Student Abstracts: Biology at PNNL

The Toxic and Carcinogenic Potential of a 1.6 GHz Wireless Communication Signal: In Vivo Two-Year Bioassay. SEAN ADHIKARI (University of Washington, Seattle, WA 98195) LYLE B. SASSER (Pacific Northwest National Laboratory, Richland, WA 99352) .
Years ago, the possible carcinogenic effect of radio frequency (RF) radiation from cell phones was brought to the public's attention. It is believed to increase the risk of brain cancer. The purpose of this study was to assess the carcinogenic effects of Motorola's 1.6 GHz Iridium Signal on Fischer 344 rats. Four groups of male and female rats were tested: one cage-control (kept in cages and not loaded and unloaded into exposure chambers), one sham-exposed (loaded into exposure chambers without exposure to the signal), and two fully exposed to the signal at different doses. According to data collected based on statistical analysis, no significant differences existed in survival rates or body weights between the groups of males and females. Also, no significant differences existed in birth performances between the four treatment groups of pregnant females. Therefore, the study has not yet established a clear connection between RF radiation and cancer. Nevertheless, much is left to be completed of this study, including necropsy and histopathology of all animal tissues. Afterwards, it still may not successfully prove the harmful effects of cell phone radiation.

Deletion of hsdR gene from Shewanella oneidensis MR-1 genome. . STEPHANIE CHU (Sacramento City Community College, Sacramento, CA 95822) MARGIE ROMINE (Pacific Northwest National Laboratory, Richland, WA 99352) .
Shewanella oneidensis MR-1 has the ability to respire a large variety of compounds, including radionuclides and metals. Respiration of radionuclides, such as U and Tc, leads to the precipitation of relatively insoluble metal oxides, thereby limiting their mobility in aquifers where they can pose a health risk. MR-1 is poorly receptive to "foreign" DNA and therefore it is more difficult to transfer DNA that has been genetically engineered in E. coli into MR-1. Exclusion of foreign DNA is in part due to the hsdR gene, which is part of a restriction and modification system (RMS). HsdR encodes an enzyme that cleaves unmethylated, "foreign" DNA. By deleting this gene we hope to eliminate the restriction activity, thereby making MR-1 more receptive to genetically engineered DNA. A clone in which DNA flanking either side of the hsdR gene were fused together was generated by PCR. The resulting PCR fragment was cloned into pcrII-TOPO and transformed into E.coli. Preliminary results indicate we were successful in cloning the PCR product. The insert will next be transformed into a suicide vector, which will then be transferred to MR-1 to promote replacement of the genomic region containing hsdR with our genomic segment lacking hsdR via a process known as homologous crossover. Once a strain lacking hsdR is constructed through this approach we can test whether this mutation produces a variant that is more successful in taking up and maintaining DNA isolated from E. coli.

Deletion of hsdR gene from Shewanella oneidensis MR-1 genome. Valerie Crusselle (University of Utah, SLC, UT 84112), Stephanie Chu, Amber Alford, Margaret Romine (Pacific Northwest National Laboratory, Richland, WA 99355). VALERIE CRUSSELLE (University of Utah, SLC, UT 84112) MARGARET ROMINE (Pacific Northwest National Laboratory, Richland, WA 99352) .
Shewanella oneidensis MR-1 has the ability to respire a large variety of compounds, including radionuclides and metals. For some radionuclides, such as U and Tc, this respiration leads to the precipitation of relatively insoluble metal oxides, thereby limiting their mobility in aquifers where they can pose a health risk. As the genome of this organism has just been sequenced, much research is being done to determine the pathway of this unique respiration. However, it has been found that S. oneidensis MR-1 is not very receptive to "foreign" DNA, which is a necessary characteristic in order for genetic manipulation of the bacteria. This is due to the hsdR gene, which is part of a restriction and modification system (RMS). The RMS system enables the bacteria to distinguish "foreign" DNA from its own. HsdR codes for a restriction activity which cleaves unmethylated, "foreign" DNA. Therefore, deletion of this gene would eliminate the restriction activity. This deletion is created through a series of techniques, including polymerase chain reaction (PCR), cloning, and homologous crossover. Hypothetically, the deletion of hsdR from the S. oneidensis MR-1 genome would therefore make the bacteria more receptive to DNA from other species of bacteria.

Efficacy of surface coatings in prevention of microbial infection: an in vitro study. BROOKE DEATHERAGE (Washington State University, Pullman, WA 99163) DR. ALLISON A. CAMPBELL (Pacific Northwest National Laboratory, Richland, WA 99352) .
The increasing incidence in the medical field of post-implant infection has prompted further investigation into possible coatings to alleviate this problem. This study explores using a "scaffold" system to carry antimicrobial substances and prevent infection. Hydroxyapatite (HAP; Ca5(PO4)3OH), was used as a protective coating for bone bonding and as a delivery system. Two polymers also explored as delivery systems were PMMA (polymethyl-methacrylate) and PLGA (poly-lactide-glycolide). The two antimicrobial agents analyzed were chlorhexidine and silver nitrate. Chlorhexidine has antimicrobial effects on both gram-positive and gram-negative bacteria with little resistance. Silver nitrate also inhibits growth of a wide range of microorganisms. The surfaces of the metal substrates were coated with HAP-based systems via surface induced mineralization (SIM), and with polymer systems through dip-coating. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction spectroscopy (XRD), and scanning electron microscopy (SEM) were used to characterize the coatings. The efficacy of each coating in inhibiting microbial growth was tested in culture plates inoculated with Staphylococcus aureus, a common cause of the targeted infections. Rods coated with HAP/chlorhexidine and HAP/silver nitrate both showed inhibition of Staphylococcus aureus, whereas the uncoated rods, HAP-only coated rods, and polymer coated rods exhibited no antimicrobial effects. A coating containing both HAP and chlorhexidine has the most potential for reduction of infection rates due to its in vitro display of the largest zones of inhibition, and would be the best choice for use in medicine.

Monitoring of Groundwater Microbial Community. Alison Eakin (Eastern Washington University, Cheney, WA 99004) H. Kostandarithes (Pacific Northwest National Laboratory, Richland, Washington 99355).. ALISON EAKIN (Eastern Washington University, Cheney, WA 99004) HEATHER KOSTANDARITHES (Pacific Northwest National Laboratory, Richland, WA 99352) .
This was the initiation of a study for monitoring the groundwater microbial community at the Oyster Site in Virginia. This analysis is only a small part of a more extensive research program for developing a potential remediation strategy for leakage of underground storage. Two groundwater flow cells were installed in which microbial transport experiments have been performed under induced and natural flow-gradients. This study was conducted on samples that came from both within and outside the flow cell. Samples from each location were "static" or under natural flow-gradients, and "post-gradient" came from induced gradients. Samples will continue to be collected and tested in the continued induced current state. Plates were made to test for the presence of total coliforms (including E-coli) and streptococcus. If a sample showed the presence of coliforms it was also tested to see if the coliforms were of fecal descent. The second set of samples (post gradient after time with the induced current) showed that no fecal coliforms were present. The second set of samples also contained fewer samples that showed positive for iron related bacteria and sulfate reducing bacteria. Spread plates of the samples were made to observe the general growth and morphology. A Biolog assay was done using 31 of the most useful carbon sources for soil and groundwater community analysis. Further studies will be done to compare the continued post-gradient samples to the initial post-gradient and static samples.

PETRI NET REPRESENTATION OF THE KREB CYCLE. DEAN GULL (Southern Utah University, Cedar City, UT 84720) DR. JOE OLIVEIRA (Pacific Northwest National Laboratory, Richland, WA 99352) .
We have developed a computational model which accurately depicts sequences of enzyme-catalyzed reactions as specialized directed graphs. We hypothesize that creation of network models for biochemical systems will allow elucidation and quantification of the system response to a given perturbation. Our model is a first step toward a goal of facilitating manipulation and study of a complete biochemical system. Graphical network models provide a computational framework for identifying key circuits, oscillatory behaviors, and response to biochemical perturbation. The model presented here represents a first approximation of the set of all mass-flux balance conserving pathways or circuits for a given biochemical reaction sequence. The size and complexity of the problem of identifying all such paths and combinations of paths requires enormous computational resources. We have extended previous approaches to this problem by formulating a combinatorial geometric model referred to as an oriented matroid. The interested reader is referred to our previous work and to the Mathematics section of this paper.

Spatially Resolved Single Cell Irradiator to Study Bystander Responses to Low LET Radiation. BROOKE HOLBEN (Washington State Univeristy, Pullman, WA 99163) MARIANNE SOWA RESAT (Pacific Northwest National Laboratory, Richland, WA 99352) .
The bystander effect refers to the observation of a biological response in the absence of direct irradiation. To examine this for low LET radiation, we are using a novel single cell irradiation device to deposit energy in a pre-selected subset of cells for which the un-irradiated neighbors can be easily identified. Using this device we investigated the presence of a calcium flux following exposure to ionizing radiation. Transient calcium levels were measured using visible wavelength calcium sensitive dye, Flou 4, which exhibits an increase in green fluorescence upon binding to calcium. This research is on going and control results are presented here. To monitor another aspect of the biological response to ionizing radiation, a p53 reporter system has been developed where CHO and D2XRII cells were transfected with p53 and fluorescence reporter EGFP. Characterizations of this system were made by exposing various cell lines to wide field Gamma radiation. We measured p53 localization within the cell as well as stabilization (accumulation) of p53 after DNA damage. Survival curves were obtained for both wild-type and transfected CHO cells following Cobalt-60 radiation. Transfection of cells had no significant effect on cell survival. Using the western blot technique, we were able to analyze protein expression levels of irradiated D2XRIIp53(15X)EGFP cells. The time response following 5.0 Gy irradiation did not conclusively show an increase in the p53 expression and further experiments are necessary.

Data Analysis for Ecological Risk. KATHRYN HYLDEN (University of Notre Dame, South Bend, IN 46556) TERRI MILEY (Pacific Northwest National Laboratory, Richland, WA 99352) .
For four weeks I have worked at PNNL as an intern on a data analysis team. We run a FORTRAN model to assess the effects of Hanford activities on the local ecology. We use a Visual Basic data extraction program to select the results of interest, then we put the data into graphs so the project ecologist can determine where trouble spots are both currently and under potential future conditions. The scope of the larger project is to ascertain what cleanup of the nuclear waste still needs to happen and how much of the waste is already below harmful levels. I personally had the opportunity to work with various computer programs and languages to produce these results.

The Process of DNA Sequencing. CALVIN KWAN (University of California, Riverside, Riverside, CA 92507) KWONG-KWOK WONG (Pacific Northwest National Laboratory, Richland, WA 99352) .
The process for DNA sequencing has improved greatly over the last few years. With the ever increasing pressure to discover cures to genetic diseases and ailments, one must be able to first determine the sequence of nucleotides that codes for various genes and proteins. The methods of DNA sequencing have become more efficient, allowing for DNA strands of 500 base pairs to be determined within a matter of hours. Larger fragments of DNA can be elucidated in days. With the process becoming more common, it is important for many researchers, particularly those involved in biosciences to understand the procedures involved in DNA sequencing. It is important that accurate results are achieved during DNA synthesis as the procedure can be quite costly. However, once a gene sequence has been determined, genes functions can be studied easily.

Exposure of Aquatic Biota to Uranium Groundwater Contamination. KYLE LARSON (Columbia Basin College, Pasco, WA 99301) BRETT TILLER (Pacific Northwest National Laboratory, Richland, WA 99352) .
Conclusive results were sought in our field research by attempting to control as many of the experimental variabilities as possible. In past studies, biota was sampled at random in known areas of contamination and tested for possible effects. However, these studies do not accurately represent the natural accumulation of contaminants in biota since they are mobile and probably do not live their entire lives in the contaminated areas. By creating "exposure" cages, we will make crayfish, sculpin, and native cubicula live within known contaminated areas for a set period of time at several different controlled densities. These cages are constructed of 100% non-toxic materials, thus eliminating the possibility of co-contamination. To date, the cages have been constructed and attached to the river bottom and have 3 different densities of cubicula living in them. Crayfish and sculpin must be tagged with an identification tool (PIT tags and iridescent tabs) so that they are not confused with any others that may enter the cages by accident. All sample biota used in our experiment will be collected from the river near the exposure test site so there is minimal risk of cross-contamination from other possible sites. The success of this experiment will help to determine how aquatic biota accumulate low levels of contamination and will hopefully launch similar experiments with terrestrial biota as well.

Determining Activation of Epidermal Growth Factor Receptor and Extracellular-Signal Regulated Protein Kinase. EDWARD MEYER (Pasadena City College, Pasadena, Ca 91106) BRIAN D. THRALL (Pacific Northwest National Laboratory, Richland, WA 99352) .
Cells communicate extra-cellular signals through activation of protein kinase-signaling cascades. MAP kinase signaling pathways are known to regulate many different cellular responses, such as proliferation and cell death. One class of MAPK pathways is the extra-cellular signal regulated kinase (ERK) pathway, which is commonly activated by growth factors, such as epidermal growth factor (EGF), and is thought to be involved in cell proliferation and pro-survival responses. In the case of EGF, binding of EGF causes phosphorylation of the EGF receptor, which ultimately leads to phosphorylation and activation of the downstream kinases, Raf, MEK and ERK. Phosphorylation of ERK leads to stimulation of its kinase activity, translocation to the nucleus, which results in changes in gene expression. While growth factors such as EGF activate the ERK pathway through specific receptors, many environmental contaminants such as radiation can also activate this pathway. Our ultimate goal is to understand how the MAPK pathways are activated by environmental contaminants

An Electron Microscope Examination of Iron Reducing Bacteria. PENELOPE POWELL (Truckee Meadows Community College, Reno, Nev 89502) DR. YURI GORBY (Pacific Northwest National Laboratory, Richland, WA 99352) .
Dissimilatory metal reducing bacteria couple the oxidation of reduced organic compounds to the reduction of multivalent metals. In natural, anaerobic environments ferric iron, Fe (III), is the most abundant electron acceptor. At typical neutral pH values, Fe (III) is very insoluble and forms ferric oxide minerals. Enzymatic reduction transforms these mineral phases to more soluble Fe (II). Other multivalent metals, such as uranium, technetium, and chromium, can be reduced by metal reducing bacteria. In contrast to iron, these metals are soluble in their oxidized state and poorly soluble in their reduced state. Hence enzymatic reduction of heavy metals and radionuclides provides potential for stopping the migration of these contaminants in groundwater and for removing them from aqueous contained wastes. Demonstrates an electron microscope approach for determining the location and composition of precipitated heavy metals and radionuclides in and around iron reducing bacteria. The work presented here, demonstrates an electron microscope approach for determining the location and composition of precipitated heavy metals and radionuclides in and around iron reducing bacteria.

Toxicity of 2,3,7,8-TCDD, PCB-77, PCB-126 and 1,2,4,5,7,8-HCX to Ictalurus punctatus during ELS testing. HEATHER ROBINSON (Portland State University, Portland, OR 97207) HEIDA DIEFENDERFER (Pacific Northwest National Laboratory, Richland, WA 99352) .
Battelle's Marine Science Laboratory was contracted to perform early life stage (ELS) tests to observe the toxic effects of 2,3,7,8 TCDD, PCB 77, PCB 126 and 1,2,4,5,7,8-hexachloroxanthene (HCX) on the channel catfish, Ictalurus punctatus. The purpose of the study was to replicate potential exposure at Centredale Manor, a NPL Superfund site in Providence, Rhode Island. TCDD and PCBs, along with VOCs, semivoloatile organic compounds, pesticides and metals, were previously found in the Woonasquatucket River. The fertilized eggs were exposed to the chemicals for 24-hours then allowed to hatch in a flow through system. After a 10-day range-finding test the LC-50 of TCDD was calculated to be 0.01 ng/ml. A second set of concentrations with the same mixtures of TCDD, PCB-77 and PCB-126, included hexachloroxanthene (HCX), a little-studied chemical found in large quantities at the site. The LC-50 for the HCX series was also 0.01 ng/ml. Data including mortality rates, growth rates and physical abnormalities from a 32-day definitive test is currently being analyzed. Noted abnormalities include hemorrhaging, craniofacial skeletal deformities and cardiac and yolk sac edemas.

Satellite Imagery Analysis. LORENA SANCHEZ (Columbia Basin College, Pasco, WA 99301) JANELLE L. DOWNS (Pacific Northwest National Laboratory, Richland, WA 99352) .
Satellites have become a recent form of technology that has allowed us humans to view the continuous changes of our earth. As a Community College Initiative (CCI) intern with the PNNL Ecology Group, I was allowed the opportunity to use this technology. My projects involved ground truthing or verifying analysis products derived from satellite imagery of the Hanford Site in Eastern Washington and sites near Grand Junction in Western Colorado. Satellite imagery was used to verify areas on the Hanford Site that burned and did not burn. Collecting and analyzing data at sites on our model were chosen to improve the Hanford Site ash/vegetation index and allow us to improve the calibration of our image and better identify areas of the Hanford Site that did and did not burn. In Colorado, satellite imagery was used to verify areas of high and low vegetation for the Bureau of Land Management (BLM) lands. Remote sensing data provided us with an understanding of vegetation conditions of the areas important for managing grazing lands. Part of this data collection was to identify areas that are anomalous or areas of concern because they did not have the vegetation we expected.

Theoretical Determination of Rate Constants for VOC's + OH: A DFT Study. NICHOLAS SCAIEF (WSU, Pullman, WA 99163) SHAWN KATHMANN (Pacific Northwest National Laboratory, Richland, WA 99352) .
Radical reactions are very important to atmospheric chemistry, but are very difficult to study experimentally and theoretically. In principle, it is possible to determine rate constants for these reactions by ab initio methods. In the present work the reactions of some volatile organic compounds (VOC) with hydroxyl radical were examined. Minimum energy structures and ground state energies for the reactants, transition states, and products of reactions were found at various levels of theory including MP2/cc-pvdz and UCCSD(T) levels, using the Gaussian 98 electronic structure software package. DRDYGAUSS (Direct Dynamics with Gaussian) was used to trace out the minimum energy path along various reaction channels. Variational transition state theory (VTST) was used to obtain a rate constant from the topology of this energy surface. Changes in the level of theory used (selection of basis set and treatment of electron correlation) produce small changes in ground state energies and structures; the resulting pot ential energy surface becomes altered in the process. It was found that the theoretical rate constant depends exponentially on small variations in the potential energy surface topology. Thus, the level of theory has tremendous effect on the theoretically determined rate constant.

Determining the activation of EGF receptors depending on the availability of the ligand.. MELISSA SILVA (western washington university, bellingham, wa 98225) LEE OPRESKO (Pacific Northwest National Laboratory, Richland, WA 99352) .
Cells communicate in many different ways. One way in particular is through ligands. The ligands in the epidermal growth factor receptor are membrane anchored. The epidermal growth factor (EGF) receptor can be turned on and off by different stimuli in a cell's environment. In our research we are trying to determine which stimuli do just that and why, how the ligands are regulated, and why these stimuli have different outcomes in a given cell. We also want to look at how the cell signaling pathways are affected. We transfected Chinese hamster ovary cells (CHO cells) with six different chimeras. These chimeras were constructed in blue script and were cut out and put into the expression vector, pIRES puro. The six different chimeras are EGF: amphirigulin (ACT) (CT= cytoplasmic tail), EGF: betacellulin (BCT), EGF: EGF (ECT), EGF: heparin binding (HCT), and EGF: TNFalpha (TCT). Once the chimeras were successfully transfected into the cells, we put the cells into selection using puromyicen and lipofectamine. We then screened these cells using a LC antibody. In doing this we weeded out the cells that were not expressing the ligand. Once we did this, we grew up the positive colonies and froze them down. We will take the frozen cell and quantitate the rate of the release of a particular ligand using an EGF ELISA assay. This will allow us to see how the signaling pathways are affected.