Ligusticum porteri is a plant that grows in the higher altitudes (6,000 to 10,000 feet above sea level) of the southwestern and rocky mountain states. The root of this plant has been used for centuries to treat a variety of ailments. The common name for this plant is osha. The purpose of this study was to see if extracts of this plant had antimicrobial, allelopathic, or antiviral activity. Alcoholic extracts of the plant were used to determine any antibacterial, antifungal, or antiprotozoan activity. These extracts were inhibitory to many bacteria including Mycobacterium sp. and Bacillus sp. There was some antifungal and antiprotozoan activity detected. The hot water extract of Ligusticum porteri inhibited the growth of bean sprouts. This assay is used as a screening test for allelopathy as well as for anticancer activity. To determine what was occurring microscopically, onion root tip squashes were prepared and fixed onto slides. Altered cell morphology and differences in number of mitotic cells were observed in the hot water extract of Ligusticum porteri. Hot water extracts were also tested for antiviral activity against T4 phage. The extracts did not lower the number of plaque forming units per ml. In order to attempt to determine the solubility of the biologically active compounds, a differential solvent extraction was performed using six different solvents varying from non-polar to polar. Antibacterial activity was used to determine which extracts had biological activity. The most active fractions were petroleum ether and ethanol extracts.

[Index]

Evaluation of Ticks Collected from Selected Eastern States for the Presence of Borrelia Species.

Collins, Sarah L.* and Wright, Stephen M. Department of Biology.

Lyme disease, caused by the bite of Borrelia burgdorferi-infected Ixodes scapularis, is the most commonly reported vector-borne disease in the United States. This bacterial infection can result in serious diseases of the nervous system, the musculoskeletal system, or the heart if left untreated. Recently, a new species, B. lonestari, was discovered in Ambylomma americanum and determined to be the causative agent of a novel Lyme-like disease, Southern Tick Associated Rash Illness (STARI). This study surveyed 200 ticks from eight eastern states to evaluate the epidemiology of B. lonestari and of its association with B. burgdorferi. Polymerase chain reaction was performed using specific primers determined from the B. lonestari flagellin gene that target both species of Borrelia. Species-specific labeled probes were then used to differentiate between B. lonestari and B. burgdorferi. Ten percent of the ticks were positive for Borrelia species. Positive samples were detected from ticks collected from Kentucky (4 B. lonestari, 1 B. burgdorferi), Virginia (3 B. lonestari, 11 B. burgdorferi), and Massachusetts (1 B. burgdorferi). Particularly noteworthy, one B. burgdorferi sample was detected in the dog tick, Dermacentor albipitus. This connection has never been reported and may trigger re-evaluation of current belief regarding vectors of Borrelia.

[Index]

Construction and Evaluation of Green’s Functions for Exterior Problems.

Darrell, Robert M.* and Melnikov, Yuri A. Department of Mathematical Sciences.

In the field of differential equations the development and implementation of Green’s (influence) functions for solving boundary value problems (BVPs) is well established for ordinary differential equations. However this is not the case for BVPs for partial differential equations (PDEs). One reason is the difficulty in their development, and more important is the fact that they are usually expressed as infinite sums which have rather poor rates of convergence. Thus, when they are truncated, the results are not accurate enough for practical use. However, using Melnikov’s method, which is a modified version of the classical eigenfunction expansion method, the rates of convergence for these sums is vastly improved. The purpose of this project is to extend this method to solve PDEs in polar coordinates for exterior problems of homogeneous and piece-wise homogeneous regions over the half-plane. Problems from mathematical physics associated with potential fields (Laplace’s equations) are explored. The singular components of Green’s functions are analytically obtained, while the regular components are represented in terms of infinite Fourier series. Some examples with numerical results are illustrated.

[Index]

Preparation and Analysis of Biodiesel from Plant Lipids and Beef Tallow

David C. Galloway, Dr. Ngee Sing Chong Chemistry Department Middle Tennessee State University

Biodiesel has moved to the forefront as an alternative fuel for diesel engines as petroleum supplies worldwide begin to diminish. This biofuel is commonly prepared from plant lipids such as canola (rapeseed) oil using methanol and potassium hydroxide catalyst. Compared to conventional petroleum diesel fuel, biodiesel has a much higher flashpoint and is thus much safer to store. Harmful emissions are substantially reduced in Diesel engines operating with biodiesel fuel. The energy value of biodiesel is only slightly less than that of petroleum diesel. Biodiesel extends engine life due to its lubricating ability (lubricity). The effect that feedstock type has on biodiesel yield on a laboratory scale has been examined. Corn oil and canola (rapeseed) oil gave the highest yields while waste vegetable oil furnished the lowest yield, presumably due to the presence of polymeric substances formed by prolonged heating. Increasing the volume of methanol reactant beyond a certain point caused a reduction in biodiesel yield due to dilution effects. Cosolvents such as THF and diethyl ether augmented biodiesel yield but were difficult to remove in a satisfactory fashion at the end of the reaction period. Although additional work needs to be done to establish benchmark biodiesel yields, future research efforts will focus on analysis of biodiesel samples using GC/MS and IR. An intensive search of the chemical literature revealed that these analytical techniques have been infrequently employed to evaluate biodiesel composition. Chemometric software will be used to deduce the physical properties of biodiesel samples from their IR spectra.

[Index]

Quantifying Organic Constituents of Urban Runoff in Murfreesboro, TN: Potential Impact on Spring Water Chemistry.

James, Rebecca R.*, Ogden, Albert E.and DiVincenzo, John P. Department of Chemistry, Department of Geography and Geology.

The Stones River Watershed is identified by the Tennessee Department of Environment and Conservation (TDEC) as an impacted watershed due to rapid urban growth. The four springs in this study, Murfree, Oakland, Black Fox, and Guy James, are located within the Upper Stones River Watershed. The first phase of the project was to delineate the recharge areas for the springs using dye tracing techniques. The springs were then sampled two to three times a month beginning in September 2002. Field parameters such as pH, temperature, nitrate, ammonia, phosphate, and conductivity were measured to compare chemical characteristics of each spring. Dissolved oxygen (DO), chemical oxygen demand (COD), and biochemical oxygen demand (BOD₅) were measured in the lab. All springs showed low nitrate, ammonia, phosphate, BOD₅, and COD levels (< 34 mg/L). Conductivity and DO measurements were highest after storm events. Samples of stormwater runoff within the specified drainage basins were taken during rainfall events and analyzed for COD in addition to oil and grease (OG) and total petroleum hydrocarbons (TPH) using EPA Method 1664. A solid phase extraction (SPE) approach was developed as an alternative to the liquid-liquid extraction approach used in the traditional method. Runoff samples showed COD ranges (30-1000 mg/L) substantially higher than spring samples. Oil & grease values were found to be between 6 – 405 mg/L. Total petroleum hydrocarbon levels ranged from 5 – 49 mg/L. This type of runoff degrades the quality of natural water resources and is in line with the findings of the Nationwide Urban Runoff Program (NURP).

[Index]

Effects of Histone Deacetylase-1 on Topoisomerase II Activity.

Kaur, Niger* and Burden, D. Andrew. Department of Chemistry.

DNA topoisomerase II is an essential nuclear enzyme in DNA replication and transcription, chromatin segregation, and cell cycle progression.¹ DNA gets tightly packed in cells, and for it to replicate and carry out its normal function, it has to be able to untangle. Topoisomerase II unknots and decatenates DNA, thereby enabling DNA to change its topology and carry out its normal functions.² Protein-protein interactions between topoisomerase II and several other proteins have been shown to modulate the activity of the enzyme. Histone deacetylases (HDACs) are proteins involved in gene expression and in the packing of DNA.³ One of these proteins, histone deacetylase-1 (HDAC-1), has been previously shown to inhibit catalytic activity of topoisomerase II.^4,5 HDAC-1 was expressed as a GST fusion protein and was isolated from Escherichia coli cells. Optimal conditions for purifying the protein were also determined. The aim of this research is to determine the mechanism by which HDAC-1 inhibits topoisomerase II activity. It is known that HDAC-1 has an effect on topoisomerase II activity, but little is known about how this occurs. DNA binding and DNA relaxation assays were performed in order to achieve this goal. The results from this study can give further insight into the in vivo regulation of topoisomerase II.

[Index]

The Effects of the Inflammatory Cytokine, TNF-alpha, on Transformed Rat Beta-Cells of the Pancreas.

LeBoeuf, Jared P*, Jetton, Amy E. Department of Biology.

Recently, the incidence of type II diabetes mellitus (NIDDM) has increased drastically. Obesity and increased levels of inflammatory cytokines are associated with NIDDM. New treatments for NIDDM are designed to block effects of tumor necrosis factor-a (TNF-a), an inflammatory cytokine, on insulin target tissues. Inflammatory cytokines are also known to kill insulin-producing, b cells in type I diabetes; death of these cells is also a consequence of uncontrolled NIDDM. Our study explored possible interactions between TNF-a and glucose stimulation of INS-1 cells in culture. INS-1 cells are derived from a rat pancreatic tumor and produce insulin in response to glucose stimulation in a dose-dependent manner. INS-1 cells were cultured in 5.5 mM glucose in RPMI 1640 prior to exposure to high or low doses of TNF-a with or without increased glucose (3 x 2 design; n=6 wells/group; total of 36 wells). After 24 hrs. exposure to TNF-a (0, 0.1 or 100 ng/ml) and glucose (5.5 or 16.5 mM), media was collected for insulin assay by ELISA, and cells were visually assessed for apoptosis using Hoescht and propidium iodide dyes. Results were analyzed with ANOVA. In contrast to similar, previous work with INS-1, we were unable to demonstrate a correlation between TNF-a exposure and insulin production or to demonstrate a correlation between TNF-a or level of glucose exposure and apoptosis rates. Additionally, we saw no definite correlation between level of glucose exposure.

[Index]

Isolation and Identification of Lipase-Producing Microorganisms.

Logan-Dunn, Kimber * and Zamora, John M. Department of Biology.

Microbial enzymes are used in the baking, brewing, distilling, and textile industries. Lipases are the enzymes that break down lipids. The purpose of this study was to isolate and identify lipase-producing organisms. Several laboratory strains of organisms were plated onto spirit blue agar and minimal salts agar containing peanut oil to determine if these organisms produced lipase. Most organisms grew on spirit blue agar and the determination of lipase production was based on changes in the media. Only organisms capable of degrading peanut oil could grow on the minimal salts agar containing peanut oil. Soil samples were inoculated onto minimal salts agar containing peanut oil. Bacteria capable of breaking down the peanut oil were isolated and identified to species. The organisms were grown in liquid culture and lipid breakdown was determined using a titrimetric assay. Results were obtained and bacterial lipase units were calculated for each organism. Statistical analysis was used to determine the best lipid degraders. These environmental isolates may be useful in the production of lipase.

[Index]

Analysis of Polar Organic Compounds and Their Degradation By-products by GC-MS and FTIR

Chun U Park, Dr. Ngee-Sing Chong Chemistry Department

Analytical methods based on gas chromatography-mass spectrometry (GC-MS) and Fourier Transform infrared spectrometry (FTIR) are used to analyze polar organic compounds in environmental matrices and physiological fluids. Hexachlorocyclopentadiene and organophosphate compounds were sampled with gas cells, thermal desorption, and solid phase microextraction (SPME). These techniques allow the analysis of compounds such as chemical warfare agents, pesticides, and those from organic chemical manufacturers or hazardous waste incinerator. Different sorbent materials including Carbotrap 300, Tenax, C18, and XAD-2 were tested for their suitability for GC-MS analysis via thermal desorption. It was found that Carbotrap 300 had the cleanest or lowest background but was only useful for the detection of moderately polar compounds such as C10-C12 fatty acid methyl esters. C18 sorbent was found to be effective for the analysis of octanol, nonanal, 2,6-dimethylphenol, and 2,6-dimethylaniline but it gave undesirable background species such as trimethylsilanol, octanal, 2-ethyl-1-hexanol, and diethylbenzenes. SPME as an extraction technique for GC-MS was investigated with polydimethylsiloxane, polyacrylate, and Carbowax sorbents and its results will be reported with regard to extraction efficiency, equilibration time, sampling mode. The performance of FTIR with gas cells was studied for the measurement of dimethyl methyl phosphonate, diethyl ethyl phosphonate, and hexachlorocyclopentadient. It was found that maximum sensitivity was obtained at a cell temperature of 50 degree Celsius and detection limits at the low parts-per-million levels could be easily obtained. The use of sorbent tubes in conjunction with FTIR analysis has been investigated for achieving greater sensitivity of measurements at the parts-per-billion levels.

[Index]

Drink or Swim: Bacteriological Water Quality of Areas on the Stones River and J. Percy Priest Lake.

Rogers, Jamie L. *and Zamora, John M. Department of Biology.

The use of water for recreation, such as swimming, boating, fishing, is increasing. People who use these waters take it for granted that they are safe to use. A safe and abundant supply of drinking water is also taken for granted. Water supply is a matter of both quality and quantity. The demand for water is so great that often one city’s sewage water is reused as another city’s drinking water. The Stones River and J. Percy Priest Lake contain areas that are considered by the Tennessee Department of Environment and Conservation impacted and highly impacted by pollution. The purpose of this study was to test several areas of the Stones River and J. Percy Priest Lake for the presence of coliform bacteria as well as total bacteria using two methods. The first method was a standard aerobic plate count where water samples were collected, diluted, and plated onto tryptic soy agar as well as MacConkey agar. Tryptic soy agar was used for the enumeration of bacteria. MacConkey agar was used for the enumeration of coliforms. The second method was an agar dip slide method that allows for the estimating of total bacteria as well as enteric bacteria. This second method is much simpler and allows for routine monitoring of sewage, rivers, and lakes. Both methods showed elevated numbers of bacteria as well.

[Index]

Synthesis and Biological Analysis of m-Hydroxyaniline Derivatives of Podophyllotoxin

Ian Romaine, Department Of Chemistry Middle Tennessee State University

Since the early 1900s, when podophyllotoxin was isolated as the active ingredient from the Native American medicine podophyllum emodi, the science of chemotherapy has made great advances. Many podophyllotoxin derivatives have been made and used as everything from topoisomerase II poisons and microtubule inhibitors, to anti-HIV drugs. In the 1970s, a renewed interest in podophyllotoxin was experienced because of the podophyllotoxin analogs etoposide (VP-16) and teniposide (VM-26). Etoposide (VP-16) is one of the most widely used anti-cancer drugs in the world. Podophyllotoxin derivatives are used to treat small-cell lung cancer, testicular cancer, and leukemia. The target of these drugs is the topoisomerase II / DNA complex, where the drugs act by stabilizing the enzyme / DNA cleavage complex. Most recent synthetic studies have been aimed at changes in the C-4 position of podophyllotoxin and also at the 4′ methoxy group.⁵ These are produced through halogenated intermediates. Several series have been evaluated as to their inhibition of topoisomerase II and their cytotoxic effects on cells. Structure-activity relationships have shown the importance of the 4′ group and the importance of the C-4 group for cyctotoxicity and DNA cleavage. This research focuses on the synthesis of several C-4 and 4′ analogs, with the attempt to develop a DNA / drug binding assay. Previous assays have focused on the inhibition of topoisomerase II rather than on the binding of the drug to DNA. These studies will provide information on how the interaction between DNA and the drug may result in poisoning of topoisomerase II.

[Index]

Synthesis and Biochemical Analysis of p-Nitroaniline Derivatives of Podophyllotoxin

Moh’d Shadid, Department of Chemistry Middle Tennessee State University

Podophyllum peltatum, commonly known as the American mandrake or mayapple, and the related Indian species podophyllum emodi have been used medicinally for centuries. Podophyllotoxin is a bioactive lignan isolated from these plant sources and this compound has been the focus of extensive chemical modifications leading to clinically useful anticancer drugs. Semi-synthetic analogs of the naturally occurring podophyllotoxin have drawn much renewed interest in recent years as a result of the development of etoposide (VP-16) and teniposide (VM-26) as anticancer drugs.Etoposide reached clinical study in the early 1970s and its clinical efficacy has stimulated a renewed interest in the medicinal chemistry of the podophyllotoxin derived antitumor agents.³ Etoposide is used in the clinic for treatment of small-cell lung cancer, testicular cancer, lymphoma and leukemia. It has been shown that etoposide and related compounds are potent poisons of DNA topoisomerase II. These drugs act by stabilizing an enzyme-DNA complex in which the DNA is cleaved and covalently linked to the enzyme. Recent synthetic studies on podophyllotoxin have been focused on the synthesis of C-4 substituted analogs, which show improved topoisomerase II inhibition and cytotoxicity.⁶ A series of C-4 hydroxylated and halogenated anilino derivatives of epipodophyllotoxin and 4-demethylepipodophyllotoxin have been synthesized and evaluated for their inhibitory activity against human DNA topoisomerase II. Structure-activity relationship studies have demonstrated a direct correlation between cytotoxicity and topoisomerase II poisoning among the podophyllotoxin analogs. The focus of this research is the synthesis of p-nitro aniline derivatives of epi-podophyllotoxin. Previous biological assays of these derivatives have focused on cytotoxicity rather than enzyme binding. In this project we are attempting to gain information on the capability of various analogs to poison.

[Index]

“I’m dying. How do you think I feel?”

Shin, Chul M.,* Greer, Karalesa, and Brinthaupt, Thomas M. Department of Psychology.

Many experiments have studied the effects of mortality salience on mood; however, the results have been contradictory. We proposed that religion serves as a possible mediator for these findings such that non-religious individuals’ moods would be lowered through thoughts of death while religious people’s moods would be buffered by the thoughts of a better place. Analyses of the data revealed no significant differences between groups but the means showed the predicted trends.

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SYNTHESIS OF NUCLEOSIDE ADDUCTS OF meso-BUTADIENE DIEPOXIDE

Leah Wong, Department of Chemistry Middle Tennessee State University

Butadiene is a chemical commonly used in the industrial production of synthetic plastics and rubbers. It is also an environmental pollutant found in car exhaust and cigarette smoke. When butadiene is metabolized, it forms a diepoxide that has been found to be carcinogenic and mutagenic in laboratory mice and rats. The stereoisomers of adducts of the diepoxide have been studied, and it was found that adducts of the meso-butadiene diepoxide (BDO₂) are more cytotoxic than the (S,S) and the (R,R) stereoisomers. Analogs of the (S,S) and (R,R) isomers of BDO₂ have been synthesized previously; however, those syntheses are not amenable to the synthesis of the meso-analogs. The purpose of this research is to develop a synthetic route to pure stereoisomers of the meso-diepoxide adducts. The resulting nucleosides will ultimately be incorporated into oligonucleotides and tested for mutagenicity. The synthesis begins with 4-6-O-benzylidene-D-glucopyranose, which can be purchased relatively inexpensively. Oxidative cleavage followed by reductive amination gives a protected amine. Removal of the protecting groups results in (2S,3R)-1-amino-2,3,4-butanetriol. Coupling with nucleosides affords the adducts. A synthetic route to both enantiomers of the nucleoside adducts of meso-butadiene has now been developed. Access to these stereochemically pure isomers will aid in the study of the mutagenicity of butadiene.

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