Biopanning of the Single Chain Fragment Variable T7 Phage Display Library for Immunoprecipitation of Target Proteins. PAVEL BOGOMIAKOV (Johns Hopkins Baltimore, MD 21218) DIANA CONNOLLY (Hofstra University Hempstead, NY 11549) JOHN DUNN (Brookhaven National Laboratory, Upton, NY, 11973)

Antibody-antigen binding interactions can be exploited to selectively bind to specific proteins or other molecules of interest. One potentially rapid and efficient method for screening for reactive antibodies is antibody phage display, which relies on affinity selection of antibodies displayed on the surface of bacteriophage. We are developing a chicken Single chain Fragment variable (ScFv) T7 phage display system as a powerful method for immunoprecipitation of target proteins. ScFv antibodies are attractive for phage display because of their small fragment size, easy expression, and high clonal diversity. DNA sequencing verified that a previously constructed chicken ScFv T7 phage library exhibits considerable antibody diversity. To further develop protocols for using this phage display system, we are screening this library for antibodies specific for the E. coli cAMP receptor protein (CRP), which we crosslinked to a solid support for affinity biopanning. Four rounds of selection for CRP-specific antibodies have been completed and confirmed by immunoblotting of plaque lifts. Once specific phage clones are isolated, the single chained antibodies they encode will be expressed and used in chromatin immunoprecipitation (ChIP) experiments to examine CRP-DNA interactions that mediate gene expression changes. Long term implications of this research include the rapid production of antibodies to target molecules for disease research, pathogen detection, and protein structure studies.

Comparisons of Solvent Effects on Horseradish Peroxidase Stability. JONATHAN SALAZAR (New Jersey Institute of Tech Newark, NJ 07102) A.J. FRANCIS (Brookhaven National Laboratory, Upton, NY, 11973)

Enzymes can function as catalysts in non-aqueous solvents containing little or no water. This provides many advantages, such as increasing substrate solubility, shifting of thermodynamic equilibria, and improving enzyme stability. Ionic liquids have been considered potentially environmentally friendly solvents because of their unique properties, and more importantly, have been shown to be effective co-catalysts. This research explores the different solvent effects (hydrophilic and hydrophobic properties) on enzyme stability, and compares the effects of ionic liquids with a conventional organic solvent. It also provides data that can lead to new opportunities of bio-catalysis in ionic liquids. Horseradish peroxidase (HP) was chosen in this study for its broad applications in industry and organic synthesis. HP was incubated in different solvent systems, containing different concentrations of acetone, 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIM]PF6), or ethylpyridinium tetrafluoroborate ([EtPy]BF4) Enzyme activities were monitored by spectrophotometric methods in 48 hours.

Imaging Compositional Changes in Mouse Bone at High Resolution. THEODORE FELDMAN (State University of New York - Stony Brook Stony Brook, NY 11776) LISA MILLER (Brookhaven National Laboratory, Upton, NY, 11973)

Understanding the changes in material properties of bone during formation is critical for diagnosing and treating bone disease. Mechanical properties can be studied using assays such as nanoindentation (NI), whereas synchrotron-based infrared microspectroscopy (SIRMS) provides information on the chemical makeup of bone. In order to correlate the mechanical and chemical properties, these two techniques must be performed on the same sample. Typically, NI is performed on an embedded and polished thick specimen, whereas SIRMS data are collected by measuring the transmission of infrared light through thin, microtomed sections. We have developed a new method for collecting SIRMS data in a reflection geometry, such that NI and SIRMS can be performed on the same sample. This study validates the accuracy and quality of reflection SIRMS, while investigating spatially-resolved chemical changes in developing bone with respect to the sagittal axis. The tibia of two male BALB/cByJ mice, 10 to 21 days of age, were dehydrated and embedded in poly-methyl-methacrylate (PMMA). A 3µm-thick sagittal section was cut from the surface of each embedded bone. IR microspectroscopic imaging was performed on both the thin section and the surface of the sample block in transmission and reflection geometry, respectively. Transmission data were converted to absorbance using Beer's Law. Reflection data were converted to absorbance through a Kramers-Kronig (KK) transformation. The KK transformation will also be validated theoretically. The data from the two imaging methods were compared using common visible landmarks. Peak area ratios were performed on data from both the thin section and sample block to provide quantitative measures of mineral and matrix content and stoichiometry (i.e. phosphate-to-protein ratio; carbonate-to-phosphate ratio). Data analysis indicates that there are disparities between transmission and reflection data, particularly within the global phosphate-to-protein ratio. Chemical indices of collagen cross-linking and the carbonate-to-phosphate ratio appear consistent in trend and value across both time points and data collection modes. Future studies will be necessary to determine the source(s) of these differences.

Investigation of Cell Signal Transduction Pathways that Account for Anti-Apoptotic Effects of Basic Fibroblast Growth Factor in Radiation-Induced Apoptosis Models II: Bis-Benzimide Staining in Human Umbilical Vein Endothelial Cells. ALICIA WILLIAMS (Fort Valley State University Fort Valley, GA 31076) LOUIS PENA (Brookhaven National Laboratory, Upton, NY, 11973)

In cell biology, apoptosis is a process of deliberate suicide by an unwanted cell in a multicellular organism. Apoptosis can be initiated in a number of ways, including ionizing radiation. In cells, this process results in DNA damage and activation of signaling pathways that lead to cell death. Basic fibroblast growth factor (bFGF), known to cause cell proliferation, can inhibit apoptosis in irradiated cells. LY294002 hydrochloride is a Phosphatidylinositol 3 Kinase (PI3K) inhibitor. It blocks the action of PI3K thereby inhibiting the Protein Kinase B (AKT/PKB) pathway. This pathway functions to promote cell survival by inhibiting apoptosis. U73122 is a toxin that inhibits the hydrolysis of polyphosphoinositide (PPI) to inositol triphosphate (IP3). It blocks the action of the Phospholipase C- (PLC- ) hydrolase enzyme thereby inhibiting the Protein Kinase C (PKC) pathway. This pathway functions to promote cell differentiation. Endothelial cells can be used as a model system in which to study radiation-induced apoptosis. About two hours prior to irradiation, cells were serum starved from 10% fetal bovine serum (FBS) to 0.5% FBS. After irradiating the cells at 0.5 Gy/min with the Philips RT 100 X-ray machine, the samples were placed back in the incubator for varying lengths of time, harvested, stained with bis-benzimide (Hoechst 33258), and finally the cells were examined with fluorescent microscopy for changes in nuclear morphology (condensation and fragmentation) associated with apoptosis. In this project, several parameters were tested: time period after irradiation (4 - 24 hours), radiation doses (0 - 24 Gy), and harvesting methods (in situ fixation versus trypsinization). The best time interval to score apoptosis appeared to be about 18 hours after irradiation. For radiation doses, the range between 8 and 16 Gy were found to be adequate amounts of radiation for the samples because too much radiation can damage the cells and it would be difficult to determine whether a cell was apoptotic or not. Trypsinization was found to be the best harvesting method because the cells are easy to recover. The overall results indicate that there is still some work to be done on what inhibits apoptosis the most. Therefore, we are continuing working with other inhibitors to see which one blocks apoptosis without killing the cells.

Investigation of Signal-Transduction Pathways Accounting for Anti-Apoptotic Effect of bFGF in Radiation Induced Apoptosis I: Growth and Toxicity Assays in Murine Clonal Myoblast (MC3T3) and Human Umbilical Vein Endothelial (HUVEC) Cells. JEREMIE WILLIAMS (Fort Valley State University Fort Valley, GA 31030) DR. LOUIS PENA (Brookhaven National Laboratory, Upton, NY, 11973)

Apoptosis is a programmed cell death in which cells use specialized cellular machinery to kill themselves. Basic Fibroblast Growth Factor (bFGF) is a growth factor that causes cell proliferation, but can also inhibit apoptosis from occurring in cells. There are specific signal transduction pathways that are activated by bFGF. These include the AKT/PKB pathway and the PKC pathway. The Phosphatidylinositol 3 Kinase (PI3K) acts upon the protein kinase AKT which functions to promote cell survival by inhibiting apoptosis. The Protein Kinase C (PKC) pathway controls cell differentiation. There are also fungal-derived toxins that can inhibit these pathways. LY294002 blocks the action of the PI3K and thus blocks the AKT/PKB pathway. U73122 inhibits the hydrolysis of PPI to IP3, by PLCg and thus blocks the PKC pathway. The goal of this project is to find the optimum assay conditions in which we can test the anti-apoptotic culture effects of bFGF and to determine which the most critical pathway is. The XTT assay is used to measure bFGF effects in a growth assay and in a toxicity assay to assess the potency of the inhibitors. We tested LY294002 and U73122 at a concentration range from 1-1,000 µM. The results revealed that over 5 µM the fungal toxins kill the cells, but below 5 µM no toxicity was measured. We then irradiated the cells to see if they underwent apoptosis. The results revealed that they indeed did undergo this process and that the specific pathways that were targeted for this experiment were indeed correct.

Microbeam Radiation Therapy. JASMINE ESCALERA (Pace University New York, NY 10038) DR. AVRAHAM DILMANIAN (Brookhaven National Laboratory, Upton, NY, 11973)

Conventional radiation therapy has proven to have clinical limitations due to the overall risk of radiation-induced tissue damage. One of the new forms of radiation therapy used to treat a wide variety of cancers is Microbeam Radiation Therapy (MRT) which was first initiated at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (Slatkin et al., PNAS 1995). MRT uses a parallel array of microscopically thin planar slices of synchrotron-generated X-rays (microplanar beams, or microbeams). The unique feature of MRT is that the radiation exposure comprises thin, single-fraction, unidirectional microbeams. These microbeams have been shown in animal models to spare normal tissues, including the normal central nervous system, while damaging malignant tissue at therapeutic doses. This 'sparing' effect has been attributed to the rapid and selective regeneration of the normal-tissue capillary blood vessels damaged in the direct paths of the microbeams. Dr. Avraham Dilmanian has originated the novel use of microbeams in a special geometry called "interlaced bidirectional microbeams." This procedure produces a solid beam at the target from two interlacing arrays of microbeams. The method was used to irradiate targets in two animal models a) the normal rat brain, and b) a subcutaneous carcinoma tumor implanted in mice. The interlaced microbeams consist of two arrays, one array is shifted vertically with respect to the other by half the beam spacing, causing the two beams to interlace at the target, producing a solid beam there. In a study conducted while interning at Brookhaven, Balb/C mice were subcutaneously inoculated with the EMT-6 mammary carcinoma tumors behind their neck. They were then exposed in an upright position to a) bidirectional interlaced microbeams, or b) bidirectional broad beams. The neck site was chosen to use salivary glands as the normal tissue model. Results are still pending on this study. But earlier similar studies have concluded that a) "thick" microbeams (0.68 mm) spaced twice the beam thickness on-center retain their sparing effect on the normal brain, and b) interlaced microbeams combine the sparing effect of microbeams in the normal tissue around the tumor with the tumoricidal effect of broad beams. It is expected for the same conclusions to be seen in this new study.

Microbeam Radiation Therapy. ANN LAYVEY (Lehman College CUNY Bronx, NY 10453) AVRAHAM DILMANIAN (Brookhaven National Laboratory, Upton, NY, 11973)

Microbeam Radiation Therapy (MRT) is a new experimental form of radiation treatment for cancers. We studied irradiation of normal and tumor-bearing rats with arrays of parallel thin, planes of synchrotron-generated x-rays at the National Synchrotron Light Source (NSLS). The rats were monitored after the irradiations, and their brain was dissected upon euthanasia for histology. Previous studies suggest that MRT seems to be less damaging to healthy tissue than conventional radiation therapy. The aim of this study was to compare the efficacy of MRT to the conventional radiation therapy. First, we anesthetized the rats using a ketamine-xylazine solution and inoculated them in their brain with 9LGS tumor cells. The cells are injected into the striatum at the level of bregma 4 mm left of the midline, and 4mm deep into the brain. After fourteen days, the rats were irradiated at the NSLS; two groups of rats were irradiated at 60 Gy incident dose using two different array sizes. After the Light Source irradiation, the rats are weighed on a weekly basis. We also checked them daily for their general health, and euthanized them at the first sign of sickness due to the tumor growth. Euthanasia was carried out using either tissue perfusion with paraformaldehyde, or by subjecting the rats to a mixture carbon dioxide and oxygen. Few of the tumor bearing rats also had subcutaneous tumor and have to be euthanized. The brains of the euthanized rats were then preserved in formaldehyde for twenty-four hours and then placed into sucrose solution until they are needed for histology analysis. Behavior studies were conducted using an Open field test and RotoRod on the normal rats, irradiated with similar beams in their brain earlier, to determine the possible central nervous system effect produced by radiations. The study is inconclusive because some animals are still being followed up.

New Insights into the Cellular Actions of the Plant Defense Hormone, Methyl Jasmonate. ABIGAIL FERRIERI (College of the Holy Cross Worcester, MA 01610) RICHARD FERRIERI (Brookhaven National Laboratory, Upton, NY, 11973)

Studies have shown that exposing a plant to insect herbivory or to exogenous methyl jasmonate (MeJA), a known defense hormone linked to insect herbivory, will cause increased sucrose loading in phloem sieve tube elements with subsequent long-distance transport to sink tissues. Little is known, however, about the early actions of MeJA at the cellular level which can impact leaf sugar synthesis, its active loading, or long-distance transport. Recent in vitro cellular studies suggest that MeJA increases lipid membrane polarization enhancing the proton electromotive force which drives synthesis of adenosine triphosphate (ATP), the primary energy currency for cellular function . If true, changes in sugar synthesis and active loading could be a consequence of this action. The objectives of this project were, first, to establish a direct association between MeJA and lipid membrane polarization at whole-organism levels; and second, to test for correlations between chemical structure and membrane polarization for future radioligand design for in vivo imaging of membrane function. We hypothesized that exogenous jasmonate treatment in cut leaf studies will counter the inhibitory effects of treatment using an uncoupling agent such as carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Such agents prevent oxidative phosphorylation of adenosine diphosphate (ADP) back to ATP with consequences to sugar synthesis and its transporters. Sucrose loading was quantified by administering11CO2 to cut leaves, and measuring vascular loading of [11C]sucrose in leaf tissue using phosphor plate imaging. Tobacco leaves, treated with 40 uM CCCP through the cut petiole for 15 minutes displayed 0.2 ± 0.2% (n=3) loading in comparison with 7.0 ± 3.1% (n=4) loading in control cut leaves. Similar inhibitory effects were observed using another uncoupling agent, 2, 4-dinitrophenol (DNP). We also noted that cut and intact control leaves exhibited the same loading. Furthermore, cut leaves pre-treated with 1 mM MeJA prior to CCCP treatment displayed 3.4 ± 1.6% (n=3) loading. Our results provide additional evidence supporting the hypothesis that MeJA increases membrane polarization. Subsequent studies compared dose responses for [11C]sucrose loading between DNP and 4-fluoro-2-nitrophenol (FNP) with an intent to assess efficacy for using [18F]FNP in subsequent imaging studies. Although less acidic, FNP was found to be a more potent uncoupling agent than DNP.

Number of Active, Active-Sites in the SARS CoV Main Proteinase Homo-dimer. JOYCE LAI (University of California, Berkeley Berkeley, CA 94720) WALTER MANGEL (Brookhaven National Laboratory, Upton, NY, 11973)

During the first SARS epidemic, 10% of patients died. The infection was contained by quarantine. There still is no vaccine available; therefore, anti-viral agents are needed urgently. The SARS CoV main proteinase is an attractive target for antiviral agents as it is an enzyme required for viral replication. We have shown that the monomeric form of the enzyme is inactive; the active form of the enzyme is the homo-dimer (Figure 1). Are the two active sites in the homo-dimer functional or is only one of them active? To answer this question, the specific activity of dimers made from a mixture of potentially active monomers and inactivated monomers will be measured. If the specific activity of the active homo-dimer is twice that of the hetero-dimer, then both active sites in the homo-dimer are indeed functional. On the other hand, if the specific activity of the active homo-dimer is equal to that of the hetero-dimer, then only one of the active sites in the homo-dimer is functional. As a control, small angle x-ray scattering will be used to confirm that hetero-dimers form under the conditions used to measure enzyme activity. In order to do these experiments, a series of preliminary experiments were performed. Conditions were found under which a preparation of SARS CoV main proteinase dimers was completely inactivated by treatment with dithiodipyridine (dTdP). About 66% of the inhibition could be reversed by TCEP, a strong reducing agent. The mono-dimer equilibration rate was shown to be extremely fast. Finally, equations were derived to quantitate the distribution of monomers and dimers at equilibrium. Eventually the results of these experiments will be used to design antiviral agents.

Optimal Lengths For Genome Sequence Tags. LOGAN EVERETT (Binghamton University Binghamton, NY 13902) SEAN MCCORKLE (Brookhaven National Laboratory, Upton, NY, 11973)

A Genome Sequence Tag (GST) is a short (less than 50 base pairs) sequence of nucleotides which is extracted from a DNA fragment of interest and used to identify the same position in a known gene sequence. GSTs are experimentally useful because fragmentation processes often provide no indication of where in the genome each fragment came from. GSTs provide a method for determining the original location of the fragment with minimal sequencing. Shorter GSTs can lead to higher throughput experiments because more GSTs can be concatenated and sequenced as a group. However, GSTs below a certain length become useless because they are less likely to be unique. This work studied the theoretical effectiveness of GSTs over a range of lengths in order to identify the optimal lengths for various experiments. The method of studying GSTs was in silico analysis of known genomes, specifically: simulating the fragmentation and tag sequencing processes and comparing all potential GSTs of a fixed length to calculate how many are unique. The comparison is computationally intensive due to the large amount of GSTs to be considered (tens of millions for mammalian genomes). Several methods of optimizing the comparison were used, including a hash table, a 4-ary tree, and a sorted array. This work produced several computationally feasible methods for predicting the effectiveness of GSTs in experiments using specific splicing enzymes on mammalian DNA. Predicting GST effectiveness for sonicated DNA (randomly fragmented) has proved to be more complex; however, statistical approximations have shown to be equally effective and far less time consuming than an exhaustive analysis. These results have been used by other lab teams to design in vitro experiments using GSTs. In conclusion, in silico analysis is a powerful tool for designing a GST-based in vitro method because it can predict the effectiveness of any restriction enzyme. Sonication-based methods can also be predicted in silico, although a statistical, rather than exhaustive analysis, is recommended.

Purification of the Phospholipase A2 Domain from the B19 Parvovirus. MATTHEW ENGEL (State University of New York - Stony Brook Stony Brook, NY 11790) MARC ALLAIRE (Brookhaven National Laboratory, Upton, NY, 11973)

The human parvovirus B19 is a 5.6 kb single-stranded DNA containing virus. Its structure and genetics are similar to other parvoviruses, including the adeno-associated virus (AAV), a promising vector for gene therapy. The genetic material of B19 is contained in the capsid which governs infectivity, receptor binding, immune response, host cell range and tissue tropism. The capsid of B19 is composed of two structural proteins named VP1 and VP2. VP1 contains the 554 amino acids (aa) of VP2 but stretches for an additional 227aa at the N-terminus. This unique overhanging portion of VP1 (VP1up) exhibits phospholipase A2 (PLA2) activity and is essential for infectivity. To further understand the PLA2 interfacial enzymology and its role in the viral life cycle, a high resolution three-dimensional structure is necessary. In collaboration with Z. Zádori and P. Tijssen (INRS-Institut Armand-Frappier, Laval, QC Canada), the PLA2 domain of B19 was expressed in E.Coli as a fusion protein flanked by thioredoxin and a histidine tag. The 399 aa fusion protein was digested to remove these fragments and the region of interest was purified with high performance liquid chromatography (HPLC). This method relies on intrinsic biochemical and physical properties of the protein fragments for separation. The purification method consisted of anion exchange chromatography followed by gel filtration. The size of VP1up was verified by mass spectroscopy during HPLC to correlate each fraction with its corresponding fragment. Results have yielded samples pure enough for crystallization studies of the B19 PLA2 domain. We expect the high resolution atomic structure of VP1up will contribute to the further understanding of B19 PLA2 enzymology and its role in infectivity.

PyMOL Made EZ - A Tool for Simplifying Molecular Viewing in PyMOL. LAURA GRELL (Rochester Institute of Technology Rochester, NY 14623) CHRISTOPHER PARKIN (Rochester Institute of Technology Rochester, NY 14623) LEN SLATEST (Brookhaven National Laboratory, Upton, NY, 11973)

PyMOL is a molecular modeling program that can be used in a wide range of studies within the scientific community. Its ability to produce informative, detailed, stereoscopic images makes it a very powerful tool both in the laboratory and in the classroom. However, the PyMOL user interface is difficult to use and its "development has been focused on capabilities, not on ease-of-use for new users." (PyMOL Users Manual) To resolve this, we set out to develop a new interface for PyMOL that can be installed as a plugin and helps to eliminate the need for command line interactions, making it a much more user-friendly interface. Using tools from Python's Tkinter and PMW toolkits, we created a tabbed interface called PyMOL Made EZ. The interface contains a series of buttons and entry fields that allow users to select given attributes of a molecule and make changes to it with the click of a button as opposed to issuing highly syntactical commands. Other features of the interface include a Chime/PyMOL command converter, nine preset molecular views, four molecular movies, and molecular sequence and hetero atom information all at the click of a button. The ongoing and continued development of PyMOL Made EZ could help to further the use of PyMOL which we believe to be one of the most powerful molecular viewing programs currently available.

Quad-Buffered Stereoscopy for Collegial Education Programs. IAN LUND (Jamestown Community College Jamestown, NY 14701) JOHNATHAN LUNDGREN (Jamestown Community College Jamestown, NY 14701) ROBERT BENNET (Brookhaven National Laboratory, Upton, NY, 11973)

Quad-buffered stereoscopic visualization involves generating left and right eye images of an object and delivering each image to its designated eye. This is accomplished through denoting each image with light of opposite polarization. Special glasses are needed to allow light of specific polarization through so each eye receives its respective image. Stereo viewing produces depth (z-axis) in an object, which is not seen in a flat two-dimensional image, or even in a traditional three-dimensional image. As students of math and science often have difficulty visualizing fundamental concepts due to the limits of traditional 3D, stereoscopic rendering has firm academic uses. The goal of this project describes in detail potential uses of stereoscopy in a college curriculum. Some of the most beneficial topics to view in stereoscopy include atomic orbitals, chemical mechanisms, biochemistry, human anatomy, microchemistry, and engineering tools. PyMOL and VMD (Visual Molecular Dynamics) are both molecular viewers. These programs open up .pdb files (among others), which can easily be downloaded from the protein data bank. The primary molecules viewed are proteins, of which many are stored in the PDB. Atomic electron orbitals can be viewed using Orbital Viewer. Chemistry mechanisms, biology images, and many other things can be found by searching for VRML (Virtual Reality Modeling Language) files. A player can view any VRML file in stereoscopy and be able to rotate the object.

Stereoscopic Imaging of Scanning Electron Micrographs. TIMOTHY SMITH (Nassau Community College Garden City, NY 11530) JOHN SPILETIC (Brookhaven National Laboratory, Upton, NY, 11973)

Scanning electron micrographs exhibit three-dimensional qualities due to the high depth of field inherent in the use of the scanning electron microscope (SEM). Using the program StereoPhoto Maker, we have attempted to render these micrographs viewable in three dimensions. For this to be accomplished, a routine procedure was developed that permitted movement from trial-and-error to a method that yields consistent results. As in conventional photography, creation of a stereoscopic image using the SEM requires a left-eye and right-eye image. SEM stage controls permit sample movement in the X and Y axis, 360 degree rotation, and a tilt of 90 degrees toward and 15 degrees away from the signal detector. Images were captured using successive 2.5 degree increments, horizontal and vertical shifting, and sample rotation. Since rotation could not be measured directly, the SEM-generated scale bar was used to measure increments depending upon magnification. The resultant two-dimensional images were opened in a software application known as Stereo Photo Maker and combined to form a stereoscopic image based on two images of differing perspectives. In addition to the routine method of generating stereo pairs through sample tilting, other methods were also tested. It was determined that lateral shifting does not allow for the production of a visually satisfying stereo image. Rotation, however, produced three dimensional images of equal quality to that of tilted samples. We find that the greater variation of surface structure a sample has, a higher initial degree of tilt is required. However, the study of many more samples will be needed to verify this hypothesis.

Teaching Students About Evolution and Genetics: Analyzing DNA Polymorphisms to Test Competing Hypotheses. AMY DEMARCO (Columbia University New York, NY 10027-6902) SCOTT BRONSON (Brookhaven National Laboratory, Upton, NY, 11973)

People often get caught up in comparing one another based on broad, macro, and external criteria, such as skin color or ethnicity. However, this can be extremely misleading, and it overlooks the fact that genetically, we are all very similar. The present experiment was performed to teach thirty high school students to think of themselves on a micro, genomic level. Two regions of the genome were analyzed. First, each student isolated and amplified by PCR a 440 nucleotide sequence from the control region of the mitochondrial DNA of their cheek cells. This region of the mitochondrial DNA is also known as the hypervariable region, because it accumulates point mutations at a very high rate. The students sequenced this region and then compared their sequences to each other and to the sequences of other people of various backgrounds all around the world. Assuming that mutations occur at a constant rate, the number of mutations is proportional to the length of time that two groups have diverged. Thus, by comparing different sequences, it is possible to analyze relative relatedness. It was stressed to the students that so-called race is actually not genetically visible, and that members of the same background may have relatively more sequence differences than members of different backgrounds, which adds further evidence to the Out of Africa, or Displacement Hypothesis. Second, each student amplified a region of chromosome 16 in order to determine the presence or absence of an Alu jumping gene. Alu insertions represent ten percent of the human genome, and since these transposable DNA sequences are inherited in a Mendelian fashion, they can be used to track lineages. The ancestral state of chromosome 16 does not include an Alu insertion. This fact tells us that two people with an Alu insertion at the chromosome 16 locus have inherited this additional DNA from a common ancestor. Together, these two experiments not only provide students with basic knowledge concerning evolution and genetics, as well as laboratory skills such as PCR, DNA isolation, and gel electrophoresis, but they also allow students to realize how genetically similar people are even despite large phenotypic differences.

Thermodynamics of the Activation of the Adenovirus Proteinase by an 11-Amino Acid Peptide. RICHARD LIU (Stanford University Stanford, CA 94305) WALTER MANGEL (Brookhaven National Laboratory, Upton, NY, 11973)

The adenovirus proteinase (AVP) is unusual in that it requires cofactors for maximal enzyme activity. One cofactor is pVIc, the 11-amino acid peptide from the C-terminus of the precursor to adenovirus protein VI; the other cofactor is the viral DNA. The thermodynamics of the binding of AVP to pVIc was investigated by isothermal titration calorimetry (ITC). Before those experiments could be done, both the solubility of AVP and the binding constant for pVIc had to be increased. Lower pH increased the solubility of AVP to at least 50 mM; a mutant of pVIc had an increased binding constant. In one ITC experiment the measured change in enthalpy ( H) was -6000 cal/mol, the calculated change in entropy ( S) was 6.38 cal/deg/mol, and the calculated change in free energy ( G) was -7902.2 cal/mol. This implies the binding reaction was driven by the formation of hydrogen bonds and ion pairs. The ITC titration curve was complex, indicating several types of reactions were releasing heat. Understanding the thermodynamics of the binding of pVIc to AVP should offer profound insights into the activation mechanism of the proteinase by the cofactor. Ultimately, this information will be used to develop drugs that will act as antiviral agents for treating medical ailments caused by adenoviruses.

Use of the Program DISTANCE to Assess Population Size of the Eastern Hognose Snake (Heterodon platirhinos) at the Brookhaven National Laboratory. CHRIS CAMACHO (Wesleyan University Middletown, CT 06459) JEREMY FEINBERG (Brookhaven National Laboratory, Upton, NY, 11973)

The eastern hognose snake (Heterodon platirhinos) is a species of particular interest on Long Island and throughout the northeast, where habitat loss and declines in reported sighting have become a cause for concern. The program DISTANCE, a Windows based software package that aids in the design and analysis of wildlife population surveys, was used in an attempt to determine the population size of H. platirhinos at Brookhaven National Laboratory (BNL). Line transects were generated with the DISTANCE software and walked from early May to early July. Only one hognose snake was found in approximately 114 hours of walking transects, whereas six hognose snakes were found in 29 hours when conducting random pedestrian surveys in preferred H. platirhinos habitat. Thus, it is likely that DISTANCE is inefficient for generating a population estimate of hognose snakes at BNL and may not be appropriate for surveying other cryptic animals overall.

Using furfural derivatives for quantification of Detergent Concentration During Dialysis. JOSEPH GONCALVES (State University of New York - Stony Brook Stony Brook, NY 11743) DAX FU (Brookhaven National Laboratory, Upton, NY, 11973)

Monitoring detergent concentration during dialysis is a difficult task, primarily due to hydrophobic effects causing monomeric units to aggregate at or above the critical micellular concentration, an intrinsic property unique to specific detergents. A convenient procedure for quantifying the concentration of glycosidic detergents exploits the saccharide head group of detergents in this class. The concerted reaction of these molecules with phenol in the presence of concentrated sulfuric acid results in a furfural derivative, an aromatic compound capable of absorbing light with a wavelength of 490nm. Varying concentrations of n-dodecyl Beta-D maltoside were added to 20mM Hepes pH7.5, 100mM NaCl, 20% Glycerol and dialyzed against one another. A series of 50 ul fractions were collected from both external and internal dialyzing solutions for each trial daily over the course of 14 days. Upon collection of the final sample, a calibration curve was constructed using differing detergent concentrations in otherwise identical solutions. When analyzed as a function of dialysis time, detergent concentrations closely correlate to theoretical exponential decay, which allow for the calculation of a rate constant indicating that dialysis is virtually completed after four days.