Isolation and profiling of integral membrane proteins from developing Brassica napus [abstract]

Abstract only availableFaculty Mentor: Dr. Jay Thelen, BiologyDuring seed development the concentration of starch, oil, and protein fluctuates. At the early stages of seed filling in oilseeds starch is the principal component. Oil (triacylglycerol) and protein concentrations do not reach a maximum until the later stages of seed development. This metabolic shift within the seed, from production of starch to production of oil and protein, indicates that seed metabolism is regulated temporally. To better understand these metabolic changes it is useful to examine the cognate changes in protein expression. Integral membrane proteins represent one class of proteins which are important for inter-organellar metabolic flow. Current two-dimensional electrophoresis techniques are unsuitable for the profiling of hydrophobic membrane proteins. To specifically characterize this class of proteins, this project was focused on developing a reproducible protocol for membrane protein isolation that can be used with standard sodium dodecyl sulfate polyacrylamide gel electrophoresis. Three different techniques for the isolation of integral membrane proteins were compared, and the resulting gels will be presented

Protoemics of integral membrane proteins from developing Brassica napus

Abstract only availableAs plant seeds develop the accumulation of natural products, starch, oil, and protein undergo dramatic changes. At the early stages of seed filling in oilseeds starch is the principal component. Oil (triacylglycerol) and protein concentrations do not reach a maximum until the later stages of seed development. This metabolic shift within the seed, from production of starch to production of oil and protein, indicates that seed metabolism is regulated temporally. To better understand these metabolic changes it is important to examine the cognate changes in protein expression. Integral membrane proteins represent one class of proteins which are important for inter-organellar metabolic flow. Current two-dimensional electrophoresis techniques are unsuitable for the profiling of hydrophobic membrane proteins. To specifically characterize this class of proteins, a reproducible protocol for membrane protein isolation that can be used with standard sodium dodecyl sulfate polyacrylamide gel electrophoresis needed to be developed. Alkaline sodium carbonate washing of membranes followed by ultracentrifugation appeared to yield washed membrane fractions distinct from total protein fractions. To quantify relative volume and molecular weights of individual bands, Coomassie stained gels were analyzed with ImageQuant software. Identification of these bands was performed by trypsin digesting each protein (in-gel) and obtaining accurate peptide mass 'tags' using Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry. Peptide mass fingerprinting resulted in twelve conclusive identifications (out of 25 analyzed). Of these, six proteins were involved in the glucosinolate-myrosinase defense pathway. These proteins are suspected to be membrane associated, and are involved in a defense system that protects plant tissues from herbivory and fungal, viral, and bacterial infections. Other proteins were identified as: the RuBisCO large subunit, histone H3, NADH dehydrogenase subunit, pyruvate dehydrogenase E1 alpha subunit, and two types of cruciferins which are seed storage proteins. Of these, only NADH dehydrogenase is an integral membrane protein. Based on this data, the alkaline sodium carbonate wash method did not effectively enrich for integral membrane proteins. This may be due largely to the fact that certain proteins, especially cruciferin seed storage proteins, RuBisCO and myrosinases, are expressed at much higher levels than integral membrane proteins and are not quantitatively removed from membrane fractions by salt washing alone. Future work will include alternative approaches to membrane protein isolation including organic extraction.MU Monsanto Undergraduate Research Fellowshi

Effects of prenatal exposure to xenobiotic estrogen and the development of endometriosis in adulthood

Abstract only availableEndometriosis is an estrogen-dependent disease that affects millions of women worldwide, causing pain and infertility. While it is known that retrograde menstruation places endometrial tissue in the peritoneal cavity, it is unclear why it invades and proliferates in women with endometriosis. Studies have shown that other hormone-dependent diseases have a fetal basis (e.g. breast cancer), suggesting that the presence of different hormones before birth may alter the incidence of endometriosis in adulthood. For example, women whose mothers took the synthetic estrogen diethylstilbestrol (DES) during pregnancy had an eighty percent increased incidence of endometriosis. Thus, our hypothesis is that prenatal exposure to xenobiotic estrogen will increase the severity of endometriosis in adulthood in a mouse model of surgically-induced endometriosis. To test this hypothesis, mice were time mated and dosed with vehicle control, 100 ng/kg DES or 10,000 ng/kg DES from days 11-17 of gestation. Surgical induction of endometriosis was performed in adulthood by autotransplantation of one uterine horm. The horn was removed, opened, divided into three pieces, and sutured to the arterial cascade of the intestinal mesentery. The implants became vascularized and formed endometriotic lesions. The mice were then collected at 2 or 4 weeks post-surgery, and the following endpoints were measured: 1) uterine weight; 2) implant size; and 3) implant weight. Additionally, implants were set aside for further analysis of 1) histology; 2) estrogen receptor indicator reporter gene activity; and 3) endometriosis-related gene expression. At the conclusion of this ongoing study, we expect to show whether there is an estrogen-mediated fetal component to endometriosis.Life Sciences Undergraduate Research Opportunity Progra

Neonatal exposure to xenobiotic estrogen alters the adult immune response and exacerbates endometriosis in mice [abstract]

Faculty Mentor: Dr. Susan C. Nagel, Obstetrics/Gynecology, and Women's HealthAbstract only availableEndometriosis is a common medical condition affecting 5-10% of women worldwide, and results in severe cramps, pelvic pain, and infertility. The cause of the disease is still unknown. Endometriosis occurs when endometrial tissue, which escapes into the peritoneal cavity via retrograde menstruation, adheres to other tissues in the cavity and causes irritated, inflamed lesions. Studies have suggested that the risk of developing endometriosis increases in women who have been exposed to xenobiotic (foreign to the body) estrogens during developmental stages of life. Thus, it is our hypothesis that programming of the immune system by xenoestrogens during development could potentially exacerbate endometriosis. This could occur by altering the peritoneal environment and/or the invading endometrial tissue. Therefore, it is our goal to study the effects of neonatal xenoestrogen exposure on the immune system; and ultimately, on the establishment of endometriosis in adulthood. In order to study this response, we dosed two strains of mice (CD1 and C57) with xenobiotic estrogens on postnatal days 2-14. In experiment A, CD1 mice were dosed with vehicle control (corn oil), 20 µg/kg/day, or 200 µg/kg/day bisphenol A. In experiment B, C57 mice were dosed with a vehicle control (corn oil) or 0.1 µg/kg/day diethylstilbestrol. At 8 weeks of age, endometriosis was induced in each strain via both a surgical induction and an injection technique. At 12 weeks, the endometriotic implants were counted and weighed to determine which mice had a greater susceptibility to the condition. Our next objective will be to analyze peritoneal fluid from the treated mice to identify key immune functions (for example, the release of certain cytokines) that may have been programmed by developmental xenoestrogen exposure.Endometriosis is a common medical condition affecting 5-10% of women worldwide, and results in severe cramps, pelvic pain, and infertility.  The cause of the disease is still unknown.  Endometriosis occurs when endometrial tissue, which escapes into the peritoneal cavity via retrograde menstruation, adheres to other tissues in the cavity and causes irritated, inflamed lesions.  Studies have suggested that the risk of developing endometriosis increases in women who have been exposed to xenobiotic (foreign to the body) estrogens during developmental stages of life.  Thus, it is our hypothesis that programming of the immune system by xenoestrogens during development could potentially exacerbate endometriosis.  This could occur by altering the peritoneal environment and/or the invading endometrial tissue.  Therefore, it is our goal to study the effects of neonatal xenoestrogen exposure on the immune system; and ultimately, on the establishment of endometriosis in adulthood.  In order to study this response, we dosed two strains of mice (CD1 and C57) with xenobiotic estrogens on postnatal days 2-14.  In experiment A, CD1 mice were dosed with vehicle control (corn oil), 20 µg/kg/day, or 200 µg/kg/day bisphenol A.  In experiment B, C57 mice were dosed with a vehicle control (corn oil) or 0.1 µg/kg/day diethylstilbestrol.  At 8 weeks of age, endometriosis was induced in each strain via both a surgical induction and an injection technique.  At 12 weeks, the endometriotic implants were counted and weighed to determine which mice had a greater susceptibility to the condition.  Our next objective will be to analyze peritoneal fluid from the treated mice to identify key immune functions (for example, the release of certain cytokines) that may have been programmed by developmental xenoestrogen exposure

Neonatal exposure to xenobiotic estrogen may alter the adult immune response and exacerbate endometriosis in mice [abstract]

Abstract only availableEndometriosis is a common medical condition affecting 5-10% of women worldwide and often results in severe cramps, pelvic pain, and infertility. The condition occurs when endometrial tissue, which escapes into the peritoneal cavity via retrograde menstruation, adheres to peritoneal cavity tissues and causes irritated, inflamed lesions. Studies have suggested that the risk of developing endometriosis increases in women who have been exposed to xenobiotic (foreign to the body) estrogens during development. This could be due to developmental programming of the peritoneal environment, and specifically, an altered immune function within this environment. Therefore, it is our hypothesis that developmental programming by xenoestrogens alters the immune response to shed endometrial tissue and exacerbates endometriosis. To better understand the role of xenoestrogens in immune programming, we are conducting our studies using a mouse model of surgically induced endometriosis. In particular, we are concentrating on two major aspects of immunity: 1) the presence of immune cells and 2) the function of those cells. Our study of the former is being largely performed using methods of immunohistochemistry (IHC). IHC allows us to quantify the macrophages present in the peritoneal fluid of experimental mice (exposed to diethylstilbestrol) versus control mice (no xenoestrogen exposure). In order to study our second focus, immune cell function, we are using a cytokine antibody array to determine the relative cytokine concentrations in the peritoneal fluid samples. By identifying the degree to which certain cytokine concentrations differ, we hope to better understand the effect of xenoestrogen exposure on immune cell function.Life Sciences Undergraduate Research Opportunity Progra

Proteomic Analysis of Seed Filling in Brassica napus. Developmental Characterization of Metabolic Isozymes Using High-Resolution Two-Dimensional Gel Electrophoresis

Brassica napus (cultivar Reston) seed proteins were analyzed at 2, 3, 4, 5, and 6 weeks after flowering in biological quadruplicate using two-dimensional gel electrophoresis. Developmental expression profiles for 794 protein spot groups were established and hierarchical cluster analysis revealed 12 different expression trends. Tryptic peptides from each spot group were analyzed in duplicate using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry. The identity of 517 spot groups was determined, representing 289 nonredundant proteins. These proteins were classified into 14 functional categories based upon the Arabidopsis (Arabidopsis thaliana) genome classification scheme. Energy and metabolism related proteins were highly represented in developing seed, accounting for 24.3% and 16.8% of the total proteins, respectively. Analysis of subclasses within the metabolism group revealed coordinated expression during seed filling. The influence of prominently expressed seed storage proteins on relative quantification data is discussed and an in silico subtraction method is presented. The preponderance of energy and metabolic proteins detected in this study provides an in-depth proteomic view on carbon assimilation in B. napus seed. These data suggest that sugar mobilization from glucose to coenzyme A and its acyl derivative is a collaboration between the cytosol and plastids and that temporal control of enzymes and pathways extends beyond transcription. This study provides a systematic analysis of metabolic processes operating in developing B. napus seed from the perspective of protein expression. Data generated from this study have been deposited into a web database (http://oilseedproteomics.missouri.edu) that is accessible to the public domain