Publication: Georgia Journal of Science
Publication Date: 22-MAR-07
Delivery: Immediate Online Access
Author:
Article Excerpt
Section I: Biological Sciences
ACAD 141
Mark S. Davis, presiding
9:00 INVESTIGATIONS OF CELL WALL STRUCTURE AND EASE OF CHLOROPHYLL EXTRACTION IN SUB-AERIAL MICRO-ALGAE**, J. Griner*, J. Trull, and J. A. Nienow, Valdosta State University, Valdosta GA 31698. Subaerial algae are a diverse group of microorganisms able to survive on open surfaces in environments where liquid water is scarce. The physiological adaptations that enable them to live in these environments are not clear. Two factors that might be important have been suggested. First, many subaerial algae have hydrophobic cell walls, a fact which seems in conflict with a need to obtain water quickly. Second, chlorophyll is difficult to extract from many subaerial algae using standard methods. We want to see if these two features are correlated by simultaneously testing different strains of subaerial algae and cyanobacteria for hydrophobicity and ease of chlorophyll extraction. To determine hydrophobicity, we are using the standard microbial adherence to hydrocarbons (MATH) method, whereby cells suspended in buffer are mixed vigorously in the presence of hexadecane. Hydrophobic cells adhere to the hexadecane droplets and remain in the upper phase. The degree of adherence is determined photometrically. Chlorophyll is extracted either using cold acetone (the standard method) or hot DMSO followed by cold acetone. The amount of chlorophyll in the extract is determined using standard equations. Our preliminary results indicate that hot DMSO can extract more than twenty times the amount of chlorophyll from some subaerial algae than can be extracted by cold acetone.
9:15 PhIP INDUCED DNA DAMAGE AND INHIBITORY EFFECT OF NAC AND GHS. Ashok Jain*, Department of Natural Sciences, Albany State University, Albany, GA. Breast cancer is the second leading cause of cancer-related deaths for women. There are many environmental chemicals that are estrogenic and are believed to be associated with increased risk of developing breast cancer. The diet of highly industrial nations, such as the United States, consists heavily of meats. However, Asian diet consists of more fruits, vegetables, and soy products. Heterocyclic amines (HCAs) are formed when meat products such as beef, chicken, pork, and fish are cooked at high temperatures. These amines have been shown to be carcinogenic and are considered major risk factors of cancer initiation. 2-Amino-l-methyl-6-phenylimidazol [4, 5-b]-pyridine (PhIP) is the most abundant HCA found in well-done and grilled meats Therefore, the objective of this study was to understand the beneficial effect of antioxidants, present in fruits and vegetables, to prevent the toxic effects of PhIP. The culture of breast epithelial cells (MCF 10A) was initiated and treated with PhIP for 3 hours. After treatment cells were harvested and DNA strand breaks was quantified by measuring the mean olive tail movement following single cell gel electrophoresis (comet assay). To investigate a possible role of antioxidants in the protection from PhIP toxicity, the ability of three antioxidants (ascorbic acid [AsA -- 10mM], Glutthione [GHS -- 10mM], and N-acetyl-cysteine [NAC -- 10mM]), were examined. GHS and NAC show protective effects in MCF 10A cells as the olive tail movement was short, indicating GHS and NAC are capable of repairing DNA strand breaks. On the other hand, AsA did not show protective effect in MCF 10A cells. Further studies in this area will uncover the interaction of genotoxic/carcinogenic heterocyclic amines and chemopreventive effects of dietary supplements.
9:30 MYCOPLASMA AND SALMONELLA IN GOPHER TORTOISES FROM MOODY AIR FORCE BASE, GEORGIA, J. Mitchell Lockhart* (1) and Gregory W. Lee (2), (1) Department of Biology, Valdosta State University, Valdosta, Georgia, 31698 and (2) Department of the Air Force. Since 1999, we have studied a population of gopher tortoises at Moody Air Force Base, Lowndes and Lanier Counties, Georgia for the presence of Upper Respiratory Tract Disease. This study has subsequently branched into studies of plasma chemistry/protein electrophoresis, the analysis of approximately 100 plasma samples for 20-25 plasma chemistry variables, genetic relatedness of subdivided populations. Salmonella and Mycoplasma presence, and basic demographics in regard to the military mission. Our findings indicated five tortoises were suspect and one positive for Mycoplasma, and 3 tortoises were positive for Salmonella. Recently, we have begun tracking tortoises using a relatively novel remote frequency identification technique, which will be presented.
9:45 RESTORATION OF THE FEDERALLY ENDANGERED GEORGIA ENDEMIC BAPTISIA ARACHNIFERA (FABACEAE) AT THE LAKE LOUISE BIOLOGICAL STATION, LOWNDES COUNTY, GA, John B. Pascarella, Valdosta State University, Valdosta, GA 31698. An experimental ex situ population of the federally endangered Georgia endemic legume Baptisia arachnifera (Fabaceae) exists at the Lake Louise (LL) Biological Field Station in Lowndes County, GA. I investigated the restoration success of B. arachnifera using seeds and transplants in enhancement plantings. While greenhouse germination typically averages over 70%, seed germination in the wild was 15.5% (fall 2004) and 16.5% and 19% in two trials (spring 2005). Seed germination was highest in open, no litter treatments followed by buried, no litter treatments. Seedlings in unburned areas from 2005 had 0% survival in a shaded woody area (unburned since 1998) and 6% survival in a previously burned area (2005). The 256 five-month-old seedlings transplanted in spring 2005 had 86.2% survival over the next year (2005-2006). No plants flowered in either 2005 or 2006. The 49 two-year-old plants planted in the fall 2004 had 85.7% survival during the 1st year following transplant and 97.6% survival the second year. Percent flowering increased from 14% the first year to 38% the second year. To maximize population size in minimal time, this study suggests collecting seeds from multiple plants, germinating them in a greenhouse, and planting either <1 yr or 1-2 year old seedlings.
10:00 Section business meeting
10:30 Poster session
POSTERS
INTRASPECIES VARIATION IN ASPERGILLUS FLAVUS IN GEORGIA PEANUTS, Deaton Thomas* and Premila N Achar, Kennesaw State University, Kennesaw, GA 30144. Aspergillus flavus can invade peanuts in the field before harvest, and during harvest, storage, and transportation. Aflatoxins are secondary metabolites produced by Aspergillus species and are carcinogenic in humans. Several strains of A. flavus exist in peanut growing areas in Georgia, however, their virulence varies with geographical distribution. A number of molecular techniques are currently available for studying genetic relationships between fungal populations. In the present study we used polymerase chain reaction (PCR) to establish genetic diversity among isolates of A. flavus from peanut growing areas in Georgia. For cultural characterization, isolates were transferred onto PDA plates and incubated at 30YC. After 7 days of incubation, potential colonies were screened for A. flavus based on morphology and colony character. Monoconidial colonies were checked under stereomicroscope, then transferred onto PDA plates and incubated at 25YC in the dark. All strains were stored on either PDA slants or Petri dishes. Fungal DNA was isolated using standard protocol with slight modification whenever necessary. PCR amplification of genomic DNA was performed using universal (ITS) 1 and (ITS) 4 primers. PCR amplification of ribosomal DNA from A. flavus revealed one common band of approximately 600 bp for all the isolates, although 2 of the isolates showed a slight variation. This variation appeared only in the toxigenic forms of A. flavus used in this study. Restriction digestion of PCR products with specific enzymes and sequencing of the same may give additional information about the molecular relatedness of different isolates of toxic and the non-toxic forms of A. flavus in Georgia peanuts.
DEVELOPMENT OF MDCC-PBP AS A REAL TIME SENSOR FOR CHROMATIN REMODELING**, Rosa Wright* (1) and A. Nagaich (2), (1) Albany State University, Albany GA 31705 and (2) National Institutes of Health, Division of Protein Therapeutics, FDA, Bethesda, MD 20892. ATP hydrolysis was used to carry out nucleosome remodeling. The exact molecular mechanism regarding the coupling of ATP hydrolysis to nucleosome remodeling is not clear. The first step in devising a real time sensor for chromatin remodeling is through development of a biosensor protein N-{2-(1-maleimidyl)ethyl}-7-(diethylamino)coumarin-3-carboxamide labeled phosphate binding protein (MDCC-PBP) that can be used to monitor the rate of ATP hydrolysis during the process of chromatin remodeling reaction in vitro. The biosensor will be utilized to develop a kinetic framework of chromatin remodeling reaction in real time. To develop this biosensor, an E. coli strain (PSN518/7) carrying phosphate binding protein (PBP) was inoculated in Luria Broth in the presence of tetracycline. The plasmid DNA was extracted using a Hi-Speed Plasmid Midi Kit. The presence of plasmid was confirmed using agarose gel electrophoresis. The purified plasmid was digested with Pstl and EcoRI to liberate cDNA of full length. The pET 28a expression vector was grown in LB media and purified using an Qiagen plasmid purification kit. The pET vector was digested with appropriate restriction enzymes, purified on the gel and ligated with the insert DNA. The recombinant vector was then induced with IPTG to overproduce the recombinant protein and transformed into E.coli. The purified PBP was then derivatized with MDCC. By coupling the MDCC-PBP assay with an Ultrafast UV laser test, a real time assay for chromatin remodeling will be developed.
A COMPARATIVE STUDY ON BOVINE LIVER AND ONION CATALASE WITH SPECIAL REFERENCE TO THE EFFECT OF pH ON ENZYME ACTIVITY, John Lattier* and William Said, Georgia State University, Atlanta, GA 30303. Several methodologies available to estimate the kinetic parameters of catalase are mostly laborious and costly. We used a simple, inexpensive, and efficient method to investigate catalase kinetics. The enzymatic activity of onion pulps, as a source of catalase, was compared to the activity of purified bovine liver catalase. We used the Lineweaver-Burk plot to extrapolate the kinetic parameters, Km and Vmax, for computer-generated Michaelis-Menten graphs. Blended and centrifuged onion pulp separates into three distinctive layers. The "foamy" layer yielded a Vmax = 0.009 ml [O.sub.2].[sec.sup.-1], a Michaelis Constant, Km = 0.349 M. The supernatant layers provided almost no measurable catalase activity, but the precipitant, "whole-cell" layer provided relatively high catalase activity (Vmax = 0.033 ml [O.sub.2].[sec.sup.-1]; Michaelis Constant, Km = 0.151 M). Purified catalase from bovine liver, in comparison, exhibited a substantially greater catalase activity (Vmax = 0.518 ml [O.sub.2].[sec.sub.-1]; Michaelis Constant, Km = 0.29 M). The pH dependence of catalase activity was tested over a wide pH range. A pH of 6.34 and [[H.sub.2][O.sub.2]] of 0.11M, gave a velocity of catalase reaction of 0.122 ml [O.sub.2].[sec.sup.-1]. Higher or lower pH values resulted in reduced rates of the enzymatic reaction.
PREVALENCE OF GNATHOSTOMA PROCYON IS (NEMATODA: GNATHOSTOMIDAE) IN RACCOONS IN NORTH FLORIDA AND SOUTH GEORGIA, J. Mitchell Lockhart, Valdosta State University, Valdosta, GA 31698. From 2001-2006, the United States Department of Agriculture-Wildlife Services, in coordination with the University of Georgia, Auburn University, Tall Timbers Research Station, and The Jones Ecological Research Center, lethally removed mesomammalian nest predators of bobwhite quail from four sites in north Florida and south Georgia. Valdosta State University participated in this project from 2003-2006. I began examining collected raccoons for the presence of Gnathostoma procyonis, a nematode found in the stomach of its definitive host, from 2004-2006. A total of 511 raccoons was examined and 141 (27.6%) were infected with G. procyonis (2004-29.0%, 2005-37.7%, 2006-17.2%). Male and female raccoons were infected equally (males - 27.5%, n = 335, females - 27.8%, n = 176), and worm burdens did not differ significantly (males - mean = 2.88, range 1-17; females - mean = 2.78, range 1-10). Infections peaked in spring with maximum prevalence occurring in March (43.4%, n = 120) and declined to a minimum prevalence in September (5.4%, n = 37).
ASPECTS OF LAKE FERTILIZATION IN SOUTH GEORGIA, J. A. Nienow, Valdosta State University, Valdosta, GA 31698. Fertilization is one practice used to control the growth of filamentous benthic algae and submerged macrophytes in shallow lakes and ponds. A local lake manager was concerned that the addition of fertilizer to control algal mats was having an impact on the pH of Mission Lake. To test this hypothesis, algal growth and pH over the course of a growing season were monitored, while normal lake-management practices, including fertilization, were continued. A temperature/pH recorder was placed in shallow water at one end of the lake, set to record at 30-minute intervals. Water samples were collected at roughly two-week intervals, from which chlorophyll concentration and dominant phytoplankton species were determined. Algal mats were sampled to determine the major component species whenever macroscopic changes were observed. Fertilizer was applied in mid-March, mid-April, and mid-May. During this period a dense growth of the planktonic species Aphanizomenon flos-aquae developed, and chlorophyll concentrations in excess of 300 [micro]g/L were recorded for April. Phytoplankton numbers declined in May and remained low for the rest of the growing season. Floating mats of filamentous algae, composed primarily of Plectonema (Lyngbya) wollei, but also including Rhizoclonium hieroglyphicum, developed during the summer. Lake pH showed a marked diurnal fluctuation throughout the growing season, with diurnal fluctuations of 3 pH units not uncommon. The pH fluctuations continuing after fertilization was halted in mid-May were apparently related to photosynthetic activity, not the fertilization regime.
COMPARISON OF THE EFFECTS OF TWO DIFFERENT TREADMILL TRAINING STRATEGIES ON THE REGENERATION OF AXONS IN INJURED PERIPHERAL NERVES, Natalie Redmon* (1) and Arthur English (2), (1) Albany State University, Albany GA 31705 and (2) Emory University School of Medicine, Atlanta, GA 30322. Poor functional outcomes after peripheral nerve injuries result from slow growth of regenerating axons in the injured nerves. Prolonged treadmill training (one hour, 10 m/min) leads to enhanced growth, but mice do not exercise in this way, voluntarily. The effectiveness of short duration, high intensity (interval) treadmill training, which mimics the natural pattern used by mice, was compared to that of prolonged training. We used mice carrying the thy-1-YFP-H transgene, marked by the presence of yellow fluorescent protein (YFP) in a sample of axons in peripheral nerves, to study the growth of individual regenerating axons with confocal microscopy. We cut common fibular nerves and repaired them using nerve grafts from non-fluorescent littermates. After two weeks, we checked for regenerating YFP+ axons in images of optical sections through nerves harvested from euthanized mice. We measured lengths of fluorescent axon profiles in mice trained for four, ten, and 15 two-minute intervals at a treadmill speed of 20 m/min, and compared them to lengths of axon profiles from mice exposed to prolonged training, as well as to a group of unexercised (control) mice. In mice exercised in four intervals daily, the lengths of regenerating axons were significantly longer than those in unexercised controls, and similar to those axons in mice exercised continuously, more than four times longer each day. In mice whose exercise consisted of more than 4 intervals daily, no significant enhancement was found. Thus, interval training could be an effective way of promoting axon regeneration in peripheral nerves, but the number of intervals used must be considered carefully. This project was support in part by the MBRS-RISE program at Albany State University, NIH Grant #GMO71415.
INTERSPECIES VARIATION IN THREE DIFFERENT SPECIES OF ASPERGILLUS IN GEORGIA PEANUTS, Ibijoke Akinjobi* and Premila N Achar, Kennesaw State University, Kennesaw, GA 30144. Detection of fungal infection is important before peanuts reach market, especially the detection of aflatoxin producing Aspergillus. Although several methods have been developed for the detection of fungal infection in peanuts, most have limitations and can result in misidentification. Molecular approaches are now being developed to provide a more rapid and objective identification of fungi compared to traditional phenotypic methods. Protocols for the DNA-based diagnosis of Aspergillus species have recently been developed. The polymerase chain reaction (PCR) has revolutionized the detection of certain pathogens from field samples, including potentially aflatoxin producing Aspergillus species. In the present study our aim was to develop PCR based protocols in order to investigate interspecies variation at the molecular level among three species of Aspergillus: A. flavus, A. niger, and A. parasiticus from contaminated peanuts in Georgia. DNA was isolated by use of a commercial kit. A comparative analysis at the molecular level between aflatoxin vs. non-aflatoxin producing strains of A. flavus and A. parasiticus was also performed. The utility of the large-subunit RNA gene D1-D2 region, and internal transcribed spacers 1 and 4 (ITS1 and ITS4) as targets for the molecular comparison of A. flavus, A. niger, and A. parasiticus was assessed. Our results indicate that interspecies variation among the three isolates is minimal in their ITS1 and ITS4 sequences, and at the molecular level there is no significant difference between aflatoxin vs. non-aflatoxin producing strains of A. flavus and A. parasiticus.
EFFECT OF SHORT-TERM EXPOSURE TO CIGARETTE SMOKE ON BAKER YEAST CATALASE**, Kieu-Nhi Bui*, Vladislav Zima, Elizabeth Stanford and William Said, Georgia State University, Atlanta, GA 30303. Cigarette smoke (CS) has long been recognized as a major environmental pollutant and is considered the leading cause of preventable death in the U.S. Worldwide, greater than 90% of lung cancers (the leading cause of cancer death) are CS-related. Although much is known about the type of the damage caused by CS, little is known about the effect of short-term exposures to CS on kinetics of cellular anti-oxidant enzymes such as catalase and superoxide dismutase. The present work attempts to employ a simple, inexpensive yet reliable, method to investigate kinetics of yeast catalase in relation to CS exposure. Yeast cells pre-exposed to 15 minutes of CS exhibited a higher rate of catalase activity compared to that of the control [mean = 0.51 + 0.012 mM [O.sub.2] x [s.sup.-1] (n = 3 trials)] and [0.44 [+ or -] 0.046 mM [O.sub.2] x [s.sup.-1] (n = 3 trials)], respectively (P < 0.05). Although deactivation of yeast catalase was observed over time in both CS-treatment and controls, CS-pre-exposed yeast cells (for 30 min.), as well as yeast cells dispensed in glass vials pre-exposed to CS (for 15 min.) exhibited lower percent reduction of catalase activity compared with those of the controls. Our preliminary data suggest that short-term exposure to cigarette smoke caused both activation and a "prolonged deactivation" of yeast catalase. The initial results suggest that early stages of CS-induced cellular events can be elicited using the yeast as model organism and should contribute to the understanding of the molecular basis of CS-induced damage.
IMPACT OF ESTROGEN AND SOY ON BEHAVIORAL RECOVERY FROM STROKE IN FEMALE RATS, Danielle Butler* (1), Tara L. Swan (2) and Derek A. Schreihofer (2), (1) Paine College and (2) Medical College of Georgia, Augusta, GA. Phytoestrogens, naturally occurring compounds that mimic some of the effects of estrogen, are often used as an alternative to treat menopausal women. This lab has shown that a high soy diet, high in phytoestrogens, reduces stroke size 24 hours after blockage of the middle cerebral artery (MCAO) in female rats. The present study was designed to establish measures of long term functional recovery from stroke and determine whether estrogen or a high soy diet improves stroke outcome. Adult female SD rats were placed on a soy-free diet and ovariectomized to remove their endogenous source of estrogen (E). Rats were divided into two groups: soy free + placebo (SFP), soy free + E (SFE). These groups underwent sham surgery or left MCAO (stroke) for 90 minutes. Three, 7, and 14 day post surgery, rats were tested for behavioral recovery. Preliminary experiments (n=4/group) suggest that the Cylinder test (sensory/ motor) and the Open Field test (anxiety/activity) may be most sensitive in detecting improvement in behavior during the recovery period. During the Cylinder test, stroked SFP rats favored their left paw on day 3, but had no lateral bias by day 14. In the Open Field test, both groups increased total movement over time, but only the SFE rats increased the number of vertical movements. These preliminary data show that estrogen improves the functional recovery following stroke. Ongoing studies are evaluating the ability of a high soy diet to mimic estrogen's functional neuroprotective effects.
THE EFFECTS OF PETROLEUM PRODUCTS ON PLANTS, Ayala Gray* and Ashok Jain, Department of Natural Sciences, Albany State University, Albany, GA 31705. The 2005 hurricane season caused water and soil contamination. Petroleum spills were of particular concern on the Gulf Coast following hurricanes Katrina and Rita. An experiment was conducted...
NOTE: All illustrations and photos have been removed from this article.
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