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.
With numerous bioassessment methodologies available, a regional protocol needs to be developed to ensure that results are comparable. A regional assessment protocol was developed that includes collecting five benthic macroinvertebrate samples, identifying organisms to genus, and calculating the following metrics: Number of Taxa, Total Number of Individuals, Simpson's Diversity Index, Shannon's Diversity Index, Percent Contribution of Dominant Taxa, Hilsenhoffs Biotic Index, and Percent Contribution of Dipterans. Once the protocol was developed, it was used to assess the Bayou Chico tributaries and watershed. All three tributaries had been significantly impacted by human activity as had the watershed as a whole. This study indicates that a regional protocol could be developed and is appropriate for biomonitoring at the watershed scale.
An in vitro system of generating protoplasts from their callus cultures was established. The friable callus was more productive in terms of producing protoplasts than the green compact callus. The concentration of the various cell wall degrading enzymes had an effect on the viability of the protoplasts in the medium. The protoplast system developed from the experiments was stable and could be used for the transformation experiments of Albizia lebek and for other plant improvement practices.
In the United States, biodiversity impact assessment has historically received little attention. Responding in 1993, the Council on Environmental Quality (CEQ) released guidelines on incorporating biodiversity into environmental impact assessment under the National Environmental Policy Act of 1969. The objectives of the study here were to identify the level of documentation of biodiversity impact assessment in sample Environmental Impact Statements (EISs); identify whether in the years following the release of 1993 CEQ guidelines any significant changes have taken place in assessment of biodiversity; identify deficiencies, and if the need exists, formulate appropriate recommendations and approaches for addressing biodiversity in EISs. The study involved a systematic review of 30 EISs published since the release of CEQ guidelines, and five EISs published prior to it. The review involved answering a series of standard questions, which attempted to ascertain the level of biodiversity impacts included in each impact statement. Trends in approaches to biodiversity impact assessment were investigated and deficiencies summarized. The analysis resulted in a series of recommendations for improving the manner in which biodiversity impact assessment can be approached.
Since the introduction of management practices by the Forest Service to stabilize red-cockaded woodpecker (RCW) populations, the number of cavity trees killed by southern pine beetles (SPB) has increased. A model of the landscape ecology of RCW and SPB in the Homochitto National Forest was created using data collected from the Forest Service and Global Atmospherics. The conclusions of the study were that the RCW and SPB utilize the same type of habitat and the stand hazard maps are an accurate means of determining the locations of SPB infestations. The functional heterogeneity maps created for the SPB and RCW would be useful predictors of future occurrences of either species if complete data were obtained.
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.
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.
Spontaneously active primary cultures obtained from dissociated embryonic medial medulla tissue were grown on microelectrode arrays for investigating burst patterns and pharmacological responses of respiratory-related neurons. Multichannel burst rates and spike production were used as primary variables for analysis. Pacemaker-like neurons were identified by continued spiking under low Ca++/high Mg++conditions. The number of pacemakers increased with time under synaptic blocking medium. Sensitivity to CO2 levels was found in some neurons. Acetylcholine changed activity in a complex fashion. Curare, atropine and gallamine modified ACh effects. Eserine alone was ineffective, but potentiated ACh-induced responses. Norepinephrine caused channel-specific increases or decreases, whereas dopamine and serotonin had little effect at 30 μM. GABA and glycine stopped most spiking at 70 μM. Developmental changes in glycine sensitivity (increasing with age) were also observed. It is concluded that pacemaker and chemosensitive neurons develop in medial medulla cultures, and that these cultures are pharmacologically histiotypic.
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.
Cyanide was rapidly removed when added to culture supernatants of seven different Pseudomonas. The ability to remove cyanide was correlated with the accumulation of α-keto acids (pyruvate and α-ketoglutarate). These compounds react with cyanide forming less toxic cyanohydrins, thus conferring a mechanism for bacterial cyanide tolerance. When added to growth media the α-keto acids were shown also to serve as effective cyanide antagonists. While all bacteria tested accumulated α-keto acids, only those capable of utilizing cyanide as a nutritional nitrogen source were able to metabolize cyanohydrins. In P. fluorescens NCIMB 11764, the same enzyme (cyanide oxygenase) shown previously to be involved in cyanide metabolism appears responsible for cyanohydrin transformation. Keto acid excretion is believed to represent a new mechanism of bacterial cyanide detoxification with further enzymatic metabolism of the cyanohydrins helping to explain how cyanide can satisfy the nitrogen requirement in cyanide-utilizing bacteria.
The underwater optical climate of Lake Texoma was measured at eleven fixed stations from August 1996 to August 1997. Secchi transparency and submarine photometry characterized seasonal and spatial values of secchi depth (SD), vertical attenuation coefficient (η''), and depth of euphotic zone (Zeu). Indices of Zeu:SD and η'' × SD were compared with universally applied values derived from inland and coastal waters. Turbidity explained 76% of the variation (p = 0.0001) of η'' among water quality parameters, including chlorophyll-α. Using a spectroradiometer, spectral signatures of chlorophyll-α and turbidity were located. Stations with low turbidity exhibited a distinct green reflectance peak around 590-610 nanometers, indicating presence of chlorophyll-α. Stations with high turbidity exhibited a reflectance peak shift towards the red spectrum, making it difficult to detect the chlorophyll signature. Derivative analysis of the reflectance signal at 590-610, and 720-780 nanometers allowed discrimination of this chlorophyll signature from those of turbidity (0.66 ≤ r^2 ≤ 0.99).