2,5-Diphenyloxazole (PPO) was tested as a potential alternate inducer for the aryl hydrocarbon hydroxylase (AHH) system. Its apparent lack. of carcinogenicity and toxicity provide a possible system for investigation of enzyme systems related to chemical carcinogenesis without exposure of the researcher to potent carcinogenic compounds. These studies found PPO to be an inducer of AHH in cultured human lymphocytes. When PPO was utilized as a substrate for the AHH assay system, the major metabolites produced were strongly fluorescent. A simple fluorometric assay was developed which employed PPO as the substrate and which measured constitutive activity more efficiently than similar assays using benzo(a)pyrene as the substrate. Quantitation of both basal and induced lymphocyte AHH metabolism of PPO may be applicable to human population studies and may provide a tool to determine possible genetic variables with respect to carcinogen metabolism related to cancer risk.

Bacteria classified in the family Methylomonadaceae must derive their carbon from one-carbon compounds. They are characterized by the possession of internal membranes of two types. Type I membranes are layered and fill the middle of the cells while type II membranes form concentric layers around the periphery of the cells. Also, there are two metabolic pathways by which the methylobacteria assimilate one-carbon compounds. Further evidence of this dichotomy was sought by DNA-DNA saturation hybridization of DNAs from both types of methylobacteria. Very low DNA-DNA homology was seen between types I and II or within the types. It was not possible, therefore, to correlate the degree of genetic relatedness with either the nature of the internal membranes or the pathway of carbon assimilation.

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 …

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.

Six reagent waters, eleven diets, and two culture sizes were evaluated for culturing C. dubia. Different filtration techniques were used to prepare the reagent waters. The eleven diets were comprised of two algae augmented with eight supplements. Reproduction and growth were assessed to discern differences among C. dubia raised in mass cultures and cultured in individual cups, during which, bacterial population densities, lipid, protein, and carbohydrate concentrations of the diets were measured. Results showed that a glass-distilled, carbon filtered, deionized reagent water and a Selenastrum capricornutum- Cerophyl® diet were optimum for culturing. Mass culturing supported the highest reproduction and growth, while no correlation was found between nutritional measurements and production.

Pseudomonas aeruginosa is a Gram-negative rod, aerobic, non-fermenting, oxidase positive, pigment producing, and nutritionally versatile bacterium. Infections by P. aeruginosa are the most important cause of morbidity and mortality in immunocompromised patients, given virulence factor production that suppresses antibiotic therapy and promotes persistent infection. This research is the first comprehensive report of the pyrimidine biosynthetic pathway for all phases of growth in minimal and rich media coupled with the evaluation of virulence factor production of P. aeruginosa in comparison to four other bacterial species (Pseudomonas putida, Pseudomonas fluorescens, Burkholderia cepacia, and Escherichia coli wild-type strains). Cellular growth and passing genetic information to the next generation depend on the synthesis of purines and pyrimidines, the precursors of DNA and RNA. The pyrimidine biosynthetic pathway is essential and found in most organisms, with the exception of a few parasites that depend upon the pyrimidine salvage pathway for growth. Both the pyrimidine biosynthetic and salvage enzymes are targets for chemotherapeutic agents. In our laboratory, research on pyrimidine auxotrophic mutants showed the role of the pyrimidine biosynthetic pathway and its intermediates on P. aeruginosa metabolism and impaired virulence factors production. The present research shows that pyrimidine enzymes are active in all phases of growth, …

Plant estrogens have long been associated with health benefits. The potential of tissue culture techniques for the production of several secondary metabolites has been known for many years. Tissue cultures stimulate the production or induce the biosynthesis of novel compounds not found in the mature plant. Tissue culture of Morus alba, family Moraceae, is known to contain phytoestrogens, was established on plant-hormone supplemented Murashige and Skoog (MS) medium. Petiole and the stem tissue from mature trees were the best explants for initiation and proliferation of calli. The best callus proliferation was obtained on MS medium containing 1-napthalene acetic acid (1mg/ml) and benzylaminopurine (0.5mg/ml) for M. alba. Comparison of phytoestrogens of Moraceae species from in vivo and in vitro tissue isolation were carried out. The estrogenic activities of callus extracts were assayed in an estrogen-responsive yeast system expressing the human estrogen receptor alpha. Male callus extracts had higher estrogenic activity than male and female extracts from in vivo and in vitro tissues. Isolation and characterization of phytoestrogens from above tissues were carried out using solid phase extraction, high perfomance liquid chromatography and mass spectrometry techniques. Biochanin A, an isoflavonoid, was isolated as one of the compounds in male callus extracts. Biochanin …

Pyrimidine salvage pathways are essential to all cells. They provide a balance of RNA synthesis with the biosynthetic pathway in pyrimidine prototrophs and supply all the pyrimidine requirements in auxotrophs. While the pyrimidine biosynthetic pathway is found in almost all organisms and is nearly identical throughout nature, the salvage pathway often differs from species to species, with aspects of salvage seen in every organism. Thus significant taxonomic value may be ascribed to the salvage pathway. The pyrimidine salvage pathways were studied in 55 microorganisms. Nine different salvage motifs, grouped I-IX, were identified in this study based on the presence of different combinations of the following enzymes: cytidine deaminase (Cdd), cytosine deaminase (Cod), uridine phosphorylase (Udp), uracil phosphoribosyltransferase (Upp), uridine hydrolase (Udh), nucleoside hydrolase (Nuh), uridine/cytidine kinase (Udk), 5'-nucleotidase and CMP kinase (Cmk).

Sirtuins (SIRT) regulate the transcription of various genes involved in the development of diet-induced obesity and chronic disease; however, it is unknown how they change acutely following a high-fat meal. The purpose of this study was to determine the effect of a high-fat meal (65% kcals/d; 85% fat recommendation), on SIRT1-7 mRNA expression in blood leukocytes at 1, 3, and 5-h post-prandial. Men and women (N=24) reported to the lab following an overnight fast (>12H). Total RNA was isolated and reverse transcribed prior to using a Taqman qPCR technique with 18S rRNA as a normalizer to determine SIRT1-7 mRNA expression. An additional aliquot of serum was used to measure triglycerides. Data was analyzed using a RM ANOVA with P<0.05. Triglycerides (P<0.001; 124%) peaked at 3-h. SIRT 1 (P=0.004; 70%), and SIRT 6 (P=0.017; 53%) decreased expression at 3-h. SIRT4 (P=0.024) peaked at 5H relative to baseline (70%) and 3-h (68%). To our knowledge, this is the first study to report that consumption of a high-fat meal transiently alters SIRT mRNA expression consistent in a pattern that mirrors changes in serum triglycerides. Decrease in expression of SIRT1 and SIRT6 combined with an increased SIRT4 would be consistent with an increase in …

The American Kestrel (Falco sparverius) is the smallest and most abundant falcon in North America with a wide geographic range. Unfortunately, surveys have suggested that some kestrel populations have been in decline since the 1950s, though the nominal causes of this decline are unknown. Migratory movement patterns and connectivity have yet to be established for any population of migratory kestrels. In Chapter 2, I investigated methods for attaching migration trackers to kestrels. Specifically, I showed that leg-loop style harnesses may have negatively affected return rates whereas backpack harnesses did not. Based on these results, I recommend that backpack-style Teflon harnesses is the safest and most effective method for attaching tracking devices to small raptors. In Chapter 3, I quantified survivorship for kestrels wintering in north Texas to identify the timing of kestrel mortality. Notably, I found that juvenile kestrels had similar annual survival rates as adults (81.6% versus 79.5%). High overwintering survival in north Texas indicated that once kestrels arrived on their wintering grounds, they were highly likely to survive to spring migration. In Chapter 4, I investigated pairing behaviors previously undocumented in wintering kestrels. I found that winter pairing was relatively common, but more prevalent in urban environments than …

The effects of single injections and daily oral administration of ethanol on plasma levels of ACTH, beta endorphin, and corticosterone in response to cold stress were examined. The long-term experimental animals were given 0.25 ml of 28% ethanol or water orally once a day, five days a week, for fourteen months. Plasma levels of ACTH, beta endorphin, and corticosterone were lower in alcohol-treated rats as compared with water-treated rats when exposed to cold stress. The effects of a single injection of ethanol significantly elevated plasma levels of all three hormones. Mortality in sham-treated males was higher than ethanol-treated.

Mercury (Hg) is a ubiquitous environmental contaminant, which is capable of global atmospheric transport. As a result, even the most pristine aquatic ecosystems are affected by atmospheric Hg deposition, following which microbial transformation yield organic Hg forms, the most concerning of which is methylmercury (MeHg). Methylmercury is capable of bioaccumulation and biomagnification in food webs, resulting in potentially toxic body burdens due to regular dietary exposure in long-lived organisms at higher trophic levels. It is also a molecular mimic of some endogenous amino acids, providing a route of transfer from mother to offspring via large amino acid transporters. Exposure during neurodevelopment can lead to serious, irreversible neurological dysfunction, associated with a variety of cognitive and motor abnormalities across species. The present studies evaluate the effects of maternally-transferred dietary MeHg, at environmentally relevant concentrations on early life stage fathead minnows (Pimephales promelas). Embryos were collected from adult fatheads exposed to one of three diets with varying concentrations of MeHg for 30 days. Adult reproductive metrics were also monitored over the course of the study, with results indicating no effects on spawning frequency, clutch size, or total egg output. In embryos, Hg concentration was a function of female diet and the duration …

Proteomic techniques were used to evaluate the protein profile of the earthworm, (Lumbricus terrestris), following a bacterial challenge. One control group received no injection; a second control group received injections of phosphate buffer solution (PBS). The experimental group received injections of PBS containing (Aeromonas hydrophila). After incubation for 12 hours at 20°C, coelomic fluid was collected from each group for analysis by 2-D electrophoresis. There were significant differences in spot appearance and density between control and experimental groups. Sixteen spots showed a two-fold increase in density and 63 showed at least a two-fold decrease in density between samples from control and bacteria-challenged earthworms, respectively, suggesting up- and down-modulation of proteins potentially involved in the earthworm's response to bacterial challenge.

To examine if a rhythm can be entrained in either heart rate or oxygen consumption in late stage embryos (days 17-19.5) with light as a zeitgeber, chicken embryos were incubated in complete darkness (D:D) and 12:12 light:dark cycle (L:D). Light had no impact on oxygen consumption (390 µL O2∙min-1∙egg-1) but increased heart rate for non-internally pipped embryos (260 to 270 beats∙min-1 during light cycle). Oxygen consumption increased independent of pipping while heart rate increased (255 to 265 beats∙min-1) in D:D embryos due to pipping. A light-induced rhythm or effect occurred in heart rate but not oxygen consumption, suggesting heart rate and oxygen consumption may be uncoupled.

The purpose of this study was to examine the hormonal response to acute ethanol ingestion following a bout of heavy resistance exercise in women. Eight resistance trained women completed two identical acute heavy resistance exercise tasks (AHRET). From 10-20 minutes post-AHRET, participants consumed either a grain ethanol or a placebo beverage. Blood was collected before (PRE) and immediately after the AHRET (IP) and then every 20 minutes for five hours. Blood collected after beverage ingestion was pooled into 3 batches (phases: 20-40 minutes, 60-120 minutes, and 140-300 minutes post-exercise) and analyzed for serum total testosterone (TT), free testosterone (FT), insulin-like growth factor-I (IGF-I), human growth hormone (GH), cortisol (COR), and estradiol (E2) concentrations. Circulating concentrations of TT were significantly greater at P20-40 than at PRE, P60-120, and P140-300. Circulating concentrations of FT were significantly greater at P20-40 than at all other times. Circulating concentrations of GH were significantly greater at IP than at PRE, P60-120, and P140-300. Circulating concentrations of COR were significantly greater at P20-40 than at all other times. Additionally, COR concentrations at P140-300 were significantly lower than at all other times. Circulating concentrations of IGF-1 were significantly greater at P20-40 than at P60-120 and P140-300. Circulating concentrations …

Lactate dehydrogenase was isolated in pure form from crude extract of the cestode Hymenoleois diminuta by heat treatment and column chromatography. The purified enzyme has a specific activity of 106 units per mg protein. The molecular weight of the purified protein was 75,000 as determined by Sephadex gel filtration and analytical ultracentrifugation. An equilibrium ultracentrifugation study suggests a subunit molecular weight of 39,000. From these data, a dimer form of the native enzyme is proposed.

Plants have many nitrate transporters; in the model legume Medicago truncatula, MtNPF1.7 is among them. MtNPF1.7 is important for M. truncatula growth and it has been established that MtNPF1.7 is a high affinity nitrate transporter. M. truncatula plants with mutations in MtNPF1.7 gene show defects during plants growth, with striking abnormalities in nodule development and root architecture. Nitrogen fixation is an energy expensive process; when legumes have sufficient bioavailable nitrogen like nitrate available, it suppresses nodulation and nitrogen fixation. Previous preliminary results in our lab showed that plants constitutively expressing MtNPF1.7 have a growth phenotype in the absence of nitrate, but no data was available on how M. truncatula plants constitutively expressing MtNPF1.7 are affected by the presence of nitrate. For my research, I confirmed the preliminary results on the growth of M. truncatula plants overexpressing NPF1.7 and examined these plants' phenotypes when nitrate was not provided in the growth media and when it was provided at two different concentrations. Compared with wild type A17, plants constitutively expressing MtNPF1.7 gene grow larger, have more lateral roots and more nodules when grown in the absence of nitrate and when 0.2 mM KNO3 was provided. At 1 mM KNO3, there are fewer …

Changes in trophic status are often related to increases or decreases in the allocthonous inputs of nutrients from changes in land use and management practices. Lake and reservoir managers are continually faced with the questions of what to monitor, how to monitor it, and how much change is necessary to be considered significant. This study is a compilation of four manuscripts, addressing one of these questions, using data from six reservoirs in Texas.

Electrophoretic variation at twenty gene loci, patterns of behavioral thermoregulation, and genotype-specific malate dehydrogenase kinetics were investigated among populations of the red shiner, Notropis lutrensis, and the blacktail shiner, N. venustus, collected from thermally altered and thermally unaltered portions of their ranges. Genic variation was found to be high among red shiners and low among blacktail shiners. The behavioral response of the blacktail shiner to temperature was fixed among the populations sampled, whereas the response of the red shiner was mutable. Finally, blacktail shiners have incorporated into their genome an Mdh-B allele which functions well at low temperatures; red shiners, displaying high levels of Mdh-B polymorphism, maintain a more complex set of allozymes which function well over a wide range of environmental temperatures. These data are consistent with reported ecotypic distributions of the species in Texas waters; i.e., blacktail shiners occur in cool, thermally static habitats, and red shiners are tolerant of wide temperature ranges.

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 …

Sertraline is a selective serotonin reuptake inhibitor (SSRI) that is widely used for the treatment of depression and anxiety. Due to the abundant therapeutic use of sertraline, low levels have been detected in municipal wastewater effluents suggesting that aquatic organisms may be exposed. The purpose of this study was to evaluate the steroidogenic effects of sertraline on larval (FHM) and adult female fathead minnows (FFHM), Pimephales promelas. Larval FHM were exposed to 0.1, 1, and 10 µg/L sertraline for 28 days and analyzed via RT-qPCR for differential expression of 11β-Hydroxysteroid dehydrogenase (11β-HSD), 20β-Hydroxysteroid dehydrogenase (20β-HSD), aromatase (CYP19), and nuclear thyroid receptor alpha (TRα). FFHM were exposed to 3 or 10 µg/L sertraline for 7 days with the brain and ovary excised at exposure termination. Juvenile FHM exposed to 0.1 μg/L sertraline had a significant upregulation of both 20β-HSD and TRα. FFHM exposed to 10 µg/L sertraline had a significant upregulation of 11β-HSD expression in brain tissue, while no steroidogenic changes were observed in the FFHM ovary. Similarly, in FFHM brain tissue, CYP19 and 20β-HSD expression levels were significantly higher in fish exposed to 10µg/L sertraline compared to control. The significance of these findings with respect to survival, growth and reproduction …

The purpose of this research was to determine the relationship of the slime layer antigen(s) to the "101" or LPS antigens and to attempt to evaluate the role of antibodies against the latter in protection against experimental infections in mice with the homologous strain of Pseudomonas aeruginosa. Results from agglutination tests, chromatographic separations, passive protection tests, and characterizations of the antigens by gel double diffusion do not support the concept that LPS is a necessary portion of the immunogenic material. The immunogenicity of LPS can be attributed to co-purification of residual amounts of slime layer antigens on the washed cells from which LPS was extracted.

As part of a pre-impoundment study for the Ray Roberts Reservoir Area, Landsat-5 multi-spectral scanner (MSS) imagery was used for cover type interpretation. This research was concerned with analysis techniques for MSS images and a comparison of results obtained using computer assisted interpretatin of MSS images and a comparison of results obtained using computer assisted interpretation of MSS images with those obtained using manual interpretation of false color infrared (CIR) photographs.

The classification of the Azotobacteraceae to the level of genus and species has been uncertain since the studies of Beijerinck in 1901. This dissertation represents an effort to establish a system of classification more complete than the one now in use. In this study, both qualitative and quantitative taxonomy were used in order to establish a well founded classification scheme. Qualitative methods included certain important morphological and physiological characteristics, isoenzyme patterns, and immunological reactions. Quantitative methods included numerical taxonomy (based on total morphological and physiological characteristics) and numerical analysis of protein profiles. All the data from these experiments were subject to comparison with other genotypic and phenotypic data.