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Behavioral Effects of Glucocorticiod Excess
Charles Chambers
Department of Biology, Towson University
Whether you are a gazelle on the savanna or a commuter in bumper-to-bumper traffic stress is a part of life. A major component of the stress response is activation of the hypothalamic-pituatary-adrenal (HPA) axis. HPA axis activation starts a cascade that culminates in Glucocorticiod secretion from the adrenal cortex. Glucocortiods increase the chance of survival in potentially life-threatening situations by shifting bodily resources to meet the demands of an immediate threat. While beneficial in the short-term, excessive glucocortiod secretion in response to a real or perceived threat can have detrimental effects on the overall perceived health of an individual and can cause behavioral and /or physical manifestations that re directly observable in clinical or experimental settings. The physiological effects of chronic stress can be induced in rats by artificially increasing their principle glucocortoid, corticosterone. The behavioral effects of artificially elevated corticosterone levels, however have not been as extensively studied. We hypothesize that chronically elevated corticosterone levels will increase anxiety-like behavior in rats. We will test this hypothesis by implanting subcutaneous pellets containing either 250 mg/kg of corticosterone or cholesterol. After a week-long recovery period the rats will be put in an anxiety-provoking situation, an elevated plus maze (EPM). The EPM consist of two arms that bisect each other; one is open sided while the other has walls. Rats have a tendency towards exploratory behavior, which is in direct conflict with an inherent reluctance to enter open area. The EPM exploits this conflict. Exploration has been validated experimentally, indicating that reduced open area exploration is related to increased anxiety-like behavior. By determining the behavioral effects of chronically elevated glucocorticoids, the neural mechanisms that mediate increased anxiety during Glucocorticiod excess can be better understood.
An analysis on the compatibility of DNA barcoding and morphological taxonomy using Pheidole Ants and the COI gene
Kathleen Ketchum, Dr. Colleen Sinclair, Dr. John LaPolla
Biological conservation efforts require an in-depth analysis and understanding of the biodiversity and species richness of an ecosystem. Ants can be used to assess the health of an ecosystem, due to their importance and relative affluence. Pheidole, a dominant genus of ant, is found across the globe. Morphological analysis on specimens has identified several abundant species within the genus. Using DNA barcoding of cytochrome oxidase subunit I, several important observations have been made. The abundant species appear to be composed of several similar yet distinct species (possibly subspecies), supported by the evolutionary tree developed in Paup. Data analysis supports the hypothesis that DNA barcoding is compatible with morphology-based taxonomy on the ability to distinguish between species. This compatibility suggests that using both morphology and DNA barcoding could increase the number of species known within the Pheidole genus, as well as help identify and classify morphospecies and cryptic species. A more accurate knowledge of the diversity of ants within an ecosystem could then be applied to conservation efforts, helping to determine what areas would best benefit becoming wildlife sanctuaries. Development of Specific, Primer-based Identification of Spiroplasma sp. Using the Fibrillin Gene
Brigid O’Flaherty, Dr Gail Gasparich
Department of Biological Sciences & Molecular Biology, Biochemistry and Bioinformatics Towson University
The genus Spiroplasma is comprised of a group of wall-less prokaryotes belonging to the Kingdom Monera and Class Mollicutes, and to date 34 serogroups have been identified. Upon discovering a new species proper identification and classification involves a costly and time consuming process which utilized molecular, biochemical, serological, and morphological analyses. To provide a strategy for rapid identification of this organism a polymerase chain reaction (PCR) based analysis for the identification of spiroplasma, using regions of the fibrillin gene, is being explored. PCR reactions were set up to test strain amplification with selected primers of the fibrillin gene and products analyzed by gel electrophoresis. A nested primer method, using primers 818F and 2413R as an initial amplification and 1035F and 1981R as a secondary amplification step, has had the most amplification. To date amplification products of approximately 946 base pairs were obtained from 14 out of the16 strains tested. Current work is being done to determine the efficacy of this primer set for the remaining 30 strains and to optimize the reaction. The final goal of this project is to have a PCR reaction that amplifies all spiroplasma group or subgroup type strains and does not amplify closely related bacterial genera. Ginkgo biloba extracts induce the expression of hepatic drug-metabolizing enzymes through activation of PXR and AhR
Stanton JD, Li L, Tolson AH, Luo Y, and Wang H
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy,
20 Penn Street, Baltimore Maryland 21201 hwang@rx.umaryland.edu
Ginkgo biloba extract (GBE) is extensively used as an herbal remedy for enhancing mental focus and delaying onset of age-related loss of cognitive function. Potentials of related drug-herbal interaction have been highlighted by recent reporters, which indicated GBE causes enhancement of several P450 expression in rodent animals. However, data pertaining to GBE induction of human metabolism is limited, and the underlying mechanisms are not known. In the present study, we aimed to evaluate the metabolism induction profile of EGB761, ginkgolide A (GA), ginkgolide B (GB), and bilobalide (BB) using human primary hepatocyte cultures. Real-time PCR assay was employed to assess mRNA levels of CYP3A4, CYP2B6, CYP1A2, UGT1A1, and MDR1. To assess the potential for GBE activation of transcription factors, cell-based reporter assays for PXR, CAR, and AhR were conducted in HepG2 cells. Results demonstrated that GA and GB strongly induced CYP3A4 and CYP2B6 in human hepatocytes, and activated PXR but not CAR or AhR; EGB761 and BB only resulted in marginal changes in CYP2B6 expression, without activation of either PXR or CAR. In contrast, EGB761 but not GA or GB activated AhR in reporter assays, indicating EGB761 may contain components other than GA or GB, which mediated the activation of AhR. Current results revealed that GBE induces hepatic expression of CYP3A4 and CYP2B6 through the activation of PXR, and GA/GB is the major component responsible for this induction. EGB761 activation of AhR may through an unknown component other than GA. Human CAR is not involved in GBE mediated CYP inductions. Has Evolution occurred in Common Ragweed in response to Urban Environments?
Tiha Long, Department of Biological Sciences, Towson University
Faculty Sponsor: Dr. Larry Wimmers
Atmospheric CO2 levels have risen since the pre-industrial era and are expected to continue rising through the next century. This increase in CO2 levels correlates with a predicted increase in temperature caused by the greenhouse effect. An established urban environment is a model for the global changes that are predicted because the average CO2 levels and the average temperatures are higher in these environments. Common ragweed has been shown to respond to increased CO2 levels with an increase in overall biomass and a greater increase in reproductive biomass. However, it has also been shown that within that species there is variability between genotypes in their response to elevated CO2 levels. It is expected that selective pressures will favor these genotypes that show an increase in reproductive biomass because they will be better adapted for competition in the changing environment. The genotypes of rural and urban populations of A. artimisiifolia will be analyzed to find evidence of evolution in the urban population. To detect evolution in the genotypes of the populations, the overall genetic variability will be analyzed. The urban population is expected to have less genetic variability than the rural population because of the strong selective pressures acting on that population. Microsatellite analysis will be used to detect variability in specific genomic locations in the populations. Five sets of previously published primers will be used. Results of the microsatellite analysis will be reported. Development of Amplified rDNA Restriction Analysis (ARDRA) for the Identification of Spiroplasma species
Alyssa Chamberlain, Department of Biological Sciences, Towson University
Faculty Sponsor: Dr. Gail E. Gasparich
Spiroplasmas are wall-less descendants of Gram-positive bacteria that maintain some of the smallest genomes known for self-replicating organisms. These helical prokaryotes are motile and exploit numerous habitats, they are found in association with ticks and higher order invertebrates, but most often found in association with insects. Currently 36 Spiroplasma species have been completely characterized and given binomial names. Among the hundreds of isolates partially described, there are undoubtedly many new Spiroplasma species represented.
The current protocol for the characterization of new spiroplasmas can be long and tedious, including molecular, morphological, biochemical and serological analyses. The goal of this project is to develop amplified rDNA restriction analysis (ARDRA) in order to differentiate all described Spiroplasma species. This would provide a technically feasible screen for the presence of spiroplasmas by individuals in fields other than microbiology and without access to antisera. This technique has been successful in classifying and differentiating Mycoplasma species, which are phylogenetically closely related to spiroplasmas. In ARDRA, the 16s rDNA gene is amplified by PCR and then cut with restriction enzymes (MwoI, AluI and BfaI). The resulting digests are then analyzed for distinct banding patterns using 8% polyacrylamide gel electrophoresis.
Preliminary results indicate that digestion with MwoI produced some differentiation between the different Spiroplasma sp., however, in order to see greater differentiation, the amplified DNA was also digested with AluI. If we are unsatisfied with the differentiation achieved by AluI, the DNA will be digested using the restriction enzyme, BfaI. Final results will be recorded by identification of the specific sizes of the fragments observed in each digest to determine if spiroplasmas can be differentiated.
A Rapid Extraction Method for the Identification of Clinically Relevant Gram Negative Bacteria by Cellular Fatty Acid (CFA) Analysis
Louis Schwartzman, Mark Romagnoli, Willie Stevens
BACKGROUND:
The accurate and rapid identification of medically important gram negative bacteria is a critical component of the clinical microbiology laboratory and is required for the correct assessment of clinical significance and appropriate therapy.
Gas-chromatographic (GC) analysis of CFA has been used for many years as an identification tool in the clinical laboratory. This technique however has been primarily restricted to isolates difficult to identify with conventional methods. Widespread implementation for routine diagnostic use has been hindered by the relatively large cell mass required (20-40mg) and a lengthy extraction process. We evaluated a novel protocol developed by MIDI, Inc. (Newark, DE) that requires approximately 2 mg of cells and can be completed in less than 2 minutes.
METHODS:
Recent representative isolates of Gram negative bacteria were selected. CFA were converted to their corresponding methyl esters using a proprietary reagent. Extraction of the CFA for subsequent GC analysis was performed with hexane. Chromatographic analysis and organism identification was performed by the MIDI SherlockÒ software using a beta test version of the database.
RESULTS:
Pending
Detection of misidentified plants in Theobroma cacao germplasm collections in Trinidad
Sara E. Campbell, Roland P. Roberts, Larry Wimmers, Kaumudi Jonnalagadda, David Butler, Darin Sukha, Frances Bekele and James A. Saunders
Theobroma cacao an important crop plant grown commercially in numerous tropical countries originating from the Amazonian basin of South America. Diverse germplasm is maintained in various living collections of trees due to the poor viability of seeds in storage. Multiple trees are usually grown for each clonal accession; however, management of these germplasm collections is complicated by the likelihood of misidentification or introduction of mislabeled individual trees into a clonal germplasm group. Towson University and the University of West Indies have initiated a collaborative research project aimed at determining the limits of detection of contaminating DNA samples from non-identical clones in pooled genomic extracts of all members of selected cacao accessions. DNA genotyping by SSR profiling is a useful procedure for determining the true genetic identity of trees that have been mislabeled or misidentified. Pooled DNA extracted from multiple trees will be analyzed and correlated with extensive morphological characterizations of T. cacao that have been ongoing in Trinidad for many years. Our second goal is to train new scientists in tropical biology and the use of molecular techniques in agricultural research. Students at both institutions will participate in this research. Through international research and training of this type, we hope to improve the practical economic realities of growing cacao as a commodity, while we also train new scientists in the challenges of tropical agriculture. Investigating factors that affect chromosomal localization of essential chromosomal proteins
Yidnekachew Ejamo, Dr.Yoshimitsu Takahashi, and Dr. Alexander V. Strunniko
The transmission of genetic information from one generation to the next requires the accurate replication of the DNA during S phase and the faithful partitioning of chromosomes during mitosis. Many proteins are known to change localization during cell cycle. For example condensin protein is concentrated into the nucleolus during anaphase. Little is known about how individual proteins are trans-located in the nucleus. One of the common features of essential chromosomal proteins is modification by SUMO protein. SUMO is a ubiquitin-like modifier that regulates many proteins by direct conjugation. E1, E2 and E3 enzymes are needed to transfer SUMO to a specific target. Our previous trial using SUMO fusion to Top2 protein suggested that SUMO modification targets protein to the surface of nucleolus. We are testing our hypothesis that SUMO affects global localization of nuclear proteins.
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