High performance liquid chromatography was used to measure ribonucleoside triphosphates in microbial samples. Anion exchange columns in a radial compression module were used to separate and quantify purine and pyrimidine ribonucleotides. Endogenous ribonucleoside triphosphates were extracted from Escherichia coli and pseudomonas aeruginosa using three different solvents, namely trifluorocetic acid (TFA; 0.5M), trichloroacetic acid (TCA; 6 per cent w/v) and formic acid (1.0M) Extracts were assayed for uridine 5'-triphosphate (ATP), and guanosine 5'-triphosphate (GTP) by using anion exchange radial compression high performance (pressure) liquid chromatography. The three extraction produres were compared for yield of triphosphates. E. coli, the TFA extraction procedure was more sensitive and reliable than TCA and formic acid extraction procedures, but , in P. aeruginosa, the best yields of ATP and GTP were obrained following extraction with TFA. Yields of UTP and CTP increased when extraction was performed in TCA. These data illustrate that different extraction produres produce different measures for different triphosphates, a point often overlooked.

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 …

Polyaromatic hydrocarbons (PAHs) are contaminants synthesized through incomplete combustion of carbon based substances. PAHs are known to be photodynamic and toxicity increases exponentially when in contact with ultraviolet radiation (UV). The effect of UV absent recovery periods and potential for latent toxicity during photo-induced toxicity are previously unknown and are not included within the toxicity model. Results of equal interval tests further support the current reciprocity model as a good indicator of PAH photo-induced toxicity. Interval test results also indicate a possible presence of time-dependent toxicity and recovery thresholds and should be included into toxicity risk assessments. Moreover, results of latent effects assays show that latent mortality is a significant response to PAH photo-induced toxicity and should be included into toxicity risk assessments. The present research demonstrates that UV exposure time rate is a significant driving force of PAH photo-induced toxicity.

Bioconcentration factor testing serves as the most valuable surrogate for the assessment of bioaccumulation. The assessment of potentially harmful chemicals is crucial to not only the health of aquatic environments, but to humans as well. Chemicals that possess the ability to persist in the environment or that have the potential to bioaccumulate, pose a greater risk to organisms that are exposed to these chemicals. The Organization for Economic Cooperation and Development Guideline 305 outlines specific protocols to run an accurate and reliable aquatic flow-through test. However, since its adoption in 1996, very few changes have been made to accommodate the endeavor to lowering the amount of test species to run one of these said tests. Running an aquatic flow-through test, according to 305, takes much time and money as well as numerous amounts of fish. Such burdens can be eliminated through simple modifications to the standard protocols. In this study, we propose an abbreviated study design for aquatic bioconcentration testing which effectively alleviates the burdens of running a flow-through test. Four chemicals were used individually to evaluate the usefulness of the proposed shortened design; 4-Nonyphenol, Chlorpyrifos, Musk Xylene, and DDT. The study consisted of exposing Cyprinus carpio for 7 days …

The value of the adenylate energy charge [(ATP)+1/2(ADP)/(ATP)+(ADP)+(AMP)] in Azotobacter vinelandii cells was monitored during growth and germination in flask cultures. The miximal value of 0.88 was attained during mid-log phase; this declined gradually to 0.50 by late stationary phase. When these cultures were transferred to encystment media, the adenylate energy charge decreased to an average value of 0.40 as the vegetative cells encysted and remained unchanged during the next 20 days. Encystment cultures wre composed of vegetative cells, encysting cells and mature cysts but the proportionate value of the energy charge could be assigned. Viability of the total population remained 95% or higher during the entire period studied. Azotobacter vinelandii cysts cultivated on phosphate-sufficient media. Although cell protein and nucleic acids were unaffected by phosphate deficiency, cell wall structures, oxygen uptake and sncystment were significantly affected. Phosphate-limited cysts contained much larger amounts of poly-beta-hydroxybutyric acid but had a lower adenylate energy charge than did control cysts. The ATP/ADP ratio was much lower in phosophate-deficient cysts than in the control cysts. The data indicate a "substrate saving" choice of three metabolic pathways available to cells of Azotobacter under different growth conditions.

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.

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 …

Crude oil commonly enters freshwater aquatic ecosystems as thin sheens forming on the water surface. Oil contains mixtures of toxic compounds called polycyclic aromatic hydrocarbons (PAHs), some of which are known to be photodynamic, increasing toxicity when combined with ultraviolet radiation. Benthic macroinvertebrate communities are commonly utilized as bioindicators, and as such rely on abundant data in literature concerning benthic macroinvertebrates' relative tolerances to a wide range of pollutants. A series of 10 plastic traps, half of which were filtered from UV radiation, were deployed in an urban pond for 27 days to determine colonization preferences of benthic macroinvertebrates to UV exposure. Results of this in situ experiment indicated that the majority of aquatic insects collected from traps inhabited the UV exposed treatment group, particularly the nonbiting midge, Chironomidae. A series of bioassays were then completed to investigate the sensitivities of a Chironomidae species to thin sheens of crude oil in the presence and absence of UV radiation. All bioassays were conducted using 10 day old Chironomus dilutus larvae cultured in the lab. The series of C. dilutus bioassays were all conducted under the same water quality parameters, temperatures, and oil sheen dosing methods, under a 16:8 photoperiod and exposed …

Spontaneously active monolayer neuronal networks cultured on photoetched multimicroelectrode plates (MMEPs) offer great potential for use in studying neuronal networks. However, there are many problems associated with frequent, long-term use of MMEPs. The major problems include (1) polysiloxane insulation deterioration and breakdown, (2) and loss of gold at the gold electroplated indium-tin oxide (ITO) electrodes. The objective of this investigation was to correct these major problems. Quality control measures were employed to monitor MMEP fabrication variables. The phenotypes of polysiloxane degradation were identified and classified. Factors that were found to contribute most to insulation deterioration were (1) moisture contamination during MMEP insulation, (2) loss of the quartz barrier layer from excessive exposure to basic solutions, and (3) repetitive use in culture. As a result, the insulation equipment and methods were modified to control moisture-dependent insulation deterioration, and the KOH reprocessing solution was replaced with tetramethylguanidine to prevent damage to the quartz. The problems associated with gold electroplating were solved via the addition of a pulsed-DC application of gold in a new citrate buffered electroplating solution.

Cannabinoid (CB) receptors have been found in most vertebrates that have been studied. The location of various CB receptors in the body and brain are known, but their physiological functions are not fully understood. The effects CBs have on the cardiovascular system have been of growing interest in recent years. Increasing reports from emergency departments and law enforcement agencies detail acute cardiovascular and psychological effects from synthetic CB intoxication, such as JWH-018. This major health concern is substantiated by governmental agencies like the CDC and NIDA. This pilot study investigates the acute toxic effects of the synthetic CB, JWH-018, on the cardiovascular and neuroendocrine systems in Ictalurus punctatus (channel catfish). Research in organisms besides the traditional mammal models can provide new insights into CB function and physiology. Ictalurus punctatus lend multiple benefits as a model organism that permits researchers to investigate in vivo effects of both cardiovascular and neuroendocrine systems without much influence from traditional sampling methods, and further more provide ample size and tissue to perform specific cardiovascular experiments. Multiple methods were used to assess cardiovascular function and sympathetic nervous system activation. Two different doses, low (500 µg/kg) and high 1,500 µg/kg, of JWH-018 were evaluated in the study. …

Water quality of a stream or river is influenced by the surrounding landscape and vegetation. The Normalized Difference Vegetation Index (NDVI) is commonly used to characterize landcover and vegetation density. Benthic macroinvertebrates are ubiquitous in freshwater streams and are excellent indicators of the quality of freshwater habitats. Data from one NDVI remote sensing flight and one macroinvertebrate sampling event for eight wadeable stream study sites in the National Ecological Observatory Network (NEON) were acquired. Proportions of high, moderate, and sparse vegetation were calculated for each stream watershed using ArcGIS. Functional feeding groups and tolerance values were assigned to macroinvertebrate taxa. The Fourth-corner and RLQ methods of analysis, available in the ade4 package for R software, were used to evaluate the relationships of macroinvertebrate traits with environmental variables. Hypothesis testing using Model 6 in the ade4 package resulted in p-values of 0.066 and 0.057 for global (overall) significance. Mean NDVI values of moderately vegetated areas and proportion of sparse vegetation were found to be significant to percent shredders at alpha ≤ 0.05. Results of these methods of analysis, when combined with traditional macroinvertebrate sampling metrics, show that NDVI can be a useful, additional tool to characterize a watershed and its effects …

Acrolein is produced endogenously after traumatic brain injury (TBI) and is considered a primary mechanism for secondary damage occurring after TBI. We are using frontal cortex networks derived from mouse embryos and grown on microelectrode arrays in vitro to monitor the spontaneous activity of networks and the changes that occur after acrolein application. Networks exposed to acrolein exhibit a biphasic response profile. An initial increase in network activity, followed by a decrease to 100% activity loss in applications ≥ 50 µM. In applications below 50 µM, acrolein was not toxic but generated activity instability with coordinated but irregular population busts lasting for up to 6 days. The increase in activity preceding toxicity may be linked to a decrease in free spermine, a free radical scavenger that modulates Na+, K+, Ca+ channels as well as NMDA, Kainate, and AMPA receptors. Action potential wave shape analysis after 20 and 30 µM acrolein application revealed a concentration-dependent 15-33% increase in peak to peak amplitude within minutes after exposure. For the same concentrations of acrolein (50 µM), the time required to reach 100% activity loss (IT100) was longer in serum-free medium than in medium with 5% serum, in which IT100 values were reduced by …

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.

Myosin subfragment-2 (S2) is an intrinsically unstable coiled coil. This dissertation tests if the mechanical stability of myosin S2 would influence the availability of myosin S1 heads to actin thin filaments. The elevated instability in myosin S2 coiled coil could be one of the causes for hypercontractility in Familial Hypertrophic Cardiomyopathy (FHC). As hypothesized FHC mutations, namely E924K and E930del, in myosin S2 displayed an unstable myosin S2 coiled coil compared to wild type as measured by Fluorescence Resonant Energy Transfer (FRET) and gravitational force spectroscopy (GFS). To remedy this, anti-S2 peptides; the stabilizer and the destabilizer peptides by namesake were designed in our lab to increase and decrease the stability of myosin S2 coiled coil to influence the actomyosin interaction. Firstly, the effectiveness of anti-S2 peptides were tested on muscle myosin S2 peptides across MYH11 (smooth), MYH7 (cardiac), and MYH2 (skeletal) with GFS and FRET. The results demonstrated that the mechanical stability was increased by the stabilizer and decreased by the destabilizer across the cardiac and skeletal myosin S2 isoform but not for the smooth muscle isoform. The destabilizer peptide had dissociation binding constants of 9.97 × 10-1 μM to MYH7 isoform, 1.00 μM to MYH2 isoform, and no …

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.

The present study sought to determine some morphological and physiological critical windows during very early development in zebrafish exposed to crude oil. I hypothesized that exposed zebrafish would present a decrease in survival rate and body mass, and an increase in routine oxygen consumption (ṀO2), and critical oxygen tension (PCrit). To test these hypotheses, zebrafish were acutely exposed (24 h) during different days of development (1 to 6 days post-fertilization, dpf) to different concentrations of high-energy water-accommodated fractions (HEWAFs). The endpoints of survival, body mass, routine oxygen consumption, and critical oxygen partial pressure were measured at 7 dpf. Survival rate decreased based on the exposure concentration but not as a function of the day of crude oil exposure. No significant effects were found in PCrit. Body mass was reduced by the different concentrations of HEWAF, with the size of the effect varying with exposure day, with the effect strongest on when exposure occurred at 2 and 3 dpf. Oxygen consumption (ṀO2) differed significantly depending upon the day of exposure in fish exposed to crude oil. Specifically, HEWAF exposure significantly increased ṀO2 in larvae exposed at 3 dpf (9.081 µmol O2/g/h, ±0.559) versus 2 dpf (6.068 µmol O2/g/h, ±0.652) and 6 …

Current energy and environmental challenges are driving the use of cellulosic materials for biofuel production. A major obstacle in this pursuit is poor ethanol tolerance among cellulolytic Clostridium species. The first objective of this work was to establish a potential upper boundary of ethanol tolerance for the cellulosome itself. The hydrolytic function of crude cellulosome extracts from C. cellulolyticum on carboxymethyl cellulose (CMC) with 0, 5, 10, 15, 20 and 25% (v/v) ethanol was determined. Results indicated that the endoglucanase activity of the cellulosome incubated in 5% and 10% ethanol was significantly different from a control without ethanol addition. Furthermore a significant difference was observed in endoglucanase activity for cellulosome incubated in 5%, 10%, 15%, 20% and 25% ethanol in a standalone experiment. Endoglucanase activity continued to be observed for up to 25% ethanol, indicating that cellulosome function in ethanol will not be an impediment to future efforts towards engineering increasing production titers to levels at least as high as the current physiological limits of the most tolerant ethanologenic microbes. The second objective of this work was to study bioethanol production by a microbial co-culture involving Clostridium cellulolyticum and a recombinant Zymomonas mobilis engineered for the utilization of oligodextrans. The …

Environmental air pollution is one risk factor associated with the onset and progression of multiple sclerosis (MS). In this project, we investigated the effects of ubiquitous traffic-generated pollutants, namely a mixture of gasoline and diesel vehicle exhaust (MVE), on signaling pathways associated with the pathophysiology of MS in the central nervous system (CNS) of either ovary intact (ov+) or ovariectomized (ov-) female Apolipoprotein (Apo) E-/-. Specifically, we investigated whether a subchronic inhalation exposure to MVE (200 PM μg/m3; 6 hr/d, 7d/wk, 30d) vs. filtered air (FA) controls altered myelination, T cell infiltration, blood-brain barrier (BBB) integrity, or production of reactive oxygen species (ROS) and expression of neuroinflammation markers in the CNS ov+ and ov- Apo E-/- mice. Our results revealed that inhalation exposure to MVE resulted in increased demyelination and CD4+ and CD8+ T cell infiltration, associated with alterations in BBB integrity. Disruption of the BBB was evidenced by decreased tight junction (TJ) protein expression, increased matrix metalloproteinase (MMPs) activity, and increased permeability of immunoglobin (Ig) G, which were more pronounced in the MVE ov- group. Moreover, MVE-exposure also promoted ROS and neuroinflammatory signaling in the CNS of ov+ and ov- mice, compared to FA groups. To analyze mechanisms that …

A common measurement of body temperature during exercise in a hot, humid environment is mean skin temperature collected from 3-12 sites on the body. However, such an approach fails to demonstrate localized differences in skin temperature that are likely to exist as a function of gender. The purpose of this study was to examine potential differences in skin temperature between men and women at 17 different locations on the body. Young women (21 ± 1 y; n = 11) and men (23 ± 3; n = 10) were recruited to complete a 60-min walk/jog interval protocol in a hot (34 ± 1 °C), humid (64 ± 8%) environment while skin temperature was measured. Data was analyzed using a repeated-measures ANOVA (p < 0.05) and location of interaction effects determined using a Fisher’s least squares difference test. We observed a higher change (p < 0.05) from baseline skin temperatures (ΔTsk) for women in three locations: left upper back (women: avg. ΔTsk = 4.12 ± 0.20 °C; men: avg. ΔTsk = 2.70 ± 0.10 °C), right upper back (women: avg. ΔTsk = 4.19 ± 0.07 °C; men: avg. ΔTsk = 2.92 ± 0.05 °C), and right mid-back (women: avg. ΔTsk = 4.62 …

N-Acylethanolamines (NAEs) are bioactive acylamides that are present in a wide range of organisms. Because NAE levels in seeds decline during imbibition similar to ABA, a physiological role was predicted for these metabolites in Arabidopsis thaliana seed germination and seedling development. There is also a corresponding increase of AtFAAH (fatty acid amide hydrolase), transcript levels and activity, which metabolizes NAE to ethanolamine and free fatty acids. Based on whole genome microarray studies it was determined that a number of up-regulated genes that were responsive to NAE were also ABA responsive. NAE induced gene expression in these ABA responsive genes without elevating endogenous levels of ABA. It was also determined that many of these NAE/ABA responsive genes were associated with an ABA induced secondary growth arrest, including ABI3. ABI3 is a transcription factor that regulates the transition from embryo to seedling growth, the analysis of transcript levels in NAE treated seedlings revealed a dose dependent, inverse relationship between ABI3 transcript levels and growth, high ABI3 transcript levels were associated with growth inhibition. Similar to ABA, NAE negatively regulated seedling growth within a narrow window of early seedling establishment. When seedlings are exposed to NAE or ABA within the window of sensitivity, …

The applicability of an isotopically labelled assay system to determine the lipase production in Pseudomonas aeruginosa was evaluated. Supernatant from cultures of Pseudomonas aeruginosa grown in a medium containing olive oil was incubated with a substrate containing labelled trioleate. Fatty acids were isolated by means of a liquid-liquid partition system. Enzyme activity was determined by measuring the amounts of free fatty acid by liquid scintillation counting. Findings indicate that the isotopicallylabelled, liquid-liquid partitioning assay is reliable, sensitive and adaptable to rapid assay conditions. It was also determined that different strains of Pseudomonas aeruginosa produce varying amounts of lipase. Partial purification of supernatant by gel filtration produced two protein peaks showing enzymatic activity.

Olestra is, a sucrose polyester, a noncaloric fat substitute, made from sucrose and several fatty acid esters. It has been approved by the FDA as a food additive used in preparing low-fat deep-frying foods such as savory snacks. Available literature on olestra was evaluated that had both positive and negative connotations. Clinical trials in numerous species of animals including humans were conducted to determine if olestra would affect the utilization and absorption of macro- and micronutrients; the effects of olestra on growth, reproduction, or its toxicity were also examined. The roles of olestra as a fat substitute, how it could effect on humans and the environment, and the potential impacts from its use in large amounts were assessed. Olestra can be removed from the environment by aerobic bacteria and fungi which may be isolated from activated sludge and soils.

Hypoxia as a stressor has physiological implications that have been a focal point for many physiological studies in recent years. In some studies, hypoxia had large effects on the organ tissue degeneration, which ultimately effects multiple ecological processes. These organ tissue studies played a part in the development of new fields like neurocardiology, a specialty that studied the relationship between the brain and the heart. This thesis focuses on how hypoxia-induced cardiac arrest alters the amounts of GLYT2, a glycine reuptake transporter, in the central nervous system of zebrafish, Danio rerio. At 7 days post-fertilization (dpf), zebrafish were exposed to acute, severe hypoxia until they lost equilibrium, and minutes later, subsequent cardiac arrest occurred. Zebrafish were then placed into recovery groups to measure the GLYT2 levels at multiple points in zebrafish recovery. Fish were then sacrificed, and their brains dissected. Using immunofluorescence, the outer left optic tectum of the zebrafish was imaged, and mean image pixel fluorescent intensity was taken. There were significant changes (one-way ANOVA) in the levels of GLYT2 compared to that of the control groups during the course of recovery. GLYT2 levels continued to rise through the 24-hour recovery mark but did not show significant difference after …

To better understand the nature and function of N-acylethanolamines (NAEs) in Camelina sativa, we used mass spectrometry analysis to identify and quantify NAE types in developing seeds, desiccated seeds and seedlings. Developing seeds showed a differential increase in individual NAE species and an overall increase in NAE content with seed development and maturation. The NAE composition in mature, desiccated seeds mostly reflected the total fatty acid composition in the seed tissues, except for a noted absence of 11-eicosenoic (20C monounsaturated) fatty acid in the NAE pool. During seed stratification and seedling growth, individual NAE species were depleted at similar rates. Simulated drought treatments during seedling development resulted in a significant rise in NAE levels for the major 18C NAE types compared with untreated seedlings. Arabidopsis and Camelina mutants with reported altered fatty acid profiles were analyzed for their NAE compositions; both Arabidopsis and Camelina had relatively similar changes between compositions of total seed fatty acids and NAEs. Furthermore, seeds were analyzed from transgenic Arabidopsis and Camelina with engineered, non-native, long-chain polyunsaturated fatty acids (18C, 20C and 22C), and the results showed the production of novel N-acylphosphatidylethanolamines (presumed precursors of NAEs) and NAEs with the same long acyl chains. These results …