Aspartate transcarbamoylase (ATCase) of Streptomyces was characterized and its interaction with other pyrimidine enzymes explored.

Bacillus subtilis (ATCC 19659) were damaged by exposure to various concentrations of glutaraldehyde, as shown by decreased germination rates. The damage caused was repaired or otherwise obviated by the presence of sodium lactate in the holding medium. When two different salts of lactic acid were compared for ability to overcome the effect of glutaraldehyde, it was found that calcium salt of lactate was more effective than the sodium salt. The damage repair system involved l-alanine, lactate and either the sodium or calcium ions. The study involved in determining the difference in efficiency of spore repair was due to an organic or an amino acii snowed that the presence of two carboxylic functional groups did not effectively alter the reactivity.

Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in pyrimidine biosynthesis. Bacterial ATCases are divided into three classes, A, B and C. Class A ATCases are largest at 450-500, are. dodecamers and represented by Pseudomonas ATCase. The overlapping pyrBC' genes encode the Pseudomonases ATCase, which is active only as a 480 kDa dodecamer and requires an inactive pyrC'-encoded DHOase for ATCase activity. ATCase has been studied in two non-pathogenic members of Mycobacterium, M. smegmatis and M. phlei. Their ATCases are dodecamers of molecular weight 480 kDa, composed of six PyrB and six PyrC polypeptides. Unlike the Pseudomonas ATCase, the PyrC polypeptide in these mycobacteria encodes an active DHOase. Moreover, the ATCase: DHOase complex in M. smegmatis is active both as the native 480 kDa and as a 390 kDa complex. The latter lacks two PyrC polypeptides yet retains ATCase activity. The ATCase from M. phlei is similar, except that it is active as the native 480 kDa form but also as 450,410 and 380 kDa forms. These complexes lack one, two, and three PyrC polypeptides, respectively. By contrast,.ATCases from pathogenic mycobacteria are active only at 480 kDa. Mycobacterial ATCases contain active DHOases and accordingly. are placed in class A1 . …

A recent chemical spill of 4-Methylcyclohexanemethanol (4-MCHM) in West Virginia left 300,000 people without water. Officials claimed that this compound is not lethally toxic, but potentially harmful if swallowed or inhaled, and can cause eye and skin irritation. Sittig's Handbook of Toxic and Hazardous Chemical Carcinogens reports high exposures from skin contact or inhalation may cause damage to the heart, liver, kidneys, and lungs, and may result in death. However, no quantitative data seem to exist and no references can be found on neurotoxicity. We have investigated the neurotoxicity of 4-MCHM using mammalian nerve cell networks grown on microelectrode arrays. Network spontaneous activity from multiple units (range 48 – 120 per network) were used as the primary readout. Individual units were followed based on spike waveforms digitized at 40 kHz (Plexon MNAP system). Dose response curves show the effective inhibitory concentration at 50 percent decrease (EC50) to average 27.4 microM SD±6.17. However, in the presence of 40 microM bicuculline, a competitive GABAA antagonist, the EC50 shifts to 70.63uM SD ±4.3; implying that early, low concentration exposures to 4-MCHM involve GABA activation. Initial activity loss occurs without active unit loss (defined as 10 or more template threshold crossing per min), indicating …

The Cardiac Profile, a pattern of serum enzyme changes seen within seventy-two hours after an AMI, is diagnostic aid for detecting occurrence of infarcts. The effects of exercise stress on the Cardiac Profile aid clinicians in avoiding diagnostic errors in patients immediately after exercise. Five male volunteers ran from six to ten miles. Serum enzyme levels were monitored serially three days before and five days after stress. Enzyme activity was determined spectrophotometrically and electrophoretically. Significant increases in total CPK and LDH were seen. An LDH 'one-two flip' occurred eight hours after exercise. No MB-CPK was found following the run.

Bacterial aspartate transcarbamoylases (ATCase's) are divided into three classes that correspond to taxonomic relationships within the bacteria. The opportunistic pathogen Moraxeila catarrhalis has undergone several reclassifications based on traditional microbiological criteria. The previously uncharacterized ATCase from M. catarrhalis was purified to homogeneity and its chemical properties characterized. The ATCase from M. catarrhalis is a class C ATCase with an apparent molecular mass of 480-520 kDa. The M. catarrhalis ATCase is a dodecomer composed of six 35 kDa polypeptides and six 45 kDa polypeptides. The enzyme has an unusually high pH optimum of greater than pH 10. The enzyme exhibited hyperbolic kinetic with a Km for aspartate of 2 mM. A single, separate 78 kDa dihydroorotase from M. catarrhalis was identified and it was not associated with ATCase. These data support the reclassification of M. catarrhalis out of the Neisseriaceae family.

A commonly prescribed contraceptive, the synthetic progestin norethindrone (NET) inhibits ovulation in humans. However, ecotoxicological data are lacking. Preliminary tests produced an LC50 for NET of > 1.0 mg/L (96-hour, fathead minnow (FHM) and medaka) and a NOEC of 242.0 µg/L, a LOEC of 485.0 µg/L (7-day, growth for FHM and medaka). Reproductive testing revealed a LOEC for fecundity of 24.1 ng/L (21 days, medaka). Further testing confirmed the LOEC of 24.1 ng/L while defining a NOEC of 4.7 ng/L (28 days, medaka). Effect of NET in medaka life-cycle exposure at concentrations exceeding 4.7 ng/L was evident. Few females were present in the 24.7 ng/L exposure concentration, with none in the 104.6 ng/L. Egg production was significantly reduced at concentrations exceeding 4.7 ng/L. Additionally, weight, condition factor and somatic indices were significantly different in males exposed to concentrations exceeding 4.7 ng/L. For fecundity and sexual differentiation; the NOEC was 4.7 ng/L, the LOEC 24.6 ng/L; growth and somatic indices, the NOEC was more appropriately 0.9 ng/L, with effect evident at 4.7 ng/L. Sexual differentiation of the F1 population was similar to the F0. A defining result of this test was development of exceptionally large ovaries in NET- exposed female medaka, …

This work explores the metabolism of mercury in liver and spleen tissue of fish from a methylmercury contaminated wetland. Wild-caught bass, catfish, bowfin and gar were collected. Macrophage centers, which are both reactive and primary germinal centers in various fish tissues, were hypothesized to be the cause of demethylation of methylmercury in fish tissue. Macrophage centers are differentially expressed in fish tissue based on phylogenetic lineage, and are found primarily in the livers of preteleostean fish and in the spleen of teleostean fish. Histology of liver and spleen was examined in both control and wild-caught fish for pathology, size and number of macrophage centers, and for localization of mercury. Total mercury was estimated in the muscle tissue of all fish by direct mercury analysis. Selenium and mercury concentrations were examined in the livers of wild-caught fish by liquid introduction inductively coupled plasma mass spectrometry (ICP-MS). Total mercury was localized in histologic sections by laser ablation ICP-MS (LA-ICP-MS). Mercury speciation was determined for inorganic and methylmercury in liver and spleen of fish by bas chromatography-cold vapor atomic fluorescence spectroscopy (GC-CVAFS). Macrophage center tissue distribution was found to be consistent with the literature, with a predominance of centers in preteleostean liver and …

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.

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 …

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.

Putative cotton cDNA clones and cognate genomic clones for a palmitoyl-acyl carrier protein (ACP) thioesterase (PATE) and an osmotin-like pathogenesis-related 5 (PR5) protein have been isolated and characterized. PATE is a class B fatty acid thioesterase with specificity for saturated long-chain fatty acids such as palmitate, and is implicated as a key enzyme to be targeted for regulation of fatty acid synthesis in order to alter cotton seed oil profiles. A nearly full-length 1.7-kb cDNA clone was isolated using a hybridization probe derived from an Arabidopsis PATE cDNA clone designated TE 3-2. A 17-kb genomic segment encompassing the PATE gene was also isolated, which has six exons and five introns with high sequence identity with other FatB cDNA/gene sequences. The deduced PATE preprotein amino acid sequence of 413 residues has putative signal sequences for targeting to the chloroplast stroma. PR5 proteins called osmotins are made in response to fungal pathogen stress or osmotic stress (water deprivation or salt exposure). Osmotins may actually form pores in fungal membranes, leading to osmotic rupture and destruction of the fungal cells. A cotton osmotin-like PR5 cDNA insert of 1,052 base-pairs was isolated and shown to encode a preprotein of 242 amino acids and is …

Concern over sediment toxicity has increased the need for toxicity test information with organisms that inhabit sediments. Oligochaetes are exposed to toxicants through feeding and direct body contact with aquatic sediments. Chronic testing with oligochaetes has historically focused on tubificids with test lengths of one year or more to encompass several generations. Most naidid oligochaetes have generation times of three to seven days and could provide chronic information in a matter of weeks. The cosmopolitan distributed naidid, Pristina leidyi, was evaluated for use as a toxicity test organism. Results of research conducted includes culture methods, effects of temperature on reproduction, growth rates in a reference sediment, acute toxicity tests, and chronic toxicity tests.

This research is designed to test how sublethal exposure to copper affects tadpole predator-escape behavior and how quickly tadpoles recover. After exposure, tadpoles were separated. Escape behavior was recorded for two-thirds of exposed tadpoles while one-third of the exposed population was measured weekly to determine growth and recovery. Control tadpoles were consumed within 15 minutes whereas those exposed to higher concentrations were consumed at a slower rate, which does not support the hypotheses. Although the rate of predation was lower, tadpoles exposed to higher Cu concentrations were on average, 1.47 cm in total body length. Those exposed to 0.93 mg/L averaged 0.86 cm. After being placed into clean water, treatment tadpoles recovered after 20 days.

Acclimation temperature had a significant effect on the lethality of copper to fathead minnows in replicated 96-hour lethality tests. Lowest median lethal copper concentrations (LC50) were recorded at 12 and 22°C, with LC50s at 5 and 32°C at least 140 μg/1 higher. This research found LC50 copper concentrations in the 300 to 500 μg/1 range and a polynomial relationship between LC50s and acclimation temperature. Following a 24-hour exposure to three sublethal concentrations of copper, critical thermal maxima (CTMax) were tested in minnows acclimated to four temperatures. Sublethal exposure to copper significantly decreased the CTMaxs relative to controls at three of the four temperatures. Control CTMaxs ranged from 28.6 to 40.4°C and increased 0.46°C for each 1°C increase in acclimation temperature.

Mefloquine hydrochloride is an antimalarial agent that has been used for the past 40 years. Numerous reports of neurological side effects have recently led the FDA to issue a strong warning regarding long-term neurological effects. This warning lead to the U.S. Army’s Special Forces and other components to discontinue its use in July of 2013. Despite reported adverse side effects, mefloquine remains in circulation and is recommended to travelers going to specific Asian countries. Mefloquine has been used as a treatment for those already infected with the malaria parasite (blood concentrations ranging from 2.1 to 23 µM), and as prophylaxis (blood concentrations averaging 3.8 µM) (Dow 2003). The purpose of this study was to quantify Mefloquine’s toxicity using spontaneously active nerve cell networks growing on microelectrode arrays in vitro and to identify compounds that alleviate or reduce toxic effects. The current literature on mefloquine toxicity is lacking electrophysiological data. These data will contribute to research on the mechanism of adverse side effects associated with mefloquine use. Sequential titration experiments were performed by adding increasing concentrations of mefloquine solution to cultured neurons. Network responses were quantified and reversibility was examined. In each network, activity decreases were normalized as a percent of …

In this project, the effects of dietary polyphenols on exercise-induced muscle damage and vascular health are examined. Dietary polyphenols exert well-known anti-inflammatory effects; however, how these effects are realized with respect to vascular health and EIMD is relatively unknown. I begin by reviewing the available literature surrounding the impact of three dietary polyphenols (curcumin, catechins, and quercetin) on inflammation associated with EIMD. It is well established that their primary means of anti-inflammation is through alterations of NF-κB and AP-1 transcription activities. Given this, their inclusion into training strategies seems reasonable. Consistent evidence is presented making a case for the anti-inflammatory effects of dietary polyphenols following EIMD. I follow this review up by completing an in-depth study on the consumption of curcumin prior to EIMD. I found curcumin (1000 mg/day) can reduce subjective soreness and decrease inflammation compared to placebo controls. To further understand the effects of dietary polyphenols on health, I investigate the effects of a four-week supplementation period of cocoa (catechins) on vascular. I concluded that atherogenic risk in obese women is reduced after consumption of cocoa. In addition to these experimental projects, I developed two novel methods that can be used to investigate vascular health (EMP concentration) and …

Research estimated toxicity of refuse-derived fuel fly ash (RDF-FA) on two earthworms species, Lumbricus terrestris and Eisenia foetida. Specific objectives were to: (1) Compare their 14-day LC50s under light and dark conditions; (2) separate toxicity due to osmotic, pH and physical factors from that of heavy metal contaminants; (3) compare relative differences of artificial soil and commercial soil as exposure media for evaluating toxicity to earthworms. The 14-d LC50s for L. terrestris in dark and light were 57.0 and 48.34 % RDF-FA, and 59.25 and 41.00 % RDF-FA for E. foetida using artificial soil. All of the toxicity resulted from heavy metals within the RDF-FA. Using L. terrestris, the LC50s for artificial soil and commercial soil were 52.30 and 64.34%.

I assessed fish community data collected by the Oklahoma Conservation Commission from 82 minimally impaired wadeable reference streams in eastern Oklahoma to determine whether existing aquatic ecoregions provide the best framework for spatial classification for the development of biological assessment methods and biocriteria. I used indirect ordination and classification to identify groups of sites that support similar fish communities. Although correspondence was observed between fish assemblages and three montane ecoregions, the classification system must be refined and expanded to include major drainage basins and physical habitat attributes for some areas to adequately partition variance in key measures of biological integrity. Results from canonical correspondence analysis indicated that substrate size and habitat type were the primary physical habitat variables that influenced the fish species composition and community structure.

Pseudomonas fluorescens does not appear to regulate the enzymes of de novo pyrimidine biosynthesis at the level of gene expression. Little or no apparent repression of pyr gene expression is observed upon addition of exogenous pyrimidines to the growth medium. The Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) were sized down and cloned into the broad host range plasmid, pKT230. Upon introduction into a P.fluorescenspyrB mutant strain, ATCase showed repression in response to exogenously fed pyrimidine compounds. Thus, it was possible to bring about changes in pyrimidine nucleotide pool levels and in transcriptional regulation of gene expression at the same time.

Superbursts (SBs) are large, seemingly spontaneous activity fluctuations often encountered in high density neural networks in vitro. Little effort has been put forth to define and analyze SBs which are paroxysmal bursting discharges. Through qualitative and quantitative means, I have described specific occurrences of superbursting activity. A complex of paroxysmal bursting has been termed a "superburst episode," and each individual SB is a "superburst event" which is comprises a fine burst structure. Quantitative calculations (employing overall spike summations and coefficient of variation (CV) calculations), reveal three distinct phases. Phase 1 is a "build up" phase of increasingly strong, coordinated bursting with an average of a 17.6% ± 13.7 increase in activity from reference. Phase 2, the "paroxysmal" phase, is comprised of massive coordinated bursting with high frequency spike content. Individual spike activity increases by 52.9% ± 14.6. Phase 3 is a "recovery phase" of lower coordination and an average of a 50.1% ± 35.6 decrease in spike production from reference. SBs can be induced and terminated by physical manipulation of the medium. Using a peristaltic pump with a flow rate of 0.4ml/min, superbursting activity ceases approximately 28.3 min after the introduction of flow. Alternatively, upon cessation of medium flow superbursting …