This study employed a new model of mild-to-moderate head trauma to specifically identify the role of dural mast cell (MC) histamine in trauma-induced increased permeability in the blood-brain barrier (BBB). A single line was scored partially through the left dorsal parietal skull. Immediately following the trauma, degranulation was seen in 39% of the MCs on the left and in 2% on the right. After a 20 min survival period, left duras showed 55% with MC degranulation (fewer with complete degranulation) compared to 34% on the right. In the other experiments two parallel lines were scored following the injection of Evan's blue. Histamine assay showed histamine increased in the left cortex to 154% at 5 min, 174% at 10 min, and 151% at 20 min. Fluorescent quantitation of extravasated Evan's blue at 20 min following the trauma gave an increase of 1385% over the value measured for the right cortex. Zolantidine, a selective histamine H2 receptor antagonist, administered at 10- and 20- mg/kg 30 min before the trauma blocked 65% of the Evan's blue extravasation compared with the control and 2.5 mg group.

Temperature effects on the development of the zebrafish embryos and larvae and adults were examined. It was found that the earlier in development a temperature change was performed on an embryo, the more significant the change in survival and/or subsequent development. Thus, viable temperature ranges for zebrafish widened significantly as development proceeded. Adults reared and bred at 25oC produced embryos that were significantly more successful at the lower range of rearing temperatures compared to embryos produced from adults reared at 28oC. The majority of this study focused on the physiological effects of swim training during development in the zebrafish. The earlier in development the zebrafish larvae were trained, the greater the mortality. Trained free swimming larvae had a significantly higher routine oxygen consumption after 11 days of training, and a higher mass specific routine metabolic rate after 8 and 11 days of training. Trained free swimming larvae consumed significantly less oxygen during swimming and were more efficient at locomotion, compared to control larvae. Training enhanced survival during exposure to extreme hypoxia in all age groups. Performance aspects of training were investigated in attempt to quantify training effects and in most cases, trained fish performed significantly better than controls. As blood …

Sensilla styloconica on the proboscis of 107 species of North American and tropical butterflies were comparatively studied using the scanning electron microscope. Focus was on 76 species of North American Nymphalidae representing 45 genera and 11 subfamilies. Nomenclature for generalized and specific types of nymphalid sensilla is proposed. Written descriptions and micrographs are presented for each species studied. Morphological features were generally consistent for all or most species within genera and sometimes within subfamilies, with specified exceptions. Statistical analysis revealed significant differences for six of eight variables tested between two distinct feeding guilds of North American Nymphalidae. Average number, density, extent of proboscis coverage with sensilla, their total length, and shoulder spine length were all significantly greater in the non-nectar feeding guild than in nectar feeders, and may indicate adaptation for greater efficiency in feeding on flat surfaces. The greater frequency of apical shoulder spines in non-nectar feeders may represent adaptation for protection of sensory pegs from mechanical abrasion during feeding, or for anchoring the flexible proboscis tip to the surface. Correlation analysis revealed 9 out of 28 positive correlations in nectar feeders and 5 out of 28 in non-nectar feeders. Results of preliminary cladistic analysis were not considered to …

Estrogen is the natural agonist of the estrogen receptor (ER). However, certain plant-derived compounds or phytoestrogens have been identified that mimic estrogens and act as agonists and/or antagonists of ERs, depending on subtype and target tissue. Understanding how phytoestrogens interact with ERs, and therefore effect the estrogenic response, may prove beneficial in hormone replacement therapy and in the prevention and treatment of hormone-related diseases. Using Thin Layer Chromatography, gas chromatography/mass spectrometry (GC/MS), and proton nuclear nagnetic resonance (HNMR), I identified 4-ethoxymethylphenol (4EM) found in Maclura pomifera. While most phytoestrogens are heterocyclic compounds, 4EM is a simple phenol that acts as an agonist of ER-alpha and -beta in HeLa and MCF-7 cells. To study the effect of 4EM on ER-alpha and -beta activity, I performed transient transfection assays and showed that 4EM activates ER dependent gene transcription in a dose dependent manner in both ER subtypes. Further, 4EM- mediated transcription in ER-alpha, like estrogen, was enhance in the presense of co-activators, SRC-1 (steroid receptor coactivator-1), CBP (CREB binding proteins), and E6-AP (E6-associated protein) and inhibited by trans-4- hydroxytamoxifen (4HT). I found that 4EM was specific for ER and did not activate transcription of the progesterone receptor in HeLa cells.

This manual has been designed for a class of twenty- four students concurrently enrolled in the lecture course. The laboratory aids in the learning process and fosters an interest in the science of genetics. This manual and the experiments contained within are both informative and fun. The manual correlates with and expands upon the genetics course. Each investigation, with the exception of the Drosophila melanogaster project, can be completed in a 3-4 hour timeframe. This manual provides a “hands on” experience of theories simply discussed in the lecture course. This manual is intended to be a one-source manual where each investigation is designed to include an adequate introduction. Special attention has been given for each investigation with both the student and instructor in mind.

A study of the life history and secondary production of Caenis latipennis, a caenid mayfly, was conducted on Honey Creek, OK. from August 1999 through September 2000. The first instar nymph was described. Nymphs were separated into five development classes. Laboratory egg and nymph development rates, emergence, fecundity, voltinism, and secondary production were analyzed. C. latipennis eggs and nymphs take 132 and 1709 degree days to develop. C. latipennis had an extended emergence with five peaks. Females emerged, molted, mated, and oviposited in an estimated 37 minutes. Mean fecundity was 888.4 ± 291.9 eggs per individual (range 239 -1576). C. latipennis exhibited a multivoltine life cycle with four overlapping generations. Secondary production was 6,052.57 mg/m2/yr.

This dissertation research explored the capabilities of neuronal networks grown on substrate integrated microelectrode arrays in vitro with emphasis on utilizing such preparations in three specific application domains: pharmacology and drug development, biosensors and neurotoxicology, and the study of burst and synaptic mechanisms. Chapter 1 details the testing of seven novel AChE inhibitors, demonstrating that neuronal networks rapidly detect small molecular differences in closely related compounds, and reveal information about their probable physiological effects that are not attainable through biochemical characterization alone. Chapter 2 shows how neuronal networks may be used to classify and characterize an unknown compound. The compound, trimethylol propane phosphate (TMPP) elicited changes in network activity that resembled those induced by bicuculline, a known epileptogenic. Further work determined that TMPP produces its effects on network activity through a competitive inhibition of the GABAA receptor. This demonstrates that neuronal networks can provide rapid, reliable warning of the presence of toxic substances, and from the manner in which the spontaneous activity changes provide information on the class of compound present and its potential physiological effects. Additional simple pharmacological tests can provide valuable information on primary mechanisms involved in the altered neuronal network responses. Chapter 3 explores the effects produced …

This study examined central mechanisms of persistent pain using an autoradiographic technique to localize phosphoinositide hydrolysis (PI) in the rat spinal cord dorsal horn. The lateral half of laminae I-II showed the highest levels of baseline PI turnover and carbachol-stimulated PI turnover in normal animals as well as after inflammation. Inflammation resulted in increased baseline PI turnover in this region of the ipsilateral (76%) and contralateral (65%) dorsal horns. Carbachol increased PI turnover in this region in normal rats (55%) and following inflammation (ipsilateral: 46%, contralateral: 45%). The absolute magnitudes of these increases were 1.85, 2.71, and 2.51 nCi/mg, respectively. The results of this study demonstrate the involvement of PI turnover in neural mechanisms of persistent pain, and provide evidence for the involvement of cholinergic systems in this process. Because spinal cholinergic systems have been reported to be anti-nociceptive, the present results appear to reflect an upregulation of anti-nociceptive activity in response to inflammation. Thus, the spinal cholinergic system may be a regulatory site within the anti-nociceptive pathway, and may provide an attractive target for the development of new therapeutic agents.

Embryonic murine neuronal networks cultured on microelectrode arrays were used to quantify acute electrophysiological effects of fluoxetine and ethanol. Spontaneously active frontal cortex cultures showed highly repeatable, dose-dependent sensitivities to both compounds. Cultures began to respond to fluoxetine at 3 µM and were shut off at 10-16 µM. EC50s mean ± S.D. for spike and burst rates were 4.1 ± 1.5 µM and 4.5 ± 1.1 µM (n=14). The fluoxetine inhibition was reversible and without effect on action potential wave shapes. Ethanol showed initial inhibition at 20 mM, with spike and burst rate EC50s at 52.0 ± 17.4 mM and 56.0 ± 17.0 mM (n=15). Ethanol concentrations above 100 -140 mM led to cessation of activity. Although ethanol did not change the shape and amplitude of action potentials, unit specific effects were found. The combined application of ethanol and fluoxetine was additive. Ethanol did not potentiate the effect of fluoxetine.

Remote sensing and GIS have become standard tools for evaluating spatial components of wildlife habitats. These techniques were implemented to evaluate Rio Grande wild turkey (Meleagris gallopavo) poult-rearing habitat in the Western Cross Timbers region of Texas. Texas Parks and Wildlife (TPWD) random roving turkey counts for 1987-1989 and 1998-2000 were selected, indicating locations where hens with poults were observed. Satellite imagery from 1988 and 1999 was classified and then processed with Patch Analyst. To add robustness, stream, road and census population densities were also evaluated for each turkey location. Analysis of the 1988 canopy cover image, comparing observed locations with randomly-selected habitat cells (N = 20) indicated significant differences (p <.05) for patch edge variables. Mean patch edge was significantly greater for habitat locations where hens with poults were observed than for those selected at random. Spatial data for 1999 did not indicate a significant difference (p < .05) between sampling groups (observed vs. random, N = 30). Significant differences (p <.05) did occur for turkey locations observed in both 1988 and 1999 (N = 7). This demonstrates the adaptability of wild turkey hens, as habitats change over time, hens continued to visit the same locations even though the …

A life history and productivity study of Enallagma civile was conducted in a playa that was located in the southern High Plains of Texas. Other odonates were also studies to identify their contributions to the habitat.

Development of a college level freshman biology course was undertaken in response to government reports that American students have fallen behind students of other countries in the area of the sciences. Teaching strategies were investigated to accomplish two objectives, to define essential academic material to include in the course and to investigate teaching techniques that would increase deep processing of the information. An active process that consisted of applying the cognitive information to solving problems or developing answers to questions was defined as critical thinking. Critical thinking was incorporated into the course by the use of case studies.

Methanococcus jannaschii is a thermophilic methane producing archaebacterium. In this organism genes encoding the aspartate transcarbamoylase (ATCase) catalytic (PyrB) and regulatory (PyrI) polypeptides were found. Unlike Escherichia coli where the above genes are expressed from a biscistronic operon the two genes in M. jannaschii are separated by 200-kb stretch of genome. Previous researchers have not been able to show regulation of the M. jannaschii enzyme by the nucleotide effectors ATP, CTP and UTP. In this research project we have genetically manipulated the M. jannaschii pyrI gene and have been able to assemble a 310 kDa E. coli like enzyme. By using the second methionine in the sequence we have shown that the enzyme from this organism can assemble into a 310 kDa enzyme and that this enzyme is activated by ATP, CTP and inhibited by UTP. Thus strongly suggesting that the second methionine is the real start of the gene. The regulation of the biosynthetic pathway in Pseudomoans aeruginosa has previously been impossible to study due to the lack of CTP synthase (pyrG) mutants. By incorporating a functional uridine (cytidine) kinase gene from E. coli it has been possible to isolate a pyrG mutant. In this novel mutant we have …

A bacterial challenge assay was developed utilizing the earthworm, Lumbricus terrestris, in order to assess potential immunotoxic effects from exposure to specific polychlorinated biphenyl congeners. Earthworms were inoculated with Aeromonous hydrophila, establishing a 10-day LD50. In vitro assays for effects of PCBs on phagocytosis agreed with mammalian studies, demonstrating potent suppression of phagocytosis by the non-coplanar PCB congener 138 and no suppression by the coplanar congener 126. However, when the effects of the two PCB congeners were evaluated for suppression of resistance to a whole animal infection challenge assay, coplanar PCB 126 decreased the ability of L. terrestris to withstand infection while non-coplanar PCB 138 did not.

Superior swimming stamina in adult fish is presumably passed on to their offspring, but the ontogeny of the appearance of superior stamina and the requisite enhanced cardio-respiratory support for locomotion in larval fishes has not been determined. Is the expression of the suite of parental traits enabling superior swimming stamina in their offspring dependent upon their achieving juvenile/adult morphology, or does it appear earlier in their larvae? To answer this, adults were classified into three groups based on swimming stamina, followed by measurement of length, mass, and width. Larval offspring from the two parental groups -high stamina larvae (HSL) and low stamina larvae (LSL)- were reared at 27°C in aerated water (21% O2). Routine and active heart rate, routine and active mass specific oxygen consumption were recorded through 21dpf, and cost of transport (COT) and factorial aerobic scope were derived from oxygen consumption measurements. Routine heart rate at 2dpf of LSL was 164 ± 1 b·min-1, compared to only 125 ± 2 b·min-1 for HSL. Routine heart rate subsequently peaked at 203 ± 1 b·min-1 at 5dpf in the HSL group, compared to 207 ± 1 b·min-1, at 4dpf in the LSP larvae. Active heart rate at 5 dpf of …

Established interspecific metabolic allometric relationships do not adequately describe the complexity and variable physiological states of developing animals. Consequently, intraspecific allometric relationships of oxygen consumption and carbon dioxide production as a function of body mass; the respiratory quotient; the function of the silk cocoon; and body composition were investigated for each distinct developmental stage of the silkworm, Bombyx mori. Whole animal O2 consumption in Bombyx ranged from 0.00064 + 0.000047 ml O2 .hr-1 at larval instar I to 0.77 + 0.06 ml O2 .hr-1 in pre-pupal, falling to 0.21+ 0.01 ml O2 .hr-1 in the pupae. Those instars having a significant relationship between O2 consumption as a function of body mass, the slope of the line relating O2 consumption to body mass varied between 0.99 and 1.02, while across all instars the slope was 0.82. Developmental allometry should be presented for individual developmental stages because the individual allometric exponents of the stages can be significantly different from the overall allometric exponent throughout development and in some cases, the overall allometric exponent can be a statistical artifact. The first larval instar of Bombyx mori has the lowest cross sectional area of high metabolic tissue of the midgut (27%) and had one …

The black-tailed prairie dog (Cynomys ludovicianus) has experienced dramatic declines over much of its historical range due to habitat loss, plague, poisonings, and shootings. Many populations now occur as isolated genetic relicts. A multiple locus genetic profile was obtained using microsatellite analyses of six polymorphic nucleotide repeats from 319 black-tailed prairie dogs collected from 16 colonies throughout the state of Texas. This assessment revealed that existing populations have sufficient variation at all six loci to verify the usefulness of this approach as a primary genetic tool in conservation and preservation. The data reveals regional-dependent frequency patterns as well as support for founder/bottleneck effects for several of the 16 sites. Although the prairie dog population in Texas as a whole may appear genetically diverse, considerable genetic divergence has already occurred among the subpopulations (FST = 0.164). Isolation by distance is supported by genic differentiation analysis (P < 0.001) and pairwise correlation analysis between genetic distance and geographic distance (P < 0.001). Prairie dogs from six (COC, LUBA, LUBC, LUBD, LUBE, and TAR) of the original 16 sites have been relocated or exterminated, or were in the process of being relocated. Results indicated the following colonies (COT, DAL, HOW, and HUD) are …

The pyrimidine salvage enzyme, nucleoside hydrolase, is catalyzes the irreversible hydrolysis of nucleosides into the free nucleic acid base and D-ribose. Nucleoside hydrolases have varying degrees of specificity towards purine and pyrimidine nucleosides. In E. coli, three genes were found that encode homologues of several known nucleoside hydrolases in protozoa. All three genes (designated yaaF, yeiK, and ybeK) were amplified by PCR and cloned. Two of the gene products (yeiK and ybeK) encode pyrimidine-specific nucleoside hydrolases, while the third (yaaF) encodes a nonspecific nucleoside hydrolase. All three were expressed at low levels and had different modes of regulation. As a comparative analysis, the homologous genes of Pseudomonas aeruginosa and P. fluorescens (designated nuh) were cloned. Both were determined to encode nonspecific nucleoside hydrolases. The nucleoside hydrolases of the pseudomonads exhibited markedly different modes of regulation. Both have unique promoter structures and genetic organization. Furthermore, both pseudomonad nucleoside hydrolase were found to contain an N-terminal extension of 30-35 amino acids that is shown to act as a periplasmic-signaling sequence. These are the first two nucleoside hydrolases, to date,that have been conclusively demonstrated to be exported to the periplasmic space. The physiological relevance of this is explained.

An anti-G"11 antibody was used to label neuronal cilia throughout the rat central nervous system. Immunoreactive cilia were observed in every examined region of the rat CNS, but not in monkey or mouse tissue. Antibodies to G"q and G"q/11 failed to label cilia. Immunoreactive cilia were observed as early as postnatal day 0 in spinal tissue, and postnatal day 3 in hypothalamic tissue. There was a statistically significant negative correlation between a region's mean cilium length and that region's distance to the nearest ventricle; regions nearest ventricles were those with the longest cilia. This correlation suggests neuronal cilia may function as chemosensors, detecting substances as they move out from the cerebrospinal fluid and into the extracellular space of the brain.

This study describes macroinvertebrate community structure and assesses its potential in detecting point and non-point sources of disturbance associated with rural and urban areas in the Upper Trinity River Basin. Geospatial techniques were used to quantify landuse within the watershed in a GIS. At rural sites near the headwaters of the Trinity River, collector-gathering burrowers that are adapted to minimal flow comprised the majority of taxa. Destinies of taxa compositions at downstream sites increased and shifted toward psammophilic and rheophilic invertebrates, including primarily collector-filtering clingers, that are characteristic of shifting sand habitats in large prairie rivers. Benthic community structure generally benefited from point source impacts including wastewater treatment plant effluents that maintained higher flow. Community indices were negatively associated with forest landuse and positively associated with urban landuse. Partial CCA determined that flow and landuse contributed equally to species dispersions. Comparisons with historical biomonitoring studies in upper Trinity River Basin indicate improved watershed health.

The effects of three oral dosages (0.4 mg, 0.8 mg, and 1.6 mg) of quercetin on Colon-25 tumors implanted in Balb-c mice were studied. The data in this study show that: (1) certain dosages of quercetin in alcohol solutions, reduces the weight, and size of implanted Colon-25 tumors in Balb-c mice, (2) these same dosages of quercetin all produce a profound neutrophilia combined with a significant lymphopenia at day 20 post-implantation, and (3) there was relatively little evidence of histological changes in the quercetin-treated tumor section which would indicate that the action(s) of quercetin is primarily at the subcellular level probably within the nuclei of the tumor cells.

Condom trace evidence must not be overlooked in sexual assault cases; understanding the chemical and physical characteristics of condoms is imperative if condoms are to be useful evidence. Previous research shows that condom identification is possible, but it is equally important to evaluate durability of condom residues versus time. Using FT-IR, this study examined vaginal swabs from subjects who self-sampled at intervals for up to 72 hours after having intercourse with a condom. This study investigated whether age and the stage of the menstrual cycle affected the durability of residues in the vagina over time. This study revealed that condoms containing nonoxynol-9, silicone-based lubricants, and particulates provide valuable information for identification, and that nonoxynol-9 specifically withstands the vaginal environment for up to 72 hours. Additionally, age and menstrual cycle both appeared to have an effect on the durability of residues although larger sample size is desirable.

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.

Earthworms, Lumbricus terrestris, were challenged orally and intracoelomically with two bacterial species, Aeromonas hydrophila and Pseudomonas aeruginosa, and mortality rates were observed. Neither were found to be particularly pathogenic at injected doses of up to 108 bacteria per earthworm. The influence of Cu++ (as CuSO4) on the earthworm's response to bacterial challenge was investigated by exposing earthworms to sublethal levels of Cu++ prior to bacterial challenge. Exposure at sublethal concentrations up to 3 m g/cm2 did not have a pronounced influence on host resistance to challenge as measured by earthworm mortality. Cu++ increased the earthworm's ability to agglutinate rabbit erythrocytes, indicating that Cu++ exposure caused coelomocyte death, autolysis and release of agglutinins into the coelom, possibly explaining resistance to bacterial challenge.