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Biomedical Sciences Seminar Series

Winter 2007 Poster Presentation

January 26, 2007 at 1 pm
Physical Sciences Lobby

Poster #1-Renee Williams

Complexation of Aβspecies with Fe3+

Renee T. Williams, Dianlu Jiang, Feimeng Zhou*
Department of Chemistry and Biochemistry
California State University, Los Angeles
5151 State University Drive
Los Angeles, CA 90032 

In collaboration with

Lijie Men and Wang Yishen* 

Department of Chemistry
University of California, Riverside
900 University Avenue
Riverside, CA 92521

Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder characterized by the deposition of amyloid plaque.  The plaque is primarily composed of insoluble beta-amyloid (Aβ) protein fibrils and metals at elevated concentrations.  In a human brain, the neurotoxicity of Aβ plaque has been suggested to originate from a Fenton-like reaction wherein the peptide-metal complex(es) is(are) involved in the generation of  reactive oxygen species.  To date, the majority of studies have focused on Aβ-copper and Aβ-zinc interactions, whereas Aβ-iron complexes have not been extensively investigated.  As such, the objective of this research is to 1) show that an Aβ-iron complex is formed, 2) determine if the complex is redox-active, and 3) establish a binding constant for the complex.  We will correlate our results obtained in vitro to in vivo observations and attempt to provide insight to the understanding of the Aβ aggregation process and its possible link with oxidative stress.

Poster #2-Shandee Dixon

In-vivo Pathogenicity studies of Sendai Virus Reverse Genetics Variants Containing Different Combinations of Mutations in the M and F Genes

Shandee Dixon, Marysol Navarro, Joseph T. Seto and Nancy L. Mc Queen
Department of Biological Sciences,
California State University Los Angeles

Wild-type (wt) Sendai virus causes a localized respiratory tract infection while its variant, F1-R, causes a systemic infection. Two phenotypic differences in F1-R have been correlated with its pantropic phenotype. The first difference is the enhanced cleavability of the fusion (F) protein of F1-R that has been attributed to one or more of the amino acid substitutions in the F protein. The second difference is the microtubule disruption and subsequent bipolar budding of F1-R that has been attributed to two amino acid substitutions in the M protein. We have hypothesized that specific combinations of the amino acid substitutions in the F and M proteins of Sendai are responsible for the pantropic behavior of the virus. To confirm this hypothesis a reverse-genetics viral recovery system was used to generate three variant viruses, RGV-0, RGV-1 and RGV-7. RGV0 has all six F1-R F gene mutations and both F1-R M gene mutations, RGV1 only has one of the F1-R F gene mutations, and RGV7 has the same F1-R F gene mutation as RGV1 plus both of the F1-R M gene mutations.

Intranasal infections were used to test the pathogenicity of the reverse genetics viruses in mice. Wt and F1-R Sendai viruses were used as controls for pneumotropic and pantropic viruses, respectively. Viruses collected from lungs, kidneys, and livers of the infected mice were amplified in 10-day–old embryonating chicken eggs and viruses recovered from the allantoic fluid of the infected eggs were titered using a hemagglutination (HA) assay. Viral RNA recovered from HA positive samples were transcribed into cDNA by means of Reverse-Transcriptase PCR followed by sequencing to confirm that the viruses had the appropriate mutations. The results indicate that wt, RGV1 and RGV7 were all pneumotropic, while F1-R and RGV0 both caused a systemic infection. Based on these studies we can say conclusively that the mutations in genes other than M and F genes of F1-R do not contribute to the systemic infection caused by F1-R. Studies to determine the specific combination of F1-R F and M gene mutations that contribute to F1-R’s ability to cause a systemic infection are underway.

Acknoledgements: Funding for this project was provided by MBRS Score Grant # F06GM8101-32 to N. L. McQueen, NIH MBRS-RISE MS TO PHD Grant # GM 61331 to C. Gutierrez and LSAMP-BDIII NSF Grant # HRD-0331537.

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Poster #3- Gilson Sanchez

Bioinformatic and Molecular Biological Approaches to Identifying Id2 Promoter Sequences Required for Down-Regulation

 G. J. Sanchez# and S. B. Sharp 
Department of Biological Sciences
California State University, Los Angeles

Background: Id helix-loop-helix (HLH) proteins play a key role in the regulation of cellular growth and differentiation.  Specifically, Id proteins serve as negative regulators of gene expression and differentiation by sequestering basic HLH transcriptional regulatory factors. Indeed, Id2 has been found to be up-regulated in some cancers.  Both Id1 and Id2 are expressed in cycling pre-muscle C2C12 cells, but are down-regulated to about 800 % at the level of mRNA accumulation in differentiating cells.  Interestingly, we have not observed a significant down-regulation using a reporter for the mouse Id2 promoter spanning -2250 to +32 relative to the transcription start site. Therefore, we sought to identify sequences upstream of -2250 that might contribute to more substantial down-regulation. 

     Methods: Distal promoter regions of the Id2 gene were compared using FamilyRelations (http://family.caltech.edu) software.  CisTematic (http://cistematic.caltech.edu) was used to perform a genome-wide search of the human genome for the presence of conserved motifs in the Id2 upstream region.  Based on results from these comparisons, a DNA fragment including sequence from -4331 to -2206 of the mouse Id2 promoeter was isolated from a genomic clone and used to replace sequence upstream of -2206 in our -2250 reporter construct, thereby adding additional upstream sequence.  C2C12 mouse myoblast cells were transiently co-transfected with Id2 promoter-firefly constructs and the SV40 driven renilla luciferase plasmid.  The transfected cells were induced to withdraw from the cell cycle and to differentiate by serum deprivation.  Extracts from the transfected cells under cycling and differentiated phases were used to perform luciferase assays to compare the transcriptional regulatory efficiencies of the mouse Id2 promoter in the presence or absence of the additional upstream sequence. 

     Results: Comparison with FamilyRelations of Id2 gene promoters across species from human to sea urchin revealed a set of four highly conserved motifs located between -2846 and -2629 in the mouse Id2 gene.  CisTematic showed that two of these motifs are also found upstream of the human Id1 gene at -1147 and -937.  Transient transfections with the use of the -4331 Id2 promoter construct revealed a 5 fold increase in expression efficiency in comparison to the -2250 Id2 construct.  However, our results failed to identify sequences that contribute to down-regulation under the conditions of our assay.    

     Conclusion: Transfection results suggest that addition of the 5’ promoter region expanding from -4331 to -2250 including the four conserved motifs, contributes to efficiency of expression in cycling cells but not to the differentiation-dependent down-regulation of Id2 expression in the test system.  We will further test whether each of the four conserved regions is required to confer the increase in expression efficiency using site directed mutagenesis.  The fact that the region of four conserved motifs in Id2 is partially conserved in Id1 suggests both common and unique mechanisms for regulation of these two proteins.

     Acknowledgement: Supported by CSU-LSAMP from NSF Grant HRD-0331537 and the CSU Chancellor’s Office.

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Poster #4- Marysol Navarro

Is the amino acid change at position 104 in the fusion protein of F1-R essential for Sendai virus variant F1-R’s ability to cause a systemic infection?

Marysol Navarro, Joseph T. Seto, and Nancy L. McQueen

Abstract: Sendai virus (SeV) is a murine negative-strand RNA virus. Wild type (wt) virus causes a localized respiratory tract infection in mice. A variant, F1-R, causes a systemic infection. We have identified two determinants that correlate with the systemic infection caused by F1-R. One of these determinants is the enhanced cleavability of the fusion (F) protein of F1-R that we have attributed to two or more of the six mutations in the F gene. Cleavage is required for virus infectivity. Wt F is only cleaved by Tryptase Clara, a protease restricted to the lungs, while F1-R F is cleaved by ubiquitous proteases. Thus, wt Sendai can only undergo multiple rounds of replication in the lungs while F1-R can undergo multiple rounds of replication in many different organs.

The second determinant is the differential budding behavior of F1-R that we have attributed to two mutations in the matrix (M) gene of F1-R. Wild type SeV buds from the apical domain of epithelial cells into the lumen of the respiratory tract where it subsequently infects new cells in the respiratory tract to cause a localized infection. F1-R buds from both the apical and basolateral domains, thus releasing virus into the basement membrane through which it can gain quick access to the bloodstream for dissemination. Based on studies of a revertant of F1-R that has lost its pantropic phenotype, we originally hypothesized that the two mutations in the M gene of F1-R and a mutation in the F gene that results in an amino acid substitution adjacent to the cleavage site of F (F115) were the only mutations needed for F1-R to cause a systemic infection. To test this hypothesis we used reverse genetics to make Sendai viruses with various combinations of the F1-R F and M gene mutations. RGV0 contains all six F1-R F and both M mutations, while RGV1 contains only the F1-R F mutation that encodes F115 and RGV7 contains the F1-R F mutation encoding F115 and both M mutations. Much to our surprise only RGV0 had an F protein with enhanced cleavability and only RGV0 caused a systemic infection in mice. These results mean that in addition to the F1-R F mutation adjacent to the cleavage site of F that had previously been shown to be critical for the systemic infection caused by F1-R, other mutations in F contribute to the enhanced cleavability of F1-R F and the pantropism of F1-R.

Wt F has a glycosylation site at amino acid 104 that is lacking on F1-R F because of an amino acid change at that site. A loss of an oligosaccharide side chain on an influenza virus strain and functional analyses of glycosylation sites on F suggests that fusion activity and cleavability of F is increased when the glycosylation site at F104 is lost. Therefore, my project is to generate a variant SeV, designated RGV18, with the F1-R amino acid changes at F104 and F115, plus both M amino acid changes. The mutant will be studied in both tissue culture and mice to determine its affects both in vitro and in vivo. As a first step in this process I have amplified pRGV7, a cDNA plasmid of SeV that contains the F1-R F mutation encoding F115 and both M mutations. Site directed mutagenesis of pRGV7 was done to introduce a mutation encoding the F1-R amino acid change at F104. The resulting plasmid, pRGV18, still remains to be confirmed through sequencing. If it contains the desired mutation it will subsequently be used to generate the virus, RGV18, and to study its’ in vitro and in vivo pathogenicity.

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Poster #5-Joleen Bourne



The Effects of Gang Affiliation on Juror Verdicts

Poster: Mitchell Eisen, Patti Arrendando, Joleen Bourne,
Orbelin Montes, Amber Ritter

This study was designed to examine the effects of introducing evidence related to the defendant's Gang Affiliation on juror decision-making. Two hundred and nine participants viewed a 35 minute video of staged a fictionalized trial of a man charged with stabbing a patron at a bar. Participants saw one of two versions on the video. In the no gang condition, no mention of the defendant’s gang affiliation was mentioned. In the gang condition, the investigating officer testified that the defendant was a known gang member and had the tattoo of the gang on his right arm. Otherwise the trial videos were identical. The Staged trail included 4 elements: (1) Brief opening arguments by the defense and prosecution; (2) Direct and cross of the eye-witness; (3) Direct and cross of the investigating officer; and (4) Closing arguments for both sides. Chi Sqare Analyses revealed that participants in the gang condition were significantly more likely to convict the defendant than participants in the no gang condition. Specifically, 62.5% of the participants in the gang condition found the defendent guilty of the crime compared to 43.8% of participants in the non gang consition. Results will be discussed in terms of the current literature and case law related to the biasing influence of introducing gang evidence at trial.

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Poster #6- Lucinda Robledo

Metapopulation Model of Predator-Prey Mussel Bed Dynamics

A relatively recent theoretical development in ecology is the study of ecological dynamics on multiple scales.  In the area of intertidal ecology, recent research has focused on population-scale models of intertidal mussel beds.  In these models the effects of predators on the stability and spatial distribution of these populations have been studied. One model consists of three coupled ordinary differential equations (ODE) for predator-prey mussel bed dynamics.  The present study extends this one-bed population-scale model for predator-prey mussel bed dynamics of intertidal mussel beds to a regional scale “metapopulation” of mussel beds linked by predation.  The initial model consists of two mussel beds linked by a common predator population.  The dynamics of the two-bed model were studied for varying levels of predation.  For very low levels of predation both mussel beds had a high biomass and for high levels of predation both mussel beds had a low biomass.  For intermediate values of predation there are multiple stable equilbria and the final state of the metapopulation will depend on initial conditions.  The two-bed metapopulation model was extended to any arbitrary number of metapopulation models.  The topological arrangement must be considered when modeling metapopulations of several mussel populations.  Two types are being studied: the harbor model and the coastal model.  The harbor model consists of n mussel beds which share a common pool of predators that move randomly among the mussel beds.  The coastal model consists of a linear array of n mussel beds which act as “stepping stones;” predators can move to and from adjacent mussel beds only.  These models will be analyzed using linear stability analysis and numerical simulation.  The analyses will focus on how changes in one subpopulation will effect other surrounding populations (e.g. a decrease in recruitment or growth rates of a mussel bed).  Using a multi-scale spatial model will provide a better understanding the ecology of predator-prey dynamics at a regional scale and may suggest field experiments.

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Poster #7-Victor Galvan

Single Nucleotide Polymorphism (SNP) Discovery in the Pantropical Spotted Dolphin, Stenella attenuata: Using a Targeted-Gene Approach

 

The advent of the polymerase chain reaction (PCR) sparked an enormous growth in the use of molecular markers for evolutionary, taxonomic, phylogeographic, and population studies. Among the most widely used genetic markers are mitochondrial DNA sequences (mtDNA) and microsatellites. Both of these markers have been used in a wide variety of studies and each has its preferred application.  However, these genetic markers also show several analytical and technical limitations.  A newly emerging type of marker, Single Nucleotide Polymorphisms (SNPs), possesses many of the identified characteristics of an ideal marker.  Here, we will attempt to discover, characterize, and establish genotyping assays for SNPs in the pantropical spotted dolphin, Stenella attenuata, using a targeted gene approach. Through the use of PCR, we will identify CATS loci that produce a single amplification product.  Successful loci will then be sequenced and compared across several individuals to identify SNPs.  Genotyping assays for the discovered SNPs will be designed and optimized for high throughput. Initial PCR amplification of 59 CATS loci has proven successful in ~51% of primer sets used. To date, 12 SNPs have been identified in approximately 6 loci. This study will provide a new and powerful type of molecular marker that will enable marine mammal scientists to answer more complex questions, as well as help reduce future costs associated with SNP discovery in other non-model species. 

 

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Poster #8- Grace Masangkay

An Alternative Road for the Expression of Human Sulfiredoxin

 

Abstract: Hydrogen peroxide is a reactive oxygen species that can produce hydroxyl radicals (•OH) that will damage DNA.  This damage can lead to mutations, causing diseases such as cancer, neurodegenerative diseases and aging.  Organisms have developed defense mechanisms, such as antioxidants, to counteract the oxidation process.  Peroxiredoxins are ubiquitous enzymes that reduce H2O2 and other peroxides to H2O.  However, when the level of H2O2 is too high, the peroxidatic cysteine thiol groups of some isoforms of 2-Cys peroxiredoxins become oxidized to form sulfinic acid (-SO2H).      

       In this state, peroxiredoxins cannot reduce H2O2 to H2O.  The conversion of thiols to sulfinic acids was believed to be irreversible; however, studies have shown that a yeast enzyme called sulfiredoxin can reduce the cysteine-sulfinic acid of yeast peroxiredoxin TsaI to its thiol form.  The reduction of sulfinic acid by sulfiredoxin requires ATP for activation and Mg2+.  Our goal and hypothesis is to express human sulfiredoxin in E. Coli cells and demonstrate that human sulfiredoxin can reduce sulfinic acid back to its thiol form in a variety of protein substrates.

       Our previous approach to express human sulfiredoxin produced 15 frameshift mutations that led to a premature stop codon, preventing the protein from being expressed.  An alternative road to express sulfiredoxin is to clone the human sulfiredoxin cDNA (codons 32-137) into pET-19b and express it in BL21(DE3)pLysS E. coli cells.  To achieve our goal, we designed primers and purchased them from Integrated DNA Technologies (IDT), Inc.  The truncated sulfiredoxin was amplified by polymerase chain reaction (PCR) technique.  Sticky ends on pET-19b and sulfiredoxin were created by double digestion technique.  Double digested sulfiredoxin and pET-19b were ligated using T4 DNA ligase and the reaction was transformed into TOP10 E. coli cells and plated on LB medium supplemented with ampicillin.  Ligation of fragments was not successful; therefore, the ligation reaction will be modified to build the construct, transform it into E.coli cells, and induce its expression with Isopropyl-ß-D-thiogalactopyranoside.

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Poster #9- Carlos Hernandez

 Dynamic Capsules made from Quinoxaline cavitands

We are proposing the development of a large synthetic host to serve as a drug delivery device or as a chamber for molecular reactions. These molecules might be able to mimic the natural actions taking place in biological recognition of cells or other natural molecular events. 

The dynamic capsules presented here are being synthesized from diquinoxaline cavitand by protecting either one of both pairs of hydroxyls of the diquinoxaline cavitand.

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Poster #10- Maritza Hernandez

ALTERATION OF LECITHIN:CHOLESTEROL ACYLTRANSFERASE AND CHOLESTERYL ESTER TRANSFER PROTEIN ACTIVITIES IN CHOLESTEROL AND JOJOBA OIL FED RABBITS 

Authors: Maritza Hernandez, Aileen Ariosa and Raymond E. Garcia, PhD.

 Abstract:  Elevated concentrations of serum cholesterol conduces the formation of atherosclerotic plaques in arteries leading to cardiovascular disease. New Zealand White (NZW) rabbits fed a cholesterol-rich diet (1% w/w) exhibit a decrease in high density lipoprotein cholesterol concentration ([HDL-C]), whereas those fed a cholesterol-rich (1% w/w) diet supplemented with jojoba oil (3% w/w) maintain normal (HDL-C). The objective of this project is to determine the mechanism behind these observations by quantifying the activities of enzymes involved in cholesterol metabolism when rabbits are fed these diets. Our hypothesis is that dietary jojoba oil, in the presence or absence of dietary cholesterol, stimulates lecithin:cholesterol acyltransferase (LCAT) and inhibits cholesteryl ester transfer protein (CETP). Blood samples were obtained at 0 and 7 days. NZW rabbits are fed either a normal (N), 3% jojoba oil (J), 1% cholesterol (C), or 1% cholesterol + 3% jojoba oil (CJ) diet. Total cholesterol (TC) and free cholesterol (FC) concentrations were measured using enzymatic assays. Cholesteryl ester (CE) concentrations were calculated from TC and FC concentrations. LCAT activity was determined by measuring the disappearance of FC over time and CETP via a fluorometric assay. Rabbits showed greater [HDL-CE] in J- and CJ-fed rabbits compared to N- and C-fed rabbits correspondingly. LCAT activities were determined in µmol/dL/h: CJ=217.8±14.2 > C=157.3±22.8; J=37.9±2.3 ≈ N=40.4±2.9. CETP rates were measured in nmol/mL/h: C=22.5 > (CJ=20.25 ≈ J=18.87 ≈ N=18.92). Results indicate that dietary jojoba oil stimulates LCAT and inhibits CETP whenever cholesterol is present in the diet.

 

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Poster #11- Sharonne Herbert

Predictors of Anxiety and Depression Among Sri Lankan School Children: A Regional Comparison

Elizabeth Mota, Sharonne Herbert, Kalani Makanui, Laura Petersen, & Gaithri A. Fernando

Ethnopolitical armed conflict which lasted almost 20 years in Sri Lanka is likely to have resulted in mental distress in ordinary school children, militant, and civilian adults. The current study seeks to understand how demographic variables such as gender, age, religious affiliation, and region of residence, as well as indices of exposure to war-related violence (e.g bomb blasts) and non-war-related violence (e.g. domestic violence) may combine to predict symptoms of anxiety and depression among Sri Lankan school children. A sample of 917 school children (girls = 580) from grades 6-13 (mean age =13.56, SD = 2.03) from Jaffna (north) and Colombo (south) completed a survey assessing the variables of interest. Regression analyses were conducted on the outcome variables (anxiety and depression scores) with violence exposure and demographic variables as predictors.  Results revealed that non-war-related exposure to violence and religious affiliation were significant independent predictors of anxiety in Jaffna (R = 0.22, p < .001) and Colombo (R = 0.21, p < .001).  For the outcome depression, non-war-related exposure to violence and religion were significant predictors in Jaffna (R = 0.4, p < .001), while non-war-related exposure to violence, gender, and age were significant predictors in Colombo (R = .36, p < .001). Results suggest that assessment and interventions for mental distress in Sri Lankan school children should not overlook the toll taken by daily exposure to different types of violence, including domestic violence; and religio-cultural factors must also be taken into account if interventions are to be successful with these children.

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Poster #12- Andrea Salazar

Comparative Analysis of anti-Asian
Prejudice Among People of Color

Andrea Salazar

Abstract: The current study explored the extent to which different socioracial groups endorsed stereotypes about Asian Americans including Asian Americans themselves. Using racial identity and color-blind racial attitudes as theoretical frameworks for analyzing anti-Asian prejudice, a comparative analysis examined these attitudes among People of Color and Asian Americans. Empirical studies have demonstrated that higher levels of color-blind racial attitudes were related to higher levels of racial prejudice (e.g., Neville, 2000).

Multiple regression analyses were performed to examine the influence of participants’ racial identity attitudes on anti-Asian prejudice among racially diverse participants and Asian Americans.  To explore how racial identity statuses predicted anti-Asian prejudice and color-blind racial attitudes and how color-blind attitudes predicted anti-Asian prejudice, 7 multiple regression analyses were conducted for each sample group.

Poster #13-Annie Mirsoian

Interferon-g Mediated Regulation of Antimicrobial Peptide Human Defensin 5 Expression in Urogenital Epithelial Cells

Annie Mirsoian and Edith Porter

Department of Biological sciences

California State University, Los Angeles

Sexually transmitted diseases (STDs) are a group of infections that are have now become the most common types of notifiable infectious diseases in most countries. The worldwide incidence of these types of diseases is estimated at 125 million new cases every year worldwide. A crucial mechanism of antimicrobial defense employed by epithelial cells the production of antimicrobial peptides, including Human Defensin 5 (HD5). We have previously shown that HD5 is up-regulated in STDs and pilot experiments suggested that the proinflammatory cytokines IL1 and IFNg increase HD5 production in urogenital epithelial cells.  Through Real-Time Polymerase Chain Reaction (RT-PCR) analysis and western immunoblot assays we are mapping out the regulation of HD5 expression in various urogenital epithelial cell lines and present here our first data. Up-regulation of epithelial defensin HD5 by IFNg has not been described before. Our result may suggest a novel interaction between IFNg producing cells, including natural killer cells, and epithelial cells in innate immunity.

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