THE APPALACHIAN STUDENT RESEARCH FORUM, April 5, 2012
Our Undergraduate Students
CHARACTERIZATION OF THE MICROVASCULATURE OF MOUSE UTERINE TISSUE USING VASCUALR CORROSION CASTING AND SCANNING ELECTRON MICROSCOPY
Steven Kyle Fleener and Dr. Allan Forsman
The vessels that supply blood to the endometrial lining of the uterus are of considerable interest due to their ability to constantly undergo changes throughout the reproductive life of the female. The purpose of this study is to characterize the changes in the microvasculature that supplies the endometrium of mice during different stages of the estrous cycle. To accomplish this, a technique known as vascular corrosion casting is used to produce a replica of the uterine vasculature that can then be examined with scanning electron microscopy. The success of this study relies heavily on being able to determine the stage of the estrous cycle that each test subject is in at the time of the casting procedure. A procedure known as a vaginal wash was performed to approximate the stage of each animal's estrous cycle. Our initial goal is to create and evaluate uterine vascular casts from 3 animals for each stage of the estrous cycle (diestrous, proestrous, estrous, and metestrous,). By using scanning electron microscopy, the casts can be viewed at the ultra structural level to detect any changes in the vasculature. It is important to fully understand the changes in this vasculature since the mouse is a commonly used research animal. The information gained from this study should be useful to future researchers when designing their individual experiments.
DOSE-DEPENDEN EFFECT OF ESTROGEN ON INTESTINAL LIPOPROTEIN SECRETION
Forrest J. Longway and Dr. Andromeda M. Nauli
Background: Studies on intestinal lipid absorption has been hampered by the lack of a cell culture model. Although it has been suggested that female rats have a different intestinal lipoprotein profile than male rats, it is not known whether this difference is caused by estrogen. AIM: The purpose of our studies is to determine if estrogen has a dose-dependent effect on intestinal lipoprotein secretion using our in vitro cell culture model. Methods: Two-week postconfluent Caco-2 cells were incubated with a lipid solution (4 mM oleic acid, 0.8 mM mono-olein, 0.68 mM lecithin, and 1mM sodium taurocholate in 15% FBS in DMEM) containing 0 pg/ml, 40 pg/ml, 160 pg/ml, or 400 pg/ml of beta-estradiol (estrogen; physiological range) (n=2 each). As a negative control, cells were incubated with lipid solution above but without the oleic acid and estrogen. After 16 hours of incubation, the cells were washed twice, and the lipoprotein secretions were collected for 2 hours in DMEM. To isolate the lipoprotein particles from the collected media, samples were spun at 30,000 rpm for 2.5 hours with ultracentrifugation (Thermo T-867 rotor), and the lipoprotein fractions were analyzed by triglyceride (TG) and phosphatidylcholine (PC) colorimetric assay. Results: Based on our preliminary results, the TG concentrations were 9.06 mg/dl, 10.45 mg/dl, 12.94 mg/dl, and 14.33 mg/dl for 0, 40, 160, and 400 pg/ml estrogen, respectively. The PC concentrations were 23.36 mg/dl, 25.59 mg/dl, 31.76 mg/dl, and 26.25 mg/dl for 0, 40, 160, and 400 pg/ml estrogen, respectively. The negative control (without oleic acid and estrogen) had 1.94 mg/dl TG and 26.72 mg/dl PC. Conclusions: Our preliminary study suggests that there is a dose-dependent effect of estrogen on TG secretion. The negative control displayed a very low TG concentration, confirming that Caco-2 cells utilize the oleic acids for TG secretion. The high PC in the negative control was expected since the cells were supplied with lecithin, which contain PC. PC secretion did not seem to show a dose-dependent effect probably due to constant secretion of very small lipoprotein particles that are known to have very high PC to TG ratio. We will analyze further the size/type of lipoproteins, the relative amount of apolipoproteins, and the effect of other sex hormones on intestinal lipoprotein secretion. Our studies suggest that estrogen regulates intestinal lipoprotein secretion, which may consequently lead to gender differences in cardiovascular diseases and body adiposity.
EFFECTS OF MICROGRAVITY ON MUCIN PRODUCTION AND MUCIN PRODUCING CELLS OF MOUSE UTERINE TUBES
Gorica Svalina and Dr. Allan D. Forsman
Numerous studies have indicated that the microgravity environment of space has harmful effects on several tissues throughout the body. Although this phenomenon is well documented, research in this area is still in its relative infancy. This study investigates the effects of spaceflight on mucin production and the mucin producing cells of the uterine tubes of mice. To determine the possible effects of microgravity on mucin production, we examined the epithelium of the uterine tubes from female mice that were flown on the space shuttle Endeavour for 12 days in August, 2007. This study utilized three sets of female mice, with each set consisting of 12 animals. The three sets were designated as Flight, Ground Control, and Baseline. The flight animals were flown in the Commercial Biomedical Testing Module-2 (CBMT-2), which was housed in the shuttle's mid-deck locker area. Ground control animals were also housed in CBTM-2 units, which were kept in environmentally controlled rooms at the Space Life Sciences Lab at Kennedy Space Center. These units were on a 48 hour delay in relation to the flight CBTM-2 units. Baseline animals were also housed at the Space Life Sciences Lab but were housed in standard rodent cages with ambient temperature and humidity, with a 12/12 light dark cycle. The tissue was paraffin embedded, sectioned, mounted, and histologically stained using an Alcian Blue Periodic Acid Schiff staining procedure. The tissue was qualitatively analyzed for the amounts of mucins produced by measuring the width of the mucin layer in micrometers for each section of the uterine tube: isthmus, ampulla, and infundibulum. The ANOVA single factor test was used to compare these measurements between all three sets of animals. Preliminary analysis of three flight animals, one ground control animal, and seven baseline animals showed no significant difference in the thickness of the mucin layer at an alpha level of 0.05. Further analysis will need to be conducted for a proper conclusion to be made. If it is determined that spaceflight causes a decrease in mucin production in the uterine tubes it could indicate that reproduction in a spaceflight environment would be greatly impaired. Along with the microgravity of spaceflight comes a high exposure to cosmic radiation. To date it has been virtually impossible to determine if some of the deleterious effects attributed to microgravity may actually be caused by radiation exposure. If this is the case, extensive ground based studies would have to be designed to test that hypothesis. If radiation does have adverse effects on mucin production in uterine tubes, this could have a large impact on the use of radiation therapy in women who hope to become pregnant after therapy.
INDUCING CHYLOMICRON PRODUCTION WITH A MIXTURE OF LIPIDS AND BILE ACIDS
Roger W. Thompson Jr., Peter Laska, Seif Atyia, and Dr. Andromeda Nauli
Background: About 95% of our dietary fat is triglycerides (TGs), while the remaining 5% consists of phospholipids (PLs), cholesterol, and fatty acids (FAs). In the presence of bile acids, dietary TGs are hydrolyzed into monoglycerides (MGs) and FAs; and PLs into lyso-PLs and FAs. These lipid digestion products are then transported by chylomicrons (>80 nm in diameter) from the intestine to the circulation. Currently, there is an absence of a satisfactory in vitro model to study chylomicrons. AIM: The purpose of our study is to determine the optimal lipid mixture that induces chylomicron production. Methods: Two-week post-confluent Caco-2 cells, which were grown in DMEM containing 15% fetal bovine serum (FBS), were incubated with varying lipid mixtures for 16 hours, followed by 2-hour collection of lipoprotein secretion (minimum n=3). The collected lipoproteins were isolated by ultracentrifugation, which were analyzed by TG and PL colorimetric assays, as well as transmission electron microscopy (TEM) using negative staining method. The varying lipid mixtures tested contained: oleic acids (0, 1.6, 4, or 4.8 mM), mono-olein (0, 0.4, 0.8, or 1.6 mM), egg-lecithin (0, 0.34, 0.68, or 1.34 mM), and 1 mM sodium taurocholate. Results: One-way ANOVA shows that there was a dose-dependent effect of OA on TG secretion (P=0.0073; n=3) but not on PC (P=0.133; n=3) secretion. MG (P=0.069; n=4) and lecithin (P=0.094; n=2) appeared to have a dose-dependent effect as seen by the upward trend of TG secretion. However, in regards to PL secretion both MG (P=0.64; n=4) and lecithin (P=0.60; n=2) appear to have negligible effect. Importantly, the presence of numerous chylomicrons was confirmed by TEM. Conclusions: Our results suggest that OA is the most important type of lipid in a mixture for inducing chylomicron production relative to MG and lecithin. PL secretion did not seem to be affected by any of the components of the lipid mixture probably due to the presence of other minute lipid particles. Our in vitro chylomicron model will be valuable for studying the molecular mechanisms of chylomicron metabolism and their atherogenic properties.
INTESTINAL MORPHOLOGY IS ALTERED IN STRESSFUL CONDITIONS
Ksenia Zhuravleva and Dr. Edward M Onyango
The protective mucosal barrier of the gastrointestinal tract consists in part of heavily glycosylated proteins called mucins. This study investigated the effects of psychological stress on mouse intestinal morphology especially that of mucin-producing cells. Intestinal tissues were obtained from two groups of mice one group (test group) was exposed to psychological stress (social defeat) for 10 days and the other group (the control group) was not exposed to stress. Intestinal portions were removed and fixed in Methacarn, embedded in paraffin, sectioned and stained using Alcian Blue-Periodic acid Schiff to visualize the mucin-producing goblet cells. Goblet cells in the intestinal villi of the different groups were counted. The heights of villi were also determined. Histology showed a decrease in the number of goblet cells in stressed mice compared to control mice. Villi length was lower in stressed mice compared to control animals. The findings suggest that stress may affect the dynamics of mucin production and the overall absorptive surface in the intestinal tract of mice.
THE APPALACHIAN STUDENT RESEARCH FORUM, April 5, 2012
Our Graduate Students
DETECTION AND PURIFICATION OF A NATURAL INHIBITORY COMPOUND FROM AN ISOLATED STRAIN OF RHODOCOCCUS USING AN AGAR EXTRACTION METHOD
Megan Carr and Bert Lampson
The soil bacterium Rhodococcus has a wide array of secondary metabolic pathways such as production of pigments, siderophores, and antibiotics which makes it an organism of interest for the production of novel natural products. Analysis of the genome sequence of Rhodococcus indicates the presence of 24 non-ribosomal peptide synthases and 7 polyketide synthases possibly involved in production of secondary metabolites. The use of a solid agar extraction method to screen soil isolates of Rhodococcus for compounds with inhibitory activity against other bacteria resulted in the discovery of a promising candidate molecule. The Rhodococcus strain KCHXC3, previously isolated from eastern Tennessee soil, produces a substance that inhibits the growth of several Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei and Gram-positive bacteria such as Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, among others. After bulk extraction of this compound with ethyl acetate from agar plates, the material was partially purified through different combinations of LH20 column chromatography, silica gel column chromatography, high pressure liquid chromatography and thin layer chromatography.
THE EFFECTS OF INTRACEREBROVENTRICULAR LEPTIN ON MILK AVAILABILITY IN LACTATING RATS
Brittany Moore and Effiong Otukonyong
Leptin, a hormone secreted from white fat cells, has a well established role in the regulation of energy balance. In addition, leptin and energy balance have been linked to improved fertility in rodents and humans. In cultured rat endometrial cells, exogenous leptin increases the secretion of prolactin, a hormone which stimulates milk production. Prolactin, in turn, has a reciprocal effect on leptin by enhancing leptin secretion from white adipose tissue. Recent evidence indicates that leptin hormone is present in breast milk and leptin receptors are well expressed in mammary epithelial cells. This suggests that leptin could be involved in stimulating the growth of mammary epithelial cells and thus improving milk yield in lactating breast. The hypothesis was tested that insufficient levels of leptin in the brain feeding center, the hypothalamus, may underlie infertility in rodents and humans and the failure of lactating breast to express an adequate amount of milk. If enough leptin can be administered to the hypothalamus, fertility and milk yield could improve.
All animals used in this study were stereostaxically implanted with permanent stainless steel cannula into the third cerebroventricle and kept in a controlled, specific pathogen-free room. Food and water were made available ad libitum. Female Sprague-Dawley rats of the same age weighing 200-250 grams were divided into two groups. One group, leptin treated, was injected with 5 l of pure recombinant leptin for 7 days while the control group was injected with 5 l saline for the same period. The estrous cycle was determined and monitored for each female rat. The females were mated with male Sprague Dawley rats upon the proestrus cycle of estrous. The females were observed throughout the pregnancy until delivery. Beginning four days after delivery, the dams and pups were then observed during a one hour period of suckling in the morning and evening for a total of 7 days. The body weights of both the dam and litter were measured before and after each period of suckling. At the end of this observation period, both the dams and the litter were sacrificed by decapitation. Blood was collected and brains were harvested for use in further analysis.
Serum levels of prolactin were found to be increased in the leptin treated dams. Body weight and litter size were also increased in the leptin treated group when compared to the control rats treated with saline. Results from this study suggest that leptin treatment plays a positive role in reproduction and milk yield. These findings could lead to the improvement of future clinical treatment of women with infertility and deficiency of lactation.
DETERMINING THE OPTIMAL CONDITIONS FOR IN VITRO CHYLOMICRON PRODUCTION
Yuxi Sun, Roger W. Thompson, and Andromeda M. Nauli.
BACKGROUND: There has not been a good in vitro model for producing chylomicrons, the largest lipid particles (diameter > 80 nm). Our lab has determined the types and amounts of lipids that will efficiently induce chylomicron production. Other conditions, such as the differentiation stage of the cells, the incubation time of the lipids, the collection time of the secreted lipoproteins, and the amount of fetal bovine serum (FBS) have not been determined. AIM: To determine the above conditions that are optimal for chylomicron production. METHODS: Based on our preliminary studies, the lipid mixture that efficiently induces chylomicron production consists of 4mM oleic acid, 0.8mM mono-olein, 0.68mM egg-Lecithin and 1mM sodium taurocholate. Differentiated Caco-2 cells were incubated with the above lipid mixture, followed by the collection of lipoprotein secretion. The collected media was then spun with an ultracentrifuge. The triglyceride (TG) and phosphatidylcholine (PC) concentrations of the top layer were measured by using colorimetric assays, and chylomicron presence was determined by transmission electron microscopy (TEM). Higher TG to PC ratio would suggest more efficient chylomicron production since chylomicrons have TG-rich core surrounded by phospholipid monolayer. To determine the optimal conditions for chylomicron production, we tested the effects of the differentiation stage of the cells (11, 14, 15, 18, and 21-day postconfluent), incubation time of lipid mixture (16, 19, 22, 25, and 28 hours), collection time of lipoprotein secretion (2, 6, 10, 18 hours), and optimal % FBS during lipid incubation (5, 15, 35, and 45%) and lipoprotein collection (5, 15, 25, 35, and 45%). RESULTS: One-way ANOVA shows that there was a significant effect of cell differentiation on the PC secretion (P=0.0071) but not TG secretion (P=0.15); the PC secretion was significantly lower in 15-day postconfluent cells. On the other hand, lipid incubation time had a significant effect on TG secretion (P=0.0034) but not PC secretion (0.087). The 22-hour lipid incubation time was optimal in inducing TG secretion. The lipoprotein collection time was found to be critical for both TG (P=0.017) and PC (P=0.011) secretion with 6 hours being the optimal for TG; and both 2 and 6 hours equally optimal for PC. The % FBS during lipid incubation did not show any significant effect on both TG (P= 0.12) and PC (P=0.22) secretion. However, the % FBS during lipoprotein collection had a significant effect on both TG (P=0.018) and PC (P=0.003) secretion with 15% or more being the optimal for both TG and PC secretion. CONCLUSIONS: Our cumulative data suggest that 15-day postconfluence, 22-hour lipid incubation, 6-hour lipoprotein collection, 15% FBS during lipid incubation resulted in optimal chylomicron secretion, which was confirmed by TEM. These optimal conditions will allow us to produce large amount of chylomicrons, which can be used for obesity and cardiovascular studies.
EAST TENNESSEE STATE UNIVERSITY BOLAND UNDERGRADUATE RESEARCH SYMPOSIUM, April 3, 2012
EFFECTS OF SPACEFLIGHT ON MUCIN PRODUCTION AND MUCIN PRODUCING CELLS IN THE URINARY BLADDER OF MICE
Brandon Farmer and Allan Forsman
The effects of the microgravity of spaceflight are largely unexplored with regard to biological tissues. One particular area of interest is the possible effects microgravity could have on the production of mucins. To determine the possible effects of microgravity on mucin production in the urinary bladder, we examined the transitional epithelium of the urinary bladder from female mice that were flown on the space shuttle Endeavour for 12 days in August, 2007. The flight tissue was compared to tissues from two control groups of animals, ground control and baseline. This study utilized three sets of female mice, with each set consisting of 12 animals. The three sets were designated as Flight, Ground Control, and Baseline. The flight animals were flown in the Commercial Biomedical Testing Module-2 (CBMT-2) which was housed in the shuttle's mid-deck locker area. Ground control animals were also housed in CBTM-2 units which were kept in environmentally controlled rooms at the Space Life Sciences Lab at Kennedy Space Center. Baseline animals were also housed at the Space Life Sciences Lab but were housed in standard rodent cages with ambient temperature and humidity, with a 12/12 light dark cycle. Bladder tissue was paraffin embedded, sectioned, mounted, and histologically stained using an Alcian Blue Periodic Acid Schiff staining procedure. The bladder tissue from the three treatment groups is being qualitatively analyzed for mucin thickness and types of mucins produced. To date the study indicates that the mucin layer of the Flight tissue is thinner than that of the Baseline or Ground Control tissue, but only significantly thinner than the Baseline tissue.
ISOLATION OF algZ
Ima Isang and Ranjan Chakraborty
Pseudomonas aeruginosa, a gram negative bacillus, commonly infects individuals that are immunocompromised such as patients who have AIDS, burns, or more specifically cystic fibrosis. A mutation in the gene for cystic fibrosis transmembrane conductance regulator (CFTR) results in faulty chloride ion transport across epithelial cells in the lungs.A consequence to this defect is the buildup of thick dehydrated mucus that the mucocilliary escalator of the respiratory tract is unable to clear. Pathogens, especially Pseudomonas aeruginosa, thrive in this dense mucus and contribute to the chronic pulmonary disease that is resistant against the body's immune system as well as antibiotic treatment. The ability of P. aeruginosa to evade host defenses is due to the production of alginate, a polymer of mannnuronic acid and guluronic acid. Alginate has the ability to obstruct phagocytosis, to prevent complement activation, to prevent the migration of immune cells to site of infection, among other functions. Antibodies to alginate have proven to be ineffective in cystic fibrosis patients possibly due to the aforementioned factors. Furthermore, biofilms, colonies of bacteria enclosed within alginate produce a synergistic effect towards the body. Mucoidy, when these microorganisms overproduce alginate, precedes the formation biofilm. It has been observed that cystic fibrosis patients who sustain chronic infections contain within their sputum P. aeruginosa in the mucoidy. If the production of alginate can be targeted, possibly remedies for the pulmonary disease attributed to P. aeruginosa may be uncovered. The gene that encodes for the synthesis of an enzyme that aids in the production of guluronic acid, algD, is a target in cessation of alginate production. AlgD is regulated by a two-component kinase transduction system, composed of a response regulator, AlgR, and sensor AlgZ. Although the mechanism of the interaction of AlgR and AlgZ is unknown, it has been reported that in mucoid strains of P. aeruginosa, inactivation of algZ results in the increase of production of alginate. In this research project, the algZ gene is isolated in order to be later purified and crystallized. With the crystal structure of AlgZ determined, the interaction between AlgZ and AlgR may be understood, and thus the mechanism of signal transduction may be derived.