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 Department: Department of Biological Sciences
Characterization of Triclocarban, Methyl- Triclosan, and Triclosan in Water, Sediment, and Corbicula Fluminea (Müller, 1774) Using Laboratory, in Situ, and Field Assessments
In the last decade emerging contaminants research has intensified in a bid to answer questions about fate, transport, and effects as these chemicals as they get released into the environment. The chemicals of interest were the antimicrobials; triclocarban (TCC) and triclosan (TCS), and a metabolite of triclosan, methyl triclosan (MTCS). This research was designed to answer the question: what is the fate of these chemicals once they are released from the waste water treatment plant into receiving streams. Three different assessment methods; field monitoring, in-situ experiments, and laboratory studies were used to answer the overall question. TCS, TCC, and MTCS levels were measured in surface water, sediment and the Asiatic clam Corbicula fluminea. Field studies were conducted using four sites at Pecan Creek, Denton TX. Levels of all three chemicals in clams were up to fives orders of magnitude the water concentrations but an order of magnitude lower than in sediment. Highest sediment levels of chemicals were measured in samples from the mouth of Pecan Creek (highest organic matter). TCC was the most and TCS was the least accumulated chemicals. In-situ and lab studies both indicated that uptake of these chemicals into the clams was very rapid and measurable within 24hours of exposure. The after clams were transferred into clean water most of the compounds were depurated within 14 days.
Comparison of Bare Root vs. Potted Plants, Species Selection, and Caging Types for Restoration of a Prairie Wetland, and Quantitative Analysis and Descriptive Survey of Plant Communities and Associations at Lewisville Lake Environmental Learning Area (LLELA), Lewisville, TX
Lewisville Lake Environmental Learning Area (LLELA) is an 809-hectare property in Denton County, TX. A study of the vegetation community identified 466 species in 104 families, with 25% of the species from only two families, Asteraceae and Poaceae. The property demonstrates the characteristics of an early successional community, dominated by weedy species. Prairie communities are dominated by Johnson grass and ragweed, with climax tall grass prairie communities only in areas that have been planted with native grass seed. Forest communities are similarly in an early successional stage, dominated by the hackberry-elm-ash alliance, with small remnants of native Cross Timbers found in isolated patches. Species richness and diversity were highest in the forests and lowest in the wetlands; evenness, though not different across ecosystems, demonstrated a strong seasonal component. The species list was compared with previously reported lists for Denton County, and 256 species identified had not been previously reported for the county. A wetland restoration study was conducted to determine if there was a difference in survival and growth between potted transplants with intact root systems and bare-root transplants. Two different mesh sizes were used for protection, and the success of the different caging was evaluated. Of eight species, only four survived through the second growing season. There was no significant difference in the success of the propagule types for Sagittaria latifolia. The treatments planted with intact root systems showed significantly higher growth and reproduction than the bare-root treatments for Eleocharis quadrangulata, Heteranthera dubia, and Vallisneria americana. There was no survival recorded in the coarse mesh cages, likely due to the presence of crayfish that are able to get through the coarser mesh and feed on the transplants.
Concentrations of Triclosan in the City of Denton Wastewater Treatment Plant, Pecan Creek, and the Influent and Effluent of an Experimental Constructed Wetland
The Pecan Creek Waste Reclamation Plant in Denton, Texas, an activated sludge WWTP, was sampled monthly for ten months to determine seasonal and site variation in concentrations of triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol), an antibacterial additive. SNK separation after the highly significant ANOVA on ranked data were: summer = fall > winter = spring and influent > downstream = effluent = wetland inflow > wetland outflow (a=0.05). After the plant converted to ultraviolet disinfection, measurements were made before and after the UV basin to determine if significant amounts of triclosan were converted to dioxin. Percent loss at each of the treatment steps was determined. Concentrations of triclosan in the downstream site were below the published NOEC for the most sensitive species.
Determination of Habitat Preferences of Pronghorn (Antilocapra americana) on the Rolling Plains of Texas Using GIS and Remote Sensing
The Rocker b Ranch on the southern Rolling Plains has one of the last sizeable populations of pronghorn (Antilocapra americana) in Texas. To investigate habitat utilization on the ranch, pronghorn were fitted with GPS/VHF collars and were released into pastures surrounded by a variety of fences to determine how fence types affected habitat selection. Habitat parameters chosen for analysis were vegetation, elevation, slope, aspect, and distances to water, roads, and oil wells. Results showed that pronghorn on the ranch crossed modified fencing significantly less than other types of fencing. Pronghorn selected for all habitat parameters to various degrees, with the most important being vegetation type. Habitat selection could be attributed to correspondence of vegetation type with other parameters or spatial arrangements of physical features of the landscape. Seasonal differences in habitat utilization were evident, and animals tended to move shorter distances at night than they did during daylight hours.
Effects of Sublethal Copper Exposure on Escape Behavior and Growth of Rana pipiens Tadpoles
This research is designed to test how sublethal exposure to copper affects tadpole predator-escape behavior and how quickly tadpoles recover. After exposure, tadpoles were separated. Escape behavior was recorded for two-thirds of exposed tadpoles while one-third of the exposed population was measured weekly to determine growth and recovery. Control tadpoles were consumed within 15 minutes whereas those exposed to higher concentrations were consumed at a slower rate, which does not support the hypotheses. Although the rate of predation was lower, tadpoles exposed to higher Cu concentrations were on average, 1.47 cm in total body length. Those exposed to 0.93 mg/L averaged 0.86 cm. After being placed into clean water, treatment tadpoles recovered after 20 days.
Gene Expression Profiling of the nip Mutant in Medicago truncatula
The study of root nodule symbiosis between nitrogen-fixing bacteria and leguminous plant species is important because of the ability to supplement fixed nitrogen fertilizers and increase plant growth in poor soils. Our group has isolated a mutant called nip in the model legume Medicago truncatula that is defective in nodule symbiosis. The nip mutant (numerous infections with polyphenolics) becomes infected by Sinorhizobium meliloti but then accumulates polyphenolic defense compounds in the nodule and fails to progress to a stage where nitrogen fixation can occur. Analysis of the transcriptome of nip roots prior to inoculation with rhizobia was undertaken using Affymetric Medicago Genome Array microarrays. The total RNA of 5-day old uninoculated seedlings was analyzed in triplicate to screen for the NIP gene based on downregulated transcript levels in the mutant as compared to wild type. Further microarray data was generated from 10 days post inoculation (dpi) nip and wild type plants. Analysis of the most highly downregulated transcripts revealed that the NIP gene was not identifiable based on transcript level. Putative gene function was assigned to transcripts with altered expression patterns in order to characterize the nip mutation phenotypically as inferred from the transcriptome. Functional analysis revealed a large number of chaperone proteins were highly expressed in the nip mutant, indicating high stress in the mutant prior to infection by rhizobia. Additionally, a database containing the information regarding the nip expression profile at both 0 days post inoculation (dpi) and 10 dpi were created for screening of candidate genes as predicted from sequence in the genomic region containing NIP.
Stream water quality corridor assessment and management using spatial analysis techniques: Introduction, evaluation, and implementation of the WQCM model.
The rapid development of once-rural landscapes often produces detrimental effects on surface water quality entering local reservoirs through vulnerable stream channels. This study presents a methodology that incorporates geographic information systems (GIS) and remote sensing techniques for the creation of a stream corridor evaluation mechanism, coined the water quality corridor management (WQCM) model. Specifically, the study focuses on determining the viability of the WQCM model in assessing the stream corridor conditions within a northern Denton County pilot study region. These results will aid in the prediction and evaluation of the quality of stream water entering reservoirs that serve as the primary drinking water source for local municipalities.
Fluorescence labeling and computational analysis of the strut of myosin's 50 kDa cleft.
In order to understand the structural changes in myosin S1, fluorescence polarization and computational dynamics simulations were used. Dynamics simulations on the S1 motor domain indicated that significant flexibility was present throughout the molecular model. The constrained opening versus closing of the 50 kDa cleft appeared to induce opposite directions of movement in the lever arm. A sequence called the "strut" which traverses the 50 kDa cleft and may play an important role in positioning the actomyosin binding interface during actin binding is thought to be intimately linked to distant structural changes in the myosin's nucleotide cleft and neck regions. To study the dynamics of the strut region, a method of fluorescent labeling of the strut was discovered using the dye CY3. CY3 served as a hydrophobic tag for purification by hydrophobic interaction chromatography which enabled the separation of labeled and unlabeled species of S1 including a fraction labeled specifically at the strut sequence. The high specificity of labeling was verified by proteolytic digestions, gel electrophoresis, and mass spectroscopy. Analysis of the labeled S1 by collisional quenching, fluorescence polarization, and actin-activated ATPase activity were consistent with predictions from structural models of the probe's location. Although the fluorescent intensity of the CY3 was insensitive to actin binding, its fluorescence polarization was notably affected. Intriguingly, the mobility of the probe increases upon S1 binding to actin suggesting that the CY3 becomes displaced from interactions with the surface of S1 and is consistent with a structural change in the strut due to cleft motions. Labeling the strut reduced the affinity of S1 for actin but did not prevent actin-activated ATPase activity which makes it a potentially useful probe of the actomyosin interface. The different conformations of myosin S1 indicated that the strut is not as flexible as several other key regions of myosin as determined by the application of force constraints to elastic portions of the myosin structure.
Expression analysis of the fatty acid desaturase 2-4 and 2-3 genes from Gossypium hirsutum in transformed yeast cells and transgenic Arabidopsis plants.
Fatty acid desaturase 2 (FAD2) enzymes are phosphatidylcholine desaturases occurring as integral membrane proteins in the endoplasmic reticulum membrane and convert monounsaturated oleic acid into polyunsaturated linoleic acid. The major objective of this research was to study the expression and function of two cotton FAD2 genes (the FAD2-3 and FAD2-4 genes) and their possible role in plant sensitivity to environmental stress, since plants may increase the polyunsaturated phospholipids in membranes under environmental stress events, such as low temperature and osmotic stress. Two FAD2 cDNA clones corresponding to the two FAD2 genes have been isolated from a cotton cDNA library, indicating both genes are truly expressed in cotton. Model yeast cells transformed with two cotton FAD2 genes were used to study the chilling sensitivity, ethanol tolerance, and growth rate of yeast cells. The expression patterns of the two FAD2 genes were analyzed by reverse transcription polymerase chain reactions (RT-PCR) and Western blot analyses in cotton plants under different treatment conditions. The coding regions of both FAD2 genes were inserted downstream from the CaMV 35S promoter in the pMDC gateway binary vector system. Five different FAD2/pMDC constructs were transformed into the Arabidopsis fad2 knockout mutant background, and multiple potential transgenic Arabidopsis plant lines harboring the cotton FAD2 genes were generated. The cotton FAD2 genes were amplified by the polymerase chain reaction (PCR) from the genomic DNAs isolated from the transgenic Arabidopsis T1 plant lines. Complementation of the putative transgenic Arabidopsis plants with the two cotton FAD2 genes was demonstrated by gas chromatography analyses of the fatty acid profiles of leaf tissues. The cellular localization of cotton FAD2-4 polypeptides with N-terminal green fluorescence protein (GFP) was visualized by confocal fluorescence microscopy. The phenotype of transgenic Arabidopsis plants transformed with the cotton FAD2-4 gene was compared to Arabidopsis knockout fad2 mutant plants and wild type Arabidopsis plants regarding their sensitivity to low temperature, and the size and height of the plants.
Evaluation of zinc toxicity using neuronal networks on microelectrode arrays: response quantification and entry pathway analysis.
Murine neuronal networks, derived from embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate zinc toxicity at concentrations ranging from 20 to 2000 mM total zinc acetate added to the culture medium. Continual multi-channel recording of spontaneous action potential generation allowed a quantitative analysis of the temporal evolution of network spike activity generation at specific zinc acetate concentrations. Cultures responded with immediate concentration-dependent excitation lasting from 5 to 50 min, consisting of increased spiking and enhanced, coordinated bursting. This was followed by irreversible activity decay. The time to 50% and 90% activity loss was concentration dependent, highly reproducible, and formed linear functions in log-log plots. Network activity loss generally preceded morphological changes. 20% cell swelling was correlated with 50% activity loss. Cultures pretreated with the GABAA receptor antagonists bicuculline (40 mM) and picrotoxin (1 mM) lacked the initial excitation phase. This suggests that zinc-induced excitation may be mediated by interfering with GABA inhibition. Partial network protection was achieved by stopping spontaneous activity with either tetrodotoxin (200 nM) or lidocaine (250 mM). However, recovery was not complete and slow deterioration of network activity continued over 6 hrs. Removal of zinc by early medium changes showed irreversible, catastrophic network failure to develop in a concentration-dependent time window between 50% and 90% activity loss. Investigation of entry routes suggested the L-type but not N-type calcium channels to be the main entry pathway for zinc. Data are presented implicating the chloride channel to be an additional entry route.
Tobacco Phospholipase D β1: Molecular Cloning and Biochemical Characterization
Transgenic tobacco plants were developed containing a partial PLD clone in antisense orientation. The PLD isoform targeted by the insertion was identified. A PLD clone was isolated from a cDNA library using the partial PLD as a probe: Nt10B1 shares 92% identity with PLDβ1 from tomato but lacks the C2 domain. PCR analysis confirmed insertion of the antisense fragment into the plants: three introns distinguished the endogenous gene from the transgene. PLD activity was assayed in leaf homogenates in PLDβ/g conditions. When phosphatidylcholine was utilized as a substrate, no significant difference in transphosphatidylation activity was observed. However, there was a reduction in NAPE hydrolysis in extracts of two transgenic plants. In one of these, a reduction in elicitor- induced PAL expression was also observed.
Multiple Activities of Aspartate Transcarbamoylase in Burkholderia cepacia: Requirement for an Active Dihydroorotase for Assembly into the Dodecameric Holoenzyme
The aspartate transcarbamoylase (ATCase) was purified from Burkholderia cepacia 25416. In the course of purification, three different ATCase activities appeared namely dodecameric 550 kDa holoenzyme, and two trimeric ATCases of 140 kDa (consists of 47 kDa PyrB subunits) and 120 kDa (consists of 40 kDa PyrB subunits) each. The 120 kDa PyrB polypeptide arose by specific cleavage of the PyrB polypeptide between Ser74 and Val75 creating an active polypeptide short by 74 amino acids. Both the 40 and 47 kDa polypeptides produced active trimers. To compare the enzyme activity of these trimers, an effector assay using nucleotides was performed. The 140 kDa trimer showed inhibition while the 120 kDa polypeptide showed less inhibition. To verify the composition of the pyrBC holoenzyme complex, B. cepacia dihydroorotase (DHOase, subunit size of 45 kDa) was purified by the pMAL protein fusion and purification system and holoenzyme reconstruction was performed using purified ATCase and DHOase. Both the 140 kDa and the 120 kDa trimers could produce holoenzymes of 550 kDa and 510 kDa, respectively. The reconstructed ATCase holoenzyme from cleaved ATCase showed better reconstruction compared to that from uncleaved ATCase in the conventional ATCase activity gel assay. To characterize the relationship between pyrimidine pathway and virulence factor production, motility tests and biofilm assays were conducted using pyrC- mutant. Even though no significant difference in growth rates was observed, there were significant differences between the wild type and mutant in the production of biofilm and virulence factors. This study will help us to understand the structure and regulation of ATCase holoenzyme with DHOase, and facilitate the use of B. cepacia as an applicable bio-tool. Additionally, we can potentially pursue more efficient drug targets for B. cepacia.
Functional Characterization of Plant Fatty Acid Amide Hydrolases
Fatty acid amide hydrolase (FAAH) terminates the endocannabinoid signaling pathway that regulates numerous neurobehavioral processes in animals by hydrolyzing a class of lipid mediators, N-acylethanolamines (NAEs). Recent identification of an Arabidopsis FAAH homologue (AtFAAH) and several studies, especially those using AtFAAH overexpressing and knock-out lines suggest that a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, I provide evidence to support this concept by identifying candidate FAAH cDNA sequences in diverse plant species. NAE amidohydrolase assays confirmed that several of the proteins encoded by these cDNAs indeed catalyzed the hydrolysis of NAEs in vitro. Kinetic parameters, inhibition properties, and substrate specificities of the plant FAAH enzymes were very similar to those of mammalian FAAH. Five amino acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the plant FAAH sequences. Site-directed mutation of each of the five putative catalytic residues in AtFAAH abolished its hydrolytic activity when expressed in Escherichia coli. Contrary to overexpression of native AtFAAH in Arabidopsis that results in enhanced seedling growth, and in seedlings that were insensitive to exogenous NAE, overexpression of the inactive AtFAAH mutants showed no growth enhancement and no NAE tolerance. However, both active and inactive AtFAAH overexpressors displayed hypersensitivity to ABA, suggesting a function of the enzyme independent of its catalytic activity toward NAE substrates. Yeast two-hybrid screening identified Arg/Ser-rich zinc knuckle-containing protein as a candidate protein that physically and domain-specifically interacts with AtFAAH and its T-DNA knock-out Arabidopsis was hypersensitive to ABA to a degree similar to AtFAAH overexpressors. Taken together, AtFAAH appears to have a bifurcating function, via NAE hydrolysis and protein-protein interaction, to control Arabidopsis growth and interaction with phytohormone signaling pathways. These studies help to functionally define the group of enzymes that metabolize NAEs in plants, and further will expand the knowledge-base of lipid metabolism and signaling for manipulation of various physiological processes important to plant growth and responses to environmental stress.
Thermal Identification of Clandestine Burials: A Signature Analysis and Image Classification Approach
Clandestine burials, the interred human remains of forensic interest, are generally small features located in isolated environments. Typical ground searches can be both time-consuming and dangerous. Thermal remote sensing has been recognized for some time as a possible search strategy for such burials that are in relatively open areas; however, there is a paucity of published research with respect to this application. This project involved image manipulation, the analyses of signatures for "graves" of various depths when compared to an undisturbed background, and the use of image classification techniques to tease out these features. This research demonstrates a relationship between the depth of burial disturbance and the resultant signature. Further, image classification techniques, especially object-oriented algorithms, can be successfully applied to single band thermal imagery. These findings may ultimately decrease burial search times for law enforcement and increase the likelihood of locating clandestine graves.
Endocannabinoid System in a Planarian Model
In this study, the presence and possible function of endocannabinoid ligands in the planarian is investigated. The endocannabinoids ananadamide (AEA) and 2-arachidonoylglycerol (2-AG) and entourage NAE compounds palmitoylethanolamide (PEA), stearoylethanolamide (SEA) and oleoylethanolamide (OEA) were found in Dugesia dorotocephala. Changes in SEA, PEA, and AEA levels were observed over the initial twelve hours of active regeneration. Exogenously applied AEA, 2-AG and their catabolic inhibition effected biphasic changes in locomotor velocity, analogous to those observed in murines. The genome of a close relative, Schmidtea mediterranea, courtesy of the University of Utah S. med genome database, was explored for cannabinoid receptors, none were found. A putative fatty acid amide hydrolase (FAAH) homolog was found in Schmidtea mediterranea.
Use of Geographic Information System and Remote Sensing Technologies to Describe Mosquito Population Dynamics in the Ray Roberts Greenbelt, Denton County, Texas
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A population survey was conducted from April through September 2002 on mosquito species occurring on the Ray Roberts Greenbelt, a riparian corridor used for public recreation on the Elm Fork of the Trinity River, in Denton County, Texas. ArcGIS software was used to set up a stratified random sampling design based on habitat parameters. Multivariate analyses of sampling data and climatic variables were used to describe spatial and temporal patterns of mosquito species. A total of 33 species were collected during this study belonging to the following genera: Aedes, Anopheles, Coquillettidia, Culex, Mansonia, Ochlerotatus, Orthopodomyia, Psorophora, Toxorhynchites, and Uranotaenia. Seasonal distributions of the dominant species revealed population fluctuations. Aedes vexans was the primary species collected in April and May, occurring in low numbers throughout the rest of the sampling period. Psorophora columbiae reached its highest population density in June, with a smaller peak occurring in late July. Present from May through the end of September, Culex erraticus was the most abundant species collected with major peaks in mid-June and the end of July. Abundance of Culex salinarius followed the same general trend as that for Cx. erraticus, but with smaller numbers. The specimens were tested for a variety of arboviruses by the Texas Department of Health. One pool of Cx. erraticus and Cx. salinarius, collected in August 2002, tested positive for West Nile virus. Variables that were important factors for determining dominant species abundance were temperature, wind speed, rain accumulation occurring one-week and two-weeks prior to sampling, number of day since last rain event, dew point, and average canopy coverage.
The aquatic insect communities of Holbrook Creek and Cochetopa Creek in Colorado.
The first objective for this problem in lieu of thesis project was to gather, identify to the lowest practical taxonomic level and organize all available aquatic insects collected from high altitude Colorado aquatic systems during the summers of 1994, 1996, 1998, and 2002 for the University of North Texas Environmental Science Field Course (BIOL 5650). The curated collection will be housed in the Elm Fork Natural History Museum, located at the University of North Texas. The second objective was to provide a summary and discussion of the occurrence and distribution of the aquatic insects collected from Mt. Blanca in 1994, 1996, and 1998 and to create a taxa list of aquatic insects collected from Cochetopa Creek during the summer of 2002.
Pyrimidine Genes in Pseudomonas Species
This thesis is a comparative study of gene arrangements in Pseudomonas species, and is organized into three major sections. The first section compares gene arrangements for different pathways in Pseudomonas aeruginosa PAO1 to determine if the gene arrangements are similar to previous studies. It also serves as a reference for pyrimidine gene arrangements in P. aeruginosa. The second part compares the physical, and genetic maps of P. aeruginosa PAO1 with the genome sequence. The final section compares pyrimidine gene arrangements in three species of Pseudomonas. Pyrimidine biosynthesis and salvage genes will be aligned for P. aeruginosa PAO1, P. putida KT2440, and P. syringae DC3000. The whole study will gives insight into gene patterns in Pseudomonas, with a focus on pyrimidine genes.
Comparative Bioavailability of Dietary and Dissolved Cadmium to Freshwater Aquatic Snails
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Heavy metal bioaccumulation in aquatic organisms may occur through direct or indirect uptake routes. Research indicates that the significance of uptake route varies with contaminant and organism exposed. The relative importance of different metal sources in aquatic systems was investigated by exposing freshwater snails to dietary or dissolved sources of cadmium. Snails were exposed to control, contaminated food only, contaminated water only, and contaminated food and water treatments. During the 15-day exposure, samples were taken to determine Cd concentration in snail soft tissue, snail shell, algal food, and overlying water. Analyses of snail soft tissue and shells indicate that exposure route significantly affects Cd concentrations in the tissues. In both cases, dissolved Cd is the primary contributor to metal body burden.
Applications of remote sensing and GIS to modeling fire for vegetative restoration in Northern Arizona
An accurate fire model is a useful tool in predicting the behavior of a prescribed fire. Simulation of fire requires an extensive amount of data and can be accomplished best using GIS applications. This paper demonstrates integrative procedures of using of ArcGIS™, ERDAS Imagine™, GPS, and FARSITE© to predict prescribed fire behavior on the Kaibab-Paiute Reservation. ArcGIS was used to create a database incorporating all variables into a common spatial reference system and format for the FARSITE model. ArcGIS Spatial Analyst was then used to select optimal burn sites for simulation. Our predictions will be implemented in future interagency efforts towards vegetative restoration on the reservation.
Isolation and Characterization of Polymorphic Loci from the Caribbean Flamingo (Phoenicopterus ruber ruber): New Tools for Wildlife Management
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Methods to determine genetic diversity and relatedness within populations are essential tools for proper wildlife management. Today the approach of choice is polymerase chain reaction-based microsatellite analysis. Seven new polymorphic loci were isolated from a microsatellite-enriched Caribbean flamingo genomic library and used to characterize survey populations of Caribbean and African greater flamingos. In addition, four of these loci were used to verify parentage relationships within a captive-breeding population of African greater flamingos. Parentage predictions based upon gamekeeper observations of breeding and nesting did not always agree with genetic-based parentage analyses of the nine suggested family groups. Four family groups were supported (groups I, II, III and VI) by there results. However, an analysis of the remaining five suggested groups, with a total of eight offspring/dam and eight offspring/sire suggested relationships, yielded seven exclusions of the suggested dam and six exclusions of the suggested sire. This put the overall suggested dam exclusion rate at 35% and exclusion rate for suggested sires at 29%. Although the keeper observation data for our family groups must be considered a variable of concern at this time, these findings are certainly suggestive that more carefully controlled studies may reveal that flamingos are not monogamous as long accepted, but rather socially monogamous or even promiscuous. Thus we have now been able to both characterize and demonstrate the utility of our polymorphic microsatellite loci. We hope these results will interest additional wildlife facilities in further parentage and behavioral studies that will collectively aid to improve monitoring and maintenance of genetic diversity, and as provide better insight into breeding habits of both wild and captive populations.
Impaired virulence factor production in a dihydroorotate dehydrogenase mutant (pyrD) of Pseudomonas aeruginosa.
Previous research in our laboratory showed that when knockout mutations were created in the pyrB and pyrC genes of the pyrimidine pathway in Pseudomonas aeruginosa, not only were the resultant mutants auxotrophic for pyrimidines but they were also impaired in virulence factor production. Such a correlation had not been previously reported for P. aeruginosa, a ubiquitous opportunistic pathogen in humans. In an earlier study it was reported that mutants blocked in one of the first three enzymes of the pyrimidine pathway in the non-pathogenic strain P. putida M produced no pyoverdin pigment while mutants blocked in the later steps produced copious amounts of pigment, just like the wild type. This study probed for the same connection between pyrimidine auxotrophy and pigment production applied in P. aeruginosa. To that end a knockout mutation was created in pyrD, the fourth step in the pyrimidine pathway which encodes dihydroorotate dehydrogenase. The resulting mutant required pyrimidines for growth but produced wild type pigment levels. Since the pigment pyoverdin is a siderophore it may also be considered a virulence factor, other virulence factors were quantified in the mutant. These included casein protease, hemolysin, elastase, swimming, swarming and twitching motility, and iron binding capacity. In all cases these virulence factors were significantly decreased in the mutant. Even supplementing with uracil did not attain wild type levels. Starvation of the pyrimidine mutant for uracil caused increased specific activity of the pyrimidine enzymes, suggesting that regulation of the pyrimidine pathway occurred at the level of transcription. This effect has also been reported for P. oleovorans. The present research consolidates the idea that pyrimidine auxotrophs cause decreased pathogenicity in P. aeruginosa. Such a finding may open the search for chemotherapy targets in cystic fibrosis and burn victims where P. aeruginosa is an infecting agent.
Determination of Dissociation Constants for GABAA Receptor Antagonists using Spontaneously Active Neuronal Networks in vitro
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Changes in spontaneous spike activities recorded from murine frontal cortex networks grown on substrate-integrated microelectrodes were used to determine the dissociation constant (KB) of three GABAA antagonists. Neuronal networks were treated with fixed concentrations of GABAA antagonists and titrated with muscimol, a GABAA receptor agonist. Muscimol decreased spike activity in a concentration dependent manner with full efficacy (100% spike inhibition) and a 50% inhibitory concentration (IC50) of 0.14 ± 0.05 µM (mean ± SD, n=6). At 10, 20, 40 and 80 µM bicuculline, the muscimol IC50 values were shifted to 4.3 ± 1.8 µM (n=6), 6.8 ± 1.7 µM (n=6), 19.3 ± 3.54 µM (n=10) and 43.5 µM (n=2), respectively (mean ± SD). Muscimol titration in the presence of 10, 20, 40 µM of gabazine resulted in IC50s values of 20.1 (n=2), 37.17 (n=4), and 120.45 (n=2), respectively. In the presence of 20, 80, and 160 µM of TMPP (trimethylolpropane phosphate) the IC50s were 0.86 (n=2), 3.07 (n=3), 6.67 (n=2) µM, respectively. Increasing concentrations of GABAA antagonists shifted agonist log concentration-response curves to the right with identical efficacies, indicating direct competition for the GABAA receptor. A Schild plot analysis with linear regression resulted in slopes of 1.18 ± 0.18, 1.29 ± 0.23 and 1.05 ± 0.03 for bicuculline, gabazine and TMPP, respectively. The potency of antagonists was determined in terms of pA2 values. The pA2 values were 6.63 (gabazine), 6.21 (bicuculline), and 5.4 (TMPP). This suggests that gabazine has a higher binding affinity to the GABAA receptor than bicuculline and TMPP. Hence, using spike rate data obtained from population responses of spontaneously active neuronal networks, it is possible to determine key pharmacological properties of drug-receptor interactions.
The Life History and Contributions to the Ecology of Camelobaetidius variabilis Wiersema 1998 (Ephemeroptera: Baetidae) in Honey Creek, Oklahoma
A study of the life history and ecology of Camelobaetidius variabilis was conducted in Honey Creek, OK from February 2003-April 2004. Nymph development was assessed using changes in external morphology. Laboratory reared nymphs were used to calculate number of degree days to complete development (772 degree days at 20.8° C ±.38° C), which was used to determine voltinism. Field collected nymph microhabitat distribution was used in assessing microhabitat distribution. Nymphal thermoregulation was assessed during the winter and spring by comparing nymphal numbers present in shaded and un-shaded habitats. Camelobaetidius variabilis nymphs showed preference for algal microhabitats during the spring and leaf packs in the winter. Nymphs inhabited leaf packs to increase metabolic rate during the winter. Increased temperatures aid in development of nymphs. Camelobaetidius variabilis exhibited a multivoltine life cycle with six overlapping generations.
Applications of Molecular Genetics to Human Identity.
The primary objectives of this project were: 1. to develop improved methods for extraction of DNA from human skeletal remains, 2. to improve STR profiling success of low-copy DNA samples by employing whole genome amplification to amplify the total pool of DNA prior to STR analysis, and 3. to improve STR profiling success of damaged DNA templates by using DNA repair enzymes to reduce the number/severity of lesions that interfere with STR profiling. The data from this study support the following conclusions. Inhibitory compounds must be removed prior to enzymatic amplification; either during bone section pretreatment or by the DNA extraction method. Overall, bleach outperformed UV as a pretreatment and DNA extraction using silica outperformed microconcentration and organic extraction. DNA repair with PreCR™ A outperformed both whole genome amplification and repair with PreCR™ T6. Superior DNA extraction results were achieved using the A6 PMB columns (20 ml capacity column with 6 layers of type A glass fiber filter), and DNA repair with PreCR™ A led to an overall improvement in profile quality in most cases, although whole genome amplification was unsuccessful. Rapid, robust DNA isolation, successful amplification of loci from the sample-derived DNA pool, and an elimination of DNA damage and inhibitors may assist in providing sufficient genetic information from cases that might otherwise lie on the fringe of what is possible to obtain today.
Microsatellite-based genetic profiling for the management of wild and captive flamingo populations.
Flamingo species generate tremendous interest whether they are small captive groups or wild populations numbering in the thousands. Genetic pedigrees are invaluable for maintaining maximum genetic diversity in captive, as well as wild, populations. However, presently there is a general lack of genetic data for flamingo populations. Microsatellites are loci composed of 2-6 base pair tandem repeats, scattered throughout higher eukaryotic genomes, often exhibiting high levels of polymorphism and heterozygosity. These loci are thus important genetic markers for identity, parentage and population studies. Here, six microsatellite loci were isolated from a microsatellite-enriched Caribbean flamingo partial genomic library. Two are compound complex repeats and four are perfect trinucleotide repeats. Each locus was amplified from Caribbean, African greater, Chilean and lesser flamingo genomic DNAs. Heterozygosity frequencies were calculated for Caribbean (range 0.12-0.90) and African greater flamingos (range 0.23-0.94) loci. All six microsatellite loci were found to be in Hardy-Weinberg equilibrium and linkage disequilibrium analyses did not suggest linkage for any pair of two greater flamingo subspecies (African and Caribbean) loci. At least five of the loci also exhibit polymorphism in Chilean and lesser flamingos, but due to small sample numbers, relevant allele/heterozygosity frequency calculations could not be estimated. Nucleotide sequence comparisons of the amplicons derived from the four flamingo groups reveal a high level of sequence conservation at all loci. Although small sample numbers again limit the data for lesser flamingos and to some degree for the Chilean birds, the sequences of the two greater flamingo subspecies were identical and the number of nonconserved nucleotides appears to be higher for lesser/greater comparisons than for Chilean/greater comparisons. This is consistent with Chilean flamingos being a different species within the same genus as the greater flamingos, while lesser flamingos belong to a separate genus. Parentage analyses on suggested African greater flamingo family groups from Disney's Animal Kingdom's collection were performed using microsatellite data. Results confirmed many suggested family groups but in other cases one or more of the suggested parents were clearly excluded. The six microsatellite loci isolated provide a new population management tool useful for both wild and captive flamingo populations.
Structure-Function Studies on Aspartate Transcarbamoylase and Regulation of Pyrimidine Biosynthesis by a Positive Activator Protein, PyrR in Pseudomonas putida
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The regulation of pyrimidine biosynthesis was studied in Pseudomonas putida. The biosynthetic and salvage pathways provide pyrimidine nucleotides for RNA, DNA, cell membrane and cell wall biosynthesis. Pyrimidine metabolism is intensely studied because many of its enzymes are targets for chemotheraphy. Four aspects of pyrimidine regulation are described in this dissertation. Chapter I compares the salvage pathways of Escherichia coli and P. putida. Surprisingly, P. putida lacks several salvage enzymes including nucleoside kinases, uridine phosphorylase and cytidine deaminase. Without a functional nucleoside kinase, it was impossible to feed exogenous uridine to P. putida. To obviate this problem, uridine kinase was transferred to P. putida from E. coli and shown to function in this heterologous host. Chapter II details the enzymology of Pseudomonas aspartate transcarbamoylase (ATCase), its allosteric regulation and how it is assembled. The E. coli ATCase is a dodecamer of two different polypeptides, encoded by pyrBI. Six regulatory (PyrI) and six catalytic (PyrB) polypeptides assemble from two preformed trimers (B3) and three preformed regulatory dimers (I2) in the conserved 2B3:3I2 molecular structure. The Pseudomonas ATCase also assembles from two different polypeptides encoded by pyrBC'. However, a PyrB polypeptide combines with a PyrC. polypeptide to form a PyrB:PyrC. protomer; six of these assemble into a dodecamer of structure 2B3:3C'2. pyrC' encodes an inactive dihydroorotase with pyrB and pyrC' overlapping by 4 bp. Chapter III explores how catabolite repression affects pyrimidine metabolism. The global catabolite repression control protein, Crc, has been shown to affect pyrimidine metabolism in a number of ways. This includes orotate transport for use as pyrimidine, carbon and nitrogen sources. Orotate is important because it interacts with PyrR in repressing the pyr genes. Chapter IV describes PyrR, the positive activator of the pyrimidine pathway. As with other positive activator proteins, when pyrimidine nucleotides are depleted, PyrR binds to DNA thereby enhancing expression of pyrD, pyrE and pyrF genes. When pyrimidine nucleotides are in excess, the PyrR apoprotein binds to orotate, its co-repressor, to shut down all the pyrimidine genes. Like many positive activators, PyrR is subject to autoregulation and has catalytic activity for uracil phosphoribosyltransferase inducible by orotate.
Pyrimidine Enzyme Specific Activity at Four Different Phases of Growth in Minimal and Rich Media, and Concomitant Virulence Factors Evaluation in Pseudomonas aeruginosa
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Pseudomonas aeruginosa is a Gram-negative rod, aerobic, non-fermenting, oxidase positive, pigment producing, and nutritionally versatile bacterium. Infections by P. aeruginosa are the most important cause of morbidity and mortality in immunocompromised patients, given virulence factor production that suppresses antibiotic therapy and promotes persistent infection. This research is the first comprehensive report of the pyrimidine biosynthetic pathway for all phases of growth in minimal and rich media coupled with the evaluation of virulence factor production of P. aeruginosa in comparison to four other bacterial species (Pseudomonas putida, Pseudomonas fluorescens, Burkholderia cepacia, and Escherichia coli wild-type strains). Cellular growth and passing genetic information to the next generation depend on the synthesis of purines and pyrimidines, the precursors of DNA and RNA. The pyrimidine biosynthetic pathway is essential and found in most organisms, with the exception of a few parasites that depend upon the pyrimidine salvage pathway for growth. Both the pyrimidine biosynthetic and salvage enzymes are targets for chemotherapeutic agents. In our laboratory, research on pyrimidine auxotrophic mutants showed the role of the pyrimidine biosynthetic pathway and its intermediates on P. aeruginosa metabolism and impaired virulence factors production. The present research shows that pyrimidine enzymes are active in all phases of growth, including the production of two forms of ATCase in the late log phase in P. aeruginosa. This finding may be explained by the displacement of the inactive PyrC' by the active PyrC or PyrC2 to form a new and larger pyrBC encoded ATCase. Pseudomonas aeruginosa wild-type appears to produce by far the most virulence factors, haemolysin, iron chelation, rhamnolipid, adherence, and three types of motility (swimming, swarming, and twitching) investigated in this study, when compared to the other four wild-type strains. Growth analysis was carried out as typically done in minimal medium but also in rich medium to simulate conditions in the blood and lung tissues of humans as P. aeruginosa infections develop.
Nucleotide Inhibition of Glyoxalase II
The glyoxalase system mediates the conversion of methylglyoxal, a toxic ketoaldehyde, to D-lactic acid. The system is composed of two enzymes, glyoxalase I (Glo-I) and glyoxalase II (Glo-II), and exhibits an absolute requirement for a catalytic quantity of glutathione (GSH). Glo-I catalyzes the isomerization of a hemithioacetal, formed non-enzymatically from methylglyoxal and GSH, to the corresponding a -D-hydroxyacid thioester, s-D-lactoylglutathione (SLG). Glo-II catalyzes the irreversible breakdown of SLG to D-lactate and GSH. We have observed that ATP or GTP significantly inhibits the Glo-II activity of tissue homogenates from various sources. We have developed a rapid, one step chromatography procedure to purify Glo-II such that the purified enzyme remains "sensitive" to inhibition by ATP or GTP (Glo-II-s). Studies indicate that inhibition of Glo-II-s by nucleotides is restricted to ATP, GTP, ADP, and GDP, with ATP appearing most effective. Kinetics studies have shown that ATP acts as a partial non-competitive inhibitor of Glo-II-s activity, and further suggest that two kinetically distinguishable forms of the enzyme exist. The sensitivity of pure Glo-II-s to nucleotide inhibition is slowly lost on storage even at -80° C. This loss is accelerated at higher temperatures or in the presence of ATP. Kinetics studies on the resultant "insensitive" enzyme (Glo-II-i) show that a significant reduction of the affinity of the enzyme for the substrate, SLG, occurs and further suggest that only one form of the enzyme is kinetically distinguishable after "de-sensitization". Tryptophan fluorescence studies of the two enzyme preparations suggest that a subtle conformational change in the enzyme has occurred during de-sensitization. We have also observed that Glo-II-i is "resensitized" to nucleotide inhibition after incubation in the presence of a reagent that reduces disulfide bonds. The resensitized enzyme exhibits an increased KM value similar to that of the original Glo-II-s. Kinetics studies show that ATP or GTP again act as partial non-competitive inhibitors of the resensitized enzyme and suggest that only one form of the enzyme is present. The physiological significance of the two enzyme forms is discussed.
Enallagma civile (Odonata: Coengrionidae) life history and production in a west Texas playa
A life history and productivity study of Enallagma civile was conducted in a playa that was located in the southern High Plains of Texas. Other odonates were also studies to identify their contributions to the habitat.
Autoradiographic localization of carbachol-induced second messenger response in the rat spinal cord following inflammation.
This study examined central mechanisms of persistent pain using an autoradiographic technique to localize phosphoinositide hydrolysis (PI) in the rat spinal cord dorsal horn. The lateral half of laminae I-II showed the highest levels of baseline PI turnover and carbachol-stimulated PI turnover in normal animals as well as after inflammation. Inflammation resulted in increased baseline PI turnover in this region of the ipsilateral (76%) and contralateral (65%) dorsal horns. Carbachol increased PI turnover in this region in normal rats (55%) and following inflammation (ipsilateral: 46%, contralateral: 45%). The absolute magnitudes of these increases were 1.85, 2.71, and 2.51 nCi/mg, respectively. The results of this study demonstrate the involvement of PI turnover in neural mechanisms of persistent pain, and provide evidence for the involvement of cholinergic systems in this process. Because spinal cholinergic systems have been reported to be anti-nociceptive, the present results appear to reflect an upregulation of anti-nociceptive activity in response to inflammation. Thus, the spinal cholinergic system may be a regulatory site within the anti-nociceptive pathway, and may provide an attractive target for the development of new therapeutic agents.
College Freshman Biology Two Semester Course: Integrating Deep Processing Teaching Techniques
Development of a college level freshman biology course was undertaken in response to government reports that American students have fallen behind students of other countries in the area of the sciences. Teaching strategies were investigated to accomplish two objectives, to define essential academic material to include in the course and to investigate teaching techniques that would increase deep processing of the information. An active process that consisted of applying the cognitive information to solving problems or developing answers to questions was defined as critical thinking. Critical thinking was incorporated into the course by the use of case studies.
Influence of parental swimming stamina on the cardiac and metabolic performance of larval zebrafish (Danio rerio).
Superior swimming stamina in adult fish is presumably passed on to their offspring, but the ontogeny of the appearance of superior stamina and the requisite enhanced cardio-respiratory support for locomotion in larval fishes has not been determined. Is the expression of the suite of parental traits enabling superior swimming stamina in their offspring dependent upon their achieving juvenile/adult morphology, or does it appear earlier in their larvae? To answer this, adults were classified into three groups based on swimming stamina, followed by measurement of length, mass, and width. Larval offspring from the two parental groups -high stamina larvae (HSL) and low stamina larvae (LSL)- were reared at 27°C in aerated water (21% O2). Routine and active heart rate, routine and active mass specific oxygen consumption were recorded through 21dpf, and cost of transport (COT) and factorial aerobic scope were derived from oxygen consumption measurements. Routine heart rate at 2dpf of LSL was 164 ± 1 b·min-1, compared to only 125 ± 2 b·min-1 for HSL. Routine heart rate subsequently peaked at 203 ± 1 b·min-1 at 5dpf in the HSL group, compared to 207 ± 1 b·min-1, at 4dpf in the LSP larvae. Active heart rate at 5 dpf of LSL was 218 ± 2 b·min-1 compared to 216 ± 2 b·min-1 for HSL. Active heart rate increased slightly to 227 ± 2 b·min-1 for LSL before decreasing again, while active heart rate remained relatively constant for HSL. Routine O2 consumption at 2dpf of HSL was 0.09 μmol·mg-1·hr-1, compared to 0.03 μmol·mg-1·hr-1 in LSL. Routine O2 consumption subsequently peaked at 0.70 μmol·mg-1·hr-1 at 9dpf in the HSL, compared to 0.71 μmol·mg-1·hr-1, at 9dpf in the LSL. These values dramatically decreased before leveling off at around 0.20 μmol·mg-1·hr-1 and 0.15 μmol·mg-1·h-1, respectively. Active O2 consumption at 5dpf for HSL was 0.38 μmol·mg-1·hr-1, compared to 0.57 μmol·mg-1·hr-1 for LSL. Active O2 consumption subsequently peaked at 0.97 μmol·mg-1·hr-1 at 10dpf in HSL, compared to 1.19 μmol·mg-1·hr-1 at 7dpf in LSL. These values also dramatically decreased and leveled off. Significant differences (p < 0.05) in heart rate and oxygen consumption persisted through 21dpf. The onset of differences observed in routine and active heart rate in early larvae, correlated with parent stamina, show that juvenile or adult features are not required as a precondition for the emergence of phenotypic physiological differences.
Hindrance of the Myosin Power Stroke Posed by the Proximity to the Troponin Complex Identified Using a Novel LRET Fluorescent Nanocircuit
A novel luminescence resonance energy transfer (LRET) nanocircuit assay involving a donor and two acceptors in tandem was developed to study the dynamic interaction of skeletal muscle contraction proteins. The donor transmits energy relayed to the acceptors distinguishing myosin subfragment-1 (S1) lever arm orientations. The last acceptor allows the detection of S1's bound near or in between troponin complexes on the thin filament. Additionally, calcium related changes between troponin T and myosin were detected. Based on this data, the troponin complex situated every 7 actin monomers, hinders adjacently bound myosins to complete their power stroke; whereas myosins bound in between troponin complexes undergo complete power strokes.
Analysis of the Expression Profiles of Two Isoforms of the Antifungal Protein Osmotin from Gossypium hirsutum
The expression of two cotton osmotin genes was evaluated in terms of the mRNA and protein expression patterns in response to chemical inducers such as ethylene, hydrogen peroxide, and sodium chloride. Reverse transcriptase-polymerase chain reactions (RT-PCR) indicated that osmotin mRNAs are expressed constitutively in root tissues of cotton plants, and that they are rapidly induced in leaf and stem tissues upon ethylene treatment. Real time RT-PCR indicated that osmotin transcript levels were induced 2 to 4 h after treatment with ethephon. The osmotin mRNA levels appear to increase 12 h after treatment, decrease, and then increase again. The osmotin protein expression patterns were analyzed in Western blot analyses using an anti-osmotin antibody preparation. A 24-KDa protein band was detected from cotton plants treated with the inducers. The 24-KDa osmotin proteins were induced 4 h after treatment with ethephon, while down-regulated 96 h after treatment. Multiple osmotin isoforms were observed to be induced in cotton plants upon treatment with ethephon by two-dimensional gel electrophoresis. One goal of this dissertation research was to genetically engineer two cotton osmotin genes to routinely overproduce their antifungal proteins in transgenic Arabidopsis and cotton plants as a natural defense against fungal infections, using co-cultivation with Agrobacterium tumefaciens cells harboring pCAMBIA 2301 vector constructs containing the osmotin genes. Many transgenic Arabidopsis and cotton plants were generated. However, genomic blotting analyses indicated the absence of the osmotin transgenes, but the presence of GUS genes from the vector cassette. Alkaline blot analyses of the vector DNAs from transformed Agrobacterium cells confirmed that an anomalous DNA structural rearrangement or aberrant recombination event probably occurred in the Agrobacterium cells, interdicting the integration of osmotin transgenes into the Arabidopsis and cotton plants. This research provides crucial baseline information on expression of cotton osmotin mRNAs and proteins.
Map-based cloning of the NIP gene in model legume Medicago truncatula.
Large amounts of industrial fertilizers are used to maximize crop yields. Unfortunately, they are not completely consumed by plants; consequently, this leads to soil pollution and negative effects on aquatic systems. An alternative to industrial fertilizers can be found in legume plants that provide a nitrogen source that is not harmful for the environment. Legume plants, through their symbiosis with soil bacteria called rhizobia, are able to reduce atmospheric nitrogen into ammonia, a biological nitrogen source. Establishment of the symbiosis requires communication on the molecular level between the two symbionts, which leads to changes on the cellular level and ultimately results in nitrogen-fixing nodule development. Inside the nodules hypoxic environment, the bacterial enzyme nitrogenase reduces atmospheric nitrogen to ammonia. Medicago truncatula is the model legume plant that is used to study symbiosis with mycorrhiza and with the bacteria Sinorhizobium meliloti. The focus of this work is the M. truncatula nodulation mutant nip (numerous infections and polyphenolics). The NIP gene plays a role in the formation and differentiation of nodules, and development of lateral roots. Studying this mutant will contribute knowledge to understanding the plant response to infection and how the invasion by rhizobia is regulated. Previous genetic mapping placed NIP at the top of linkage group 1 of the M. truncatula genome. A NIP mapping population was established with the purpose of performing fine mapping in the region containing NIP. DNA from two M. truncatula ecotypes A17 and A20 can be distinguished through polymorphisms. Positional mapping of the NIP gene is based on the A17/A20 genetic map of M. truncatula. The NIP mapping population of 2277 plants was scored for their nodulation phenotype and genotyped with flanking molecular genetic markers 146o17 and 23c16d, which are located ~1.5 cM apart and on either side of NIP. This resulted in the identification of 170 recombinant plants, These plants' DNAs were tested further with different available genetic markers located in the region of interest, to narrow the genetic interval that contains the NIP gene. Segregation data from genotyping analysis of recombinant plants placed NIP in the region between 4L4 and 807 genetic markers.
Bacterial challenge in Lumbricus terrestris: A terrestrial invertebrate immunotoxicity model.
A bacterial challenge assay was developed utilizing the earthworm, Lumbricus terrestris, in order to assess potential immunotoxic effects from exposure to specific polychlorinated biphenyl congeners. Earthworms were inoculated with Aeromonous hydrophila, establishing a 10-day LD50. In vitro assays for effects of PCBs on phagocytosis agreed with mammalian studies, demonstrating potent suppression of phagocytosis by the non-coplanar PCB congener 138 and no suppression by the coplanar congener 126. However, when the effects of the two PCB congeners were evaluated for suppression of resistance to a whole animal infection challenge assay, coplanar PCB 126 decreased the ability of L. terrestris to withstand infection while non-coplanar PCB 138 did not.
Classification of toolmark surfaces on zipper teeth
This study proposes the classification of the toolmark under the heads of zipper teeth as a subclass characteristic as outlined by the Association of Firearm and Toolmark Examiners (AFTE). Two separate cases in which zipper teeth were found at crime scenes prompted this study. Brass zipper teeth manufactured by YKK were taken from 20 pairs of jeans and studied using a Reichert comparison microscope at 4X power. Photographs were taken and over 750 comparisons made. It was found that the toolmarks on each side on the 20 zippers were unique and independent of all other sides. The observations made in this study indicate that classifying zipper teeth toolmarks as a subclass characteristic is valid.
The potential of coelomocyte chemotaxis as an immune biomarker in the earthworm, Lumbricus terrestris
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Coelomocyte migration responses, both random and chemotatic, were examined in the earthworm Lumbricus terrestris. Coelomocyte random migration patterns towards non-stimulatory, non-chemotatic solutions were described. Migration responses to immunostimulatory agents lipopolysaccharides (LPS), N-formly-methionyl-leucyl-phenylalanine (FMLP), sheep erythrocytes, Saccharomyces cerevisiae, Aeromonas hydrophila, Eisenia fetida and Rhabditis pellio were characterized. Chemotaxis was reported to LPS, FMLP, sheep erythrocytes, S. cerivesae and E. fetida. Bio-indicator potential of chemotaxis is discussed relative to variability in migration responses.
Syllabus Outline for Genetics Lecture and Laboratory
This work is intended to be used as a teaching tool in conjunction with the text cited. It is written in outline format, highlighting the major concepts of each pertinent chapter. In this format, the concepts can be expanded upon at the discretion of the instructor. This work is to be used as a guide for lecture. The basic concepts contained in the outline are in such a format as to be able to work in more information regarding the subject matter if needed. The instructor can work from this outline as a starting point. Major topics in the chapters are highlighted, making lecture notes for the instructor easier to do.
Syllabus for Advanced Placement Biology
The purpose of this syllabus is to provide a working copy to those teachers of the advanced placement biology course taught at the high school level. Reference materials used were the Texas Education Agency ( TEA ) approved Campbell text Biology and the College Board's, Advanced Placement Biology Laboratory Manual. The syllabus is divided into major topics with outlined notes and includes laboratory exercises as recommended by the College Board. The AP biology course is intended to be equivalent to college biology. College freshman biology courses can differ among colleges and among teachers within the same college. This syllabus is intended to serve as an aid to AP teachers, to cover the topics and experiments as set out by the College Board, and to the high school student, the necessary material to successfully complete the AP examination while providing freshman biology equivalence.
Cassette Systems for Creating Intergeneric Hybrid ATCases
Cassette systems for creating intergeneric hybrid ATCases were constructed. An MluI restriction enzyme site was introduced at the carbamoylphosphate binding site within the pyrB genes of both Pseudomonas putida and Escherichia coli. Two hybrids, E. coli pyrB polar domain fused with P. putida pyrB equatorial domain and P. putida pyrB polar domain fused with E. coli pyrB equatorial domain, are possible. The intergeneric E. coli-P. putida hybrid pyrB gene was constructed and found to encode an active ATCase which complemented an E. coli Pyr- strain. These hybrids are useful for kinetic and expression studies of ATCase in E. coli.
Structural analysis of the TOL pDK1 xylGFJQK region and partial characterization of the xylF and xylG gene products
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TOL plasmids encode enzymes responsible for utilization of toluene and related aromatic compounds by Pseudomonas putida, ultimately converting them to central metabolic intermediates. The nucleotide sequence for the 5.6 kb xylGFJQK region of the pDK1 TOL meta operon was determined. DNA sequence analysis revealed the presence of five open reading frames corresponding to xylG (1458 bp), xylF (846 bp), xylJ (783 bp), xylQ (936 bp) and xylK (1047 bp), encoding predicted protein products of 51.6, 31.3, 27.8, 32.8, and 36.6 kDa in size, respectively. The average G+C content of the xylLTEGFJQK region was 65.7%, somewhat higher than the 58.9% seen in the immediately upstream xylXYZ region and substantially more than the 50% G+C content reported for the upper TOL operon of this plasmid. Homology comparisons were made with genes and proteins of related catabolic plasmids. The dmpCDEFG and pWWO xylGFJQK regions exhibit consistently high levels of nucleotide and amino acid homology to pDK1 xylGFJQK throughout the entire region. In contrast, although the nucleotide sequence homology of the Acinetobacter atdCDE region to xylGFJ is high, the homology of atdFG to xylQK is markedly less. Such radical changes in homology between corresponding regions of different operons, combined with variable base and codon usage patterns within and between operons, provides additional support for the idea that the upper and lower operons encoding enzymes of aromatic pathways have evolved independently of one another and that these operons have continued to exchange genetic material with homologous expression units through a series of recombination events. Recombinant plasmids were constructed for individual expression of each of the xylGFJQK genes. HMSD (XylG) and HMSH (XylF) were partially purified and characterized with respect to substrate specificity and kinetic mechanism. Evidence was obtained suggesting that the HMSD reaction occurs via a steady state ordered mechanism or a random mechanism where binding of the first substrate effects the enzyme's affinity for the second substrate.
Assessing the Spatial and Temporal Distribution of MTBE and BTEX Compounds in Lake Lewisville, Texas February 1999 - February 2000
The spatial and temporal distribution of Methyl Tertiary-Butyl Ether (MTBE) and BTEX (Benzene, Toluene, Ethylbenzene, Xylenes) compounds were assessed in a multipurpose reservoir, Lake Lewisville, Texas between February 1999 and February 2000. Concentrations of MTBE ranged from 0.0 - 16.7 mg/L. Levels of MTBE in the lake were related to watercraft. BTEX concentrations were never detected above 2.0 mg/L during the sampling period. Finished drinking water from Denton and the Upper Trinity Regional Water District (UTRWD) Treatment Plants were also tested for MTBE and BTEX. MTBE and BTEX were not detected in UTRWD water samples. Denton's finished water samples never exceeded 2.2 mg/L for MTBE and BTEX was not detected except for one replicate of 1.1 mg/L toluene.
Evaluating Tree Seedling Survival and Growth in a Bottomland Old-field Site: Implications for Ecological Restoration
In order to assess the enhancement of seedling survival and growth during drought conditions, five-hundred bare-root seedlings each of Shumard oak (Quercus shumardii Buckl.) and green ash (Fraxinus pennsylvanica Marsh.) were planted each with four soil amendments at a Wildlife Management Area in Lewisville, Texas. The treatments were a mycorrhizal inoculant, mulch fabric, and two superabsorbent gels (TerraSorb® and DRiWATER®). Survival and growth measurements were assessed periodically for two years. Research was conducted on vegetation, soil, and site history for baseline data. Both superabsorbent gels gave significant results for Shumard oak survival, and one increased green ash diameter. For overall growth, significant results were found among DRiWATER®, mycorrhizae, and mulch treatments.
Effects on Survival, Reproduction and Growth of Ceriodaphnia dubia following Single Episodic Exposure to Copper or Cadmium
Effects of episodic exposures have gained attention as the regulatory focus of the Clean Water Act has shifted away from continuous-flow effluents. Standardized laboratory toxicity tests require that exposure be held constant. However, this approach may not accurately predict organism responses in the field following episodic exposures such as those associated with rain-driven runoff events or accidental pollutant discharge. Using a modified version of the 7-day short-term chronic test recommended by the US Environmental Protection Agency, Ceriodaphnia dubia were exposed to copper or cadmium for durations ranging from 1 minute to 24 hours. In addition, adult reproductive recovery and effects on second generation individuals was assessed following select copper exposures. Finally, cadmium exposures were compared in reconstituted hard water (RHW) and municipal treated wastewater effluent (TWE). Following exposure, organisms were transferred to clean RHW or TWE and maintained for the remainder of the test. No- and lowest observed effect concentrations (NO- and LOECs) increased logarithmically with respect to logarithmic decreases in duration regardless of metal, endpoint or water type. Effective concentrations of cadmium however, were usually higher than those of copper, especially in TWE. LOECs for C. dubia survival following 24-hour and 5-minute exposures to copper were 116 and 417 µg/L, respectively. LOECs for fecundity were 58 and 374 µg/L, respectively. Neonate production of first generation adult C. dubia appeared to recover from pulsed copper exposure upon examination of individual broods. Cumulative mean neonate production however, showed almost no signs of recovery at exposure durations ≥3 hours. Pulse exposure to copper also resulted in diminished fecundity of unexposed second generation individuals. Such effects were pronounced following parental exposure for 24 hours but lacking after parental exposures ≤3 hours. LOECs for C. dubia survival following 24-hour and 5-minute exposures to cadmium in RHW were 44 and 9000 µg/L, respectively. LOECs for fecundity were 16 and 5000 µg/L, respectively. In TWE, LOECs for C. dubia survival were 83 and >10,000 µg/L, respectively. LOECs for fecundity in TWE were 48 and 7000 µg/L, respectively. Runoff pollution is site and event specific, however, data presented herein may be useful as a predictive tool under various conditions.
Evaluation of a Common Carp (Cyprinus carpio L.) Exclusion and Trapping Device for Use in Aquatic Plant Founder Colony Establishment
The focus of this study was to design and evaluate a trapping system that would reduce populations of common carp within water bodies in conjunction with establishment of native aquatic macrophytes founder colonies. A pond study and field study were conducted. A pond study was performed at the Lewisville Aquatic Ecosystem Research Facility, located in Lewisville, Texas, followed by a field study within a constructed wetland located in southern Dallas, Texas. For the pond study, twelve funnel traps were constructed (four reps of each type: control, dual-walled and ring cage). Two anti-escape devices were tested with funnels including steel fingers and hinged flaps. Ring cage and dual-walled treatments were planted using native pondweeds, while controls were left unplanted (additional bait and a drift fence scenarios were also tested). Common carp were introduced into the study pond. Chi-square statistical analyses were utilized and showed ring cage treatments using fingers as well as the use of a drift fence to be most effective. Following completion of the pond study, the two most effective treatments (controls and ring cages) were tested within the Dallas, Texas wetland; no carp were caught during the field test.
Establishment and competitive ability of Nelumbo lutea in relation to Myriophyllum spicatum
Limitations from reduced light and increasing water depth on Nelumbo lutea seedlings were determined in tank experiments. Survival was high in all tested light levels. Total biomass increased significantly with increasing light. Biomass allocation shifted significantly to root production between 3 and 6 weeks in the 10 and 24% levels. Survival decreased with increasing planting depth, and biomass of survivors reduced significantly between 0.5, 1.0, and 1.5 m depths. Nelumbo lutea and Myriophyllum spicatum populations were monitored for one season in a 0.7 ha pond to track changes in species dominance. Myriophyllum spicatum dominated early, and N. lutea dominated from July through October, suppressing M. spicatum at all depths. Competitive interactions between N. lutea and M. spicatum were investigated for two seasons in a container experiment situated within a pond. Where established, N. lutea dominated in the presence of M. spicatum. However, N. lutea could not be established in depths greater than 1 meter.
Hepatotoxicity of Mercury to Fish
Tissue samples from spotted gar (Lepisosteus oculatus) and largemouth bass (Micropterus salmoides) were collected from Caddo Lake. Gar and bass livers were subjected to histological investigation and color analysis. Liver color (as abs at 400 nm) was significantly correlated with total mercury in the liver (r2 = 0.57, p = 0.02) and muscle (r2 = 0.58, p = 0.01) of gar. Evidence of liver damage as lipofuscin and discoloration was found in both species but only correlated with liver mercury concentration in spotted gar. Inorganic mercury was the predominant form in gar livers. In order to determine the role of mercury speciation in fish liver damage, a laboratory feeding study was employed. Zebrafish (Danio rerio) were fed either a control (0.12 ± 0.002 µg Hg.g-1 dry wt), inorganic mercury (5.03 ± 0.309 µg Hg.g-1 dry wt), or methylmercury (4.11 ± 0.146 µg Hg.g-1 dry wt) diet. After 78 days of feeding, total mercury was highest in the carcass of zebrafish fed methylmercury (12.49 ± 0.369 µg Hg.g-1 dry wt), intermediate in those fed inorganic mercury (1.09 ± 0.117 µg Hg.g-1 dry wt), and lowest in fish fed the control diet (0.48 ± 0.038 µg Hg.g-1 dry wt). Total mercury was highest in the viscera of methylmercury fed zebrafish (11.6 ± 1.86 µg Hg.g-1 dry wt), intermediate in those fed inorganic diets (4.3 ± 1.08 µg Hg.g-1 dry wt), and lowest in the control fish (below limit of detection). Total mercury was negatively associated with fish length and weight in methylmercury fed fish. Condition factor was not associated with total mercury and might not be the best measure of fitness for these fish. No liver pathologies were observed in zebrafish from any treatment.
Evaluation of the Developmental Effects and Bioaccumulation Potential of Triclosan and Triclocarban Using the South African Clawed Frog, Xenopus Laevis
Triclosan (TCS) and triclocarban (TCC) are antimicrobials found in U.S. surface waters. This dissertation assessed the effects of TCS and TCC on early development and investigated their potential to bioaccumulate using Xenopus laevis as a model. The effects of TCS on metamorphosis were also investigated. For 0-week tadpoles, LC50 values for TCS and TCC were 0.87 mg/L and 4.22 mg/L, respectively, and both compounds caused a significant stunting of growth. For 4-week tadpoles, the LC50 values for TCS and TCC were 0.22 mg/L and 0.066 mg/L; and for 8-week tadpoles, the LC50 values were 0.46 mg/L and 0.13 mg/L. Both compounds accumulated in Xenopus. For TCS, wet weight bioaccumulation factors (BAFs) for 0-, 4- and 8-week old tadpoles were 23.6x, 1350x and 143x, respectively. Lipid weight BAFs were 83.5x, 19792x and 8548x. For TCC, wet weight BAFs for 0-, 4- and 8-week old tadpoles were 23.4x, 1156x and 1310x. Lipid weight BAFs were 101x, 8639x and 20942x. For the time-to-metamorphosis study, TCS showed an increase in weight and snout-vent length in all treatments. Exposed tadpoles metamorphosed approximately 10 days sooner than control tadpoles. For the hind limb study, although there was no difference in weight, snout-vent length, or hind limb length, the highest treatment was more developed compared to the control. There were no differences in tail resorption rates between the treatments and controls. At relevant concentrations, neither TCS nor TCC were lethal to Xenopus prior to metamorphosis. Exposure to relatively high doses of both compounds resulted in stunted growth, which would most likely not be evident at lower concentrations. TCS and TCC accumulated in Xenopus, indicating that the compound has the potential to bioaccumulate through trophic levels. Although TCS may increase the rate of metamorphosis in terms of developmental stage, it did not disrupt thyroid function and metamorphosis in regards to limb development and tail resorption.