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- Application of Cultured Neuronal Networks for Use as Biological Sensors in Water Toxicology and Lipid Signaling.
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This dissertation research explored the capabilities of neuronal networks grown on substrate integrated microelectrode arrays in vitro to be applied to toxicological research and lipid signaling. Chapter 1 details the effects of chlorine on neuronal network spontaneous electrical activity and pharmacological sensitivity. This study demonstrates that neuronal networks can maintain baseline spontaneous activity, and respond normally to pharmacological manipulations in the present of three times the chlorine present in drinking water. The findings suggest that neuronal networks may be used as biological sensors to monitor the quality of water and the presence of novel toxicants that cannot be detected by conventional sensors. Chapter 2 details the neuromodulatory effects of N-acylethanolamides (NAEs) on the spontaneous electrical activity of neuronal networks. NAEs are a group of lipids that can mimic the effects of marijuana and can be derived from a variety of plant sources including soy lecithin. The most prominent NAEs in soy lecithin, palmitoylethanolamide (PEA) and linoleoylethanolamide (LEA), were tested individually and were found to significantly inhibit neuronal spiking and bursting activity. These effects were potentiated by a mixture of NAEs as found in a HPLC enriched fraction from soy lecithin. Cannabinoid receptor-1 (CB1-R) antagonists and other cannabinoid pathway modulators indicated that the CB1-R was not directly involved in the effects of NAEs, but that enzymatic degradation and cellular uptake were more likely targets. The results demonstrate that neuronal networks may also be a viable platform for the elucidation of biochemical pathways and drug mechanisms of action.
- 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.
- 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.
- 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.
- Aspartate Transcarbamoylase of Aeromonas Hydrophila
- This study focused on the enzyme, aspartate transcarbamoylase (ATCase) from A. hydrophila, a Gram-negative bacterium found in fresh water. The molecular mass of the ATCase holoenzyme from A. hydrophila is 310 kDa. The enzyme is likely composed of 6 catalytic polypeptides of 34 kDa each and 6 regulatory polypeptides of 17 kDa each. The velocity-substrate curve for A. hydrophila ATCase is sigmoidal for both aspartate and carbamoylphosphate. The Km for aspartate was the highest to date for an enteric bacterium at 97.18 mM. The Km for carbamoylphosphate was 1.18 mM. When heated to 60 ºC, the specific activity of the enzyme dropped by more than 50 %. When heated to 100 ºC, the enzyme showed no activity. The enzyme's activity was inhibited by ATP, CTP or UTP.
- Assembly of Pseudomonas putida Aspartate Transcarbamoylase and Possible Roles of the PyrC' Polypeptide in the Folding of the Dodecameric Enzyme
- Aspartate transcarbamoylase (ATCase) of Pseudomonas putida consists of two different polypeptides, PyrB and PyrC' (Schurr et al, 1995). The role of the PyrC' and the assembly of PyrB and PyrC' have been studied. The ATCase made in vitro of P.putida PyrB with P.putida PyrC', and of E.coli PyrB with P.putida PyrC ' were generated under two different conditions, denaturation and renaturation, and untreated. It was found that PyrC' plays a role in the enzymatic regulation by ATP, CTP and UTP. In addition to playing a role in substrate binding, the PyrB polypeptide is also involved in effector binding (Kumar et al., manuscript in preparation). The most energetically preferred form of the P.putida WT is a dodecamer with a molecular mass of 480 kDa. The ratio between the PyrB and the PyrC' is 1:1. In studies of nucleotide binding, it was discovered that the P.putida PyrB was phosphorylated by a protein kinase in the cell extract. In the presence of 20 mM EDTA, this phosphorylation was inhibited and the inhibition could be overcome by the addition of divalent cations such as Zn2+ and Mg2+. This result suggested that the phosphorylation reaction required divalent cations. In the CAD complex of eukaryotes, phosphorylations of the CPSase and the linker region between ATCase and DHOase did not occur in the presence of UTP and it was hypothesized (Carrey, 1993) that UTP and phosphorylation(s) regulated the conformational change in the enzyme complex. Therefore, the same idea was approached with P.putida ATCase, where it was found that 1.0 mM UTP inhibited the phosphorylation of PyrB by more than 50%. These results suggested that the regulation of the conformational change of the P.putida ATCase might be similar to that of CAD. Furthermore, peptide mapping for phosphorylation sites was performed on P.putida ATCase WT, WT --11 amino acids and WT --34 amino acids from the N-terminus of the PyrB polypeptide. The results showed that the phosphorylation sites were located on the fragment that contained amino acid number-35 to amino acid number-112 from the N-terminus of the PyrB polypeptide.
- Assessing Effects of an Environmental Education Field Science Program Fostering Responsibility at an Urban Middle School
- The study investigated the ability of an extracurricular program to influence environmental responsibility of sixth and seventh graders. The Children's Environmental Attitude and Knowledge Survey (CHEAKS) was evaluated for appropriateness in assessing the worth of this particular environmental education strategy emphasizing water quality fieldwork and technology. CHEAKS is designed with psychometric reliability and validity that may be used in comparing disparate programs. Wilcoxon two sample tests were used to analyze data gathered from two student groups; one participated in an "Enviro-Mentals Club"; the other received no treatment. Analysis showed no significant change in environmental attitudes between groups, but did show significance (p <= 0.05) in environmental knowledge growth. Therefore, the investigated program had marginal success in influencing environmental responsibility.
- Assessing Outcomes of a Recycling Education and Service Program within an Elementary School
- During the spring 2004 a pilot school recycling program was implemented within Robert E. Lee Elementary. The primary goal of the program was to determine how recycling education in the school would affect curbside recycling rates within the surrounding community. The program was a cooperative effort between the University of North Texas, City of Denton Solid Waste Department and Keep Denton Beautiful. Throughout the first months of the study during the spring 2004, an increase in curbside recycling within the Robert E. Lee Elementary attendance zone was observed, with a dramatic decrease in participation over the summer and a rapid increase once again during the second full semester of the study. In a survey conducted with 3rd and 5th grade students at the pilot project school, most students expressed positive attitudes about recycling. Students whose survey responses indicated a high level of knowledge about what could be recycled were 37% more likely to claim to recycle regularly, than those students that scored low on the knowledge portion of the survey. Although the total amount of waste generation (recyclable and non-recyclable) at Robert E. Lee Elementary did not decrease during the study, the campus was able to divert recyclable material from their trash at a much higher rate than two other local elementary campuses with paper-only recycling and no associated recycling education program. Based upon the success of the recycling program at Robert E. Lee Elementary, the City of Denton Recycling Division has agreed to move forward with offering recycling to more schools within the Denton Independent School District during the 2005-2006 school year.
- Assessing the Effects of a Municipal Wastewater Treatment Plant Effluent on Zooplankton, Phytoplankton and Corbicula Flumina in a Constructed Wetland
- Wetland wastewater treatment offers low-cost, energy efficient alternatives to conventional wastewater technologies. In this study, an artificial wetland was constructed at the City of Denton, Texas Pecan Creek Water Reclamation Plant to facilitate diazinon removal from treated effluent.
- 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.
- An Assessment of Storm Water Toxicity from the Dallas/Fort Worth Metroplex and Denton, Texas
- With the advent of national storm water regulations, municipalities with populations greater than 100,000 are required to obtain National Pollutant Discharge Elimination System Permits (NPDES) for storm water discharges. In addition to the sampling required for the permit process, the City of Fort Worth contracted with the University of North Texas' Institute of Applied Sciences to conduct acute toxicity testing using Pimephales prcmelas and Ceriodaphnia dubia on storm water samples received from the Dallas/Fort Worth Metroplex. A Toxicity Identification Evaluation (TIE) was performed on four samples that exhibited acute toxicity to C. dubia. High levels of metals as well as diazinon were some of the probable toxicants found.
- An Assessment of the Use of Seeding, Mowing, and Burning in the Restoration of an Oldfield to Tallgrass Prairie in Lewisville, Texas
- An examination of the effectiveness of seeding, burning, and mowing in the reestablishment of tallgrass prairie species on overgrazed and abandoned pastureland. The study site is a 20 acre tract on U.S. Corps of Engineers land below Lake Lewisville in Denton County, Texas. The site was partitioned into thirty-nine 40 by 40 meter plots with seeding (carried out in 1996) and management treatment (burning, mowing, and no maintenance carried out in 1998) randomly applied following a two level design. For each plot, nine stratified-random 0.1 m2 subplots were examined and shoot counts for each species recorded. The effects of the treatments on individual species and species richness were analyzed with a two-way ANOVA followed by a SNK multiple range test, both on ranked data. Community level analysis was conducted with both a MANOVA on ranked data and a Canonical Correspondence Analysis on raw data. Results indicate that seeding positively affected species richness, particularly when combined with either burning or mowing in the early spring. Mowing also significantly increased species richness in areas that were not seeded, while burning negatively affected species richness on unseeded plots. Treatments significantly affected community composition with treatments having the most clear effect on spring and summer forbs.
- Attenuation of Escherichia Coli Aspartate Transcarbamoylase Expressed in Pseudomonas Aeruginosa Mutant and Wild Type Strains
- No apparent repression of pyr gene expression in Pseudomonas aeruginosa is observed upon addition of exogenous pyrimidines to the growth medium. Upon introduction of the subcloned Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) into a P. aeruginosa pyrB mutant strain, repression was observed in response to exogenously fed pyrimidine compounds. The results proved that it is possible to bring about changes in pyrimidine nucleotide pool levels and changes in transcriptional regulation of gene expression as a result. Thus, the lack of regulatory control in P. aeruginosa pyr gene expression is not due to an inability to take up and incorporate pyrimidine compounds into metabolic pools, or to an inability of the RNA polymerase to respond to regulatory sequences in the DNA but is probably due to a lack of specific regulatory signals in the promoter of the genes themselves.
- Automated Low-cost Instrument for Measuring Total Column Ozone
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Networks of ground-based and satellite borne instruments to measure ultraviolet (UV) sunlight and total column ozone have greatly contributed to an understanding of increased amounts of UV reaching the surface of the Earth caused by stratospheric ozone depletion. Increased UV radiation has important potential effects on human health, and agricultural and ecological systems. Observations from these networks make it possible to monitor total ozone decreases and to predict ozone recovery trends due to global efforts to curb the use of products releasing chemicals harmful to the ozone layer. Thus, continued and expanded global monitoring of ozone and UV is needed. However, existing automatic stratospheric ozone monitors are complex and expensive instruments. The main objective of this research was the development of a low-cost fully automated total column ozone monitoring instrument which, because of its affordability, will increase the number of instruments available for ground-based observations. The new instrument is based on a high-resolution fiber optic spectrometer, coupled with fiber optics that are precisely aimed by a pan and tilt positioning mechanism and with controlling programs written in commonly available software platforms which run on a personal computer. This project makes use of novel low-cost fiber optic spectrometer technology. A cost advantage is gained over available units by placing one end of the fiber outdoors to collect sunlight and convey it indoors, thereby allowing the spectrometer and computer to be placed in a controlled environment. This reduces the cost of weatherproofing and thermal compensation. Cost savings also result from a simplified sun targeting system, because only a small pan and tilt device is required to aim the lightweight fiber optic ends. Precision sun-targeting algorithms, optical filter selection, and software to derive ozone from spectral measurements by the spectrometer are a major contribution of this project. This system is a flexible platform which may be adapted to study other atmospheric constituents such as sulfur dioxide, nitrous oxides, and haze.
- Autonomic Reflexes of the Heart During Acute Myocardial Ischemia
- This study investigated whether acute myocardial ischemia of the anterior left ventricular wall induced an increase in cardiac sympathetic efferent nerve activity and thereby affected regional myocardial blood flow and contractile function.
- Autophosphorylation and Autoactivation of an S6/H4 Kinase Isolated From Human Placenta
- A number of protein kinases have been shown to undergo autophosphorylation, but few have demonstrated a coordinate increase or decrease in enzymatic activity as a result. Described here is a novel S6 kinase isolated from human placenta which autoactivates through autophosphorylation in vitro. This S6/H4 kinase, purified in an inactive state, was shown to be a protein of Mr of 60,000 as estimated by SDS-PAGE and could catalyze the phosphorylation of the synthetic peptide S6-21, the histone H4, and myelin basic protein. Mild digestion of the inactive S6/H4 kinase with trypsin was necessary, but not sufficient, to activate the kinase fully
- 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.
- 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.
- Bacterial Cyanide Assimilation: Pterin Cofactor and Enzymatic Requirements for Substrate Oxidation
- Utilization of cyanide as the sole nitrogen source by Pseudomonas fluorescens NCIMB 11764 (Pf11764) occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated oxygenolytically by an enzyme referred to as cyanide oxygenase (CNO), which exhibits properties of a pterin-dependent hydroxylase. The pterin requirement for Pf11764 CNO was satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 µM). These compounds included, for example, biopterin, monapterin and neopterin, all of which were also identified in cell extracts. A related CNO-mediated mechanism of cyanide utilization was identified in cyanide-degrading P. putida BCN3. This conclusion was based on (i) the recovery of CO2 and NH3 as enzymatic reaction products, (ii) the dependency of substrate conversion on both O2 and NADH, and (iiii) utilization of cyanide, O2 and NADH in a 1:1:1 reaction stoichiometry. In contrast to findings reported for Pf11764, it was not possible to demonstrate a need for exogenously added pterin as a cofactor for the PpBCN3 enzyme system. However, results which showed that cells of PpBCN3 contained approximately seven times the amount of pterin as Pf11764 (of which a significant portion was protein-bound) were interpreted as indicating that sufficient bound CNO-cofactor exists, thus eliminating any need for a supplemental source.
- A Behavioral Model for Detection of Acute Stress in Bivalves
- A behavioral model for acute responses in bivalves, was developed using time series analysis for use in a real-time biomonitoring unit. Stressed bivalves closed their shell and waited for the stressful conditions to pass. Baseline data showed that group behavior of fifteen bivalves was periodic, however, individuals behaved independently. Group behavior did not change over a period of 20 minutes more than 30 percent, however, following toxic exposures the group behavior changed by more than 30 percent within 20 minutes. Behavior was mathematically modeled using autoregression to compare current and past behavior. A logical alarm applied to the behavior model determined when organisms were stressed. The ability to disseminate data collected in real time via the Internet was demonstrated.
- Benthic Macroinvertebrates of Temperate, Sub-Antarctic Streams: The Effects of Altitudinal Zoning and Temperature on the Phenology of Aquatic Insects Associated to the Robalo River, Navarino Island (55°S), Chile
- The Cape Horn Biosphere Reserve, within the remote Sub-Antarctic ecoregion is a reservoir of expressions of biological and cultural diversity. Although it is considered one of 24 wilderness areas remaining in the world, it is not free from local and global threats, such as invasive species, and climate change. Field biologists and philosophers associated to the Sub-Antarctic Biocultural Conservation Program and the Omora Ethnobotanical Park, have worked to describe the region’s biocultural diversity, linking ecological and philosophical research into education, ecotourism, and conservation, through a methodology called field environmental philosophy (FEP), which integrates ecological sciences and environmental ethics through a 4-step cycle consisting of: 1) interdisciplinary research; 2) composition of metaphors; 3) design of field activities with an ecological and ethical orientation; and 4) implementation of in situ conservation areas. In this context, the purposes of this dissertation were to: 1) provide a comprehensive review of publications regarding the conservation status of aquatic and terrestrial insects at a global scale and with an emphasis in southern South America; 2) study the distribution of benthic macroinvertebrates through the sharp altitudinal gradient of the Róbalo River watershed; 3) describe the life histories of Gigantodax sp (Simuliidae: Diptera) and Meridialaris chiloeense (Leptophlebiidae: Ephemeroptera) in the Róbalo River and to assess the potential effects of climate change on their phenology; and 4) to apply FEP methodology in order to better understand and communicate the intrinsic and instrumental values of freshwater invertebrates in the Cape Horn Biosphere Reserve.
- Beta-adrenergic Blockade Via Atenolol and Its Effects on Blood Pressure, Heart Rate, and Renal Morphology in the Developing Chicken Gallus Gallus Domesticus
- Chicken embryos were chronically exposed to the ?1- blocker atenolol during one of three stages: mesonephros (E7-E9), mesonephros-metanephros (E11-E13), or metanephros (E15-E17). Mesonephros group hearts were larger than all other groups (P < 0.01). Mesonephros and metanephros group kidneys were larger than all remaining groups (P < 0.0001). The mesonephros group nephron number was ~40% lower than control values (P = 0.002). Glomerular areas were 26% and 18% larger than the control group in the mesonephros and metanephros groups, respectively (P < 0.001). These data suggest an E7-E9 critical window of cardiovascular and renal development for atenolol. Acute atenolol exposure in E15 embryos showed an increase in mean arterial pressure with all but the highest dose. All doses significantly decreased heart rate.
- Bioaccumulation of Triclocarban, Triclosan, and Methyl-triclosan in a North Texas Wastewater Treatment Plant Receiving Stream and Effects of Triclosan on Algal Lipid Synthesis.
- Triclosan (TCS) and triclocarban (TCC), widely used antimicrobial agents found in numerous consumer products, are incompletely removed by wastewater treatment plant (WWTP) processing. Methyl-triclosan (M-TCS) is a more lipophilic metabolite of its parent compound, TCS. The focus of this study was to quantify bioaccumulation factors (BAFs) for TCS, M-TCS, and TCC in Pecan creek, the receiving stream for the City of Denton, Texas WWTP by using field samples mostly composed of the alga Cladophora sp. and the caged snail Helisoma trivolvis as test species. Additionally, TCS effects on E. coli and Arabidopsis have been shown to reduce fatty acid biosynthesis and total lipid content by inhibiting the trans-2 enoyl- ACP reductase. The lipid synthesis pathway effects of TCS on field samples of Cladophora spp. were also investigated in this study by using [2-14C]acetate radiolabeling procedures. Preliminary results indicate high TCS concentrations are toxic to lipid biosynthesis and reduce [2-14C]acetate incorporation into total lipids. These results have led to the concern that chronic exposure of algae in receiving streams to environmentally relevant TCS concentrations might affect their nutrient value. If consumer growth is limited, trophic cascade strength may be affected and serve to limit population growth and reproduction of herbivores in these riparian systems.
- Bioavailability and toxicity of 2,4,6-trinitrotoluene in sediment.
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TNT (2,4,6-trinitrotoluene) is a persistent contaminant at many military installations and poses a threat to aquatic ecosystems. Data from environmental fate and toxicity studies with TNT revealed that sediment toxicity test procedures required modification to accurately assess sediment TNT toxicity. Key modifications included aging TNT-spiked sediments 8-14 d, basing lethal dose on measured sediment concentrations of the molar sum of TNT and its main nitroaromatic (NA) transformation products (SNA), basing sublethal dose on average sediment SNA concentrations obtained from integration of sediment SNA transformation models, avoiding overlying water exchanges, and minimizing toxicity test durations. Solid phase microextraction fibers (SPMEs) were investigated as a biomimetic chemical measure of toxicity and bioavailability. Both organism and SPME concentrations provided measures of lethal dose independent of exposure scenario (TNT-spiked sediment or TNT-spiked water) for Tubifex tubifex. Among all benthic organisms tested (Chironomus tentans, Ceriodaphnia dubia, T. tubifex) and matrixes, median lethal dose (LC50) estimates based on SPME and organism concentrations ranged from 12.6 to 55.3 mmol SNA/ml polyacrylate and 83.4 to 172.3 nmol SNA/g tissue, ww, respectively. For Tubifex, LC50s (95% CI) based on SNA concentrations in sediment and SPMEs were 223 (209-238) nmol SNA/g, dw and 27.8 (26.0-29.8) mmol SNA/ml, respectively. Reproductive effects occurred at slightly lower exposures. Median effective dose (EC50) estimates (95% CI) for Tubifex cocoon production, based on sediment and SPME concentrations, were 118 (114-122) nmol SNA/g, dw and 21.8 (21.2-22.4) mmol SNA/ml, respectively. Bioconcentration experiments with Tubifex revealed that compound hydrophobicity predicted the toxicokinetics and bioconcentration of these compounds from water, however, there was a large discrepancy between the toxicokinetics of absorbed versus metabolically-generated aminodinitrotoluenes. A large portion of bioconcentrated, radiolabeled TNT transformation products could not be identified. In addition to their ability to provide matrix-independent measures of dose, SPME concentrations were more accurate indicators of bioavailable NAs than were sediment concentrations.
- Biochemical Identification of Molecular Components Required for Cyanide Assimilation in Pseudomonas fluorescens NCIMB 11764
- Utilization of cyanide as a nutritional nitrogen source in P. fluorescens NCIMB 11764 was shown to involve a novel metabolic mechanism involving nonenzymatic neutralization outside of cells prior to further enzymatic oxidation within. Several cyanide degrading enzymes were produced by NCIMB 11764 in response to growth or exposure to cyanide, but only one of these cyanide, oxygenase (CNO), was shown to be physiologically required for assimilation of cyanide as a growth substrate.
- Bioconcentration and Morphological Effects of Triclosan on Three Species of Wetland Plants
- Triclosan (TCS) is an antimicrobial compound found in several types of common household products. After being washed down the drain, TCS will then end up in the local watershed. Although numerous studies have been conducted to evaluate the fate and effects of TCS in aquatic environments, there have been no studies evaluating the role arbuscular mycorrhizal fungi (AM) play in a plants response to TCS exposure. Three species of wetland plants native North Texas were inoculated with AM spores and exposed to 0, 0.4 g/L and 4.0 g/L TCS concentrations. Root morphology of E. prostrata and S. herbacea showed AM and exposure responses. S. herbacea produced the greatest amounts biomass and TCS bioaccumulation, in all but one treatment. It also displayed opposing results to E. prostrata in measures of root length, root surface area, relative root mass, relative shoot mass and shoot:root ratio. TCS root tissue concentrations increased with increased exposures for both E. prostrata and S. herbacea. Even though E. prostrata had the lowest levels in each measure of biomass production, it had the highest amount of root TCS bioaccumulation in the AM inoculated 4.0 g/L treatment. H. laevis was between the other two species in terms of biomass production, and did not demonstrate any exposure or inoculation effects in morphology or TCS accumulation. Overall, no clear patterns were detected, which highlights the fact that further study is required to completely understand the effects compounds like TCS can have on plant community structure, and ultimately ecosystem function.
- Bioconcentration of Triclosan, Methyl-Triclosan, and Triclocarban in the Plants and Sediments of a Constructed Wetland
- Triclosan and triclocarban are antimicrobial compounds added to a variety of consumer products that are commonly detected in waste water effluent. The focus of this study was to determine whether the bioconcentration of these compounds in wetland plants and sediments exhibited species specific and site specific differences by collecting field samples from a constructed wetland in Denton, Texas. The study showed that species-specific differences in bioconcentration exist for triclosan and triclocarban. Site-specific differences in bioconcentration were observed for triclosan and triclocarban in roots tissues and sediments. These results suggest that species selection is important for optimizing the removal of triclosan and triclocarban in constructed wetlands and raises concerns about the long term exposure of wetland ecosystems to these compounds.
- Biodiversity and Genetic Structure of Benthic Macroinvertebrates along an Altitudinal Gradient: A Comparison of the Windhond and Róbalo River Communities on Navarino Island, Chile
- Altitudinal gradients in Sub-Antarctic freshwater systems present unique opportunities to study the effect of distinct environmental gradients on benthic macroinvertebrate community composition and dispersal. This study investigates patterns in biodiversity, dispersal and population genetic structure of benthic macroinvertebrate fauna across an altitudinal gradient between two watersheds on Navarino Island in southern Chile. Patterns in diversity, density, evenness and functional feeding groups were not significantly different across the altitudinal gradient in both the Windhond and Róbalo Rivers. Taxa richness in both rivers generally increased from the headwaters of the river to the mouth, and functional feeding group patterns were consistent with the predictions of the River Continuum Concept. Population genetic structure and gene flow was investigated by sampling the mitochondrial cytochrome oxidase I gene in two invertebrate species with different dispersal strategies. Hyalella simplex (Amphipoda) is an obligate aquatic species, and Meridialaris chiloeense (Ephemeroptera) is an aquatic larvae and a terrestrial winged adult. Contrasting patterns of population genetic structure were observed. Results for Hyalella simplex indicate significant differentiation in genetic structure in the Amphipod populations between watersheds and lower genetic diversity in the Róbalo River samples, which may be a result of instream dispersal barriers. Meridialaris chiloeense exhibited weak population structure but higher genetic diversity, which suggests this species is able to disperse widely as a winged adult.
- Biodiversity of Caddisflies (Trichoptera) of the Interior Highlands of North America
- Caddisflies (Trichoptera) were collected from over 500 different locations throughout the Interior Highlands (Ozark, Ouachita, Arbuckle, and Wichita Mountains) between March 1990 and March 1994. I systematically sampled representative lotic and lentic habitats in 131 natural watersheds that comprise the 17 different physiographic subregions of this area. From my examination of approximately 60,000 specimens, surveys of regional museum collections, and review of literature records, I document 229 species distributed in 16 families and 58 genera. Included in this total are 27 endemic species and 15 new regional records. Descriptions are provided for a species new to science (Cheumatopsyche robisoni), four larvae (Helicopsyche limnella, H. piroa, Marilia species A, Polycentropus crassicornis) and a female (Helicopsyche piroa). Hydropsyche reiseni Denning, previously known only from the Arbuckle Mountains, is reduced in synonymy with H. arinale Ross. Further, I provide illustrated family, generic, and selected species-level keys that reflect this regional biodiversity.
- Biodiversity of Dragonflies and Damselflies (Odonata) of the South-Central Nearctic and Adjacent Neotropical Biotic Provinces
- The south-central United States serves as an important biogeographical link and dispersal corridor between Nearctic and Neotropical elements of western hemisphere odonate faunas. Its species are reasonably well known because of substantial collections, but there has been no concerted effort to document the extent of biodiversity and possible geographic affinities of dragonflies and damselflies of this region. The recent discoveries of Argia leonorae Garrison, Gomphus gonzalezi Dunkle and Erpetogomphus heterodon Garrison from southern and western Texas and northern Mexico suggest that Odonata species remain to be discovered in this area, particularly from far south Texas and northern Mexico. I have documented a total of 12,515 records of Odonata found in 408 counties within the south-central U.S. A total of 73 species of damselflies and 160 species of dragonflies was revealed in the region. The 233 (197 in Texas) Odonata species are distributed among 10 families and 66 genera. Illustrated family, generic, and species-level keys are provided. Since the beginning of this work in the Fall of 1993, one species has been added each to the Louisiana and Oklahoma faunas, and 12 species have been added, previously unreported from Texas, including four new to the U.S. The area of highest Odonata biodiversity overall (161 spp.) is in the Austroriparian biotic province. The greatest degree of faunal similarity between the south-central U.S. and other intra-continental regions was observed for the eastern (64%) United States. Diversity is a function of area, and as expected, the numbers of breeding birds and Odonata, in each contiguous U.S. state are positively correlated (r=0.376, n=33, p=0.031). There is, however, no strong correlation between land area and species diversity within the region, but those natural biotic provinces (Austroriparian, Texan, Balconian) where aquatic systems and topographic heterogeneity are the greatest provide a broader spectrum of potential Odonata habitats and thus support a greater number of Odonata species.
- Biogeographic Relationships of Pocket Gophers (Geomys breviceps and Geomys bursarius) in the Southeastern Portion of Their Ranges
- This research utilized population genetic analyses (protein starch-gel electrophoresis and DNA sequencing of the cytochrome b mtDNA gene), host-parasite specificity (lice coevolution), remote sensing of satellite data, and geographic information systems (GIS) to characterize newly discovered populations of pocket gophers (genus: Geomys) in Arkansas. These populations are isolated and occur in seemingly unsuitable habitat in the Ozark Mountains of Arkansas. Analyses of electrophoretic and ectoparasite data suggested the populations in the Ozark Mountains represented isolates allied to Geomys bursarius, a species not known to occur in Arkansas. Comparison of mitochondrial DNA sequence data of the cytochrome b gene with that of other taxa and morphometric analyses confirmed that these populations are most closely allied to G. bursarius occurring to the north in Missouri. Moreover, these mtDNA sequence analyses indicated a degree of differentiation typical of that between other subspecies of pocket gophers. Therefore, these populations represent a distinct genetic entity in an intermediate stage of speciation and should be designated as a new subspecies, Geomys bursarius ozarkensis. Molecular clock analysis revealed a time of lineage divergence for this new subspecies as approximately 511,000 YBP. Due to the isolated nature and limited distribution of this subspecies, an evaluation of critical habitat needs was initiated. Remote sensing and GIS technologies were used to identify and describe suitable habitat Computerized classification of satellite imagery of suitable vegetation, integrated with ancillary digital information on soil associations, roads, and water systems, revealed that human activity had played a positive role in the establishment and dispersal of pocket gophers in this area. This research represents an initial combination of classical systematic tools with remote sensing and GIS to investigate biogeographic patterns and evolution. This project establishes a framework for using an interdisciplinary approach to studying organisms with limited distributions, determining evolutionary status, and providing recommendations for conservation.
- Biogeography of Montane Mammals on the Colorado Plateau and Adjacent Regions
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This study identifies the biogeographic factors that structure small mammal communities on mountains of the Colorado Plateau and adjacent regions. Forty six isolated ranges were characterized across a 5-state study area encompassing the Colorado Plateau, including the central high plateaus of Utah and the Basin and Range Province (i.e. the Great Basin and mountains of Arizona and New Mexico). Presence/absence data of 25 montane mammal species were used to explore the interactions between historical and ecological processes affecting local and regional diversity patterns. Multivariate analyses, such as non-metric dimensional scaling, were used to explore factors which influence community composition. Results of these analyses revealed the Colorado River as a significant biogeographic barrier that affects montane mammal community structure. MtDNA cytochrome b sequence variation was analyzed among populations of the long-tailed vole, Microtus longicaudus, sampled from five interior ranges of the Colorado Plateau- Abajo, LaSal, Henry, and Chuska Mts., and Boulder Mountain of the Aquarius Plateau-and analyzed using traditional phylogenetic approaches (parsimony and likelihood) as well as nested clade analysis. Results support previous documentation of a major east-west phylogeographic break occurring between populations southeast of the Colorado River (eastern Arizona, Colorado, Wyoming and New Mexico) and all other western populations, which include a central clade, a northwest clade, and an Alaskan island clade. Evidence also supports differentiation of a 'southern Rockies' clade and a distinct 'southwest island' clade. Populations of M. longicaudus north and west of the Colorado River (Boulder and Henry Mts.) share two haplotypes, form a well-supported subclade with populations from the Kaibab plateau, and are closely related to the Northwest clade. Past approaches to studying montane mammal communities utilizing theory based on island biogeography have overemphasized area and isolation as the only forces structuring insular communities. As a result, there has been a lack of recognition of the influences of environmental factors, species turnover, and barriers that create and maintain regional diversity on the Colorado Plateau and adjacent areas.
- BioInformatics, Phylogenetics, and Aspartate Transcarbamoylase
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In this research, the necessity of understanding and using bioinformatics is demonstrated using the enzyme aspartate transcarbamoylase (ATCase) as the model enzyme. The first portion of this research focuses on the use of bioinformatics. A partial sequence of the pyrB gene found in Enterococcus faecalis was submitted to GenBank and was analyzed against the contiguous sequence from its own genome project. A BLAST (Basic Local Alignment Search Tool; Atschul, et al., 1990) was performed in order to hypothesize the remaining portion of the gene from the contiguous sequence. This allowed a global comparison to other known aspartate transcarbamoylases (ATCases) and once deduced, a translation of the sequence gave the stop codon and thus the complete sequence of the open reading frame. When this was complete, upstream and downstream primers were designed in order to amplify the gene from genomic DNA. The amplified product was then sequenced and used later in phylogenetic analyses concerning the evolution of ATCase. The second portion of this research involves taking multiple ATCase nucleotide sequences and performing phenetic and phylogenetic analyses of the archaea and eubacter families. From these analyses, ancestral relationships which dictate both structure and function were extrapolated from the data and discussed.
- Biological Nitrogen Fixation in Two Southwestern Reservoirs
- This investigation has determined the presence of biological nitrogen fixation in two reservoirs in the southwestern United States: Lake Arlington and Lake Ray Hubbard. Subsequent tests have gathered baseline data on the effects of various biological, chemical, and physical parameters on in situ nitrogen fixation in these reservoirs. Of specific importance is the relationship between nitrogen fixation arid occasional blooms of blue-green algae which produce such problems as testes and odors in these water-supply impoundments.
- Biology and Energetics of Tropisternus Lateralis Nimbatus (SAY) (Coleoptera: Hydrophilidae) in a Playa on the Southern High Plains of Texas and Aquatic Coleoptera Diversity from Seven Playas on the Southern High Plains of Texas
- A study of the biology of Tropisternus lateralis, a hydrophilid beetle, was conducted during the flood period of a single playa on the Southern High Plains of Texas from early June 1995 through early September 1995. Mechanism of colonization, tolerance/avoidance to drought, larval density, and secondary production were analyzed. T. lateralis colonized playas from surrounding aquatic habitats and avoided drought through aerial dispersion. Once in the playa, larval density increased over time. Secondary production was 1.31 g/m2/.25 yr. In addition, aquatic Coleoptera diversity was studied in seven playas on the Southern High Plains of Texas. A total of twenty three species were identified from the study region. Nine species not reported in playa literature were identified.
- Bioreactor Landfill Cell Feasibility Study ' Reference to City of Denton Subtitle-D Permit #1590A Landfill
- The City of Denton Landfill, Permit #1590A, utilizes “Dry-Tomb” techniques for disposal and promotion of municipal solid waste stabilization, as described by the Resource Conservation and Recovery Act (RCRA) prohibition in 40 CFR. Bioreactor research suggests re-circulating leachate increases biodegradation rates and reduces long-term monitoring from fifty years to less than ten years. Current procedures that are followed at Denton's landfill, literature review and the use of the Hydrologic Evaluation of Landfill Performance (HELP) model, suggest that a bioreactor landfill cell is worthy of further research. Re-circulating leachate and augmenting it with additional liquid will increase biodegradation and the need to design and build a landfill gas collection system to capture methane for energy recovery uses.
- Biotic Factors and Temperature Tolerances via Critical Thermal Methodology in Goldfish
- CTMinimum and CTMaximum were measured in 620 goldfish to determine if biotic factors, in particular starvation, condition factor and size, affect temperature tolerance. Twenty-eight days of starvation adversely affected both upper and lower temperature tolerance. Condition factor was related to upper but not lower temperature tolerance.
- BK1 and DCD1 Act Synergistically in Subsidiary Cell Formation in Zea Mays
- Subsidiary mother cell (SMC) divisions during stomatal complex formation in Zea mays are asymmetric generating a small subsidiary cell (SC) and a larger epidermal cell. Mutants with a high number of abnormally shaped subsidiary cells include the brick1 (brk1) and discordia1 (dcd1) mutants. BRK1 is homologous to HSPC300, an ARP2/3 complex activator, and is involved in actin nucleation while DCD1 is a regulatory subunit of the PP2A phosphatase needed for microtubule generation (Frank and Smith, 2002; Wright et al. 2009). Possible causes of the abnormal SCs in brk1 mutants include a failure of the SMC nucleus to polarize in advance of mitosis, no actin patch, and transverse and/or no PPBs (Gallagher and Smith, 2000; Panteris et al 2006). The abnormal subsidiary mother cell division in dcd1 is due to correctly localized, but disorganized preprophase bands (PPBs; Wright et al. 2009). The observation that brk1 has defects in PPB formation and that the dcd1 phenotype is enhanced by the application of actin inhibitors led us to examine the dcd1; brk1 double mutant (Gallagher and Smith, 1999). We found that dcd1; brk1 double mutants demonstrate a higher percentage of aberrant SCs than the single mutants combined suggesting that these two mutations have a synergistic and additive effect on SC formation. Our observations and results are intriguing and the future step will be to quantitate the abnormal PPBs and phragmoplasts in the double and single mutants using immunolocalization of tubulin and actin as well as observations of live cells expressing tubulin-YFP.
- Blood Pressure Regulation During Simulated Orthostatism Prior to and Following Endurance Exercise Training
- Cardiovascular responses and tolerance to an orthostatic stress were examined in eight men before and after eight months of endurance exercise training. Following training, maximal oxygen consumption and blood volume were increased, and resting heart rate reduced. Orthostatic tolerance was reduced following training in all eight subjects. It was concluded that prolonged endurance training decreased orthostatic tolerance and this decrease in tolerance appeared associated with attenuated baroreflex sensitivity and alterations in autonomic balance secondary to an increased parasympathetic tone noted with training.
- Callus Development and Organogenesis in Cultured Explants of Cowpea (Vigna unguiculata (L.) Walp
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Cowpea, Vigna unguiculata (L.) Walp is an excellent source of protein, vitamins and minerals and a major food crop many parts of Africa. Optimal production levels are hampered by insect pests and diseases. Biotechnological techniques such as tissue culture and genetic engineering can aid in the development of varieties with resistance to insect pests and diseases. The objective of this study was to investigate conditions necessary for the development of a reproducible tissue culture system that can be applied to regenerate transformed cells from culture. The in vitro manipulation of cowpea using Murashige and Skoog (MS) medium, auxins and cytokinins resulted in the formation of callus and rhizogenesis. Calli that were formed were separated into six classes based on color and texture. Yellowish friable callus, yellowish compact, soft yellowish callus and green and white were composed of largely vacuolated cells and were non-regenerative. Friable green callus was the most prevalent callus type and could form of roots in some hormone combinations. Green spots were formed on hard compact green callus. The green spots became nodular, forming root primordia and ultimately giving rise to roots. None of the six calli types gave rise to the formation of shoots. Embryogenic callus was induced from cowpea explants cultured on MS medium supplemented with dicamba and picloram. Embryogenic suspension cultures were initiated from callus induced on MS supplemented with 3.0 mg/L dicamba or picloram and conditions for maintenance of embryogenic suspension cultures were evaluated. Somatic embryos were formed in suspension cultures. Attempts to convert and germinate the somatic embryos resulted in the formation of callus or formation of appendages on the somatic embryos or in the death of the embryos. The appendages formed roots on prolonged culture. Further research is needed to determine appropriate optimal conditions for embryo conversion and germination and ultimately plant recovery from culture.
- Carbachol- and ACPD-Induced Phosphoinositide Responses in the Developing Rat Neocortex
- Signal transduction via the phosphoinositide (PI) second messenger system has key roles in the development and plasticity of the neocortex. The present study localized PI responses to individual cortical layers in slices of developing rat somatosensory cortex. The acetylcholine agonist carbachol and the glutamate agonist trans-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) were used to stimulate PI turnover. The PI responses were compared to the distribution of the corresponding PI-linked receptors in order to investigate the regional ontogeny of PI coupling to receptors in relation to neural development. The method for assessing PI turnover was modified from Hwang et al. (1990). This method images the PI response autoradiographically through the localizaton of [3H]cytidine that has been incorporated into the membrane-bound intermediate, cytidine diphosphate diacylglycerol. In each age group (postnatal days 4-30), carbachol resulted in more overall labeling than ACPD. For both agonists, the response peaked on postnatal day 10 (P10) and was lowest in the oldest age group. The laminar distribution of the carbachol PI response from P4-P16 corresponded fairly well with the laminar distribution of [3H]quinuclidinyl benzilate binding (Fuchs, 1995). However, in the subplate layer the carbachol response was strong while receptor binding was minimal. The carbachol response decreased after postnatal day 10, while the overall levels of receptor binding continued to increase. From P5 - P14, PI-linked metabotropic glutamate receptors are most concentrated in layer IV (Blue et al., 1997), whereas only on P6 was there a correspondingly high ACPD-initiated PI response in this layer. Unlike receptors, the PI response was strong in upper V (P4 - P12) and within layers II/III (P8 - P16). From P4 - P21, the subplate showed relatively high PI labeling compared to receptor binding. The several differences between the distribution of PI response and receptors suggest spatiotemporal heterogeneity of receptor coupling to second messenger systems.
- Carbon Flux in Reservoir Sediments
- The central objective of the study was to fractionate sedimenting organic materials according to their source (allochthonous or autochthonous) and ultimately to determine the degree of biodegradability of contributions from either source with particular reference to activities at the mud-water interface.
- A Carcinogenic Agent Elaborated by Liver Cells from Lymphosarcoma-Bearing Mice
- Liver cells from lymphosarcoma-bearing DBA/1J mice were shown, by parabiotic culture with normal liver cells from isologous mice, to elaborate an agent which could pass a 25 mu filter and transform the normal cells to a malignant state.
- Cardio-Respiratory Ontogeny and the Transition to Bimodal Respiration in an Air Breathing Fish: Morphological and Physiological Development in Normoxia and Hypoxia.
- As selection pressures exist for not only adults, but for every life history stage, it is important to understand how environmental factors shape developing animals. Despite the significance placed on aquatic hypoxia as a driving force in the evolution of air breathing, this is the first known study to examine the effects of hypoxia on cardio-respiratory ontogeny of an air breathing fish. Blue gouramis are obligatory air breathing fish that possess a labyrinth-like structure that serves as the air breathing organ. Gouramis were reared for up to 90 d in normoxia or hypoxia, and morphological and physiological development was observed. Hypoxic larvae had increased lamellar and labyrinth organ surface areas. Bradycardia and increased gill ventilation rates were observed when larvae from either rearing group were briefly exposed to hypoxia. Hypoxic larvae also showed a reduced heart rate and gill ventilation rate in the absence of a hypoxic stimulus, possibly indicative of a more comprehensive, long-term respiratory plasticity. The similarity of routine oxygen consumption between rearing groups suggests that metabolic demand did not change for hypoxic larvae, but that they were more efficient at oxygen acquisition. This is further supported by increased resistance time of hypoxic gouramis to extreme hypoxia. The onset of air breathing was between 20 and 25 d post-fertilization, and was not affected by either rearing or exposure environment. It may be that this behavior is associated with the inability of smaller larvae to successfully overcome water surface tension, rather than with the necessity of aerial respiration at this stage. Hypoxia is commonly experienced by most air breathing fishes, and studies of hypoxia-induced developmental effects may provide critical insights into the evolution of air breathing. The studies presented here provide novel data on the plasticity of cardio-respiratory development of an air breathing fish reared in hypoxia, and can serve as a solid foundation for future studies.
- Cardiorespiratory Responses to Graded Levels of Lower-body Positive Pressure During Dynamic Exercise in Man
- Cardiorespiratory responses to incremental dynamic exercise were assessed across four different levels of lower-body positive pressure (LBPP) and, as a separate study, during constant load (i.e constant work rate) exercise below and above each subject's ventilatory threshold (VT), both with and without 45 torr of LBPP.
- 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.
- Cell-Free Recovery and Isotopic Identification of Cyanide Degrading Enzymes from Pseudomonas Fluorescens
- Cell-free extracts from Pseudomonas fluorescens NCIMB 11764 catalyzed the degradation of cyanide into products that included C02, formic acid, formamide and ammonia. Cyanide-degrading activity was localized to cytosolic cell fractions and was observed at substrate concentrations as high as 100 mM. Two cyanide degrading activities were identified by: (i) the determination of reaction products stoichiometries, (ii) requirements for NADH and oxygen, and (iii) kinetic analysis. The first activity produced CO2 and NH3 as reaction products, was dependent on oxygen and NADH for activity, and displayed an apparent Km for cyanide of 1.2 mM. The second activity generated formic acid (and NH3) pfus formamide as reaction products, was oxygen independent, and had an apparent Km of 12 mM for cyanide. The first enzymatic activity was identified as cyanide oxygenase whereas the second activity consists of two enzymes, a cyanide nitrilase (dihydratase) and putative cyanide hydratase. In addition to these enzymes, cyanide-grown cells were also induced for formate dehydrogenase (FDH), providing a means of recycling NADH utilized by cyanide oxygenase.
- Characteristics of Primary Cilia and Centrosomes in Neuronal and Glial Lineages of the Adult Brain
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Primary cilia are sensory organelles that are important for initiating cell division in the brain, especially through sonic hedgehog (Shh) signaling. Several lines of evidence suggest that the mitogenic effect of Shh requires primary cilia. Proliferation initiated by Shh signaling plays key roles in brain development, in neurogenesis in the adult hippocampus, and in the generation of glial cells in response to cortical injury. In spite of the likely involvement of cilia in these events, little is known about their characteristics. Centrosomes, which are associated with primary cilia, also have multiple influences on the cell cycle, and they are important in assembling microtubules for the maintenance of the cell’s cytoskeleton and cilia. The cilia of terminally differentiated neurons have been previously examined with respect to length, incidence, and receptors present. However, almost nothing is known about primary cilia in stem cells, progenitors, or differentiated glial cells. Moreover, it is not known how the properties of cilia and centrosomes may vary with cell cycle or proliferative potential, in brain or other tissues. This dissertation focuses first on neurogenesis in the hippocampal subgranular zone (SGZ). The SGZ is one of the few brain regions in mammals that gives rise to a substantial number of new neurons throughout adulthood. The neuron lineage contains a progression of identifiable precursor cell types with different proliferation rates. This present study found that primary cilia were present in every cell type in the neuronal lineage in SGZ. Cilium length and incidence were positively correlated among these cell types. Ciliary levels of adenylyl cyclase type III (ACIII) levels relative to ADP-ribosylation factor-like protein 13b (Arl13b) was higher in neurons than in precursor cells and glia, and also changed with the cell cycle. G-protein coupled receptors, SstR3, MCHR1, and Gpr161 receptors were only found in neuronal cilia. The levels and distribution of three centrosomal proteins, γ-tubulin, pericentrin and cenexin in neurons was different from the distributions in precursors and glia. The second focus of study is glial responses to injury in the neocortex, which has been widely studied as an injury model. This study found that in the normal adult somatosensory cortex, primary cilia were present in astrocytes and polydendrocytes but not in microglia. Following injury, the incidence of primary cilia decreased in astrocytes. Also, a new cell type expressing GFAP, NG2 and Olig2 was seen 3 days following injury, but was not present in normal mice. The characteristics of primary cilia and centrosome described here suggest that in stem cells and progenitors their characteristics may be well suited for proliferation, whereas in neurons, the cilia and centrosomes are important for other sensory functions.
- Characterization of a Human 28S Ribosomal RNA Retropseudogene and Other Repetitive DNA Sequence Elements Isolated from a Human X Chromosome-Specific Library
- Three genomic clones encompassing human DNA segments (designated LhX-3, LhX-4, and LhX5) were isolated from an X chromosome-specific library and subjected to analysis by physical mapping and DNA sequencing. It was found that these three clones are very rich in repetitive DNA sequence elements and retropseudogenes.
- Characterization of Arthrobacter Globiformis Aspartate Transcarbamylase Concentrations of Substrates
- This thesis consists of one major section with two subsections. The first subsection investigates the activity of Arthrobacter globiformis aspartate transcarbamylase's specific activity with increasing concentrations of the enzyme's substrate. Dihydroorotase (DHOase) activity was also measured with increasing concentrations of the substrate dihydroorotate. The second subsection collected data in order to classify the enzyme, resulting in a classification into the category of class A ATCases with bifunctional ATCase-DHOase complexes. The thesis provides evidence to broaden understanding of the ATCase and DHOase enzymes for members of the family that Arthrobacter belongs to.