the feed deprived group. On the fourth day post-hatch, feed deprived birds exhibited the lowest (P<0.05) satellite cell mitotic activity and muscle weight. At market age (140 days), there were no significant differences (P>0.05) between treatments in body weight and Pectoralis thoracicus muscle weight. Further evaluation of mechanistic pathways governing satellite cell mitotic activity and muscle growth are necessary to devise early post-hatch feeding strategies that would lead to improvement in Pectoralis thoracicus muscle yield in male turkeys.
Effect of Probiotic Consortium on Level and Mechanism of Intestine Function.
(2007-10-25) Chichlowski, Maciej; Matthew Koci, Committee Co-Chair; Warren James Croom, Jr., Committee Chair; Jack Odle, Committee Member; Vern Christensen, Committee Member
A probiotic consortium or direct-fed microbial (DFM) is a live-microbial food supplement that improves health and performance, in broilers and other animals. They may have potential as an alternative to supplemental feed antibiotics use within the poultry industry to prevent enteric disease. Mechanisms by which DFM operate, collectively called "competitive exclusion", includes spatial exclusion, micro-environmental alterations, production of antimicrobial substances and epithelial barrier integrity. The majority of recent research suggests the likelihood of a small but additive series of beneficial changes from the animal's exposure to DFM. The interactions between intestinal microbiota, the gut epithelium, and the immune system are important in the competitive exclusion process. Data presented in this study suggest that DFM increase metabolic efficiency via changes in intestinal physiology and metabolism, especially when compared to the traditional feed supplement prophylactic, salinomycin. Also, the effects of DFM on ileal glucose and proline absorption and their relationship to gastrointestinal energy expenditures were examined. Increases in the efficiency of nutrient absorption and decreases in intestinal fermentation with DFM may contribute to previously observed decreases in energy expenditures; however, these contributions are relatively minor, indicating that other physiological mechanisms are involved. The effects of DFM on intestinal histomorphometrics and micro-architecture were examined. Since DFM colonization can prevent attachment of the pathogens to the gastrointestinal epithelium, spatial relationships between the gastrointestinal bacteria and gastrointestinal epithelium, described in this study, confirm previous assumptions about the ability of DFM to physically exclude the colonization of bacterial pathogens, preventing enteric disease. In summary, it is likely that the beneficial effects of DFM are the result of the summation of a complex, multi-variate series of alterations in gut microbial and whole body metabolism.
Effects of dietary fiber and other nutrients on swine manure odorants
(2007-08-16) Zhang, Weifang; Jerry Spears, Committee Member; Jack Odle, Committee Member; Eric van Heugten, Committee Chair; Theo van Kempen, Committee Member; Vivek Fellner, Committee Member
Eicosapentanoic and Docosahexanoic Acids, (EPA;DHA), Increase Insulin Sensitivity in Growing Steers.
(2010-06-16) Cartiff, Sydney; Joan Eisemann, Committee Chair; Charles Whisnant, Committee Member; Jack Odle, Committee Member; Vivek Fellner, Committee Member
Evaluation of the Nutritional Value of Glycerol, a Byproduct of Biodiesel Production, for Swine
(2009-04-27) Shields, Michael Christopher; Charles Stark, Committee Member; Eric van Heugten, Committee Chair; Jack Odle, Committee Member
SHIELDS, MICHAEL CHRISTOPHER. Evaluation of the Nutritional Value of Glycerol, a Byproduct of Biodiesel Production, for Swine. (Under the direction of Eric van Heugten.) The objectives of this study were to: 1) evaluate diet manufacturing characteristics and feed mill processing data when using glycerol; 2) determine the value of glycerol in diets for newly weaned pigs relative to lactose; and 3) determine the value of glycerol in diets for nursery pigs when replacing lactose and corn. For the first objective, two experiments were performed to test the performance of glycerol in the feed mill. In experiment 1, finisher feed was mixed in a 50 kg mixer at glycerol levels of 0, 2.5, and 5%. Diets were manufactured in batches of 100 lbs in a randomized block design and this was replicated three times. In Experiment 2, finisher feed was mixed and pelleted to examine feed mill performance at glycerol levels of 0, 2.5, and 5%. Diets were manufactured in batches of 1,000 lbs in a randomized block design and this was replicated three times. In Experiment 1, flowability linearly increased (P=0.03) as glycerol levels increased. In Experiment 2, production efficiency, and pellet durability were linearly increased (P=0.005 and P=0.0001 respectively) and hot pellet temperature was decreased (P=0.05) as glycerol levels increased. For the second objective, a performance study was conducted. A total of 126 pigs (body weight was 6.68 Ã‚Â± 0.17 kg) was weaned at approximately 21 days of age, blocked by weight, and allocated to 42 pens with 3 pigs per pen. Pens were randomly assigned one of six treatments in a 2x3 factorial randomized complete block design with factors: 1) glycerol inclusion in phase 1 diets (0 or 5%), and 2) glycerol inclusion level in phase 2 diets (0, 5, or 10%). Phase 1 diets were fed for 2 weeks and glycerol was supplemented to replace lactose on a weight for weight basis. Phase 2 diets were fed for 3 weeks and glycerol was included in replacement of corn and this replacement was made on a nutrient basis (thus accounting for the nutrient composition of corn). Results suggested that replacing corn at levels of up to 10% improved ADG (P<0.002), ADFI (P<0.003), and G:F (P<0.044). Serum glycerol concentration was not impacted by the glycerol supplementation during the starter 1 phase, but a linear increase in serum glycerol concentration during the starter 2 phase (P<0.0001) was observed as dietary glycerol increased. Glycerol supplementation had no effect on serum glucose, total protein, albumin, bilirubin, creatine phosphokinase, globulin, and aminotransferase serum glutamic-oxaloacetic transaminase in the starter 1 phase. In the starter 2 phase, glycerol supplementation increased urea nitrogen (P=0.002), decreased creatinine (P=0.02), and increased the ratio of blood urea nitrogen to creatinine (P=0.0002). Glycerol supplementation in the starter 2 phase had a quadratic effect on urea nitrogen (P=0.0008), which was lower at 5% level of glycerol (4.71mg/dl ) compared to the 0 and 10% (6.86 and 7.43mg/dl respectively). Cholesterol concentration I was higher for 5% added glycerol (92.21mg/dl) then for the 0% and 10% levels (79.14 and 84.64mg/dl respectively; quadratic effect, P = 0.04). Glycerol supplementation in the starter 2 phase linearly decreased creatinine concentration (P=0.02). For the third objective, a performance trial was completed. A total of 144 pigs (body weight was 6.68 Ã‚Â± 0.17 kg) were weaned at approximately 21 days of age, blocked by weight, and allocated to 28 pens with 3 pigs per pen,. Pens were randomly assigned one of six dietary treatments; (0, 2.5, 5, 7.5, 10% glycerol added to replace 10% lactose in a basal starter 1 diet (fed for two weeks) containing 20% total lactose, and a negative control with 10% lactose and 0% glycerol). A common starter diet was fed for the remaining two weeks. Pigs were weighed and feed intake was measured weekly. Results demonstrated that glycerol supplementation at 10% compared to the negative control resulted in a greater ADG during weeks 1, 2, and the starter 1 period (P < 0.03). Glycerol also improved feed efficiency in the starter 1 period (P < 0.04). There was no impact of feeding glycerol in the starter 1 phase on subsequent performance during the starter 2 phase. Serum glycerol was linearly increased (P=0.03) as glycerol levels increased. Glycerol supplementation in starter 1 diets had no effect on glucose, urea nitrogen, total protein, albumin, alkaline phosphatase, aminotransferase serum glutamic pyruvic transaminase aspartate (ALT), aminotransferase serum glutamic-oxaloacetic transaminase (AST), cholesterol, calcium, phosphorus, sodium, potassium, chloride, albumin, globulin, the ratio of blood urea nitrogen to creatinine (buncreatinine), and creatine phosphokinase. Glycerol supplementation decreased creatinine (P=0.0004) and bilirubin (P=0.02) as dietary glycerol levels increased. Overall these studies indicate that glycerol can be an asset in feedmilling by improving pellet durability, flowability, production efficiency, and hot pellet temperature. In nursery diets, it improves ADG, and ADFI at levels up to 10%. Overall data indicated that glycerol can be added in the diets at levels up to 5% and improve feedmill performance and pig performance.
Experimentation in Replacement Lithography, Reductive Desorption, and Scanning Probe Imaging on Various Metal Substrates.
(2010-06-28) Williams, James Alfred; Christopher Gorman, Committee Chair; Edmond Bowden, Committee Member; Stefan Franzen, Committee Member; Lin He, Committee Member; Jack Odle, Committee Member
Genomic Characterization of Two Models of Obesity in Mice: Divergent Selection for Epididymal Fat and the Effects of trans-10, cis-12-Conjugated Linoleic Acid
(2008-08-18) Ceddia, Ryan Patrick; Melissa S. Ashwell, Committee Chair; Eugene J. Eisen, Committee Member; Christopher M. Ashwell, Committee Member; Jack Odle, Committee Member
Obesity is rapidly becoming a major problem in the United States and throughout the world. Polygenic models of obesity are most similar to human obesity because few humans are genetically obese due to a mutation in a single gene. Numerous studies have selected mice for body size and growth rate as models for selection of agriculturally important species. One series of selection experiments produced lines of mice having differing epididymal fat (EF) masses but similar body weights. These mice may be used as a model for adipose deposition without confounding the effects of body weight. One method of studying these mice is to examine gene expression. Expression of thousands of genes can be investigated at one time using microarrays. We used microarrays and real-time RT-PCR to compare gene expression between the high epididymal (HE) and low fat (LF) lines of mice, which have dissimilar EF mass but similar body weights. Microarray analysis identified 19 genes with differential expression between the HE and LF lines of mice with 5 of these genes differentially expressed in both liver and EF tissues. We found differential regulation of genes known to play a role in glucose uptake and lipid metabolism. In addition, we identified a differentially expressed gene, solute carrier family 22 member 4, located within the confidence interval of a quantitative trait loci associated with EF mass, making it a positional candidate. Furthermore, we identified a linked group of three genes (Sortilin 1, guanine nucleotide binding protein alpha inhibiting 3, and selenium-binding protein 2) on Mus musculus chromosome 3 (MMU3), which may represent a genomic "hot spot" for genes associated with EF mass. In this study, differential expression of several genes not previously associated with obesity or adipose deposition were identified and may represent new targets for further research. Another aspect of obesity currently being investigated is anti-obesity compounds. One such compound is trans-10, cis-12-conjugated linoleic acid (CLA). CLA has been reported to reduce body weight and adipose mass in many species. Numerous studies have reported an increase in size, cytoplasmic vacuolization, and fatty acid synthesis in liver of CLA fed mice. The livers of CLA fed mice gain mass due to lipid accumulation; however, the precise molecular mechanisms are unknown. To elucidate these mechanisms, we examined the fatty acid composition, histology, and gene expression profile of liver from a polygenic obese line of mice fed CLA. Using a Periodic acid-Schiff stain, histological evidence suggests that glycogen content is unchanged in the liver of the CLA fed mouse implying that hepatic cytoplasmic vacuolization is due to increased lipid content. Microarray analysis identified 1393 genes differentially expressed at a nominal P-value of 0.01. Following Bonferroni correction and excluding lowly expressed transcripts, 198 genes were identified as being differentially expressed with 17 genes having ≥2 fold change. Real-time RT-PCR showed up regulation of acylglycerol-3-phosphate O-acyltransferase 2 and diacylglycerol O-acyltransferase 2 in CLA fed mice, both necessary for triglyceride biosynthesis. Expression of B-cell leukemia⁄lymphoma 6, a nuclear transcriptional repressor, and signal transducer and activator of transcription 5B, a transcription factor, were shown to be greater in the liver of CLA fed mice. Both genes are associated with immunoregulation. Comparing real-time RT-PCR to microarray data suggest a Bonferroni correction to microarray data is necessary in order to eliminate false positive data. Further verification of microarray results is needed to validate microarray data after a Bonferroni correction.
Mechanisms of Prostaglandin-Stimulated Recovery of Mucosal Barrier Function in the Ischemia-Injured Porcine Intestine: Role of Intestinal Ion Transport
(2006-05-16) Moeser, Adam James; Anthony Blikslager, Committee Chair; Glen Almond, Committee Member; Jody Gookin, Committee Member; Jack Odle, Committee Member
A series of experiments were conducted to determine physiologic mechanisms of mucosal repair in the ischemia-injured intestine. The first experiment (Chapter III) investigated the contributory role of individual Cl- channels in the recovery of barrier function in ischemia-injured porcine ileum. Ischemia-injured porcine ileal mucosa was mounted in Ussing chambers. Short circuit current (Isc) and transepithelial resistance (TER) were measured in response to PGE2 and pharmacologic inhibitors of epithelial Cl- channels. Overall, results from these studies demonstrate that ClC-2-mediated intestinal Cl- secretion restores TER in ischemia-injured intestine. Chapter IV entails a study aimed at more directly investigating the role of ClC-2 in mucosal repair by evaluating mucosal repair in ischemia-injured intestinal mucosa mounted on Ussing chambers treated with the selective ClC-2 agonist, lubiprostone. Results from this suggest that activation of ClC-2 with the selective agonist, lubiprostone, stimulated elevations in TER and reduction in mannitol flux in the Ischemia-injured intestine. In Chapter V, experiments focused on the role of individual NHE isoforms in the recovery of barrier function in ischemia-injured porcine ileum. Results from this study demonstrate that inhibition of NHE2 activity, possibly via EBP50, induces recovery of barrier function in ischemic-injured intestine
Molecular Mechanisms Characterizing Skeletal Muscle Phenotype and Function.
(2010-09-15) Nierobisz, Lidia Sylwia; Paul Mozdziak, Committee Chair; Jack Odle, Committee Member; Anthony Blikslager, Committee Member; James Felts, Committee Member; Amy Grunden, Committee Member
Nutritional Interrelationships between Iron, Copper and Manganese in Domestic Livestock
(2009-03-04) Hansen, Stephanie Laura; William Flowers, Committee Member; Vivek Fellner, Committee Member; Jerry Spears, Committee Chair; Jack Odle, Committee Member
Oftentimes the diets of cattle and pigs contain levels of iron well beyond the nutritional requirement of the animal. This superfluous iron may come from feedstuffs naturally high in iron, or from the consumption of soil, though bioavailability of iron from soil is unknown. Additionally, excess iron in cattle diets has been shown to negatively impact the metabolism of manganese and copper, though the molecular mechanism behind this interaction is unclear. The purpose of this research was threefold: 1) to determine the effect of ensiling on bioaccessibility of iron from soil contamination of corn greenchop, 2) to identify proteins important in iron metabolism in bovine and swine, and 3) to determine if these proteins are affected by dietary iron concentration. The results reported herein suggest that bioaccessibility of iron from soil contamination is greatly increased when soil undergoes a prolonged exposure to a low pH environment, such as that found with fermenting forages. These data indicate that very little iron from soil is available to the animal if no prior exposure to an acidic environment occurs. Also, for the first time we report that several proteins known to be essential to iron metabolism in rodents are present in bovine small intestine and liver. Specifically, the iron importer divalent metal transporter 1, the iron exporter ferroportin, and the multi-copper ferroxidase hephaestin were all present in bovine duodenum. In the bovine, reduced iron status, as induced by a primary deficiency of copper, resulted in increased gene expression of divalent metal transporter 1 and ferroportin in duodenum and decreased expression of the ferroportin regulatory hormone hepcidin and divalent metal transporter 1 in liver. Protein expression of ferroportin and hephaestin were also increased in duodenum due to reduced iron status. The addition of excessive amounts of iron to the diets of young calves also appeared to regulate protein expression of transporters important in iron metabolism. Specifically, high iron tended to decreased duodenal protein levels of divalent metal transporter 1 and reduced ferroportin protein levels, though no effect on hephaestin was observed. We also examined iron metabolism in the young pig, in order to examine the effect of an iron deficient diet on expression of these proteins. We found that hephaestin protein in the duodenum was lowered by feeding a high iron diet, and levels of both ferroportin and divalent metal transporter 1 tended to be reduced by high dietary iron compared to pigs fed a low iron diet. Additionally, we found that feeding a high iron diet to pigs negatively impacted liver concentrations of manganese. And feeding a high iron diet to either pigs or calves reduced duodenal concentrations of manganese, suggesting that high dietary iron reduces manganese absorption. Because high iron diets fed to both pigs and calves tended to reduce duodenal levels of divalent metal transporter 1, a protein known in rodents to transport both iron and manganese, it appears that the observed reductions in duodenal manganese concentrations may be a result of reduced transporter availability. Collectively, our data suggest that high dietary iron may negatively affect manganese absorption, and because the iron content of livestock diets is often high, further research is warranted.
Reduction of Cardiovascular Disease Risk Factors and Atherosclerosis in Male Syrian Golden Hamsters by Peanuts, Peanut Oil and Fat Free Peanut Flour
(2008-08-27) Stephens, Amanda Mae; Jonathan C Allen, Committee Co-Chair; Timothy H Sanders, Committee Co-Chair; Jack Odle, Committee Member
Regulation of COX-2 and PGE2-Dependent Recovery of Intestinal Barrier Function
(2004-09-29) Shifflett, Donnie Edward; Anthony T. Blikslager, Committee Co-Chair; Samuel L. Jones, Committee Co-Chair; Jody L. Gookin, Committee Member; Jack Odle, Committee Member
The regulation of intestinal barrier function is of importance in various clinical situations. For example, in cases of ischemia/reperfusion injury, there is a disruption in the gastrointestinal barrier. This may lead to release of pro-inflammatory cytokines and neutrophil (PMN) infiltration. It has been demonstrated that COX-2, which is upregulated during inflammation, and its downstream product PGE2 are important for recovery of barrier function, yet their regulation has not been fully characterized. Study 1: To study the effects of PMNs on acutely injured mucosa, we applied PMNs isolated from circulation to ischemic-injured porcine ileal mucosa. Since COX-2 is upregulated by inflammatory mediators such as IL-1beta, which is released by PMNs, we postulated that PMNs enhance recovery of ischemia-injured mucosa by a pathway involving IL-1beta and COX-2. Application of 5x10⁶ PMNs to ischemia-injured ileal mucosa significantly enhanced transepithelial resistance (TER), an effect inhibited by the selective COX-2 inhibitor NS-398 and an IL-1beta receptor antagonist. Western blots revealed up-regulation of COX-2 in response to PMNs, which was inhibited by an IL-1beta receptor antagonist. Real time PCR revealed increased mRNA COX-2 expression, which preceded increased COX-2 protein expression in response to IL-1beta. We concluded that PMNs augment recovery of TER in ischemia-injured ileal mucosa via IL-1beta-dependent upregulation of COX-2. Study 2: Mitogen activated protein kinase (MAPK) pathways transduce signals from a diverse array of extracellular stimuli, including IL-1beta. The three primary MAPK signaling pathways are the extracellular regulated kinases (ERK 1&2), p38 MAPK, and c-Jun NH2-terminal kinase (JNK). Because COX-2 expression is regulated in part by MAPK's, we postulated that MAPK pathways would play an integral role in recovery of porcine ischemia-injured ileal mucosa. Treatment of tissues with the p38 MAPK inhibitor SB-203580 or the ERK 1&2 inhibitor, PD-98059, abolished recovery. Western blots revealed that SB-203580 inhibited upregulation of COX-2 whereas PD-98059 had no effect on COX-2 expression. Inhibition of TER by SB-203580 or PD-98059 was overcome by exogenous PGE2. The JNK inhibitor, SP-600125, significantly increased TER and resulted in COX-2 upregulation. Thus, COX-2 expression appears to be positively and negatively regulated by the p38 MAPK and the JNK pathways respectively. Alternatively, ERK 1&2 appears to be involved in COX-2-independent reparative events. Study 3: In previous studies, we had shown that PGE2 restored barrier function via a signal transduction pathway involving Cl⁻ secretion and recovery of interepithelial tight junctions (TJs). To study these mechanisms, we utilized human colonic T84 cells. We postulated that PGE2 induced chloride secretion would precede increases in TER associated with re-distribution of critical proteins to TJs. T84 cells were grown to confluence, but utilized at lower TER values (200-500 ohms.cm²) to simulate our ileal mucosal model of 'leaky' restituted epithelium. Basolateral application of PGE2 induced transient increases in Isc, indicative of chloride secretion, followed by sustained increases in TER. Basolateral application of the Na/K/2Cl cotransporter inhibitor bumetanide, abolished the PGE2-induced rise in Isc and subsequent elevations in TER. PGE2 induced a shift in claudin-1 from the Triton-X soluble to insoluble fraction, beginning 4-hour after PGE2 administration, which was prevented by bumetanide. Alternatively, there were no changes in occludin or claudins-3 and -5. Immunoflourescence demonstrated that PGE2 increased accumulation of claudin-1 at the apical lateral membrane. Additionally, we showed that PGE2 increased tyrosine phosphorylation of claudin-1 within 30 min, an effect prevented by bumetanide. Therefore, PGE2-induced chloride secretion in T84 cells is directly linked with increases in TER, and these elevations in TER are associated with phosphorylation of claudin-1 and a shift in claudin-1 to the tight junction.
Understanding Rumen Fermentation: I. Effect of high DHA algal oil on microbial biohydrogenation and II. Monitoring microbial shifts in response to antibiotics and oil using T-RFLP analysis.
(2007-11-14) Johnson, Meredith Christina; Vivek Fellner, Committee Chair; Jack Odle, Committee Member; Amy Grunden, Committee Member