A comparative study of Shewanella oneidensis cytosol proteins in cells grown in aerobic and anaerobic environments. CAMILLE SMITH (Hampton University Hampton, VA 23668) CAROL S. GIOMETTI (Argonne National Laboratory, Argonne, IL, 60439)

Shewanella oneidensis is a facultative microorganism with metal-ion reducing capabilities that function under anaerobic conditions. The microbe has the potential to remove toxic metals and pollutants from the environment and, thus, is considered a possible bioremediation agent. To further understand the capability of this microbe to live in different growth environments, an investigation was developed to analyze the protein changes in S. oneidensis grown with and without oxygen as an electron acceptor. Using the two-dimensional electrophoresis system (2DE), we evaluated the protein content after different growth conditions. We also compared sample preparations done under varying temperatures: refrigerated versus non-refrigerated. The different sample preparation methods had no effect on the appearance of proteins in the patterns of the seventeen centimeter silver nitrate gels; the patterns were indistinguishable. By comparing the images using a software program specifically designed for analysis of 2DE patterns, Progenesis, the proteins from cells grown with or without oxygen were examined for significant differences. We used mass spectrometry to identify the protein content of the cytosol samples. In classifying the proteins the metabolic shifts from oxygen to oxygen-free were recognized. By displaying the changes in intensities from the proteins their appearances decreased, increased, or disappeared and this showed a major separation between the aerobic and anaerobic growth patterns.

A comparison of different protein staining techniques in 2-dimensional electrophoresis gels: Coomassie Blue-Green, Coomassie Blue-Red, Silver and Sypro Ruby Fluorescent-stained Shewanella oneidensis proteins. NICOLE JENKINS (Hampton University Hampton, VA 23668) CAROL GIOMETTI (Argonne National Laboratory, Argonne, IL, 60439)

The goal of this study was to compare staining techniques used to visualize spots on 2DE gels in order to determine the most qualitative and time efficient staining of proteins. Mini-gels were stained with the following dyes: Coomassie Blue Red, Serva colloidal Coomassie Blue Green, Invitrogen Simply Blue Coomassie Blue Green , Silver Stain and SYPRO Ruby Fluorescent stain, and compared. A destaining time course study was performed to determine the sensitivity of CB-Green using either a green or red scanner filter. A product brand comparison was done, comparing Invitrogen CB-Green to Serva CB-Green. A concentration sample loading comparison was done to determine the most appropriate amount of sample to load for substantial staining results using CB-Red. Lastly, a total comparison of all of the staining techniques was done, which evaluated all stains for optimal results. The 6 hour destain with CB-Red, and the use of a red filter are best for spot detection. Loading 60µg, in comparison to 40µg of protein, provides a better sensitivity of spot detection for CB-Red staining. The Serva brand of CB-Green has a more pronounced staining of spots than the Invitrogen Simply Blue CB-Green. The silver stain, although having the lowest protein concentration, is most sensitive when staining, producing the greatest number of spots. However, because silver staining involves the lowest protein concentrations, specific spots are more difficult to identify using mass spectrometry. Also, silver staining takes a greater amount of time and produces the most expensive waste disposal.

Design and Development of a PDA Interface for Cloning and Purification Database Systems. RICO CARRELL (Governors State University University Park, IL 60466) DR. SOON-OK PARK (Argonne National Laboratory, Argonne, IL, 60439)

The objective of the research being conducted in the Midwest Center for Structural Genomics (MCSG) and Bioscience division is to develop, and optimize new, rapid integrated methods for highly cost-effective determination of protein structures through x-ray crystallography. Our near term goal is to improve a user application, which the biologist use, for the advancement of research data storage and mobility. In working towards are goal of advancing mobility. We are faced with the task of modifying the cloning application so that it will be feasibly useable on all PDA's. We want to provide some of the desktop functionality of the application on PDA's. While developing the application there were precautions we took. We developed the application to be user friendly and made sure we had data validation, to reduce errors on data entry that is stored in the database. There are several advantages to this application: Increases the quality and efficiency on the data collection and analysis. Minimizes data entry errors. Data is collected electronically from various sources. Scans the data directly into the database by using a PDA scanner. Increases accessibility and mobility.

Discovering Peptide Ligands for Intersectin - SH3 A1 using Phage Display and comparison with src SH3. TEISHA BARNES (Grambling State University Grambling, LA 71245) BRIAN KAY (Argonne National Laboratory, Argonne, IL, 60439)

Phage display is a tool that has many applications. It can be used for the study of molecular interactions and especially in the generation of monoclonal antibodies. (1) The main goal of phage display is to determine the properties of the target protein through its interaction with the peptides displayed on the surface of M13 phage. Some typical targets for isolation of phage displayed peptide ligands include antibodies, certain receptors and other full size proteins. Our group chose to use phage-display as a tool to isolate the peptide ligands to our target protein: ITSN SH3 A1 (Intersectin Src Homology 3). This protein has not been very much studied making this research fairly new. In order to begin the process, the protein first had to be purified. Several attempts to purify SH3 A1 did not yield a soluble protein, thereby creating a problem. After the purification process is completed Affinity Selection Experiments are performed to prepare for an ELISA. After all of this is done we performed an ELISA (Enzyme Linked Immuno-Sorbent Assay). In this procedure the target protein is immobilized in a 96 well plate where it is incubated with the peptides displayed on the phage (in this case the binders resulting from selections against libraries ANL 6 & 7). The phage is detected by anti-M13 antibody with its HRP conjugate. The ELISA shows if the phage is bound to its target protein, another protein, or the plastic of the wells. A signal is determined with ABTS + H2O2 which produce a colorimetric change upon reacting with HRP. Screening the libraries yields information about protein-peptide interactions. The major task in phage display is to determine the properties of the protein and to give the identity of the clones that were selected from the process. One identifying process used in the lab is PCR (Polymerase Chain Reaction). The PCR amplifies the DNA of the phage displaying the selected peptides. Once the amplification of the DNA has occurred, sequencing is then done to see how the peptides compare to each other, if they are similar and if so how many residues are similar. Then the common trend of the peptides is studied as to figure out the interacting partners of the target protein.

Experimental Studies on Near Frictionless Carbon Coatings for Industrial Applications. LEO GERDOV (Benedictine University Lisle, IL 60532) DR. ALI ERDEMIR (Argonne National Laboratory, Argonne, IL, 60439)

Near frictionless carbon coatings (NFC) are structurally amorphous but exhibit some of the lowest friction and wear coefficients when tested in inert environments. The main characteristics of these coatings are that due to their very impressive friction and wear properties, they can significantly increase energy efficiency and durability of many moving parts. If the coatings live up to the expectations and are indeed appropriate for certain applications, the next step will be to offer this technology for industrial exploitation. In our laboratory, we use a Plasma Enhanced Chemical Vapor deposition system to deposit these nearly frictionless carbon coatings. This system is very small and hence not appropriate for large scale production. For most industrial applications, we have been looking for new deposition systems and/or companies that can scale-up the production of these coatings. At the moment, we are working with an industrial company that is capable of producing our NFC coatings in their big deposition systems. These systems use a Pulse DC power source to grow the NFC coatings on the surfaces of metallic samples. After deposition, this company is sending the coated samples back to us and we are then testing these samples to determine if the coatings produced in their systems have the same friction and wear properties as our original NFC coatings. We are also performing structural and chemical studies on these coatings using microscopes and a micro Raman spectroscopy. So far, we have conducted a series of friction tests on these samples using a Pin-on-Disk tribometer under different environmental conditions. After the tests, we determined wear rate and studied the fine details of sliding surfaces using a MicroXAM non-contact optical profilometer. We have also studied the chemical structure of the coatings using micro Raman spectroscopy. The results obtained so far suggest that this company has come very close to duplicating our coating. After the completion of our examination of the sample coated by the outside company, we will be able to suggest further adjustments to them so that the next batch of samples will hopefully be identical to our NFC coating. The implications of this achievement are mostly aimed at the industrial market.

Expression, purification and X-ray crystallographic analysis of Hypothetical protein PG0823 from Porphyromonas gingivalis, APC 80877. JOHN BUELT (Xavier University Cincinnati, OH 45207) ANDRZEJ JOACHIMIAK (Argonne National Laboratory, Argonne, IL, 60439)

Hypothetical protein PG0823 is a protein derived from source organism Porphyromonas gingivalis, a bacterium which produces enzymes that contribute to the destruction of gum tissue and bone that supports the teeth. Hypothetical protein PG0823 has been labeled as hypothetical due to the fact that its function in the source organism is unknown. Hypothetical protein PG0823 was cloned and expressed in Escherichia coli. The protein was then purified and crystallized using the hanging-drop method of vapor diffusion. The crystal structure has been determined at a resolution of 2.2 Å using a synchrotron-radiation source, courtesy of the Advanced Photon Source at Argonne National Laboratory. The protein structure exposes a helix-turn-helix motif which is commonly associated with DNA-binding proteins. Based upon the helix-turn-helix motif and similarity to protein amino acid sequences that serve as transcriptional regulators, it can be hypothesized that hypothetical protein PG0823 may serve as a transcriptional regulator in P. gingivalis. The discovered structure and hypothesized function may be used in further research in order to develop a drug intended to inhibit the transcription of P. gingivalis.

Mapping Protease Substrates using Phage Display. JUSTIN HENRY (Grambling State University Grambling, LA 71245) MICHAEL D. SCHOLLE (Argonne National Laboratory, Argonne, IL, 60439)

Proteases comprise approximately 2 percent of the human and bacterial genome, thus making it important to map substrate specificity for known proteases. Proteases hydrolyze the peptide, or scissile bonds of proteins; a cleavage event commonly observed in cancer, inflammation, and infectious diseases. The substrate library (ANL16) produced by the Kay Lab contains a 7-mer randomized peptide sequence fused to the N-terminus of gene III of M13 bacteriophage. In this study, ANL 16 was utilized to map substrates for human, SARS, and E. coli proteases. Substrate selections yielded eight positive substrates that were cleaved by the SARS protease 3CL Pro with an efficiency of ~70 percent. A dihydrophobic amino acid motif was common from the 3CL Pro substrates whereas substrates from StcE, in comparison to a known substrate of the protease, were glutamine rich. To further characterize the ANL 16 library, phage were biotinylated in vitro to map the substrates cleaved by native host cell proteases. These results showed that the majority of the substrates contained arginine and lysine rich dibasic motifs, similar to substrates cleaved by the outer membrane protease OmpT of E. coli.

Nondestructive Decontamination of Radiological Dispersal Devices from Porous Materials. NADIA KIVENAS (Richard J. Daley Chicago, IL 60629) DR. MICHAEL KAMINSKI (Argonne National Laboratory, Argonne, IL, 60439)

Technologies are needed for non-destructive removal of radioactivity from porous surfaces such as concrete and marble. We are developing a process based on superabsorbing hydrogels. The optimized process would involve three conceptual steps: (1) penetrate the pore structure with a water-based wash solution to promote the removal of radionuclides from their sorption sites and into the pore water; (2) pull water from the pore structure with a superabsorbing hydrogel, and (3) remove the radioactivity-loaded gel by wet vacuum. Polymer gel formulations and ionic wash solutions were tested for effectiveness of 137Cs removal from concrete monoliths. During this period of study, we focused on gel formulation effectiveness for desorption of 137Cs from concrete monoliths. We also evaluated the effectiveness of various wash solutions for 137Cs sorption from coarse aggregate. We found that 137Cs loading onto crystalline silicotitanate increased as a function of time, but was independent of sorbant preconditioning. Our results demonstrate a significant effect of NH4Cl concentrations to 137Cs partitioning onto crystalline silicotitanate. At 0.05M NH4Cl concentration, Kd's are reduced due to NH4+ ions competing with 137Cs for available sites. In addition, we report a decrease of decontamination from coarse aggregate with increased aging time of contamination.

Probing the Proteomic Overlap Between Angiogenesis and Neurogenesis. MARGARET BUELL (Brown University Providence, RI 02912) DIANE J. RODI (Argonne National Laboratory, Argonne, IL, 60439)

During the past few years it has become increasingly apparent that many of the molecules first characterized in developing neurons that guide their growth and migration (here called neurogenesis) are also involved in angiogenesis, the process of blood vessel formation from pre-existing capillary networks. A growing number of studies are now beginning to explore the parallels between nervous and vascular system development; however, the majority of these studies approach the topic one protein at a time. Using in vitro models of both angiogenesis and neurogenesis our group is instead conducting an exploration of about 50 proteins implicated in both processes (only 6 could be presented here). Immunofluorescence was used to test for the presence and subcellular localizations of these proteins in human microvascular endothelial cells (HMVECs) and SH-SY5Y neuroblastoma cells cultured on Matrigel™, in which they undergo tubulogenesis and neurogenesis. The majority of the proteins investigated in this study were found during both HMVEC tubulogenesis at 7-hours post-plating and SH-SY5Y neurogenesis 6 days post-plating, but certain patterns in the data indicate that more temporal investigations of these and other proteins are needed to fully determine the nature of their participation in angiogenesis and neurogenesis. Ultimately, this study and others like it promise to advance our knowledge regarding normal and pathological angiogenesis, including tumor angiogenesis, and nerve regeneration following damage to the peripheral and central nervous systems.

Purification and Crystallization of Conserved Hypothetical Protein APC 82795 Using X-Ray Diffraction Protein Crystallography. DIAN CANADAY (University of Illinois at Urbana Champaign Urbana-Champaign, IL 61801) ANDRZEJ JOACHIMIAK (Argonne National Laboratory, Argonne, IL, 60439)

Protein crystallization is used to determine the structure, and in turn, the function, of certain proteins in specific targets. We use many systems of purification and optimization to grow these crystals and once the diffraction is above a minimum level of three angstroms, crystallographers study the crystals and determine the structure. The machines implemented in this process included AKTA Express, Hydra, Mosquito, Cartesian, and RoboDesign. I worked under Andrzej Joachimiak and Lour-Evelyn Lezondra on the protein APC 82795. This protein resulted in many crystals but none diffracted more than three angstroms. The structure of this protein, consequently, has not been solved. With more variable processes, including different temperature and screens, this protein may be solved in the near future.

Relative Comparison of mRNA Concentration of Target E.coli Membrane Proteins Through Real-Time PCR. YURI POLUEKTOV (University of Illinois at Urbana Champaign Urbana Champaign, IL 61801) PHILIP D. LAIBLE (Argonne National Laboratory, Argonne, IL, 60439)

Real-time PCR is a very efficient way to measure the amount of starting DNA template in a PCR reaction relative to another PCR reaction. It is a very sensitive method that can detect up to a 2-fold difference in the concentration of DNA template between two PCR reactions. And it can work with as much as 10pmol of DNA to produce conclusive results. This method was used to determine the relative concentrations of mRNA, upon transcription into cDNA, of membrane proteins that could be detected using western blots versus ones that could not. Although this experiment would not be able to tell us why certain membrane proteins fail to be expressed in Rhodobacter (R.) sphaeroides cells while others don't, it would be able to tell us what part of the protein expression process the problem occurs in. For this purpose 10 R.sphaeroides cultures were processed, 5 that contained plasmids with target E.coli membrane proteins that were detectible with western blots and 5 that were not. In addition to the experimental cultures 2 more R.sphaeroides cultures expressing ß,a light harvesting complexes were processed to serve as controls. One of the cultures was grown just like the experimental cultures while the other one was grown aerobically. Using the fact that the ß,a light harvesting complexes have an absorption peak at 875nm, it was determined that the culture grown aerobically did not express the light harvesting complexes and so it should probably have a very low concentration of ß,a mRNA. Strangely enough the Real-Time PCR did not register any difference in mRNA concentration between the aerobic and the unaerobic cultures, which suggests that there is some other mechanism along the lines of translation and protein degradation that inhibits the expression of the light harvesting complexes. The experimental cultures however did register a 2-fold difference in mRNA concentration between the non-expressing and the expressing membrane proteins. Although this indicates that transcription is involved in the failure of certain membrane proteins to express, it also tells us that that there is a more extensive mechanism that prevents those proteins from being expressed. Perhaps the real problem lies within translation and protein degradation.

Selecting Peptide Ligands for Intersectin SH3 A2 using Phage Display and Comparison with Src SH3. MARQUISHA WASHINGTON (Grambling State University Grambling, LA 71245) BRIAN KAY (Argonne National Laboratory, Argonne, IL, 60439)

The Src homology 3 (SH3) domain is a unique protein interaction module. The domain is approximately 50-70 amino acids in length and directs protein-protein interactions through the recognition of proline rich sequences. Intersectin (ITSN) SH3 A2 is a human protein cloned into E. Coli host cells. Our goal was to determine the properties of Intersectin (ITSN) SH3 A2, and determine the identity of the peptides that were selected to bind with it. These peptides were obtained using M13 phage display. After expressing and purifying the protein, affinity selection experiments were performed to select binders for ITSN SH3 A2. The protein was screened against libraries ANL 6 and ANL 7. After three rounds of selections, the phage were plated to obtain plaques. These single plaques were picked and used to perform an ELISA. The results of this experiment are still in progress. After initial testing of the protein, it is believed that the protein was not folded properly; however, experiments are in progress to reclone the gene, and retest for binders.

Structural Analysis of Helical RNA. CHARLES TREATMAN (Oberlin College Oberlin, OH 44074) STEPHEN R. HOLBROOK (Argonne National Laboratory, Argonne, IL, 60439)

The Structural Classification of RNA (SCOR) database is designed to provide a comprehensive understanding of RNA structure, function, and tertiary interactions. However, SCOR does not contain data for double helical RNA structures. RNA structures are obtained from the Protein Data Bank, Nucleic Acid Database, and SCOR, and are analyzed for evidence of helices. The 8534 helices identified are analyzed using existing and new software in order to identify structural preferences of RNA double helices. A strong preference for helices with lengths between 2 base pairs and 4 base pairs is discovered, as well as a preference for C-G and G-C base pairs within and at the ends of helices. Attempts to identify novel helical conformations proves ineffective, and alternate methods are suggested. Identified helices will be added to the SCOR database, and this study will be expanded and repeated in an attempt to develop a more specific classification of double helical RNA.

The Potential of Highly Stable Proteins to Act as Protein Scaffolds on M13 phage. CLARE DESMOND (University of Notre Dame Notre Dame, IN 46556) DR. JOHN KEHOE (Argonne National Laboratory, Argonne, IL, 60439)

Antibodies are very capable of obtaining affinity reagents but are limited in their scope of use due to factors such as flexibility, size, folding efficiency and stability. Protein scaffolds are emerging as a useful alternative to antibodies. A potential scaffold protein must be exceptionally stable and therefore resistant to mutation. A highly stable protein, Top 7, was tested for its potential as a protein scaffold. The protein was ligated into a SAM vector and transformed into SS320 E. Coli cells. The cells were plated in top agar that contained 0.04% X-gal and 1mM IPTG in a series of dilutions. No positive plaques showed on the plates containing the ligation of the SAM vector and Top 7 insert. The negative result was most likely due to Top 7 rendering the phage non-viable. Other methods of protein display on the M13 phage pIII will be attempted in the future to determine if this method affects the viability of the phage. Another protein, PknD, was also tested for its potential as a protein scaffold. Both a periplasmic PknD and cytoplasmic PknD were expressed. After expressing both types at 25 C and 37 C C, they were subjected to trypsin for specific amounts of time (5 min, 10 min, 20 min, 40 min, and 2 hours) and at different concentration ratios of protein to enzyme (10:1, 100:1, and 1000:1). The results of the digest displayed that only the PknD expressed at 25 C was properly folded as evidenced by the presence of a strong band in a polyacrylamide gel. PknD expressed at 37 C was presumably cut into small fragments, as evidenced by no discernible bands on the protein gel. Since phage are not efficiently viable at 25 C , the folding capacity of PknD will be tested at 30 C with the same trypsin experiment.

Water Absorption Levels of Willows (Salix alba var. tristis), Walnuts (Juglans nigra) and Oaks (Quercus alba), in a Closed System. ELIZABETH DAVIS (Kennedy King College Chicago, IL 60621) PROFESSOR ARLICIA CORLEY (Argonne National Laboratory, Argonne, IL, 60439)

Phytoremediation is the cost effective use of green plants to sequester or detoxify pollutants from groundwater or damaged land. Because of the enormous cost associated with the removal of water and soil pollutants in the environment, the use of green plants is useful as it can decrease cost. A controlled study is warranted to determine how they perform. Phreatophytic and non- phreatophytic plants were then used in controlled tree well cartridges. These tree well cartridges were designed by Argonne National Laboratory (ANL) and Applied Natural Science (ANS) who owns patented technologies (Tree Mediation® and Tree Well® systems).Trees pre-installed in these controlled cartridges are 3-Willows (Salix alba var. tristis), 3- Oaks (Quercus alba), 3-Walnuts (Juglans nigra) and 3-cartridges with no-trees are used as controls. Trees and no-tree cartridges were installed in order to evaluate root density, water absorption levels, and soil carbon balances. Water absorption was monitored during the mornings and afternoon with a Solinst Water Level Indicator, which was inserted into a piezometer tube that was installed in the cartridges and the result are measured in centimeters. Five to 15 gallons of water was added after the initial reading to the cartridges periodically as their measurement dropped below 240 cm. The height and diameter was taken on all of the trees to follow up on the data from last summer. The weather was monitored and correlated with the fluctuation of water intake of the cartridges containing the Willows (Salix alba var. tristis), Oaks (Quercus alba), Walnuts (Juglans nigra) and cartridges with No-Trees installed. Major components of Phytoremediation studies at Argonne National Laboratory, currently being conducted, is to determine which species of trees are better suited to remediate contaminated, semi-arid and marginal soil, depending on the depth and severity of the pollutant or conditions; as well as, determining various avenues unto which various soil depths can be exploited in order to increase carbon sequestion .

Western Analysis of the Effects Cadmium has on Expression of CRY61 in Osteoblast and Osteoclast cell lines: New insights into the mechanism by which Cadmium might lead to osteoporosis. MARYN VALDEZ (University of Maryland College park, MD 20742) MARYKA H. BHATTACHARYYA (Argonne National Laboratory, Argonne, IL, 60439)

Cadmium (Cd) is a natural metal that has been shown to cause bone loss, sometimes drastically, which was the case in Japan, when it was leaked into the Jinzu River and caused an extreme case of osteoporosis/osteomalacia. The mechanism by which Cd causes bone loss has yet to be deciphered. The long-term goal of our research is to determine how Cd causes bone loss. Previous research using micro array analysis methods has found that the gene for Cry61 was greatly upregulated in bone from mice treated with Cd. Cry61 is a secreted Cysteine rich, matrix associated protein. Cry61 interaction with the integrin aVß3 isoform has been shown to lead to adhesion in different cell types. We hypothesize that Cd provokes expression of Cry61 in osteoblast (OB) or osteoclast (OC) cells, which bind to aVß3 integrin located on the surface of OC precursors. The binding activates the precursors, which then can increasingly differentiate into OC, which ultimately leads to bone resorbtion, and eventually to osteoporosis. Here I report specifically on the presence of Cry61 in pre OB or OC cell lines treated with Cd. We used 7F2 cells lines and RAW cell lines for our OC and OB. We treated the RAW cells and 7F2 cells with Cd, and estrogen, or nothing. After 48 hours and 1hr, we collected the media and cells, than used immunoblotting to confirm the presence of the protein. In our immunoblots we saw very little difference in the bands for each condition in the 7f2 cells and RAW cells. Conversely, we did notice that the control band differed from the Cd band in the media of the RAW cells and 7F2 cells at 48 hours. In the 7F2 media at 48 hours, we observed an increase in band strength in the Cd vs. control samples; however the increase was not as great as that displayed in the RAW cells. Based on those observations, we tentatively concluded that Cry61 is not enhanced in 7F2 or RAW cells by Cd. On the other hand, we hesitantly conclude that Cd may be inducing Cry61 secretion in the media of 7F2 cells. Furthermore, Cd is inducing greater Cry61 secretion into the media at 48 hours for RAW cells. All of these conclusions are provisional; more trials need to be run. Additional time points would also be very enlightening. We would also like to evaluate Cry61 expression on the RNA level or see if, when we add an antagonist, the expression would cease. We need to do further research, but we feel we have made a good first step in elucidating the mechanism by which cadmium causes bone loss.