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This page last updated on March 15, 2002.  
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Biodegradable Artificial Leukocytes for Targeted Drug Delivery
A. Omolola Eniola, Stephen D. Rodgers, Daniel A. Hammer
University of Pennsylvania, Philadelphia, PA 19104

        Due to the site-specific expression of selectins on endothelia cells of the blood vessels during inflammation, they are suitable biological molecules for target delivery of anti-inflammatory drugs. Previously our laboratory has shown that artificial capsules with adhesive properties of leukocytes can be made by attaching leukocyte adhesive ligands to polystyrene microspheres. A target drug delivery system using porous, biodegradable, poly-lactic-co-glycolic-acid (PLGA) microspheres coated with leukocyte functional ligand, Sialyl Lewisx, has been developed. We attached biotinylated-Sialyl-Lewisx to avidin-linked PLGA microspheres via carbodiiamide chemistry, and our in vitro assays showed that they display rolling adhesion to selectin-coated surfaces under hydrodynamic flow. The rolling velocity of these artificial leukocytes is similar to that displayed by leukocytes rolling on P- or E-selectin coated surfaces. We can tune rolling velocity, and hence residence time of capsules on surfaces, by changing the density of Sialyl Lewis x on the microsphere surfaces, and we have also demonstrated that these capsules will release model drugs on the order of several days. In vivo testing of the activity and adhesive properties of these capsules is underway.

Detection and Characterization of Laterally Phase Separated Cholesterol Domains in Model Lipid Membranes
Gregory M. Troup and Steven P. Wrenn
Drexel University, Philadelphia, Pa. 19104

        This work utilizes the fluorescence properties of the membrane probes 1-acyl-2-[12-[(5-dimethylamino-1-naphthalenesufonyl)amino]dodecanoyl]-sn-Glycero-3-phosphocholine (DANSYL), and ergosta-5,7,9(11),22-tetraen-3b-ol (ERGO) to detect and characterize what appear to be laterally phase-separated, cholesterol-rich domains in dimyristoylphosphatidylcholine(DMPC)/cholesterol, and egg lecithin(Egg-PC)/cholesterol model membrane systems. Specifically, the DANSYL membrane probe shows evidence of lateral phase separations in DMPC/Cholesterol and Egg-PC/Cholesterol membrane mixtures and can reveal liquid ordered to liquid disordered phase boundaries. Fluorescence resonance energy transfer from ERGO to DANSYL is used to characterize the size of the putative laterally phase separated cholesterol-rich domains in model membranes systems. The agreement of the experimental determination of the Ld/Lo phase boundaries via DANSYL emission maximums to ESR and FRAP techniques, validates the use of DANSYL for the detection of laterally phase-separated domains. The DANSYL red shifting data provides evidence for the presence of a lateral phase separation in the cholesterol regime that was thought to be only in the liquid ordered phase. A fluorescence resonance energy transfer model is presented that estimates the size of putative domains in 40 mole% cholesterol dmpc MLV’s at 30°C to be 288 angstroms in diameter.

The Distribution of a Lipophilic Fluorescent Compound from a Sustained-Release Device in the Rat Eye
H. Kim, J. Baffi, G. Byrnes, N. Collins, R. Lutz, K.G. Csaky, M. Robinson, and N.S. Wang
University of Maryland - College Park, College Park, MD, 20742

        Ocular diseases such as retinitis, glaucoma, lymphoma, and macular degeneration are difficult to treat with systemic drug therapy because many drugs fail to deliver significant amounts of drug to deep eye structures. Intravitreal drug injections have been administered with some success, but repeated injections for chronic treatment are impractical for most patients. With advances in controlled-release technology, interest has increased in the development of long-term, drug-releasing ocular implants for treating chronic eye diseases.
We examined two types of sustained release implants as candidates to deliver drug to the retinal region: subconjunctival implant and intravitreal implant. Compared to surgical implantation directly in the vitreous cavity, sustained-release implants placed in the subconjunctival space may be safer and more effective in delivering drugs into the choroid and subretinal space. We investigated the ocular distribution of a fluorescein compound from a sustained-release device placed in the subconjunctival space and that from an intravitreal implant placed in the vitreous cavity.
We constructed sustained-release matrix implants by coating a lipophilic fluorescein compound (5-hexadecanoylaminofluorescein, MW=586) with either polyvinyl alcohol (PVA), hydropropyl cellulose (HPC), or silicone. The implants, made into 1.5 mm flat discs, were surgically placed in the subconjunctival space and in the vitreous cavity of Brown Norway rats. The animals were sacrificed periodically up to 1 week post-implantation in the case of a subconjunctival implant and up to 3 hours in the case of an intravitreal implant and enucleated, and frozen sections of the globes were done. The slides were examined with a fluorescent microscope.
In subconjunctival implants, only the HPC matrix, which had the highest release rate, yielded detectectable amount of fluorescein label in the choroid and subretinal region. In contrast, in intravitreal implants, significant amounts of fluorescent were detected in the vitreous and inner retinal region even 3 hours post surgery, but very little of it reached the subretinal region. The choroid presents a formidable barrier to the passage of a lipophilic fluorescein compound into the subretinal region from the subconjunctival space. The rapid clearance of the drug in the choroid by the blood flow may be the cause.

N-GLYCAN SITE OCCUPANCY PROCESSING IN MAMMALIAN AND INSECT CELLS
Karthik Viswanathan, Jullian Jones, Dr. Sharon Krag, and Dr. Michael Betenbaugh
Johns Hopkins University, Baltimore, MD, 21218

        Mammalian and insect cell lines are used for the expression of numerous secreted and membrane-bound glycoproteins used as diagnostics, therapeutics and for vaccine production. Unfortunately, some heterologous glycoproteins can be generated without full occupancy of all N-glycan attachments and the presence of an unoccupied asparagine sites. The presence of variable site occupancy leads to changes in the product composition and the absence of an N-glycan attachment can alter a protein's solubility and biological activity. In order for N-glycosylation to occur, the cell must transfer the substrate, dolichol-linked oligosaccharide (DLO), from the membrane of the endoplasmic reticulum to the acceptor polypeptide. The lack of site occupancy can be caused by the inability to generate sufficient concentrations of the lipid-linked donor substrate or by the inefficient transfer of DLO to the protein. In this study, we are examining the biochemical pathways involved with DLO synthesis and processing in insect and mammalian cell lines. Cell growth and production of DLO is being measured in normal cells and cells which have been subjected to inhibitors. Analytical techniques will be used to measure the levels of the final lipid-linked substrate as well as relevant intermediates. From these studies, it may be possible to identify potential bottlenecks in the N-glycan processing event. The effect of DLO synthesis and inhibitors on site occupancy of proteins will be considered as well as potential methodologies that may overcome any identified bottlenecks.

Overexpression of an Archaeal Protein in Yeast: Secretion Bottleneck at the ER
Jason D. Smith, Anne S. Robinson
University of Delaware, Newark, DE 19716

        Archaeal enzymes have great potential for industrial use; however, expressing them in their natural hosts has proven challenging. Growth conditions for many archaea are beyond typical fermentation capabilities, and to compound the problem, archaea generally achieve much lower biomass yields than Escherichia coli or Saccharomyces cerevisiae. To determine whether a eukaryotic host, S. cerevisiae, would be a suitable alternative for archaeal protein production we examined the expression of the tetrameric beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. We engineered the beta-glucosidase to facilitate secretion into the culture medium and have demonstrated the beta-glucosidase’s secretion and activity. We determined the dependence of beta-glucosidase secretion on gene copy number and obtained a 
transformant capable of secreting approximately 10 mg/L in batch culture. All transformants retained large intracellular fractions of beta-glucosidase, indicative of an intracellular bottleneck. Cell fractionation by sucrose density centrifugation and immunofluorescence identified the endoplasmic reticulum as the secretion bottleneck. Preliminary evidence indicates that the cause of this bottleneck is misfolding of the monomeric beta-glucosidase, rather than tetrameric association. Expression at moderately elevated temperatures (between 30-40°C) improved beta-glucosidase yields, suggesting that higher temperature expression may improve folding and secretion yields.

Protein Conformation in Reversed-Phase Chromatography: Effects of of Salt and Surface Hydrophobicity
Jennifer M. Sokol, Bryan Holmes, John P. O'Connell, Erik J. Fernandez
University of Virginia, Charlottesville, VA 22904

        Hydrophobic interaction (HIC) and reversed-phase (RPC) chromatography can be powerful tools for protein purification but their use can be limited by complex retention behavior and low recoveries in some cases. Even stable proteins have been shown to unfold when exposed to RPC and HIC surfaces ; however, the structural changes that occur during these processes are not well understood. This is due in part to the difficulty in studying proteins under adsorption conditions with conventional spectroscopic techniques. 
We have used hydrogen exchange detected by mass spectrometry (HX-MS) to detect solvent exposure as a sensitive measure of protein conformation. RPC experiments were performed on C4 and C18 alkyl-modified silica media to address the role of surface hydrophobicity. The effect of added salts from opposite ends of the lyotropic series was also investigated. HX-MS revealed the distribution of conformations present upon elution from an RPC column. Retention time and the proportion of folded vs. unfolded protein varies with salt type in an order consistent with the lyotropic series, as well as with surface hydrophobicity. The relation of these observations to other hydrophobic systems will be discussed

S. aureus Dynamic Binding Interactions in Whole Blood Over a Collagen Substrate
Lisa Mascari and Julia M. Ross
University of Maryland Baltimore County, Baltimore, MD 21250

        S. aureus is a pathogenic organism responsible for many types of infections, including infective endocarditis, and has demonstrated increasing antibiotic resistance making the underlying infection mechanisms important to understand. The initial step leading to a bacterial infection is adhesion to a host substrate via specific ligand-receptor bonds. Specifically in infective endocarditis, bacteria and platelets combine to form growing thrombi on the traumatized vasculature surface where abundant collagen is exposed. Platelets and S. aureus have both been shown to bind a collagen substrate under shear conditions, with platelets being much more reactive. In this study, we examined the ability of S. aureus cells suspended in whole blood to bind a collagen surface under physiologically low (100 s-1) and high (1000 s-1) fluid shear conditions. A parallel plate flow chamber was used to create a defined shear environment. The location (at the collagen surface or in the platelet aggregate above the collagen surface) and number of bacteria cells were quantified under fluorescence using confocal laser microscopy. The results demonstrate that under both low and high physiological shear S. aureus cells are able to bind at the collagen surface despite the highly reactive platelets/collagen interactions.

Targeted Microarray Analysis of Proteolysis in Hyperthermophilic Microorganisms
Keith R. Shockley and Robert M. Kelly
North Carolina State University, Raleigh, NC 27607

        Proteases are the enzymes responsible for breaking down polypeptides and they are known to regulate many important cellular activities. Currently, proteases produced from mesophilic hosts are used very widely in industrial processes, but the production of thermostable and thermoactive proteases from hyperthermophilic microorganisms holds great biotechnological potential. In order to take advantage of this potential, it is necessary to understand how proteases are regulated in prokaryotes, especially hyperthermophiles, which can be cultivated with less risk of contamination in continuous culture1. The recently released complete genome sequences, coupled with the advent of the cDNA microarray, provide a new means to discover proteases while examining their physiology, biochemistry, and genetic regulation. 

        We have constructed a targeted cDNA microarray chip incorporating known and putative genes relating to proteolysis2, stress response, and central metabolism from the hyperthermophilic archaeon Pyrococcus furiosus. This targeted cDNA microarray was used to follow the expression of 40 known and putative protease-related genes for this organism grown under normal and stressed conditions on peptide-based media containing elemental sulfur. From this total, 5 encode ATP-dependent proteases, 13 encode ATP-independent proteases, and 22 encode peptidases. A total of 6 proteases and 8 peptidases were differentially regulated two-fold or more in response to heat shock, while 3 ATP-independent proteases and 5 peptidases were not expressed at detectable levels during either growth condition. Although all 5 ATP-dependent protease genes in P. furiosus were expressed at relatively high levels during both stressed and unstressed growth, their change in expression did not increase in the same manner as the response of the ATP-dependent protease system in bacteria that regulates the in vivo turnover of RpoH (s32). Indeed, the expression of the sole gene encoding substrate recognition (alpha) subunit of the proteasome and the gene encoding the Lon protease decreased significantly during heat shock. This suggests that the proteolytic response to stress in archaea is accomplished by ATP-independent proteases working in concert with the ATP-dependent proteases. The results of our targeted cDNA microarray experiments have been evaluated and confirmed by bioinformatic approaches and Northern blotting experiments.

1. Pysz, M. A., Rinker, K. D., Shockley, K. R., and Kelly, R. M. (2001). Continuous cultivation methods for hyperthermophilic biomass and enzyme production. Methods Enzymol. 330: 31-40. 

2. Ward, D. E., Shockley, K. R., Chang, L. S., Levy, R. D., Michel, J. K., Conners, S. B., Kelly, R. M. (2002). Proteolysis in hyperthermophilic microorganisms. Archaea. 1: 63-74.

Using CFD to Understand E. Coli Breakage in High-Pressure Homogenizers
Mark Rogowski, Justin Miller, and Dr. William J. Kelly
Villanova University, Villanova, PA 19085

        A Computational Fluid Dynamic (CFD) model of flow in a high-pressure homogenizing valve (APV Gaulin model CD-30) was developed with the Fluenttm software. The flow (single-phase) was modeled as laminar upstream of and in the channel (gap), and turbulent downstream of the channel exit. Applying a realizable k-e turbulence model, the CFD model accurately predicted the effect of gap space on fluid dynamic conditions upstream (inlet pressure and pressure gradient) and downstream (impact pressure) of the channel for a valve with a standard (CD-0) impact distance (0.25 mm) and a 1 cp. fluid. This CFD model was then used to estimate the magnitude of the fluid dynamic parameters (except cavitation effects) presumed to be responsible for cell breakage, as a function of gap space, impact distance and fluid viscosity. By conducting experiments to isolate individual cell breakage mechanisms for a single pass, threshold values were identified for breaking Escherichia Coli cells: pressure gradient - 1.2 x 1012 Pa/m, energy dissipation rate - 1 x 1010 m3/s2 and impact pressure - 140 psig. Finally, a process control methodology was developed for varying the inlet pressure in response to a viscosity change, to maintain constant cell breakage. 

1.    Alginate Strings and their Applications in Spinal Injury
Saravanan Kanakasabai and Margaret Wheatley
Drexel University, Philadelphia, PA 19104

        We have optimized a method for producing strings of alginate bioconjugated with a laminin pentapeptide that aids in cell adhesion. These strings are 400-500um in thickness. In vitro studies have shown that rat (NB2a) and human (SHSY5Y) neuroblastoma cell lines adhere to and differentiate on these modified alginate strings. These strings are strong enough to be surgically transplanted onto the spinal cord, and may ultimately help in bridging the gap at the injury site.

2.    Characterization of Intracellular Proteolytic activity in Pichia pastoris
Dr. Tracey R. Pulliam-Holoman and Nekeisha S. Sweeney
University of Maryland, College Park, MD 20742

        Intracellular proteolytic activity was investigated during the growth and protein production stages for batch fermentations of the methylotrophic yeast Pichia pastoris and analyzed using SDS-G PAGE and X-Ray film. Comparisons were made for Pichia pastoris GS115 pPicZ/lacZ which utilizes the alcohol oxidase I (AOX 1) promoter to drive the methanol inducible expression of b-galactosidase, and GS115 without the plasmid to determine whether proteolytic activity was in response to metabolic stress associated with cloned gene overexpression or methanol feed. These host vector systems were used to study methanol feed conditions in a continuous mode vs. two pulsed modes and to investigate the most favorable feed condition for protein production and minimization of proteolytic activity. Similar evaluations were made for Pichia pastoris X33, the wild-type strain, and X33 pGapz/lacz which utilizes the glyceraldehyde-3-phosphate (GAP) promoter to allow constitutive, high level expression of Beta-galactosidase. 

3.    Characterization of Neuronal Cell Injury and Neuroprotective Effect of Poloxamer
Gulyeter Serbest, Joel Horwitz, and Kenneth A. Barbee
Drexel University, Philadelphia, 19104

        To better understand the cellular mechanisms of neuronal injury, it is very important to mimic mechanical loading conditions experienced by the cells in vitro. Mechanical force applied to cells outside may activate some downstream events inside the cells, which may further cause necrosis or apoptosis. In this study, we used a cell culture model, which is an idealized system to investigate cell injury at the cellular and molecular levels. We developed an injury model, which allows a precisely controlled mechanical stimuli and an easy quantification of cellular responses. A dynamically controlled shear stress was applied on PC2 cells and cell viability was assessed at 24 hours. Dynamic mechanical loading of cells produced graded levels of injury as assessed by long-term viability. We also used Poloxamer to demonstrate its neuroprotective effect on the injured cells. This system can be used to investigate further the mechanism of the injury and to assess various treatments of neuronal injury and subsequent degeneration.

4.    CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS NEWMAN DYNAMIC ADHESION AND DESTABILIZATION FROM FIBRINOGEN
Michael A. Johnson and Julia M. Ross
University of Maryland Baltimore County, Baltimore, MD, 21250

        Staphylococcus aureus is a major cause of infectious diseases such as septic arthritis, osteomyelitis, and endocarditis. Its adhesion to fibrinogen is an important initial step in infections to injured tissue, and with increasing bacterial resistance to antibiotics new therapeutic treatments are needed. S. aureus Newman adhesion and detachment from fibrinogen was investigated under physiological shear conditions and was quantified using a parallel plate flow chamber and videomicroscopy. It was found that, of 5 monoclonal antibodies (mAb) studied, the two mAbs to clumping factor A (ClfA) caused detachment of S. aureus from fibrinogen. Detachment was found to work best at intermediate shear rates, and at an antibody concentration of 10 micrograms per ml, where adhesion to fibrinogen was reduced to the same levels as the Newman ClfA negative strain. This data suggests that the two antibodies would be able to cause reduced virulence of S. aureus.

5.    Chitosan Softarrays Based on Combinatorial Fluorescence Hybridization Assay
Hyunmin Yi1,2, Li-Qun Wu^2,3, James J. Sumner⁴, James B. Gillespie⁴, Gregory F. Payne^2,3, and William E. Bentley^1,2
1University of Maryland, College Park MD 20742
²Center for Agricultural Biotechnology, UMBI, College Park MD 20742
³University of Maryland, Baltimore MD 21250
⁴Army Research Laboratory, Adelphi MD 20783

        A combinatorial fluorescence-based nucleic acid hybridization assay was developed using a naturally occurring biopolymer, chitosan, as a covalent binding substrate for single stranded DNA oligonucleotides. A 96-well microplate format enables high throughput, easy and fast detection of many samples of several target genes at the same time. Various fluorescein derivatives, chemical crosslinkers and oligonucleotides with 5’ or 3’ end labels were used to confirm covalent immobilization to chitosan and hybridization of ssDNA to anchored oligos. Characterization and optimization of reaction conditions at each step, as well as assessment of unspecific side reactions and stability of each covalent bond are discussed.

6.    Combinatorial Protein Engineering by Domain Insertion
Gurkan Guntas and Marc Ostermeier
Johns Hopkins University, Baltimore, MD, 21218

        Molecular evolution has proven to be a powerful approach for engineering proteins with improved and novel functions. A key step in applying molecular evolution is the creation of DNA diversity by such methods as error prone PCR or DNA shuffling. One method of creating diversity that has not been extensively explored, particularly in a combinatorial fashion, is that of domain insertion: the insertion of one protein domain into another. We have created domain insertion libraries between two proteins: TEM-1 beta-lactamase and the E. coli maltose binding protein (MalE). Two different strategies for creating the libraries have been developed that involve the random digestion of DNA with either DNaseI or S1 nuclease. Libraries created by these two methods have been found to have different distributions of deletions and tandem duplications at the insertion site. Using a selection scheme involving a malE- auxotroph, we have selected bifunctional fusion proteins of beta-lactamase inserted into MalE that confer resistance to beta-lactam antibiotics and are capable of transporting maltose in E. coli.

7.    Detection of DNA hybridization by impedance and development of an impedimetric DNA microarray for clinical classification of brain tumors
Scott Taylor, Stephanie Smith, Marin Gheorghe, Derk Bemeleit, Dietmar Blohm, Oliver Bögler, William Broaddus, and Anthony Guiseppi-Elie
Virginia Commonwealth University, Richmond VA, 23284

        Current DNA hybridization detection methods are based on fluorescence. In an effort to improve existing technology, we have developed an electrical method for detection of oligonucleotide hybridization. Our system utilizes an interdigitated microsensor electrode (IME) array fabricated on an oxidized silicon or glass substrate. With this arrangement, significant changes in electrochemical impedance values (both real and imaginary components) (11 % increase in impedance modulus at 120 Hz) have been detected resulting from hybridization of covalently immobilized oligos to its complement under hybridization conditions. To expand this paradigm, the beBiochiptm has been designed. The chip comprises 32 separate regions of interdigitation where oligonucleotide probes are spotted using a Cartesian SynQuad microarray robot and subsequently covalently immobilized via amine-to-epoxide linking chemistry. Colloidal gold labeled or unlabeled reverse transcribed mRNA can be exposed to the spotted probes on the chip. Hybridization is detected by a change in impedance between 5 micron lines and spaces. A set of ~500 genes associated with brain tumors (Grade I-IV) has been identified. We are currently engaged in selecting a subset of these genes by conventional fluorescence microarray analysis that are to be spotted on a series beBiochipstm, collectively named the B.T. Oncochiptm series, for clinical diagnosis and staging of brain tumors. 

8.    Evanescent Sensing of Biomolecules and Cells with a Biconically Tapered Fiber
Hong S. Haddock, P. Mohana Shankar, and Raj Mutharasan
Drexel University, Philadelphia, PA 19104

        Fiber optic sensing is a well-developed approach for sensing of biomolecular and intracellular parameters, as optical fibers have been increasingly employed in applications such as biochemical, biomedical, and environmental sensors. we present the measurement of NADH, NADPH and cells using evanescent absorption. We demonstrate that detection of biomolecules and cells at extremely low concentrations and volume can be achieved accurately with a thin biconically tapered fiber. The lowest concentrations that can be detected for NADH, NADPH, and cells with a fiber are 0.2 uM, 0.8 uM and 0.1 million cells/ml, respectively, while with a cuvette, the detectable concentrations are 3uM, 3uM, and 1 million cells/ml, respectively. A parameter, which is the product of the extinction coefficient and the light path of the sample, has been proposed from the beer-Lamberts Law for comparison of the sensitivity with the fiber and the cuvette. Results showed that the sensitivity of the fiber to that of the cuvette is 71.3 for NADH, 13.5 for NADPH, and 4.30 and 5.36 for cells at wavelengths of 400 nm and 450 nm, respectively.

9.    Factors effecting interface location in industrial continuous flow decanters
Debra Metzger and Dr. William J. Kelly
Villanova University, Villanova, PA 19403

        Centrifugal scroll decanters (CSDs) are an important type of chemical processing equipment which is often employed as part of the process to isolate and purify fermentation products. These mechanically complex devices separate the product-bearing solvent extract from the "spent broth" layer (waste cells and aqueous supernatant) after performing a whole broth extraction of fermentation broth. While CSDs have been marketed for many years, there is almost no scientific/technical literature available on their operation. Aside from a simple hydrostatic balance calculation to make an initial guess of the ring dam setting, there is no method currently available to predict their performance. In this research, a mathematical model was developed, based on Bernoulli's equation, for predicting how variables such as heavy phase flowrate, diameter and length of the heavy phase effluent line and heavy phase viscosity effects the height of the interface. Preliminary experimental results using a gravity decanter match the mathematical model predictions, in that the interface height varies linearly with the length of the heavy phase outlet line

10.    Fluorescence Study of the Aggregation Behavior of Lecithin-Bile Salt Micelles
Manasa V. Gudheti and Steven P. Wrenn
Drexel University, Philadelphia, PA 19104

        Lecithin-Cholesterol-Bile Salt micelles are one of the transporters of cholesterol in bile. The degree of supersaturation of cholesterol in bile, which has an effect on the formation of gallstones, depends on the concentration of these micelles. As a primary step in characterizing these micelles, it would be useful to study the aggregation behavior of a simplified system of Lecithin-Bile Salt micelles. Steady state and time-resolved fluorescence spectroscopy are used to determine the aggregation numbers. The value of the aggregation number contains information on the micelle shape and size, which plays a role in determining the stability of the system.

11.    High pressure dissociates tailspike protein aggregates and promotes native structure formation
Brian G. Lefebvre and Anne S. Robinson
University of Delaware, Newark, DE 19716

        Protein aggregation plays a major role in medicine, biochemical research, and in the biotechnology industry. The tailspike protein is an excellent model system for studying beta-sheet folding and aggregation because the structure is known, folding and aggregation pathways have been identified, and aggregation of tailspike chains occurs by specific interactions. Protein folding and aggregation are in direct competition, yet the factors that determine whether an unfolded protein proceeds to a folded protein or an aggregate remain largely unknown. Recently, hydrostatic pressure has been used to dissociate protein aggregates and promote the formation of native protein (Foguel et al., 1999). In order to identify the mechanism of this high pressure behavior, we compared the folding of freshly-denatured P22 tailspike protein to the folding of pressure-dissociated tailspike aggregates under conditions in which both folding and aggregation occurred. Tailspike aggregation at 100 µg/mL was  monitored at four temperatures using a combination of size-exclusion chromatography and native polyacrylamide gel  electrophoresis (PAGE). As temperature increased, the yield of native trimeric tailspike decreased from 26.1 µg/mL at 20 °C to 0 µg/mL at 37 °C. Pressure-treatment rescued aggregates created at 37 °C and yielded 19.8 µg/mL of native trimer following depressurization and incubation at 20 °C. The rate of refolding of “freshly-denatured” tailspike was compared to that following pressure treatment. The trimer formation rate increased by a factor of roughly five, and the aggregate rate decreased by a factor of three, following pressure-treatment. These results indicate that pressure-treatment of aggregates likely produced intermediates that contain native-like structures enabling rapid trimer assembly.

12.    A High Throughput Approach of Promoter Study using Green Fluorescent Protein
Canghai Lu and Govind Rao
University of Maryland Baltimore County, Baltimore, MD 21250 

        Green fluorescent protein (GFP) is a new reporter gene and is making an impact due to the many advantages it has over other reporter genes such as: autofluorescence, in vivo detection, no requirement for co-factors. By cloning a gene promoter upstream of gfp gene and exposing the living cells transformed the fusion to the specific inducer, gene expression can be monitored by measurement of green fluorescence emitted by GFP. GFP fluorescence is quantifiable and its first derivative is able to overcome the long folding time of GFP and sensitively show the gene expression. Our approach includes the high efficiency subcloning method using pGlow-TOPO vector and the high throughput detection system in 96-well micro plate. The selected promoter genes of oxidative stress response system in E. coli were cloned upstream of GFP gene and systematically studied in both endpoint and kinetics formats. The results showed that our approach is easily executed and could be used in quantitative and temporal promoter study in high throughput format.

13.    Intracellular pH changes in shear challenged CHO cells
Jared Simons and Raj Mutharasan
Drexel University, Philadelphia, 19104

        Changes in a cell culture environment often alters intracellular conditions. In this work we measure intracellular pH as a metabolic marker of the cellular response. The goal of this study is to investigate intracellular pH changes in response to hydrodynamic shear induced stresses in a bioreactor. Recombinant Chinese Hamster Ovary cells were cultured under a low shear environment and subsequently agitated to three higher, sublethal shear levels. Intracellular pH response was measured using the rapid measurement protocol developed previously by our lab. Results indicated a pHi alkalinization occur with increasing shear.

14.    Manipulating Recombinant Protein Yield Using RNA Interference to Host Genes
Shannon F. Kramer and William E. Bentley
University of Maryland, College Park, MD 20742

        RNA interference (RNAi) is a powerful tool using double-stranded RNA that can be used to inhibit the production of protein by causing degradation of the homologous mRNA. The effects of 16 genes, including four housekeeping genes, three proteases genes, five metamorphosis related genes, two stress genes, and two unknown genes on the production of recombinant protein using the baculovirus, AcMNPV (Autographa californica multiple nucleocapsid nuclear polyhedrosis virus), in Trichoplusia ni (T. ni) larvae was studied using RNAi. As expected, most of the genes studied showed little effect on, or a decrease in recombinant protein production, but several of the genes did increase recombinant protein production. Therefore, RNAi can be used as a metabolic controller in T. ni larvae to manipulate host functions and increase recombinant protein production.

15.    Metabolic Engineering for Sugar Nucleotide Cofactor Regeneration
Dongsheng He, Elton Wade and Ruizhen Rachel Chen
Virginia Commonwealth University, Richmond, VA 23233

        Oligosaccharides, often as glycoconjugates, are bio-recognition elements involved in processes such as fertilization, embryogenesis, metastasis, inflammation and pathogen adhesion. Medicinal applications of synthetic oligosaccharides have been hampered by the difficulties in synthesizing these multifunctional compounds in sufficient quantities that support research and clinical development leading to effective therapeutics. Chemical synthesis of oligosaccharides is a laborious, low yielding process owing to the needs of multiple protection and de-protection steps to give the required regio-selectivity and stereo-selectivity. In contrast, biocatalytic synthesis employing glycosyltransferase offers the needed selectivity and often, quantitative yield. However, glycosyltransferase-based synthesis is limited by the availability of required activated sugar (sugar nucleotide) as enzyme substrate. This research addresses this critical issue by metabolic engineering. 

        An agrobacterium strain that naturally overproduce glucan polymer was genetically modified to disable the polymer production. Further modification was introduced through heterologous protein expression. 
The presentation will highlight biochemical characterizations of the genetically engineered Agrobacterium strain and preliminary results demonstrating the utility of the regeneration system. 

16.    Molecular Diagnostics Using Colloidal Gold Nano-Particles
Tin Christopher Hang and Dr. Anthony Guiseppi-Elie
Virginia Commonwealth University, Richmond, VA 23284

        Reported here is the development and use of colloidal gold nanoparticles as impedimetric labels for DNA detection using microfabricated interdigitated electrode arrays (IDAs). More specifically, the molecular recognition via the DNA-cDNA-Au hybridization reaction results in nanoparticle aggregation at the chip surface that yields a measurable change in electrical impedance. The non-specific adsorption of colloidal gold nanoparticles that are used as detection labels of biological recognition reactions in bioelectronic devices, however, may potentially create error responses or false signals in such devices. The primary goal of this research was to examine, using impedimetry and impedance spectroscopy, the interaction of these colloidal gold nanoparticles with engineered surfaces of different chemical compositions. The colloidal gold nanoparticles (ca 30 – 40 nm diameter) were synthesized using a modified procedure of Lee and Meisel (1) wherein the reduction of hydrogen tetrachloroaurate (III) hydrate (HAuCl4.3H2O) by sodium citrate (C6H5Na3.2H2O) under stirred, reflux conditions produces nanoparticles of colloidal gold. Particles were characterized in aqueous solution by UV-VIS spectroscopy and by AFM. Engineered surfaces were produced by monolayer self-assembly of organosilanes of different w-terminal functional groups, including -SH, -COOH, -NH2, -CN, - Br, -I, and –Cl, on borosilicate glass slides and IME chips. Surfaces were characterized by contact angles measurements using deionized water and hexadecane and by the impedimetric response of 15 um line and space IDE chips that were chemically modified and immersed in colloidal gold suspensions. The engineered surfaces with amine functional groups showed the most significant impedimetric responses, while the ones with amine and thiol terminal groups displayed the most notable contact angle responses. Further, Quartz Crystal Microgravimetry (QCM) was also used to quantify the various layers of oxides, organosilanes, and ultimately DNA deposited onto the platinum surfaces.

17.    Myocyte Adhesion and the Effects of Compliant Substrates
Adam Engler, Maureen Sheehan, Cindy Newman, Lucie Bacakova, and Dennis Discher
University of Pennsylvania, Philadelphia, PA 19104

        Myocyte function is ultimately predicated on adherent contraction, a feat that is physiologically enabled by suitably strong adhesion on sufficiently compliant matrices. In one set of studies directed at Myocyte adhesion, the spreading of A7R5 rat aortic smooth muscle cells was investigated as a function of collagen pre-adsorbed to glass. Four hours after plating, the projected area plateaus at high collagen levels and is well modeled by a spreading liquid drop of constant membrane tension rather than constant surface area. At longer times, however, cell area differences were minimal which probably indicates sufficient time for cell secretion of matrix proteins. The minimal adhesive difference was directly confirmed by indistinguishable results for peeling these cells off of glass versus micro-patterned collagen, using a translating micropipette system. A7R5 cells were also plated on variably crosslinked polyacrylamide gels (PAG) and displayed a two-fold increase on stiff gels (0.3% bis-crosslinker) versus soft gels (0.03% crosslinker), but without time dependence as seen on glass. In a second set of studies, C2C12 murine skeletal muscle cells were grown on either PAG for up to four weeks or on collagen-patterned glass for up to two weeks. After four hours, C2C12 cells spread several-fold more on stiff gels versus soft gels; and, on rigid glass with high concentrations of adsorbed collagen, cells spread several-fold more still. By 24 hours, however, such spread area differences had dissipated in this system also. At 48 hours on patterned collagen, ontrolled differentiation into myotubes was induced for studies of long-term adhesiveness by cell peeling. While we generally find that C2C12 myotubes peel at rates comparable to A7R5's, preliminary results do suggest a weak dependence on the compliance of the substrate.

18.    Mathematical Modeling of Bacterial Motility Including Interfacial Interactions
Nathaniel Arlander, Dr. Margot Vigeant, Dr. James Maneval
Bucknell University, Lewisburg, Pa 17837

        A computational model of the motion of bacteria moving in a system containing solid-liquid interfaces has been developed. The model uses the cellular dynamics approach of Frymier, et al. and extends that model to include the interaction of the bacteria with solids. Specifically, the new model incorporates boundary conditions which better reflect experimental observations. In previous work, cells encountering a wall would either reflect from it or adhere to it. In experiments with E. coli (and other species), it has been observed that cells approaching a surface will turn and swim for some time along the surface. They may proceed from this position to either adhering irreversibly to the surface, leaving the vicinity of the surface, or continuing to swim along the surface. By incorporating this behavior into the program, we hope to produce a more complete model of bacterial behavior in bounded conditions. The motility of bacteria has been used to predict the behavior of bacteria in subsurface environments. By incorporating more realistic boundary conditions, we can obtain a better understanding on how the cells move through the subsurface. 

        This program is based on a three-dimensional non-bounded random walk. The movement of a bacterium is composed of two unique modes: a “run” mode where the cell moves in a specified direction and a “tumble” mode where the cell does not move but instead changes its direction of motion. Runs are characterized by a constant speed and an average duration, while tumbles are characterized by a turn-angle distribution. The model allows the run speed, average duration of the run and turn-angle distribution to be specified. From inputs of number of bacteria and number of steps, this program calculates the paths that the cells trace out in space over time. 

        To validate the unbounded program, we calculated a diffusion coefficient from the mean square displacements of the cells and compared it to a theoretical diffusion coefficient calculated from the same input information. At first the model showed inconsistent results between the diffusion coefficients calculated and those found in the literature. However, after reassessing coordinate transformations, the problem was resolved. Once a working model of the base case was developed, boundary conditions were implemented that mimicked experimental conditions and took into consideration surface and hydrodynamic interactions. By comparison of the model results to the results of bacterial adhesion experiments, the model parameters can be estimated, then used to accurately predict results of further experiments.

19.    Nanopore Enzyme Enhanced Devices
A. David, A.J. Yang, and N.S. Wang
University of Maryland, College Park, MD 20742

The biotechnology field has been growing by leaps and bounds over the past few years. Rapid developments in the biochemical field have created both the availability and demand of novel bio-molecules. Many of these new enzymes can improve current processes if it were cost effective. The immobilization of the enzymes provides tremendous cost reductions by 1) allowing for repeated uses of the enzymes and 2) reducing downstream purification needs through separation of enzymes from the products. The goal of this research is to develop an effective method for the "one pot" immobilization of bio-molecules onto silica surfaces. Such materials can find use in heterogeneous catalysis, affinity chromatography, membrane reactors, bio-sensors, and drug delivery. While the immobilization of enzymes has been studied for a number of years, it has been done mainly through the modification of a pre-fabricated gel. This method can be limited to lower enzyme loading on the gel as it depends on diffusion of the bio-molecule into the pores. Greater loadings can be achieved if the enzyme is introduced before gel formation. A "one pot" immobilization method will first be developed for alpha-amylase, which hydrolyzes starch into sugars. Preliminary results have been obtained for the physical entrapment of amylase in silica gels. The entrapment is accomplished by introducing the enzyme in a silicic acid solution and then raising the solution pH to 7. A gel is formed within minutes, trapping the enzyme in a porous network. The next step is the modification of the "one pot" process for the covalent immobilization of enzymes. This will also be done, first, with alpha-amylase and then be applied to the immobilization of penicillin acylase, an enzyme critical in the production of the semi-synthetic drug intermediate 6-aminopenicillanic acid (6-APA). 

20.    Oxygen limitation in shake flask fermentation
Atul Gupta and Dr. Govind Rao
University of Maryland Baltimore County, Baltimore, MD 21250

Small-scale cultivation of aerobic microorganisms is conventionally carried out in submerged culture using Erlenmeyer flasks. Aeration in these vessels is by the gas liquid contact aided by shaking the vessels in reciprocating or gyrotory shaking machines. Flasks used in the culturing of microorganisms are traditionally sealed with various types of closures to prevent  contamination. These closures however, are not good with regard to air permeability. They offer considerable resistance to gas transfer and consequently a low level of oxygen diffusing into the vessel and poor escape of carbondioxide. This limitation is great enough to limit seriously the usefulness of shake flasks for experimental purposes. In our study, a non-invasive fluorescence based sensor has been used to measure oxygen online. This study proves that oxygen is limiting in E. coli fermentations depending on the culture conditions. 

21.    Polymersome Solubilization by Detergent
Veena Pata¹,  Fariyal Ahmed², Dr. Nily Dan1, and Dr. Dennis Discher^2
1Drexel University, Philadelphia, PA 19104
²University of Pennsylvania, Philadelphia, PA 19104

        Liposomes and polymersomes are widely investigated as drug and gene carriers. However, their utilization as such faces many obstacles, including difficulties in drug encapsulation into the vesicle, and stability, in vivo, to various amphiphilic agents. In this work, we investigate mixtures of polymersomes with surfactant and evaluate experimentally the critical surfactant concentration required to destabilize the polymersome, as a function of system parameters. 

22.    Recombinant Expression of Human MHC on the Surface of Yeast Cells
Andrew Nields and Eric Boder
University of Pennsylvania, Philadelphia, PA 19104

        Antigenic peptide fragments bind with class II major histocompatibility molecules (MHCs) to form peptide-MHC complexes (pMHCs). MHCs are heterodimers composed of two noncovalently associated subunits, which span the cellular membrane of antigen presenting cells (APCs). The extracellular  portion of each subunit is composed of two domains. The first domains of each subunit combine to form a binding pocket for antigenic peptide fragments. However, the bound peptides are recognized by the immune system only when presented by MHCs on the surface of APCs. APCs internalize foreign and endogenous proteins, break them down into peptide fragments, and present them on their surfaces via class II MHCs. T cell receptors, expressed on the surface of T cells, recognize the presented peptides, either as foreign or self, and initiate an appropriate immune response.

        Previous attempts to determine which peptides play a role in T cell responses have been limited to in vivo studies, which have associated difficulties and limitations. In this work, MHCs have been recombinantly  expressed on the surface of yeast cells. A specific class II MHC for rheumatoid arthritis has been displayed with bound peptides. However, the loss of pMHC lateral movement across the yeast cell membrane has prevented the accumulation of sufficiently dense populations of pMHCs on the surface of yeast cells to elicit an immune response.

        Work in a rat model has shown that recombinantly expressed MHCs in E. coli, which only contain the first domain of each subunit, fold into proteins of unique secondary structure. Since the truncated MHCs retain the necessary binding pocket, peptides have been shown to continue to bind to these minimized functional MHC subunits. Subsequent work has shown that minimized functional human MHC subunits can also be expressed in this way. Our group’s proposed work combines the two methods in order to  recombinantly express minimized functional human MHC subunits on the surface of yeast cells.

        Work in this area has great possibilities in combating autoimmune dieases, such as rheumatoid arthritis and multiple sclerosis. CD4+ T cells play an important role in such diseases, and by engineering the appropriate pMHCs, it would be possible to shut off the undesired autoimmune response.

23.    RELATING AUTOLYTIC ENZYME ACTIVITIES AND THE EXTENT OF CELL WALL DEGRADATION DURING PARTIAL AUTOLYSIS IN FILAMENTOUS FUNGI
Judith Kadarusman and Mark R. Marten 
University of Maryland, Baltimore, MD 21250

        Filamentous fungi are used in the production of 40% of industrial enzymes, a market that is worth $1.6 billion (1999). It has been known for many years that mycelial morphology in fungal fermentation often leads to high viscosity, oxygen limitation, and reduced productivity. While there have been few reliable solutions for this problem, a recent study in our laboratory showed that during fed-batch fermentations of Aspergillus oryzae, the morphology of the fungal mycelia depended on the way in which a limiting nutrient solution was added to the fermentor. When the nutrient was fed continuously, mycelia were relatively large and were found in large clumps. As a result, the cell broth was very viscous and difficult to mix. In contrast, when the limiting nutrient was fed in pulses, mycelia were small and clumped less. As a result, the broth viscosity was significantly lower. We hypothesize that when cells experience cyclic periods of ?starvation,? as was the case during pulsed feeding, the cells undergo partial autolysis. When cells undergo partial autolysis, the cell walls weaken and break more frequently, which results in smaller mycelia. We would like to test this hypothesis by: (1) measuring the activities of autolytic enzymes; and (2) measuring the composition of the fungal cell wall. The enzymes of interest are beta-glucanase, chitinase and protease; these hydrolyze beta-glucan, chitin, and proteins, respectively. Understanding the relationship between activity of autolytic enzymes, changes in cell wall composition, and cell morphology will open possibilities to controlling fermentation behavior with feeding strategies. 

24.    Rolling Adhesion Kinematics of Yeast Engineered to Express Selectins
Sujata K. Bhatia1, Jeffrey A. Swers², K. Dane Wittrup², and Daniel A. Hammer^1
1University of Pennsylvania, Philadelphia, PA 19104
²Massachusetts Institute of Technology, Cambridge, MA 02139 

        Selectins are cell adhesion molecules that mediate capture of leukocytes on vascular endothelium as an essential component of the inflammatory response. Here we describe a method for yeast surface display of selectins, together with a functional assay that measures rolling adhesion of selectin-expressing yeast on a ligand-coated surface. E-selectin-expressing yeast roll specifically on surfaces bearing sialyl-Lewis-x ligands. Observation of yeast rolling dynamics at various stages of their life cycle indicates that the kinematics of yeast motion depends on the ratio of the bud radius to the parent radius (B/P). Large-budded yeast “walk” across the surface, alternately pivoting about bud and parent. Small-budded yeast “wobble” across the surface, with bud pivoting about parent. Tracking the bud location of budding yeast allows measurement of the angular velocity of the yeast particle. Comparison of translational and angular velocities of budding yeast demonstrates that selectin-expressing cells are rolling rather than slipping across ligand-coated surfaces. 

25.    S. aureus-Platelet Interactions Under Hydrodynamic Shear Conditions
Rami Bardenstein and Julia M. Ross
University of Maryland Baltimore County, Baltimore, Maryland 21250

        The recent emergence of antibiotic resistant S. aureus strains is a growing health concern. S. aureus has been implicated in a variety of blood-born infections, including infective endocarditis and sepsis. Previous studies have demonstrated that platelets may play a role in these infections either through direct receptor-ligand interactions or through soluble proteins acting as bridging molecules. Those studies, however, have not included or poorly defined the relevant physiological shear conditions which may govern S. aureus-platelet interactions. Clearly defining the physiological shear conditions will help elucidate the most important binding interactions to the blood-born infection process. In order to delineate the possible role of shear forces in S. aureus-platelet interactions a parallel plate flow chamber coupled with fluorescent microscopy was used. Initial results demonstrated the validity of using washed platelets adhered to a collagen support as a functional system for determining S. aureus-platelet interactions. 

26.    Separating Plasmid Forms by Hydrophobic Interaction Chromatography
Katarina Georgiev and Dr. William J. Kelly
Villanova University, Villanova, PA 19403

        An HPLC technique has been developed, based on Hydrophobic Interaction (TSKgel Butyl-NPR, TOSO BIOSEP LLC), that allows for the separation of the open-circular (nicked) and supercoiled forms of five plasmids ranging in size from 3-30kB. The identity of the bands was determined through light scattering and gel electrophoresis. A range of buffers, gradients, flow rates and temperatures were evaluated in determining the optimum operating conditions for the separation. For all plasmids a reverse ammonium sulfate in phosphate buffer (pH 7.1) gradient was established. The chromatographic resolution between the supercoiled and nicked peaks was found to be a function of flow rate, temperature and plasmid size. The elution order did not vary with plasmid size, with the open circular always eluting before the supercoiled. The HPLC technique developed in this research allows for a rapid separation of the supercoiled and nicked plasmid forms with excellent resolution. HIC is a useful alternative to ion exchange or size exclusion for the chromatography of large plasmids up to 30kB.

27.    Swelling Phenomenon and Physical Characteristics of the Nucleus 
Samuel Kahn, Kris Noel Dahl, and Dennis E. Discher

        The nuclear envelope, which separates nuclear and cytoplasmic compartments in eukaryotic cells, presents a unique, biological membrane system for studying intracellular transport. Nuclear pores, connecting the outer to the inner membranes within the envelope, serve as water filled channels with an effective inner radius of 5 nm. Upon isolation of Xenopus laevis oocyte nuclei into a media which has similar salt concentrations as cytoplasm, a swelling of 50% is observed. The aim of this study is to determine the significance and cause of swelling phenomenon due to diffusion through these pores and to relate these results to physical characteristics of the membrane, such as the area expansion modulus. Solutions containing large molecular weight dextran polymers unable to diffuse into the nucleus showed decreased swelling in a dose dependent fashion, suggesting that osmolarity is a significant factor in nuclear swelling. Various ionic species have been added to the nuclear isolation media including divalent cations, heavy divalent metals, and buffered and unbuffered monovalent cations. Early observations, combined with qualitative microrheology, suggest that the swelling may be due to the appearance of an additional phase inside the nucleus with the addition of some of these solutions. This suggests that there are competing effects and that the swelling of the nucleus is not due solely to osmolarity.

28.    Systematic Identification of Loci for Assisted Protein Reassembly
David Paschon and Marc Ostermeier
Johns Hopkins University, Baltimore, MD 21218

        Many proteins can have their peptide backbone cut by proteolytic or genetic means, yet the two fragments can associate to make an active heterodimer. This 'monomer to heterodimer conversion' is referred to as protein fragment complementation. However, there are many locations where such a conversion is not feasible, presumably due to inefficient assembly or improper folding of the fragments. This can be overcome by fusion of the fragments to dimerization domains to facilitate correct assembly. Such' assisted protein reassembly' can be used as a two-hybrid system to evaluate protein-protein interactions or to select for interacting proteins. Using a protein engineering technique called incremental truncation, we have systematically identified loci for assisted protein reassembly of fragments of aminoglycoside phosphotransferase APH(3')-IIa using antiparallel leucine zippers as dimerization domains.

29.    Ultrasound Induced Degradation of Hollow PLGA Microcapsules
Dalia El-Sherif, Justin Lathia, Yen Le, and Margaret Wheatley
Drexel University, Philadelphia, PA, 19104

        We have investigated the effect of ultrasound energy on the degradation pattern of hollow PLGA microcapsules. These microcapsules work as ultrasound contrast agents, giving an in vitro acoustic enhancement of up to 25 dB. The dose response at 5 and 10 MHz was compared to the degradation pattern of the polymer capsules at the same two frequencies.

30.    The use of a green fluorescent protein fusion tag to develop cellular systems for optimal expression of membrane proteins
Ronald T. Niebauer and Dr. Anne Skaja Robinson
University of Delaware, Newark, DE 19716

        The G-protein coupled receptors (GPCRs) are an important class of transmembrane proteins that mediate cellular response to diverse stimuli. Many diseases including cancer and heart disease have been linked to GPCR function but little is known about expression, folding, and interactions of these proteins. Biophysical studies to elucidate structural and functional properties are limited by this inability to produce functional protein. The goal of this research is to develop cellular systems for efficient expression of functional protein. The particular GPCR used in this study is the human A2a receptor which is believed to play a cardioprotective role in the body. The green fluorescent protein (GFP) has been used as a tool to gain insight into studying this class of proteins. GFP can be fused to a protein and used as a reporter of expression as well as to determine the location of proteins in a cell. Initial studies in the yeast Saccharomyces cerevisiae reveal that the A2a-GFP fusion does not alter the functionality of the protein. Confocal microscopy studies reveal that the protein is being correctly localized to the plasma membrane. Current studies are focused on determining the effect of gene dosage on protein levels so that an optimal gene copy number can be found. In addition, a whole cell-based fluorescent assay is being developed to optimize protein expression levels.

31.    Using 2D Gel Electrophoresis to Study Heat Shock Response in Industrially Relevant Strains of Aspergillus oryzae
Jie Shen and Mark R Marten
University of Maryland, Baltimore, MD 21250

        Heat shock response is a molecular reaction to stressful but sublethal temperatures. When organisms or cells are subjected to high temperatures, they rapidly redirect gene expression to maximize synthesis of a distinct group of proteins named heat shock proteins (Hsps). These proteins are beneficial to cells in helping them adapt to the elevated temperature or survive exposure to higher, otherwise lethal temperatures. Filamentous fungi are an important group of microorganisms involved in human health (as pathogens), agriculture (as crop pathogens) and bioprocessing (as producers of billions of dollars of pharmaceuticals and chemicals). My research project involves identification of heat shock proteins in Aspergillus oryzae, an industrially relevant fungal species, by using 2D gel electrophoresis. 

32.    Utilizing EGFP to Model Oxygen and Drug Release Profiles in Encapsulate Murine Myoblasts.
Chong Yung, Timothy Barbari, and William Bentley
University of Maryland, College Park, MD, 20742

        To study the depletion of oxygen in a hollow fiber environment, murine myoblasts (C2C12) were transfected with the enhanced green fluorescent protein (EGFP) reporter plasmid. Since EGFP production and activity is dependent on dissolved oxygen concentrations, fluorescence intensity can be used to correlate oxygen levels within a cell mass. A stable line of these attachment dependent cells were produced utilizing a novel cloning method, which combined the use of an agarose underlayer and antibiotic resistance selection. In order to model protein production in response to oxygen levels, a vector containing a secretable fusion of EGFP and human interleukin-2 (hIL2) was constructed. The vector also contains an enterokinase cleavage site between the two genes, whereby the fusion protein can be separated after translation. The secretion signal on the N-terminal of hIL2 is used to shuttle the protein out of the cell. The remaining EGFP stays inside the cell, and can be used to quantify total protein production.

33.    A Transient Expression System for the Production of Therapeutic Proteins in Plant Tissue Culture
Jason Collens and Wayne Curtis

        A plant tissue based system is being developed to produce a quantity of transgenic protein sufficient for testing and characterization. Agrobacterium tumefaciens, containing a binary plasmid with the gene of interest, is co-cultured with the plant tissue. Currently, an intron-containing GUS gene is being used as a reporter gene; the intron prevents bacterial expression of the reporter, and GUS allows for either quantitative or qualitative determination of gene expression. Several root culture and cell suspension lines have been tested, with the result showing large variability in GUS expression both between species and within different clones of the same species. In addition, factors that can be controlled in a bioreactor have been examined, such as temperature, wounding of the plant tissue, pH and phenolics, with the aim of increasing transient expression. Transgenic tissue lines that contain an inducible viral replication gene and a compatible vector have been developed, and are currently being tested to determine the extent to which these refinements augment protein production. Ongoing work also includes the generation of Agrobacterium auxotrophs, to prevent unchecked bacterial growth during the co-culture with plant tissue.