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 host eosinophil response was compared in mice infected with either T. spiralis or T. pseudospiralis by determination of levels of splenic and intestinal phospholipase B, a marker enzyme for eosinophils. Primary infection of naive mice and challenge infection of homologously sensitized mice with T. pseudospiralis resulted in significantly lower tissue phospholipase B activities than infection with T. spiralis. Mice homologously challenged with T. pseudospiralis did exhibit an anamnestic eosinophil response compared to mice given a primary T. pseudospiralis infection. This anamnestic response, however, was significantly lower than the eosinophil response seen in sensitized mice given a homologous T. spiralis challenge. Mice sensitized to T. spiralis or T. pseudospiralis and heterologous challenge demonstrated an elevated eosinophil response compared to mice given a primary infection with either parasite. The heterologous challenge response, however, was not as intense as found for sensitized mice given a homologous challenge.

Pseudomonas fluorescens NCIMB 11764 was capable of utilizing cyanide as a sole nitrogen source for growth. Cyanate (OCN") and S-cyanoalanine could also serve as nitrogenous substrates, but do not appear to play a role as intermediates in cyanide metabolism. Growth of this strain on cyanate as the sole nitrogen source led to the induction of an enzyme characterized as a cyanase (EC 3.5.5.3) based on its stoichiometric conversion of cyanate to ammonia, and dependence on bicarbonate for maximal activity. However, since cyanase activity was not elevated in cyanide-grown cells it was concluded that it serves no role in cyanide metabolism. Related studies aimed at examining a possible role for S-cyanoalanine as a cyanide-assimilation intermediate showed that while this compound also serves as a nitrogen source, it also is not important in cyanide metabolism. Studies focused on the utilization of free cyanide as a growth substrate led to the development of a fed-batch cultivation procedure greatly facilitating further experimentation aimed at the identification of cyanide metabolites. In addition to CO_2 and NH_3 as described earlier, two additional metabolites including formamide and formate were detected by using nC-NMR, HPLC, radioisotrapping methods and other analytical means. The formation of metabolites was shown to …

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.

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.

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.

The chemical fate and biological impacts of cyfluthrin in aquatic ecosystems were investigated using microcosms (1.9 m^3 concrete tanks) during 1989. Results were compared to a concurrent pesticide registration study using mesocosms (634.7 m^3 earthen ponds). Ten spray drift and five soil runoff simulations were conducted. Pesticide loadings were scaled by system volume, with the same experimental design in ponds and microcosms. Aqueous cyfluthrin concentrations and sediment residue values were generally higher in microcosms, while aqueous half-life was shorter in the smaller systems.

The evolutionary relationships of woodrats (Neotoma) were elulcidated through phylogenetic analyses of mitochondrial DNA restriction site and allozyme data. DNA samples from eleven nominal species from the genus Neotoma and two outgroup taxa, Ototylomys phyttotis and Xenomys nelsoni, were cleaved using a suite of 17 Type II restriction endonucleases. Mitochondrial DNA restriction profiles were visualized following electrophoresis of restriction digests via methods of Southern transfer and hybridization with 32P- and digoxigenin-labeled mtDNA probes. Restriction mapping resulted in the identification of 37 unique mtDNA haplotypes among the woodrat taxa examined. Proteins representing 24 presumptive structural gene loci were examined through starch gel electrophoresis. Binary-coded allozyme data and allozyme frequency data were analyzed using PAUP and FREQPARS, respectively. Phylogenetic analyses of the mtDNA restriction site data incorporated three different character type assumptions: unordered binary characters, Dollo characters, and differentially weighted unordered characters employing the STEPMATRIX option of PAUP. Proposed phylogenies for Neotoma are based on majority-rule consensus trees produced using bootstrap procedures. Phylogenetic analyses of the woodrat data sets revealed a distinct dichotomy among populations of white-throated woodrats (N. albigula) suggesting the presence of cryptic species within that taxon. MtDNA and allozyme data support the specific status of N. devia as distinct …

Immunological and ultrastructural changes in mononuclear cells exposed to Mycobacterium avium serovar-specific glycopeptidolipid (GPL) and the chemically derived R-lipid (lipopeptide fragment) were examined.

The immunomodulatory effects of Trichinella spiralis or Trichinella pseudospiralis soluble antigen extracts were examined in an effort to characterize the differences in immune responses seen during these Trichinella infections. The newborn larvae extracts of either parasite exhibited similar potency for stimulating macrophage PGE production; however, the muscle larvae extracts of T. pseudospiralis stimulated greater levels of PGE than did the muscle larvae extracts of T. spiralis. These data clearly indicate that Trichinella antigens possess immunomodulatory capabilities.

The effects of the inhibitory neurotransmitters gammaamino butyric acid (GABA) and glycine were characterized on spontaneous activity recorded from mouse spinal cord cultures. The GABA concentration which completely inhibited burst activity was chosen as a quantifiable measure of culture drug response and was used to 1) assess interculture and intraculture variability, 2) determine the influence of culture age and initial activity on GABA responses, and 3) compare the GABA responses between networks obtained from whole spinal cord and ventral half spinal cord. Results showed that 1) no significant variability existed either within or among cultures, 2) the initial culture activity directly affected GABA responses, 3) the culture age had no effect on GABA responses, and 4) there was no significant difference in GABA responses between the two spinal cord tissues.

This research compares LC50 and LD50 of earthworms, Lumbricus terrestris and Eisenia foetida exposed to cadmium nitrate and chlordane using 48-h contact filter paper (FP) and 14-d artificial soil (AS) protocols. Both LC50 and LD50 showed that chlordane was more toxic than cadmium in both species regardless of the exposure. The reference toxicant 2-chloroacetamide using the standardized 48-h FP exposure was used to assess the general response of the earthworm prior to toxicity experiments. A glucose test was developed as an internal standard to assess homogeneity of mixtures among both replicates and dilutions. Accuracy of dilutions is assessed by the slope of a regression line relating nominal dilution to observed internal standard concentration. Precision of replicate preparation is assessed by among replicate variance.

Three overlapping DNA fragments from the lower operon of Pseudomonas putida TOL plasmid pDK1, covering the xy1IH genes and downstream flanking region, were cloned into pUC19. They include a 2.8 kbp XhoI fragment, a 2.7 kbp PstI fragment and a 2.0 kbp EcoRI-HindIII fragment. They were subjected to DNA sequence analysis. The xy1I (4-oxalocrotonate decarboxylase) and xy1H (4-oxalocrotonate tautomerase) genes were found to possess coding regions of 792 and 189 nucleotides, respectively. A possible transcriptional terminator resembling E. coli rho-independent terminators was identified downstream of the translational stop of xy1H. An additional stem and loop structure was found in the intergenic region between xy1I and xy1H. The individual ORF's of the oxalocrotonate branch (xy1G, xy1I and xy1H) have been cloned into pUC18/19. The expression of the xy1G gene in E. coli was successfully assayed spectrophotometrically.

The role of individual amino acid residues of the enzyme dienelactone hydrolase was investigated. Using the polymerase chain reaction (PCR), a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHI to EcoRI clcD subclone in the plasmid pUC19. Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. Specifically modified proteins from mutated clcD genes (Arg 81 to alanine, Tyr 85 to phenylalanine and Arg 206 to alanine), were encoded by the mutant clones. Enzyme assays showed that dienelactone hydrolase activity of the mutants Arg 81 and Arg 206 was totally abolished. The DLHase enzyme activity of mutant Tyr 85 is greatly decreased by approximately two thirds.

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.

Observed and expected heterozygosity (H) levels for ten electrophoretic blood marker loci and expected H for seven red blood cell (RBC) anitgen/antibody loci were examined for 20 years in American Standardbred and 30 years in American Saddlebred horses. Standardbreds were classed by gait, Trotter and Pacer, and evaluated separately in most analyses. 4,404 Trotters and 12,271 Pacers were found to have statistically highly significant losses of mean total observed H through time for the ten electrophoretic loci (P<0.005), although in Trotters the loss was more extreme (P<<0.001). Loss of H in 5984 Saddlebreds was not significant (P=0.259). Correlations of RBC expected H through time showed decreases in all three groups.

Organisms synthesize stress proteins in response to a variety of stressors. The 68/70-kDa proteins (synonymous to the 72/73-kDa proteins) have shown to be the most promising stress proteins, and have been proposed as a biomarker of general organismal stress. The 68/70-kDa proteins were used in an antigen/antibody based approach to determine the duration of the stress protein response of Pimephales promelas following an acute exposure to copper sulphate.

Stonefly life histories were studied March, 1987 through May, 1990 in the Rio Conejos, Colorado. Adult presence phenology and intensity were monitored daily in the summers of 1988 and 1989 and were coupled with monthly benthic samples to assess nymphal growth. Eggs of several species were reared. Thirty-one species were collected, with several multi-species assemblages occurring in Capnia, Utacapnia, Taenionema, Suwallia, Triznaka, Isogenoides and Isoperla. Sufficient data were obtained to reveal partial or complete life histories of 13 species, five of which have not been previously reported. New information included the 9- to 10-mo egg diapause and semivoltine life histories of Isogenoides zionensis Hanson, Pteronarcella badia (Hagen) and Pteronarcys californica Newport. Additionally, Isoperla phalerata (Smith) had univoltine-slow growth, and L quinquepunctata (Banks) was univoltine-fast. Previously unstudied emergence periods are presented for Triznaka signata (Banks), Suwallia wardi Kondratieff and Kirchner and S. pallidula (Banks). The later two species temporally segregated emergence in both years. Isoperla fulva Claassen emerged in June, and was temporally segregated from its congeners. Regression analysis of cumulative percentage catch revealed two adult presence patterns. Eight species had slopes <7%/d (extended pattern), and only two, I. zionensis and P. cal'fornica, had slopes >7 %/d (synchronous pattern). Several stoneflies …

This research defines the morphological characteristics of the immune cells, coelomocytes, of the earthworm, Lumbricus terrestris. Such a definition of coelomocyte morphology can be correlated with immune function which will be of value in the development of a nonmammalian surrogate system to assess the immunotoxic potential of a terrestrial xenobiotics. Earthworms exposed to artificial soil dilutions of two complex xenobiotic mixtures, refuse derived fuel fly ash and a Superfund sire soil demonstrated an enhanced ability to produce hemolytic factors.

A novel bacterial expression system that is capable of producing high levels of soluble, stable, biologically active human vitamin D3 and estrogen receptors has been developed. The method utilizes ubiquitin fusion technology and a low temperature nalidixic acid induction of the lambda PL promoter. This system can produce large quantities of receptor antigen, but only a small fraction displays wild-type DNA and hormone binding properties. Therefore, the use of this system to overproduce receptors for crystallization studies is not practical. To overcome these problems, a 2 um based ubiquitin fusion system which allows regulated expression of the estrogen receptor in yeast (Saccharomyces cerevisiae) was developed. This system produces the estrogen receptor to a level of 0.2% of the total soluble protein. Moreover, this protein is undegradable, soluble, and biologically active. To test the transcriptional activity of the estrogen receptor produced in yeast, a cis-trans transcription assay was developed. This assay revealed that the transcriptional activity of the human estrogen receptor expressed in yeast was similar to that observed in transfected mammalian cells. This reconstituted estrogen transcription unit in Saccharomyces cerevisiae was utilized to examine the regulation of estrogen receptor functions by antiestrogens, to develop a random and rapid approach for …

The effect of pH upon the aquatic toxicity of cyfluthrin was determined in 48 h static acute toxicity tests using 2nd instar Chironomus tentans larvae. Tests were conducted in both dechlorinated tap water and lake water of pH 8.0, 7.2, and 6.6. After 48 h, immobilized and dead larvae were removed and replaced with 2nd instar larvae to assess the persistence of toxicity. Midges were cultured in water adjusted to the pH values used in testing. Toxicity if cyfluthrin varied inversely with pH. An increase in the pH of tap water by 2 units resulted in a 2-decrease in toxicity. Toxicity of cyfluthrin also varied between tap and lake water of the same pH. EC50 values in lake water were approximately 2-3 times lower than those in tap water. Toxicity in the lake water and tap water at every pH tested was also significantly different when regression line elevations were compared. Natural waters amended with cyfluthrin were consistently more toxi to the chironomids than tap water of the same pH. Persistence of cyfluthrin at low pH also influenced chironomid behavior. Recovery of normal behavior generally began within 24 h at pH 8.0 At pH 6.0, recovery did not begin until …