Principal objectives of my research were to: (1) report for the first time that coelomocytes are able to reduce NBT dye and confirm the presence of lysozyme-like activity in earthworm; (2) develop a standard methodology for determination of NBT reduction and lysozyme-like activity in earthworms; (3) compare NBT reduction and lysozyme-like activity in earthworms with those of murine and human cells and fluids; and (4) demonstrate the sensitivity of earthworm NBT reduction and lysozyme-like activity as the assays using matrics in refuse-derived fuel fly ash (RDFF) and CuSO4.

The effects of cannabinoid agonists on spontaneous neuronal network activity were characterized in murine spinal cord and auditory cortical cultures with multichannel extracellular recording using photoetched electrode arrays. Different cultures responded reproducibly with global decreases of spiking and bursting to anandamide and methanandamide, but each agonist showed unique minor effects on network activity. The two tissues responded in a tissue-specific manner. Spontaneous activity in spinal tissue was terminated by 1 μM anandamide and 6.1 μM methanandamide. Cortical activity ceased at 3.5 μM and 2.8 μM respectively. Irreversible cessation of activity was observed beyond 8 μM for both tissues and test substances. Palmitoylethanolamide, demonstrated that CB2 receptors were not present or not responsive. However, the data strongly suggested the presence of CB1 receptors.

The effects of short term exposure (5 minutes) to EEFs at relatively high dosages (10, 25, 39, kV/m) on the electrical activity in rat pineal glands was studied. Daytime and nighttime recordings were taken from an implanted microelectrode in the gland. The data show that (1) both the activity and frequency were enhanced when the animals were exposed to EEFs at 39 kV/m continuously and discontinuously; (2) the later condition yielded a sustained increase (36%) whereas the former a brief (10 sec) increase. This enhancement was statistically significant under both conditions (day and night). The effects observed were thought to be due to membrane alterations either in the pineal gland itself or in the neural inputs to the gland.

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.

The purpose of this project is to produce a complete and thorough notebook that may be used to supplement laboratory coursework. Its intent is to be used primarily by the students to aid them in understanding background information and the proper laboratory procedures involved in various types of experiments. The laboratory notebook is a summation of all the experiments and procedures used in the six-credit hour Advanced Microbial and Molecular Biology (BIOL 5160) course offered during the summer semester at the University of North Texas. This class is a team taught effort by Professors O'Donovan and Kunz. The course is constructed as an intensive practice exercise to teach the student about gene mutations, biosynthetic pathways, preparation and analysis of plasmid DNA, and many other topics included in the notebook.

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.

This study was designed to determine if impingement and entrainment by cooling water intake at the Comanche Peak Steam Electric Station have an adverse impact upon the Squaw Creek Reservoir fish population. The yearly impingement of fish was estimated to be 262,994 of 14 species. The threadfin shad (Dorosoma petenense) accounted for 96% of this total. Entrainment of eggs and larvae for a five month period was estimated to be 15,989,987 and 42,448,794 respectively. Two fish population studies were performed on Squaw Creek Reservoir to help assess impact. It was determined that the losses due to impingement and entrainment have no adverse impact upon the fish population of Squaw Creek Reservoir.

The flagellum of Trypanosoma brucei contains a family of antigenically related EF-hand calcium-binding proteins which are called the calflagins. Genomic Southern blots indicated that multiple copies of calflagin genes occur in T brucei. All of the copies were contained in a single 23 kb Xhol-Xhol fragment. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. Two new members of the calflagin family were found from genomic clone sequences. The deduced amino acid sequences of the genomic clones showed the calflagin genes were arranged tandemly along the genomic fragments and were similar to previously described calflagins. The calflagin genes were related by two unrelated 3' flanking sequences. An open reading frame that was unrelated to any calflagin was found at the 5' end of the 2.5 kb genomic fragment. Each encoded protein (~24,000u) contained three EF-hand calcium-binding motifs and one degenerate EF-hand motif. In general, variability among the T. brucei calflagins is greater than related proteins in T. lewisii and T. cruzi. This variability results from amino acid substitutions at the amino and carboxy termini, and duplication of internal segments.

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.

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 …

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.

Low oxygen tolerances of ten fishes were estimated using an original nitrogen cascade design, and reciprocally transformed to express responses as ventilated volume necessary to satisfy minimal oxygen demand (L·mg O2^-1). Values ranged from 0.52 to 5.64 L·mg^-1 and were partitioned into three statistically distinct groups. Eight stream fishes showed moderately high tolerances reflecting metabolic adaptations associated with stream intermittency. Juvenile longear sunfish and two mollies comprised the second group. High tolerance of hypoxia may allow juvenile sunfish to avoid predation, and mollies to survive harsh environmental oxygen regimens. The sheepshead minnow was the most tolerant species of low oxygen, of those examined, explaining its presence in severely hypoxic environments.

A contaminant fate and transport computer model, Bioplume II, which allows simulation of bioremediation in ground water systems, was used to compare the effects of 11 remediation scenarios on a benzene plume. The plume was created in three different hydrogeologic settings from the simulation of an underground gasoline storage tank leak.

To further understand the ATCase/DHOase bifunctional complex formed in Streptomyces, the genes encoding these and other pyrimidine enzymes were identified and characterized. Polymerase chain reaction (PCR) was utilized in this effort. Primers were constructed by selecting conserved regions of pyrimidine genes from known gene and protein sequences of a wide variety of organisms. These sequences were then optimized to Streptomyces codon usage. PCR products were obtained from internal sites within pyrimidine genes and also from primer combinations of different genes. The size, orientation, and partial sequence of the resulting products shows that Streptomyces has a gene organization of pyrR followed by pyrB, pyrC, carA, carB, and pyrF in an operon similar to that found in other Gram-positive bacteria.

A life history study of Callibaetis floridanus was conducted over the wet cycle of a playa on the Southern High Plains of Texas from June through September 1995.

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.

Allozyme frequencies and general protein patterns were surveyed among selected Texas marine fishes and shellfishes to illustrate the application of biochemical genetic techniques to stock and species identification in fisheries management.

This study examined the effects of replicating woodland soil surface horizonation on the nutrient status of underlying soils and the initial establishment and growth of trees. A total of 283 container grown trees were planted in a bufferzone around a future landfill site. Control amendments consisted of an 8 cm layer (0.5 m3) of wood chips applied in a circular area of 4.6 m2 around the trees' planting pit. For the treatment, a 2.5 cm layer of composted biosolids (0.15 m3 or 80 Mg/ha) was applied in a circular area of 4.6 m2 around the trees' planting pit followed by an 8 cm layer (0.5 m3) of wood chips. The results indicate that the replication of woodland soil surface attributes using composted biosolids can significantly improve the nutrient status of underlying soil. Some significant effects were seen under control conditions, too. However, the effects on tree establishment and growth parameters were, for the most part, not statistically significant.

Aerobic Gram negative bacteria such as Pseudomonas putida were reported to possess class A ATCases and to have a M.W. of 360 kD. The nucleotide sequence of the P. putida pyrBC was determined to answer this question once and for all. The expected regulatory gene was not found. It is shown that the P. putida pyrB gene is overlapped by pyrC by 4 bp. The P.putida pyrB is 1005 bp (335 aa) in length and the pyrC is 1275 bp (425 aa) long. Both of these genes complement E. coli mutants with their respective genotypes. Another finding borne out from the sequence is an effector binding site at the N-terminus of pyrB of P. putIda. The binding site shows that effectors compete with carbamoylphosphate for the active site. In this dissertation, it is shown that the ATCase of P.putida is a trimer of M.W. of 109 kD (3 x 36.4 kD) and that the gene encoding pyrB is overlapped by the pyrC gene which encodes DHOase. It is also shown that the pyrBC encoded enzymes copurify as a dodecameric complex with a M.W. of 484 kD.

There is a transition nationwide on the analysis of forensic evidentiary stains containing biological material from traditional serology to Polymerase Chain Reaction (PCR) methodologies. The increased sensitivity of PCR, the limited number of alleles at each locus, and the necessity of producing unambiguous data for entry into the FBI's Combined DNA Index System make this study of extraction procedures of utmost importance. A "single tube" extraction procedure for blood stains collected onto FTA™ paper and a modified differential nonorganic extraction method from spermatozoa containing mixed stains were analyzed and compared. The extraction success was evaluated by amplification and typing of the amplified fragment length polymorphism, D1S80. These modifications of the nonorganic method utilized gave an improved separation of the spermatozoa-containing mixed stains.

In order to gain a better understanding of this microorganismand its role in human pathogenesis, a physical map of Moraxella catarrhalis type strain ATCC25238 was constructed using pulsed field gel electrophoresis (PFGE) in combination with Southern hybridization techniques. Restriction endonucleases Not I, Rsr II, and Sma I were used to digest the chromosomal DNA. An overlapping circular map was generated by cross-hybridization of isolated radiolabeled fragments of Moraxella catarrhalis genomic DNA to dried PFGE gels. The number and location of the 16S and 23S ribosomal RNA genes were determined by digestion with l-Ceul enzyme and by Southern hybridization. Virulence-associated genes, the gene for β-lactamase, and housekeeping genes were also placed onto the physical map.

The City of Denton Pecan Creek Wastewater Treatment Facility has periodically failed effluent toxicity testing. A Toxicity Identification Evaluation has determined that Diazinon in the effluent is contributing to the observed toxicity. Chlorpyrifos is also implicated as a factor. The City of Denton constructed a half acre experimental wetland to remove Diazinon related toxicity. Results from spiking and microcosm experiments indicate that the wetland can reduce the Diazinon.

Two years before impoundment, the trophic status of Lake Ray Roberts was predicted by applying the total phosphorus input into the Organization for Economic Cooperation and Development (OECD) eutrophication model. Predicted mean summer epilimnetic (MSE) chlorophyll a of Elm Fork arm, Isle duBois arm and Main Body were in the eutrophic category of the OECD model. Observed MSE chlorophyll a two years after impoundment of Elm Fork arm, Isle duBois and Main Body had not reached their predicted means and were at the mesotrophic-eutrophic boundary of the OECD model. Six years after impoundment, observed MSE chlorophyll a for Main Body, was closer to its predicted mean and in the eutrophic category of the OECD model. Six years after impoundment, Elm Fork arm was the most productive area of Lake Ray Roberts. Observed means of chlorophyll a, total phosphates, suspended solids and turbidity were often highest in the Elm Fork arm. Wastewater effluent from Gainesville and Valley View, TX, had an impact on productivity in Elm Fork arm.

The problem of using a state adopted textbook written to apply to a large body of students with varying interests and needs was overcome by using a detailed syllabus that arranged course content in a meaningful sequence that appealed to student interest. The outlined syllabus prepared a two semester life science curriculum to be used by the teacher to guide lesson planning. Both semesters were divided into three units each. Materials included in the syllabus were given to actual student groups in real classroom settings. Since hands on learning was an important part of classroom instruction, two laboratory sections were included in the appendices to be used with the syllabus.