Beckman Award Winners

2009

Megan Kissig

Role of Junctional Adhesion Molecule-A (JAM-A) in the Progression of Non-alcoholic Fatty Liver Disease (NAFLD)

Megan E. Kissig and Ulhas P. Naik
Department of Biological Sciences

Non-alcoholic fatty liver disease (NAFLD) is characterized by an abnormal amount of fat accumulation in the liver, specifically more than 5% fat by weight. Little is known about why the fat accumulates in the liver but it has been found that intestinal permeability due to leaky tight junctions may be a contributing factor. In our lab we study junctional adhesion molecule-A (JAM-A), a protein located at the tight junctions of epithelial and endothelial cells. My aim was to find what effect JAM-A has on weight gain and the eventual development of NAFLD. To analyze this relationship, groups of Jam-A (+/+) and Jam-A (-/-) mice were put on either a high fat or low fat diet for 20 weeks. During this time the mice were weighed every two weeks and blood samples were taken every four weeks to test for plasma levels of cholesterol and triglycerides. At the end of the 20 weeks, the mice were sacrificed and the livers and fat pads were removed, sectioned, and stained to find further effects of the diet, including any progression of NAFLD. There was a significant variation between the high-fat and low-fat Jam-A (-/-) groups, whereas there was little difference between the Jam-A (+/+) groups. Also, the high-fat Jam-a (-/-) mice had significantly higher LDL-cholesterol and total cholesterol levels in the plasma than the other groups. It was also found that the livers of the high-fat Jam-A (-/-) mice showed significantly more fat droplet accumulation than the high-fat Jam-A (+/+) mice. This suggests that JAM-A regulates weight gain, cholesterol levels, and ultimately NAFLD development.

Michael Napolitano

Pathogenicity Islands Are Discrete and Ancient Integrative Elements: An Evolutionary and Functional Analysis

Michael G. Napolitano, Salvador Almagro-Moreno, and E. Fidelma Boyd
Department of Biological Sciences

The evolution and emergence of pathogenic bacteria is tightly linked to the horizontal transfer of pathogenicity islands (PAIs) and bacteriophages encoding a range of virulence factors. The current theoretical framework is that PAIs and other genomic islands (GEIs) are a combination of diverse integrative elements such as bacteriophages, integrative conjugative elements (ICE), or plasmids. In this work, we describe how integrases from phages and pathogenicity islands form distinct phylogenetic groups. In addition, we show that island-encoded integrases are not evolutionarily related to a range of integrative elements. These findings do not support the view that pathogenicity and genomic islands are a broad group of integrative elements derived from other elements. Using VPI-2 as a model for pathogenicity islands, we determined the role of the VPI-2-encoded integrase (intVPI-2) and two putative excisionases (vefA and vefB) in excision. We examined VPI-2 excision levels in V. cholerae mutant strains (delta)intVPI-2, (delta)vefA and (delta)vefB and also in strains that overexpressed these genes. We found negligible levels of excision in the (delta)intVPI-2 strain whereas overexpression of intVPI-2 showed a marked increase in excision. In the (delta)vefA or (delta)vefB strains the excision levels of VPI-2 remained the same, however, overexpression of vefB (VC1809) showed a 2-fold increase in excision. Overexpression of vefA (VC1785) in the wildtype causes complete excision of the island, however complimenting vefA with the deleted strains (delta)vefA and (delta)vefB leads to only a minor increase in excision above basal levels.

2008

Tyler Larsen

Inherently Antimicrobial Hydrogels - Altering Behavior via Tryptophan/Arginine Interactions

Tyler Larsen, Daphne A. Salick, Radhika Nagarkar, and Joel P. Schneider
Department of Chemistry and Biochemistry

Hydrogels are heavily hydrated, elastic-like, porous materials that show considerable promise as artificial extracellular matrices for use in tissue regenerative therapies. Unfortunately, not only do hydrogels provide ideal environments for cell proliferation, but for opportunistic bacteria as well. To combat the threat of infection, hydrogels are often modified to display antibacterial activity, usually by impregnating the gel with antibiotic agents or covalently attaching them to the gel surface. The development of hydrogels that are inherently antibacterial has been of great interest to the hydrogel research community. We have developed MAX1, a self-assembling, twenty amino acid peptide hydrogel whose surface exhibits inherent antibacterial activity against several gram-negative and gram-positive bacteria prevalent in hospital settings. Under physiological conditions, MAX1 folds into an amphiphilic β-hairpin and subsequently self-assembles into a highly-crosslinked hydrogel network composed of fibrils rich in β-sheet. The resultant hydrogel is mechanically rigid and cytocompatible. This study aims to investigate the possible contributions of a cation-pi interaction to the antibacterial activity of a MAX1-like peptide hydrogel. Cation-pi interactions between arginine and tryptophan pairs are a common feature of many conventional antibacterial peptides, where they appear to assist in the binding and disruption of bacterial membranes. A new peptide sequence (RWMAX1) was designed and synthesized, incorporating a cross-strand R/W pair into MAX1. Preliminary studies using circular dichroism assessed RWMAX1's folding and self-assembly kinetics. Future studies will explore RWMAX1's material rigidity and antibacterial properties against E. coli and S. aureus.

Amy Styer

Expression, Purification, and Characterization of a New Sulfhydryl Oxidase from Trypanosoma brucei

Amy Styer, Vamsi Kodali, Vidyadhar Daithankar, and Colin Thorpe
Department of Chemistry and Biochemistry

A sulfhydryl oxidase from Trypanosoma brucei was expressed, purified and studied enzymologically for the first time. The protein is homologous to ALR (augmenter of liver regeneration), an essential enzyme which catalyzes disulfide bond formation in the mitochondrial inner membrane space (IMS) of eukaryotes. Trypanosomes lack a gene for Mia40, a necessary redox partner with ALR in yeasts and mammals. Future research will determine how trypanosomes compensate for the lack of Mia40, and if this sulfhydryl oxidase has the same biological localization and function as ALR. First, however, expression and purification procedures must be optimized to prevent aggregation and degradation of the protein. Preliminary research revealed that six of seven cysteines in the 33 kDa trypanosomal ALR are in the form of disulfide bonds. In oxygen electrode assays, the enzyme catalyzed disulfide-bond formation in the model substrate dithiothreitol (DTT), but not the monothiols glutathione and cysteine. Molecular oxygen was kinetically better as a terminal electron acceptor for trypanosome ALR than for human ALR (TbALR Km = 15±1μM O2). Understanding trypanosome redox biochemistry is important because Trypanosoma brucei, the causative agent of African Sleeping sickness, kills nearly fifty thousand people annually and current treatments are expensive, toxic, and impractical. This first foray into trypanosome mitochondrial IMS disulfide-bond formation may lead to biomedical advances in the fight against trypanosome diseases.

2007

Ritika Samant

Generation of Construct for the Identification of PDZ Domain Proteins Interacting with JAM-B

Ritika Samant and Ulhas Naik
Department of Biological Sciences

A family of Junctional Adhesion Molecules (JAMs) consisting of JAM-A, JAM-B, and JAM-C has recently been discovered. These molecules are transmembrane proteins involved in tight junctions, which are often associated with PDZ domain-containing proteins. JAM-A is known to contain a PDZ domain-binding motif, which it uses to interact with cytoplasmic PDZ proteins. Since there is significant conservation between JAM-A and JAM-B, we believe that JAM-B may also interact with PDZ domain-containing proteins. To study this, protein microarrays can be used to identify protein-protein interactions between JAM-B and PDZ domain-containing proteins. A cDNA construct encoding for a polyhistidine-tagged JAM-B fusion protein using polymerase chain reaction (PCR) was first created. This construct will be used to express and purify protein, using affinity chromatography. The protein can then be used to probe a protein microarray spotted with PDZ domain-containing proteins functioning as capture molecules.

2006

Vivek Desai

CD44 and Posterior Capsular Opacification

Vivek D. Desai and Melinda K. Duncan
Department of Biological Sciences

Posterior capsular opacification (PCO) is an undesirable wound healing response in which the residual lens cells remaining in the eye after cataract surgery proliferate, migrate into the visual field, and synthesize extracellular matrix molecular similar to those found in scar tissue, damaging the patient's vision. PCO arises from epithelial mesenchymal transition (EMT) of lens epithelial cells. In other systems, CD44, a receptor for hyaluronan, has been identified to mediate changes in cellular proliferation, migration and cell identity leading to EMT. We hypothesize that the exposure of lens cells to hyaluronan based viscoelastics stimulates PCO following extracapsular lens extraction and that the hyaluronan receptor CD44 is involved in EMT of lens cells. In the adult mouse lens, CD44 is expressed in the lens fiber cells and not expressed in the lens epithelial cells, whereas in embryonic mouse lens, CD44 is not expressed in both the lens fiber cells and the lens epithelial cells. RT-PCR demonstrated that the "canonical" version of CD44 is the major CD44 splice variant in the lens. In adult mice, CD44 is not expressed in the lens epithelial cells immediately after the cataract surgery, however, its expression highly up-regulates in the lens epithelial cells 1-day following the surgery. Because of the dynamics of CD44 during the development of mouse lens and following the cataract surgery in adult mouse lens, out immediate future work is to study the distribution of CD44 ligands like hyaluronan and osteopontin.

Patrick Knerr

Metal-triggered Hydrogelation of Designed β-hairpin Peptides

Patrick J. Knerr, Christopher Micklitsch, Colin Thorpe, and Joel P. Schneider
Department of Chemistry and Biochemistry

Peptides have been designed which undergo intramolecular folding from random coil to β-hairpin conformation, triggered by specific environmental conditions. The folded, amphiphilic β-hairpins then self-assemble to form an interlaced fibrillar network, converting the material from a liquid to a self-supporting, rigid hydrogel. Such responsive materials show great promise in tissue engineering, serving as scaffolds which mimic the extracellular matrix by allowing cellular adhesion and proliferation. Past work has demonstrated that pH, temperature, ionic strength and light can be utilized to stimulate the folding event. We are currently interested in developing a new trigger: metal-induced hydrogelation. Unnatural, multidentate α-amino acids have been synthesized to chelate strongly to divalent metal ions, most notably calcium or zinc. These ligands are subsequently incorporated into the primary sequence of a 20 residue peptide composed of two strand regions connected through a four residue type II' β-turn. This affords peptides in which folding and self-assembly is prevented at physiological conditions in the absence of the metal ion, but triggered when the ion is present.

2005

Tapan Patel

SC-35 Mediated Modulation of Prox1 Function

Tapan P. Patel, Xiaoren Chen, Melinda K. Duncan
Department of Biological Sciences

Prox-1, the vertebrate homolog of Drosophila Prospero, is a homeobox transcription factor that is necessary for angiogenesis, development of the liver and lens fiber differentiation. It is highly expressed throughout the lymphatic system, both in the cytoplasm and the nucleus with varying levels of expression depending on the stage of development. Previously, mouse monoclonal antibodies against Prox1 were developed and shown to react with Prox1 by western blotting. In addition, potential Prox1 interacting proteins were identified by the yeast-two hybrid assay including splicing factor sc-35. We hypothesize that sc-35 Prox1 interactions are important for the cellular function of Prox1. Initially, the ability of the Prox1 monoclonal antibodies to detect Prox1 in the lens from diverse vertebrates was tested by immunofluorescent histochemistry. Both monoclonal antibodies tested detected Prox1 in lenses from humans, rats, chickens and lizards, with much less affinity to Prox1 in the frog and fish lens. Next, investigations were initiated into the possibility that sc-35 interacts with Prox1 in vivo and modulates its function as a transcription factor. By immunofluorescence, Prox1 and sc-35 protein are colocalized in the nucleus of lens fiber cells showing that they reside in the same location in the same cell in vivo. Next, new sc-35 constructs were prepared for use in the yeast-two hybrid system since the original sc-35 clone was not in frame with the Gal4 DNA binding domain. Future work will assess whether sc-35 and Prox1 can interact in vivo and in vitro as well as the functional significance of these interactions.

Wen Allen Tseng

The Role of Junctional Adhesion Molecule-A Homodimerization in Angiogenesis

Wen Allen Tseng and Ulhas P. Naik
Department of Biological Sciences

Junctional adhesion molecule-A (JAM-A or JAM-1) is found at the tight junctions of endothelial and epithelial cells. Our lab has shown that JAM-A is involved in basic fibroblast growth factor (bFGF)-induced angiogenesis. It has also been demonstrated that JAM-A is capable of cis-homodimerization. This study intends to test the hypothesis that cis-homodimerization of JAM-A is necessary for JAM-A-mediated angiogenic activities of endothelial cells to occur. Site-directed mutagenesis was used to create constructs coding for mutant JAM-A proteins with substitutions of residues within the homodimer interface. These constructs were transiently transfected into Chinese hamster ovary cells to determine whether these mutants have an impaired ability to form homodimers. The extent of homodimerization will be determined by Western blotting after treating the transfected cells with an extracellular cross-linker. After these assays are completed, these constructs will be stably transfected into human umbilical vein endothelial cells (HUVECs), which will be assayed to determine their ability to perform activities involved in angiogenesis as compared to mock-transfected and wild-type JAM-A-overexpressing HUVECs.

2004

Liang Kang

The Role of Junctional Adhesion Molecule 1 (JAM-1) in the Corneal Epithelium

Liang Kang, Vesselina Cooke, Ulhas Naik, and Melinda K. Duncan
Department of Biological Sciences

Junctional Adhesion Molecule-1 (JAM-1) is a ~38 kDa protein that has been implicated in a variety of roles in the body, including platelet activation and adhesion, leukocyte migration, and the structural integrity of endothelial and epithelial cell layers. Recently, our lab has begun to characterize JAM-1 function in the eye since its expression was upregulated in the lens of Pax6 transgenic mice. The presence of JAM-1, JAM-2, and JAM-3 mRNA in the wildtype lens and JAM-1 mRNA in the wildtype cornea was confirmed through RT-PCR. Using immunohistochemistry, JAM-1 protein was found in the blood vessels of the developing eye as early as 12.5 dpc (days post conception) and in the corneal epithelium by 13.5 dpc. PLAP (placenta alkaline phosphatase) reporter gene activity was detected in the JAM-1 heterozygous and homozygous cornea, indicating the insertion of the Genetrap reporter construct in these mice, and immunohistochemistry showing the absence of JAM-1 staining in knockout cornea validated both JAM-1 antibody specificity and the knockout genotype. While the eye appears to develop normally in JAM-1 knockout mice, older animals have abnormalities in the corneal epithelium. We hypothesize that JAM-1 has a role in the maintenance of the cornea.

2003

Mesha Eaton

Genes Involved in Sperm Function: Investigation of Dual Expression of Hyal5 in The Testis and Epididymis

Mesha Eaton, Hong Zhang, and Patricia A. Martin-DeLeon
Department of Biological Sciences

SPAM1, the Sperm Adhesion Molecule 1, is a highly conserved sperm membrane protein with multiple essential roles in mammalian fertilization. This protein belongs to a group of closely linked hyaluronidases residing on mouse chromosome 6A2, and is responsible for the dissolution of the cumulus cells of the oocyte during fertilization. This is necessary for penetration of the sperm. More recently a Spam1 knockout study revealed that fertility was unaffected and suggested that there was a mouse-specific hyaluronidase, Hyal5, with similar functions to Spam1 (Baba et. al., 2002). The goal of this study was to determine if Hyal5 is redundant to Spam1 in regards to its mRNA and protein expression in the epididymis. An mRNA expression analysis was performed using RT-PCR on both the testis and all three regions of the epididymis (caput, corpus, and cauda), which was rendered sperm-free after excessive washing. RT positive and RT negative reactions were performed and the results, although preliminary, suggest the presence of Hyal5 in the cauda. An attempt was made to study the protein expression in the testis with immunohistochemistry, however due to non-specificity of the antibody the results were inconclusive. A new antibody is being generated to continue these studies.

Chris amEnde

De Novo Design of Lanthanide Fingers

Chris am Ende, Mao Ye, Neal Zondlo, and Douglas Taber
Department of Chemistry and Biochemistry

A series of peptides has been synthesized using the 25 amino acid zinc finger motif as a model. The sequence has been altered to allow for lanthanide, instead of zinc, binding. The binding has been analyzed using a several methods. Circular dichroism (CD) was used to monitor the extent of protein folding. The CD data showed characteristic meta bound zinc finger CD spectra upon titration with various lanthanides. Metal binding was also analyzed using fluorescence of tryptophan. In addition, fluorophores have been conjugated to selected amino acids to monitor peptide folding using fluorescence resonance energy transfer (FRET). FRET data gave further evidence of the conformational changes associated with metal binding.

2002

Stephen Brohawn

Synthesis and Characterization of Phosphines as Novel Substrates of Sulfhydral Oxidase

Stephen Brohawn, James Psathas, and Colin Thorpe
Department of Chemistry and Biochemistry

Commercially available trialkyl phosphines such as tris-(2-carboxyethyl)-phosphine (TCEP) and tris-(2-cyanoethyl)-phosphine (TCNP) are used in biochemistry as reductants of disulfide bonds. TCEP, but not TCNP, is a good substrate for chicken egg white sulfhydral oxidase (SOX), a flavoenzyme that catalyzes the oxidation of free thiols to disulfide bonds in a variety of substrates while reducing oxygen to hydrogen peroxide. In an effort to understand these differences in reactivity with the enzyme and possibly to find versatile water soluble phosphines, mono-, di-, and trimethyl ester derivatives of TCEP have been synthesized. Kinetic studies of TCEP, TCNP, and the methyl ester derivatives have been performed on a stopped flow spectrophotometer using DTNB as a model disulfide. The di- and trimethyl ester derivatives have a second order rate constant three times higher than that of TCEP and the monomethyl esters rate constant is close to two and a half times higher than that of TCEP. TCNP was found to be much less reactive than TCEP or its derivatives. SOX activity with TCEP, TCNP, and the trimethyl ester was compared using an oxygen electrode. SOX activity with TCEP gave a Vmax of 1650/min and a Km of 43 mM. The trimethyl ester derivative, like TCNP, was found to have undetectable activity towards SOX. These studies will form the basis for the investigation of the inhibition of SOX with metal ions.

Arthur Suckow

The Ocular Expression and Regulation of Junctional Adhesion Molecule-1

Arthur T. Suckow, Vesselina Cooke, Ulhas P. Naik, William Skarnes, Bharesh K. Chauhan, Ales Cvekl and Melinda K. Duncan
Department of Biological Sciences

Junctional adhesion molecule-1 (JAM-1) is a member of the immunoglobin superfamily involved in the organization of tight junctions and the regulation of leukocyte transmigration. Recently, a cDNA microarray analysis of transgenic mice overexpressing PAX-6 in lens fiber cells revealed that JAM-1 mRNA expression was 2.5 fold elevated over normal. This data suggested that JAM-1 gene expression is regulated by PAX-6, a transcription factor essential for normal eye development. The overexpression of JAM-1 in the PAX-6 transgenic lenses of adult mice was confirmed by RT-PCR. A LacZ-Neor fusion genetrap was used to disrupt the JAM-1 gene in ES cells to create knockout mice and detect JAM-1 gene activity via B-galactosidase expression. In the lens, JAM-1 gene activity is detected in the epithelium, cells where high levels of PAX-6 are detected. Levels decrease during fiber cell differentiation coincident with the downregulation of PAX-6 expression. In the cornea, the JAM-1 gene is active in the corneal epithelium, a region that requires PAX-6 for normal morphogenesis. Analysis of JAM-1 null mice revealed a down-regulation of Jam-1 gene expression in the corneal epithelium, suggesting JAM-1 may indirectly regulate its own expression. Further, histological analysis of the mice demonstrated that the corneal epithelia is thicker than normal and lack a normal squamous layer in the outermost layer of the corneal epithelia; thus, JAM-1 is essential for normal corneal morphogenesis.