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HHMI Undergraduate Summer Research Program Amanda Achberger (HHMI, LSU), Brent Christner, LSU Biological Sciences A bacterium recovered from the Vostok ice core was found to possess an Ice Binding Protein (IBP) homologous to those described in some cold-adapted marine bacteria, molds, and diatoms (1). During the phase transition of water to ice, the presence of the secreted IBP alters the structure of the ice crystals and prevents recrystallization which is often times extremely damaging to the cell membrane. The IBP is suspected to provide a phenotypic advantage under frozen conditions by preventing the damage associated with ice formation and recrystallization as well as conserving the liquid veins within the ice. To explore the temperature-dependent regulation of the IBP, Reverse Transcription-PCR was used to examine gene expression at various temperatures. Expression of the IBP gene was detected at 4oC, but not at 10oC or warmer temperatures. These results provide evidence for a low temperature stress response that is distinct from the classical bacterial cold-shock response. Zhaleh J. Amini (HHMI, LSU), Jacqueline M. Stephens, LSU Biological Sciences Obesity is a global epidemic, affecting at least 400 million adults and 5 million children worldwide, and is the primary disease of adipocytes. Adipocytes are the main sites of lipid storage, are insulin sensitive, and secrete endocrine hormones; therefore compounds which alter these properties could be protective or causative of obesity and type II diabetes. Botanical extracts are readily available and consumed by millions of people, but insufficient data exist on the efficacy of most botanical products, so understanding the effects of botanical extracts on adipocyte development and function is worthwhile. Our study looked at various species of Artemisia (which includes wormwood and tarragon) to determine whether the extracts could alter adipocyte function. This study demonstrated that fractions of A.scoparia (Redstem Wormwood) and A. santolinifolia induce adipocyte differentiation of 3T3-L1 cells, as evidenced by increased levels of adipocyte gene markers PPARg, adiponectin (ADN) and aP2. Our results suggest that Artemisia has profound positive effects on adipocyte development and function, and these studies may be useful in developing drugs that target obesity. Allen Bui (HHMI, LSU), Kevin Kleinow and Arthur Penn, LSUSVM Comparative Biomedical Sciences PAHs are multi-ring organic compounds that are ubiquitous in the environment. Incomplete combustion of 1,3-butadiene, a 4-carbon hydrocarbon from which commercial rubber products are produced, yields a plume of black smoke composed of butadiene soot (BDS) nanoparticles. BDS nanoparticle surfaces are rich in PAHs which cause oxidative stress and inflammation in lungs of mice following inhalation of BDS. A limited number of individual PAHs have been shown to interfere with zebrafish development. Our goal here was to investigate the effects of mild BDS exposure on development of zebrafish (D. rerio, AB strain) embryos. We exposed zebrafish embryos to BDS (6-600 ug/ml) by directly sprinkling BDS onto the surface of medium without sonicating the BDS or solubilizing it in DMSO. PAHs-associated fluorescence was observed in treated embryos and larvae. Larvae showed signs of lordosis, pericardial edema, larger yolk sac, and slower heart rate. BDS-treated embryos hatched later than untreated embryos. We observed fluorescence in the yolk sac of embryos and in the gastrointestinal tract, endothelial cells of blood vessels, and eyes of larvae. PAH-associated fluorescence persisted in zebrafish eleven days after exposures ended. Future studies will include: assessing metabolism and fate of BDS-associated PAHs well after BDS-exposure ends; determining whether there are any long-term functional effects associated with brief exposure of embryos to BDS; and whether those changes correlate with altered gene expression patterns. Jennifer Bunagan (HHMI, University of Texas‐Pan American),
James E. Miller, LSUSVM Pathobiological Sciences Aimee Caillet (HHMI, LSU), Huangen Ding, LSU Biological Sciences DinG is a DNA damage-inducible protein found in E. coli and is a member of the superfamily 2 DNA helicases. When DNA undergoes repair or replication, single-stranded DNA binding protein (SSB) is present due to the existence of single-stranded DNA as an intermediate. SSB has complex roles in all aspects of DNA metabolism and interacts with ssDNA through its amphipathic C-terminus. In addition to the DNA-dependent helicase activity, DinG contains DNA-dependent ATPase activity. ATPase is an enzyme that catalyzes the hydrolysis of ATP in the following reaction: ATPà ADP + Pi. DinG protein contains a [4Fe-4S] cluster which is important for helicase activity. When this [4Fe-4S] cluster is reduced, the helicase activity is inhibited, but after re-oxidation, the helicase activity is restored. Nitric oxide can modify the [4Fe-4S] cluster causing the inactivation of helicase activity, and NO-modified DinG can be repaired causing the helicase activity to be restored. We tested to see if SSB and NO had any regulatory effect on the helicase and ATPase activity of DinG. SSB stimulates the helicase activity of DinG. DinG has an ATPase activity. Single-stranded DNA stimulates the DinG ATPase activity. In addition, DinG ATPase activity is inactivated by NO modification of the [4Fe-4S] cluster. Kevin Campbell (HHMI, LSU), Meredith Blackwell, LSU Biological Sciences The insect digestive tract is host to many fungal symbiotic organisms including yeasts that may provide enzymes for the digestion of previously intractable food sources. In order to determine if additional relationships occurred, we isolated for microbes from the plant-ingesting Cotinis nitida (Coleoptera: Scaraebiidae). Yeast associates were isolated from the gut of the beetles collected in Oklahoma and Louisiana. These yeasts were treated with a lysis buffer and heated to lyse the cell wall and obtain the yeast DNA. PCR followed by DNA purification prepared the DNA samples for sequencing. About 600 bp of the D1/D2 region of the LSU rRNA gene were aligned with BLAST searches and analyses using PAUP* were used to compare the yeasts. Previously described yeasts also were included in the database. The newly isolated yeasts included 12 cultures related to members of three clades: C. tropicalis, C. corydali, and Trichosporon sp. Bootstrap values of the most parsimonious tree showed the tree to be poorly supported. Candida corydali was present in Cotinis samples taken from both locations which suggests a close association, the basis of which is not known. Previously, C. cordyali was collected from neuropterans in the Baton Rouge region. Tyler Crosby (HHMI, LSU), Todd Monroe, LSU Biological & Agricultural Engineering This project aims to develop high performance liquid chromatography (HPLC) methods to characterize and purify caged nucleic acid oligonucleotide analogs. These small synthetic nucleic acids allow for precise control of gene expression via exposure to near ultra-violet light. While a caged oligonucleotide molecule will have little biological activity, adequate exposure to near UV light will cleave the caging compound, allowing the molecule to hybridize to its complementary RNA strand, effectively muting expression of the target gene. Caging reactions used are statistical, yielding mixtures of products that have varying percentages of caged substituents per oligonucleotide. Modifications to the phosphodiester backbone have the most effect on the retention time of synthetic oligonucleotides in an anion exchange HPLC column because the interaction between the molecule and the column is reliant on the negative charge present in the phosphodiester linkages. Caged morpholino oligonucleotides show particular promise in light mediated control of gene expression due to their stability to nucleases in vivo. However, their nonionic backbone chemistry drastically affects their HPLC profile. To date, little characterization of these morpholino oligonucleotides has been reported. This project will demonstrate methods to efficiently isolate targeted compounds from a complex reaction mixture, particularly morpholino molecules that show little hybridization activity in the caged form. These caged morpholino oligonucleotides could then be used to silence gene expression in spatiotemporal fashion. Carolina De La Garza (HHMI, University of Texas‐Pan American), James E. Miller, LSUSVM Pathobiological Sciences Resistance to anthelmintics by gastrointestinal nematodes in small ruminants has called for alternative means of control. The objective of this experiment was to determine whether copper sulfate or copper oxide wire particles is more effective in lambs. Naturally infected Katahdin lambs were separated into 3 groups. One of the groups received a 4% copper sulfate solution and another group received a 2g bolus of copper oxide wire particles. Fecal egg count (FEC) and packed cell volumes (PCV) were determined weekly. When compared, the copper oxide wire particles resulted as the most effective anthelmintic alternative for controlling gastrointestinal nematodes such as Haemonchus contortus. Hannah Fullerton (HHMI, LSU), Bryan Carstens, LSU Biological Sciences We developed a non-destructive extraction method and designed primers to amplify the CO1 gene of adult arthropods for the ultimate purpose of compiling a CO1 gene library for the common insect prey of Sarracenia alata. Katelyn Fusilier (HHMI, LSU), James V. Moroney, LSU Biological Sciences Katie Hamel (HHMI, LSU), Jeffrey M. Gimble, LSU Pennington Biomedical Research Center Circadian Rhythm, commonly known as the light-dark cycle, controls several biological and behavioral processes including thermogenesis, sleep-wake cycle, feeding, and glucose and lipid metabolism. The circadian clock, located within the superchiasmatic nucleus of the hypothalamus, produces rhythmic 24-hour cycles for each of these processes. A set of Core Circadian Regulatory Proteins (CCRP) controls this cyclic process. Evidence has shown that the mechanics of the circadian clock and synchronicity of multiple internal factors within the system can affect the physiology and pathology of human adipose tissue. Evidence has also shown that a correlation exists between circadian dysregulation and obesity, which can result in several medical issues including cardiovascular disease, diabetes, and hyperglycemia. This study was designed to evaluate the effects of body mass index on the circadian biology of human adipose tissue by evaluating the expression and function of core clock genes including BMAL, PER, CRY, DBP, and Rev-erbα in the explants. Aaron Landry (HHMI, LSU), Huangen Ding, LSU Biological Sciences Iron-sulfur proteins are abundant in organisms and are highly conserved, ranging in their presence from bacteria to humans. The diversity of function in these proteins is due to their iron-sulfur cluster centers, which exist in several configurations and are included in metabolism, DNA synthesis and repair, RNA modification, and amino acid biosynthesis. The signaling molecule nitric oxide (NO) can modify the iron-sulfur cluster, producing a dinitrosyl-iron complex (DNIC). DNICs are stable in aerobic conditions and the nitrosylation of the iron-sulfur cluster is irreversible in most cases, making NO an efficient cytotoxic agent against pathogenic bacteria and even tumors. Some bacteria can repair the iron-sulfur cluster, and the mechanism of doing so is under investigation.The repair of an iron-sulfur cluster requires the preliminary removal of the DNIC from the protein. In the following experimental data, we demonstrate that removal of the protein-bound DNIC is possible with a high concentration of L-cysteine present, which removes the DNIC to produce an L-cysteine-bound DNIC. Furthermore, we demonstrate that L-cysteine-bound DNICs can be transferred to apoprotein in anaerobic conditions. Gary LeBlanc (HHMI, LSU), Fareed Aboul‐ela, LSU Biological Sciences Currently, most of the drug-based design is focused on the targeting of proteins within the body. However, more and more research is being done to broaden the area of drug application. Specifically, the idea of RNA to ligand binding is being further developed so that one day RNA can be one of the main targets of many types of drugs. In this project, we focused on a specific type of RNA target, the SAM-I, or S-adenosylmethionine-I, riboswitch. Brittany Lorenzen (HHMI, University of Wisconsin ‐ Stevens Point), Steven A. Barker, LSUSVM Comparative Biomedical Sciences It has been previously demonstrated that three N, N-Dimethyltryptamines (DMT, 5-OH-DMT 5-MeO-DMT; See Table 1) are hallucinogenic compounds which are naturally produced in the human body as a product of the metabolism of tryptophan. The role of these compounds in humans is unknown, in part due to their apparent low concentrations and the fact that their metabolism is not completely understood. A number of studies have been performed on blood and urine in an attempt to evaluate the natural levels of these substances in the body. However, these studies have failed to take into account all of the precursors and metabolites, a complete assessment of the turnover and status of the pathways involved. The target compounds and their characteristics are shown in Table 1. Based on recent metabolic studies conducted in this laboratory and the literature on the metabolism and biosynthesis of these compounds, we describe here a LC-ESI-MS/MS (liquid chromatography-electrospray ionization/ mass spectrometry/ mass spectrometry) method for the analysis of 24 compounds in these pathways (including a deuterated internal standard). It is expected that this methodology will be used to study and assess the endogenous hallucinogen pathway in humans. Standard curves for all compounds examined were linear (r2>/= 0.995). TRP, IAA, TA, 5-OH-IAA and 5-HT were readily identified in the urine of healthy individuals. Limits of detection for these compounds were not determined due to the samples having concentrations higher than the highest standard. Preliminary examination of a limited number of urine samples did not provide data indicating the presence of detectable concentrations of the other compounds. Estimated limits of detection for the other compounds ranged from 1-100 PPB, as seen in figure 4. This method is the first to analyze samples for these twenty-four compounds related to endogenous hallucinogens simultaneously. Therefore, many more studies should be performed in order to better identify and quantify these compounds and to more fully understand the metabolic pathways of DMT, 5-OH-DMT and 5-MeO-DMT in the human body. This may require enhancing the overall sensitivity of the method. This study must also be expanded to examine levels of these compounds in different health conditions such as schizophrenia or other mental illnesses and perhaps in religious phenomena. Furthermore, possible fluctuations in concentrations related to circadian rhythms must also be assessed. Preliminary data suggest that this method is applicable to the direct analysis of blood as well. Lauren Ming (HHMI, Loyola Marymount University), James E. Miller, LSUSVM Pathobiological Sciences Haemonchus contortus infection is a major constraint to sheep production and control is no longer viable using traditional anthelmintic treatment due to resistance. Alternative control measures are needed. The objective of this study was to determine the effectiveness of the condensed tannin containing forage, sericealespedeza (SL), as a deworming paddock to decrease H. contortus fecal egg count (FEC) in sheep. The treatment group of sheep had a choice of grazing SL or bermudagrass and the Control group grazed only bermudagrass. Infection was monitored by fecal egg count (FEC) and blood packed cell volume (PCV). Results indicated that Control and SL FEC were similar for the first 4 weeks of grazing. In weeks 5 and 6, SL FEC continued to decrease and the Control FEC increased, the difference being significant (p< 0.05). Shhyam Moorthy (HHMI, LSU), Steven C. Hand, LSU Biological Sciences Embryos of the brine shrimp, Artemia franciscana, are known for their exceptional ability to survive severe stresses such as anoxia and desiccation (Clegg et al., 1997). The high stress resistance of the embryo is likely due to a synergetic action between the non-reducing sugar trehalose and a unique class of proteins called Late Embryogenesis Abundant (LEA) proteins (Hand et al., 2007). We exposed human hepatoma cells (HepG2) to subfreezing temperatures as a mammalian model for water stress, and the impact of trehalose and LEA proteins on viability after freezing was evaluated. Since trehalose does not easily cross the plasma membrane, cells were exposed to the sugar for 24 h which leads to low intracellular accumulation presumable through pinocytosis (Elliot et al., 2006). HepG2 cells show a dose dependent decrease in proliferation if exposed to trehalose concentrations between 0 mM and 150 mM (n = 9, P < 0.05). Exposure to 150 mM trehalose causes a reduction in proliferation, as manifested by 57 % fewer cells present 24 h later compared to controls. The reduction in proliferation is likely caused by osmotic stress, and the effect was still observed 24 h after removal of the sugar. However, cells treated with 100 mM trehalose showed a pronounced increase in viability after freezing. The viability increased from 2 % at 0 mM trehalose to 14 % at 100 mM trehalose. To facilitate the uptake of trehalose, we transfected HepG2 cells with a gene encoding for a trehalose transporter (TRET1) from the chironomid fly larvae Polypedium vanderplanki.Kikawada et al. (2007) showed that the expression of TRET1 facilitates the uptake of trehalose in several mammalian cell lines. Transfected cells were loaded with the sugar by exposure to 150 mM trehalose for 90 min. Under these loading conditions the cells maintained a membrane integrity of 65 ± 2.1 % and viability of 55 ± 2.9 % post freezing (± SE, n = 9). Surprisingly, the membrane integrity and viability of control cells was also markedly increased with the new loading protocol to 62 ± 3.4 % and 28 ± 1.7 % (± SE, n = 9) respectively. No additional increase in membrane integrity was found for HepG2 cells after expression of a mitochondrial targeted LEA protein (AfrLEA3m). Tatenda Mujeni (HHMI, Bennett College For Women), Shisheng Li, LSUSVM Comparative Biomedical Sciences DNA damage checkpoint is the cell surveillance mechanism triggered by DNA lesions. Rad2, a yeast structure-specific endonuclease, is required for incising the DNA at the 3’ side of a DNA lesion during Nucleotide Excision Repair. To gain insights into the mechanism as to how NER is implicated in DNA damage checkpoint response, we determined the roles of different domains and critical residues of Rad2 in UV-induced checkpoint activation. Cells expressing wild type and different mutant Rad2 were UV-irradiated and checkpoint response in the UV-irradiated cells was monitored by measuring the phosphorylation of Rad53, a hallmark of checkpoint activation in yeast. We found that Rad2 mutants that lose the 3’ endonuclease or the DNA binding activities are still proficient in rapid phosphorylation of Rad53. However, cells expressing Rad2 truncates lacking the C-terminal nuclear localization signal showed slower phosphorylation of Rad53. Our results indicate that the incision on the 3’ side of a lesion during NER and the DNA binding activity of Rad2 are not required for UV-induced checkpoint activation. However, the nuclear localization of Rad2 is critical for the checkpoint activation. Wedad Rahman (HHMI, LSU), Anne Grove, LSU Biological Sciences The functional roles of one of the HMG domains of the yeast homolog HMO2 were examined by identifying amino acid residues that are involved in DNA intercalation. Through site directed mutagenesis, a valine in the box A domain (site 19), predicted to intercalate between DNA base pairs, was substituted with alanine. A binding assay with linear DNA showed that the mutated protein has lower binding affinity compared to wild type HMO2, while both mutant and wild type proteins have similar affinity for supercoiled DNA. The ability of the mutant HMO2 to protect free DNA ends from exonuclease digestion was determined; both wild-type protein and the mutated protein were able to protect the DNA from exonuclease activity, suggesting that the substitution does not affect the preferred binding to DNA ends characteristic of HMO2. DNA binding assays further showed that both wild-type and mutant protein bound with only modest preference to DNA with one loop (tandem mismatches) and two loops that were seven base pairs apart. However, both bound with significant preference to DNA with two loops that were nine base pairs apart showing that this distance is optimal. This suggests that distance between distortions is a factor in determining whether or not HMO2 binds to DNA. A competition assay comparing the mutated protein’s ability to bind to linear and supercoiled and to DNA with 2 loops (9bp apart) was then carried out to see which construct is most suitable. Supercoiled was able to compete out DNA with 2 loops while linear was not. Evidently, the valine in HMO2 box A is important for the protein’s ability to bind to linear DNA, while its substitution does not alter the preferred binding of HMO2 to DNA ends or to DNA with distortions. Samir Rana (HHMI, LSU), Shaomian Yao, LSUSVM Comparative Biomedical Sciences Objectives: Rat Dental Follicle Stem Cells (DFSCs) are found in the rat dental follicle. Stem cells are found to survive various stresses with potential of differentiating into osteoblasts and adipocytes. Thus, the objective of this research was to study the survival and differentiation capacity of rat DFSCs. Methods: To study the DFSCs survivility, cells were treated with hyperosmotic media for 24 hrs and then allowed to grow in normal media. Alamar blue test was performed to measure the cell viability. To study the differentiation capacity of DFSCs, treated cells were grown in osteogenic and adipogenic media respectively. After some visual indication of calcium deposits and fat droplets; alizarin red staining for osteogenesis and oil-red-o staining for adipogenesis was performed. RT-PCR was performed to study gene expression for various genes. Results: DFSCs were able to survive in media of osmolality 700 osmol/kg. Beyond that, all cells died. DFSCs surviving from 621 osmol/kg showed the most differentiation into osteblasts and adipocytes. Cell size tends to increase when treated with hyperosmotic media. Conclusion: Treatment of DF cells at 621 osmol/kg for 24 hours result in death of most cells. Surviving cells possess greater potential for differentiation. In addition the cells express high level of cycil E indicating more proliferation of cells. RT PCR results show that 3 genes SAPK-β, ROSIT, IGF1 genes are increased in stress cells. Of the 3 genes, SAPK-β may be the important one. James DeWitt Stowe (HHMI, LSU), Eric Achberger, LSU Biological Sciences Escherichia coli concentration in a body of water has traditionally been used as an indication of water quality. The qualities of E. coli that allow it to serve this purpose include its high concentration in animal feces, a link between E. coli concentration and the concentration of other pathogenic bacterial species, and its inability to survive for a long period of time outside a host organisms body. In tropical and subtropical climates, some of these qualities do not hold true for all E. coli. In this study, DNA fingerprinting was used to identify specific E. coli stains isolated from an agricultural wastewater treatment system. A database of E. coli DNA fingerprints was used to select strains that demonstrated superior survivorship in the treatment system. The ability of these genetically defined strains to survive in various environments was tested in the laboratory. Those predicted to be “survivors” displayed traits consistent with continued viability in the environment when compared to a “non-survivor group” selected using the same criteria. Bao Tran (HHMI, LSU), Huangen Ding, LSU Biological Sciences Zinc is an essential trace metal. Yet, excess zinc is highly toxic to cells. Here we investigated the effect of zinc on the iron-sulfur cluster assembly in E. coli cells. We found that 1mM of zinc is sufficient to greatly reduce bacteria proliferation without complete inhibition. In the presence of 1mM of zinc in growth media, we showed that an iron-sulfur enzyme aconitase B activity is reduced by 40%. Furthermore, we found that both iron-sulfur cluster assembly proteins IscA and IscU can bind zinc in E. coli cells. We conclude that excess zinc may affect the iron-sulfur cluster assembly in E. coli cells. Noé Hernandez (HHMI EXROP, Carleton College), Isiah M. Warner, LSU Chemistry
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2009 Abstracts
