Abstracts

Mannans are hemicellulosic polysaccharides within plant cell walls with cellular functions including structure, storage, and signaling. Understanding the patterning of mannan synthesis will enhance our understanding of the roles of these polysaccharides in plants. Numerous members of the CSLA gene family encode mannan synthase enzymes involved in the synthesis of the backbones of mannans. This project aims to create an expression atlas that documents the expression pattern for each of the nine CSLA genes in the model plant Arabidopsis thaliana by using GUS-reporter gene fusions and microscopy throughout much of the plant life cycle. This will be used to identify the contribution of each CSLA throughout development.

Plasminogen activator inhibitor-1 (PAI-1), a primary inhibitor of both tissue-type and urokinase-type plasminogen activators in plasma, is a well-established risk factor in various disease conditions. Increased levels of active PAI-1 in plasma are correlated with the development of atherosclerosis, diabetes, stroke, and other maladies. In the present study, we describe the synthesis of new series of compounds that aim to reduce physiologically active PAI-1 levels. These molecules are related to a series of bis-arylsulfonimides and arylsulfonamides connected by short linking diamines. These studies resulted in the identification of small molecule inhibitors of PAI-1 that displayed in vitro IC50 values in the low micromolar range.

PAI-1 is a naturally occurring serine protease inhibitor involved in the inhibition of urokinase- and tissue-type plasminogen activators. At physiological levels, PAI-1 takes part in many processes, such as cell migration, fibrinolysis, angiogenesis, and wound healing. At pathological levels, PAI-1 has been linked to renal disease, obesity, deep vein thrombosis, type 2 diabetes, cardiovascular disease, cancer, and pulmonary disease. Therefore, synthesis of potent and specific PAI-1 inhibitors is of great importance. A variety of aryl sulfonamides and aryl sulfonimides were synthesized, and their potencies as PAI-1 inhibitors were determined. Based on the data collected, a structure-activity relationship model has been developed.

The inhibition of plasminogen activator-inhibitor-1 (PAI-1) is anticipated to increase our understanding of various human ailments including diabetes, stroke, and atherosclerosis, with which high levels of PAI-1 have been associated. Previous accounts have reported the synthesis of inhibitors that bind to PAI-1 with a low affinity and fail to inhibit PAI-1 when vitronectin, a cofactor of PAI-1, is present. Therefore, the synthesis of small-molecule inhibitors of PAI-1 that improve upon these properties has been the main goal of this research. The refinement of one of these synthesized moieties into a selective and highly active inhibitory species has been achieved. IC50 values of our synthetic inhibitors were determined in an ex vivo plasma inhibition assay.

Plasminogen activator inhibitor-1 (PAI-1) is a mammalian protein active in the regulation of fibrinolysis. Data indicates that individuals in various disease states _ namely cancer, diabetes, and cardiovascular disease _ have elevated levels of PAI-1, resulting in disordered fibrinolysis, and an increased risk of thrombosis and embolism. Reduction of excessive levels of active PAI-1 may decrease the risk of thrombosis and embolism. Several inhibitors of PAI-1 have been synthesized, however, most either compete directly with naturally occurring inhibitors in vivo or possess inhibition activity in vitro exclusively, and are not feasible drug candidates. Here we present a novel class of inhibitors designed to have improved pharmacological potential. Based on previous research, the inhibitors take advantage of functional groups that have proven inhibitor-activity relationships while making use of a novel pyrrolidine ring core. Half maximal inhibitory concentration (IC50) data indicates that the pyrrolidine core improves inhibitor binding affinity and potency. The design and synthesis of the inhibitors is discussed, and the supporting IC50 data are presented.

Human Islet Amyloid Polypeptide (hIAPP) has been implicated in Type II Diabetes development. The extent of membrane disruption and the presence of amyloidal clusters on the surface of pancreatic _-cells reflect the severity of the disease. The development of Type II Diabetes increases with age, and age effects changes in cell membrane structure. Cholesterol is believed to inhibit hIAPP permeabilization of POPG model lipid membranes. Contrary to our hypothesis, liposome dye leakage experiments on DOPC/DOPS model liposomes in our study demonstrate that with elevated membrane-incorporated cholesterol levels and increased hIAPP concentrations, membrane disruption actually increases. These results will lead to further studies to attempt to correlate other membrane changes with a cell's susceptibility to hIAPP.

Pancreatic beta cells secrete insulin, an endocrine hormone that regulates blood glucose levels and maintains normal physiological activity in humans and animals. Diabetes mellitus type II is a consequence of the gradual destruction of these important cells, likely by human islet amyloid polypeptide (hIAPP) that is co-secreted along with insulin. Increasing health care costs, coupled with the World Health Organization's prediction of a worldwide diabetic epidemic by year 2030, make experimental diabetes research a crucial prologue to future clinical trials in prevention, diagnosis, and treatment of Diabetes mellitus type II. Our experimental set-up simulates hIAPP and pancreatic beta cell membrane interactions in order to uncover factors that initiate and promote progression of beta cell death. Results from our study establish the potential role of various fragments of hIAPP in the disease process and explore the molecular mechanisms involved in pancreatic damage.

Amylin (human Islet Amyloid Polypeptide, hIAPP) is a 37 amino acid polypeptide, co-secreted with insulin from pancreatic beta cells, which plays a role in cell membrane damage by forming amyloid fibrils in Type 2 diabetes. Recent studies suggest that the N-terminal region of hIAPP (hIAPP 1-19) is responsible for initial interactions with the membrane, rather than the central amyloidogenic region (hIAPP 20-29), likely by attractive forces between the positively charged residues and the negatively charged membrane phospholipids. This research involves the synthesis of an uncharged peptide analogue of hIAPP (1-19) by replacing positively charged residues lysine and arginine with isosteric aminoheptanoic acid (Ahept) and acetylation of the N-terminus. Expected to have minimal activity and test the charge attraction theory, the peptide was synthesized, cleaved from resin, purified, and analyzed. A dye leakage assay was then employed to assess the membrane disruptive potential of the synthesized peptide.

Short-chain fatty acids (SCFA) such as butyrate and lactate are the products of colonic bacterial degradation of starch and other carbon sources important in human health. Prior work has indicated that SCFA production in the microbiota is markedly affected by interaction of the organisms in the microbial community. The time courses of butyrate and lactate production during growth of the organisms in a bioreactor are being investigated to permit a meaningful analysis of microbial biochemistry. The goal of the research is to investigate the extent to which physical contact of microorganisms and/or shared biochemical pathways for production of SCFA are responsible for the dramatic changes in lactate or butyrate concentrations in media that occur when they are co-cultured in vitro.

Pyrimidine nucleotides play a critical role in cellular metabolism by serving as activated precursors of RNA and DNA. Aquifex aeolicus encodes pyrimidine pathway proteins homologous to those found in mesophilic organisms. The aspartate carbamoylase (ATC) domain catalyzes the second step in the pathway forming carbamoyl aspartate that is then converted by the dihydroorotase domain (DHO) to dihydroorotate. The DHO and ATC domains from A. aeolicus were overexpressed in Escherichia coli and purified by affinity chromatography. The X-ray structure shows that E193 and G194 on the DHO domain are residues that might be critical for interaction with the ATC. I used enzyme kinetics to test the functional significance of this region in the presence of a peptide synthesized to mimic a loop on the DHO.

The Creative Scientific Inquiry Experience (CSIE) program uses an innovative approach to address the decreasing success rates of undergraduate students in STEM (Science, Technology, Engineering, and Mathematics) fields. In this study, we evaluate the impact of CSIE strategies on retention and graduation rates of STEM students. We identified 71 CSIE students enrolled in STEM classes in the fall of 2006 and compared them to 882 non-CSIE students enrolled in the same classes. The CSIE cohort had higher chances of remaining as a STEM major and higher graduation rates. Interestingly, CSIE students were more likely to convert to a STEM major by graduation time. Qualitative and quantitative results will be presented to support the success of the CSIE program in improving recruitment and success rates for EMU STEM students.

Metastasis is the result of the disruption of the precise balance between proliferation and apoptosis. It has been estimated that up to 90% of metastatic tumor cells are lost by apoptosis. Thus, approaches that can tip the balance in favor of apoptosis would be expected to be effective in combating proliferative disorders. FAM129B is a currently identified protein that is important for cell invasion.). It has a pleckstrin homology domain near the amino end and a proline-rich region near the carboxyl end. One crucial facet of the mechanism through which FAM129B promotes cancer cell invasion is likely to be the suppression of apoptosis. We have purified the FAM129B protein in E. coli by affinity chromatography and are characterizing its properties by gel filtration chromatography and its ability to interact with key proteins.

Bacteremia is the bacterial invasion of the blood. Bacterial proliferation in the blood requires that the organism adapt its metabolism to available nutrients. Nucleotides precursors that could be used are present at very low levels in the blood, and thus the invading bacteria must rely on de novo nucleotide biosynthesis for survival. The dihydroorotase domain is a key enzyme in pyrimidine biosynthesis and catalyzes the third step in the pathway. It was recently shown to be a promising drug target since defects in this enzyme caused an approximate 1000-fold decrease of viable cells in the blood. We cloned the genes encoding the dihydroorotase and aspartate transcarbamoylase from the bacterium, Bacillus anthracis, and expressed them in Escherichia coli. The proteins were purified by affinity chromatography and their oligomeric structures determined by gel filtration and cross-linking methods. The oligomeric structures were determined in the presence and absence of substrates.

Aquifex aeolicus, an extreme hyperthermophile, encodes proteins that are homologous to the major carbamoyl phosphate synthetase (CPSase) domains found in mesophilic organisms. The CPS.A and CPS.B homologs from A. aeolicus were overexpressed in Escherichia coli and purified to homogeneity by affinity chromatography. Previously, it was found that a stable 124-kDa complex could be reconstituted from stoichiometric amounts of CPS.A and CPS.B proteins that synthesized carbamoyl phosphate from ATP, bicarbonate, and ammonia. In this study, the purified carbamoyl phosphate synthetase components were tested for their binding and interaction with the aspartate transcarbamoylase and dihydroorotase domains, the enzymes that catalyze the next reactions in the pathway. The interaction was tested in the absence and presence of the substrates and at different temperatures. Enzymatic assays to determine the outcome of protein-protein interaction on the catalytic activity of each component were also carried out to investigate putative functional linkages.

Bacteremia is the bacterial invasion of the blood. Bacterial proliferation in the blood requires that the organism adapts its metabolism to available nutrients. Nucleotide precursors that could be used are present at low levels in the blood and thus the invading bacteria must rely on de novo nucleotide biosynthesis for survival. The dihydroorotase domain is a key enzyme in pyrimidine biosynthesis and catalyzes the third step in the pathway. It was recently shown to be a promising drug target. The genes encoding the dihydroorotase and aspartate transcarbamoylase were cloned from the bacterium, Bacillus anthracis and expressed in Escherichia coli. The proteins were purified by affinity chromatography and visualized by SDS PAGE. The activity of these enzymes was determined by using enzyme assays. Interestingly, the activity of ATC was increased by about 2-fold when mixed with DHO in a 1:1 mole/mole ratio. In addition, their oligomeric structures were determined by S-300 gel filtration chromatography and cross-linking methods.

Mammalian CAD, a multifunctional protein that catalyzes the first three steps of the pyrimidine bio synthetic pathway is known to be over expressed in several types of tumor cells. The analogues complex of CAD, found in Aquifex aeolicus, a hyperthermophilic organism, was used in this research to understand its structural organization. Gel filtration chromatography was used to study the interaction between the proteins along with chemical cross-linking with dimethyl suberimidate. Peptides designed to disrupt the interaction between specific proteins that form the DAC (dihydroorotate and aspartate transcarbamoylase) complex were used. It was found that these peptides inhibit the activities of the proteins by disrupting the complex at specific sites. Gel filtration chromatography is being used in order to unravel the interaction between carbamoyl phosphate synthetase (CPSase) and the other components of the pathway.

Plasminogen Activator Inhibitor-1 (PAI-1) plays a key role in the inhibition of fibrinolysis, the process by which blood clots are broken down. In many cases it becomes necessary to increase the rate of fibrinolysis. One way that this can be accomplished is by inhibiting PAI-1. Previous research has shown that polyphenolic compounds can be effective inhibitors of PAI-1. In this work, Density Functional Theory was used to model several inhibitors in the PAI-1 binding site in order to better understand the interaction between inhibitor and protein. A Natural Bond Orbital (NBO) analysis is used to analyze the strength of the interaction within the binding site.

The Islamic Department at the Detroit Institute of Art has in its possession a 15th-century Iranian Timurid Qur'an. This manuscript is the subject of a multidisciplinary study that includes identifying the materials used in the construction as well as its cultural and historical significance. EMU is utilizing direct analysis in real time mass spectrometry to identify the dyes, binders, pigments, and inks of the Qur'an. Mock samples were made using materials from the period and region of the manuscript. The manuscript itself is Middle Eastern in origin but with Chinese artistic influences, so materials from both regions are being used. The mock samples contain a combination of dyes, binders, and lead white. The different combinations will end up as a database of spectra that can be compared to the real sample spectra when it is run.

Archaeologists want to know if the paintings in Cueva La Conga, the only recorded painted cave in Nicaragua, were influenced by the Maya, Caribbean cultures, or were a purely indigenous development. Using radiocarbon dating to determine the age of the paintings will help to understand the possible cultural relationships between known cultures and the rock art. The prerequisite for radiocarbon dating using accelerator mass spectrometry is that an organic binder must be present in the painting samples and is extracted and dated later. We are using thermally assisted hydrolysis/methylation-gas chromatography-mass spectrometry (THM-GC-MS) to study the composition of the paints to determine if any binder material remains. Comparing the compositions of the paint and unpainted limestone will allow us to determine if a reliable date is likely to be obtained. We will describe the inherent difficulties of reconciling good analyses with preservation of these irreplaceable and at-risk cultural materials.

Chemical characterization of rock paintings is important for reliable dating using radiocarbon analysis. Identification of the sugar composition of these materials can tell about their sources, which would thus help in selecting the right candidates for dating. A GC-MS method, which involves the formation of diethyldithioacetal trimethylsilyl derivatives of monosaccharides, was employed to characterize carbohydrates in archaeological materials such as rock paintings and a stone tool residue. Carbohydrates were not detected in any of the three rock painting samples while the stone tool residue was found to contain carbohydrates. The GC-MS method of determining carbohydrates in artifacts involves complex chemical derivatization, even though it is successful. We are now developing a more rapid, efficient, and reliable method to determine sugar composition in these and other archaeological residues using thin layer chromatography coupled with DART-MS. Results from GC-MS and the ongoing TLC-DART-MS method will be discussed.