Cowpea, Vigna unguiculata (L.) Walp is an excellent source of protein, vitamins and minerals and a major food crop many parts of Africa. Optimal production levels are hampered by insect pests and diseases. Biotechnological techniques such as tissue culture and genetic engineering can aid in the development of varieties with resistance to insect pests and diseases. The objective of this study was to investigate conditions necessary for the development of a reproducible tissue culture system that can be applied to regenerate transformed cells from culture. The in vitro manipulation of cowpea using Murashige and Skoog (MS) medium, auxins and cytokinins resulted in the formation of callus and rhizogenesis. Calli that were formed were separated into six classes based on color and texture. Yellowish friable callus, yellowish compact, soft yellowish callus and green and white were composed of largely vacuolated cells and were non-regenerative. Friable green callus was the most prevalent callus type and could form of roots in some hormone combinations. Green spots were formed on hard compact green callus. The green spots became nodular, forming root primordia and ultimately giving rise to roots. None of the six calli types gave rise to the formation of shoots. Embryogenic callus was …

Spontaneously active frontal cortex and spinal cord networks grown on microelectrode arrays were used to study effects of D-methylphenidate. These central nervous system tissues have relatively low concentrations of dopaminergic and noradrenergic neurons compared to the richly populated loci, yet exhibit similar neurophysiological responses to methylphenidate. The spontaneous spike activity of both tissues was inhibited in a concentration-dependent manner by serial additions of 1-500 µM methylphenidate. Methylphenidate is non-toxic as spike inhibition was recovered following washes. The average concentrations for 50% spike rate inhibition (IC50 ± SD) were 118 ± 52 (n= 6) and 57 ± 43 (n = 11) for frontal cortex and spinal cord networks, respectively. A 3 hour exposure of a network to 1 mM methylphenidate was nontoxic. The effective concentrations described in this study are within the therapeutic dosage range. Therefore, the platform may be used for further investigations of drug mechanisms.

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 pyrimidine biosynthetic pathway is an essential pathway for most organisms. Previous research on the pyrimidine pathway in Pseudomonas aeruginosa (PAO1) has shown that a block in the third step of the pathway resulted in both a requirement for exogenous pyrimidines and decreased ability to produce virulence factors. In this work an organism with a mutation in the second step of the pathway, aspartate transcarbamoylase (ATCase), was created. Assays for pyrimidine intermediates, and virulence factors were performed. Results showed that the production of pigments, haemolysin, and rhamnolipids were significantly decreased from PAO1. Elastase and casein protease production were also moderately decreased. In the Caenorhabditis elegans infection model the nematodes fed the ATCase mutant had increased mortality, as compared to nematodes fed wild type bacteria. These findings lend support to the hypothesis that changes in the pyrimidine biosynthetic pathway contribute to the organism's ability to effect pathogenicity.

Burkholderia cepacia is a common soil bacterium of significance in agriculture and bioremediation. B. cepacia is also an opportunistic pathogen of humans causing highly communicable pulmonary infections in cystic fibrosis and immunocompromized patients. The pyrB gene encoding ATCase was cloned and ATCase was purified by the glutathione S-transferase gene fusion system. The ATCase in B. cepacia has been previously classified as a class A enzyme by Bethell and Jones. ATCase activity gels showed that B. cepacia contained a holoenzyme pyrBC complex of 550 kDa comprised of 47 kDa pyrB and 45 kDa pyrC subunits. In the course of purifying the enzyme, trimeric subunits of 140 kDa and 120 kDa were observed as well as a unique proteolysis of the enzyme. The 47 kDa ATCase subunits were cleaved to 40 kDa proteins, which still demonstrated high activity as trimers. The proteolysis site is between Ser74 and Val75 residues. To confirm this, we converted the Ser74 residue to an Ala and to an Arg by site-directed mutagenesis. After this primary sequence changed, the proteolysis of ATCase was not observed. To further investigate the characteristics of B. cepacia pyrB gene, a pyrB knock-out (pyrB-) was constructed by in vitro mutagenesis. In the assay, …

The Rio Gavilan watershed, located in Mexico 's northern Sierra Madre Occidental , has significance in conservation history. Upon visiting the remote, largely un­developed watershed during two hunting trips in the 1930s, renowned conservationist Aldo Leopold thought it was the best picture of land health he had seen. His main indicators of healthy land were slow water runoff rates regulating erosion and historical predator-prey relationships. The visits confirmed Leopold's concept of land health, inspired many of his essays, and helped shape his land ethic. Leopold proposed the area as a control site to research healthy land throughout North America . The proposal never went forward and the area has since been more intensively logged and grazed. This dissertation research used extensive literature review, archives, oral histories, citizen surveys, and rapid assessment of forest, rangeland, riparian, and socioeconomic health to assess impacts of past cultures and update the area's land health status. Projects that could restore land health, such as linked eco-tourism, forest density reduction, and rotational grazing, were assessed for feasibility. Recent critiques of Leopold's land ethic were also reviewed. Results indicate most pre-1940s impacts were light, current land health status is moderate, and local interest exists in restoring land …

TNT (2,4,6-trinitrotoluene) readily and predominantly transforms to 2ADNT (2-amino-4,6-dinitrotoluene) and 4ADNT (4-amino-2,6-dinitrotoluene) in environmental matrixes and tissues. Solid phase microextraction (SPME) was used to extract ADNTs (amino-dinitrotoluenes) from tissue as a potential method to investigate the recalcitrance of metabolically-generated ADNTs versus absorbed ADNTs. Tubifex tubifex was allowed to metabolize TNT into ADNTs in 24-hr static non-renewal exposure test followed by 24-hr depuration in clean reconstituted hard water. Polyacrylate-coated (PA) SPME fibers were then deployed and agitated in tissue homogenates containing metabolically-generated ADNTs for 48 hr to provide a measure of available ADNTs. Extractability of ADNTs from T. tubifex tissue containing metabolically-generated ADNTs was significantly less than extractability of ADNTs from T. tubifex tissue containing absorbed ADNTs: 50-60% and 81-90% of expected extractability based on fiber-water partition ratio. The lower SPME extractability of metabolically-generated ADNTs may stem from the unavailability of metabolically-generated ADNTs sequestered in tissue or bound to tissue macromolecules during metabolism of TNT to ADNT. Tissue extractions using SPMEs may be able to estimate such bound organic residues in tissue and serve as potential indicators of toxicological bioavailability and biomagnification potential of tissue-associated organic compounds.