Health sciences.

Chair: Ibrahim O. Farah, Jackson State University 

Vicechair: Hamed Benghuzzi, University of Mississippi Medical Center 

THURSDAY MORNING

Gulf Hall

10:00 3D ORIENTATION AND DYNAMICS OF PRIMARY SEMICIRCULAR CANAL AFFERENTS IN THE RHESUS MONKEY

Asim [Haque.sup.*], Dora E. Angelaki, and J. David Dickman, Alcorn State University, Alcorn State, MS 39096; Washington University, St. Louis, MO 63110; and, Central Institute for the Deaf, St. Louis, MO 63110

Neural signals from primary afferents in three rhesus monkeys were recorded during rotational motion using a 3-axis system. Each afferent was characterized as a posterior, horizontal, or anterior fiber using rotations in different planes. Next, the responses to sinusoidal stimuli at frequencies ranging between 0.02-4 Hz were obtained. Out of 72 afferents recorded, the directions of maximum sensitivity and response dynamics were determined for 13 posterior, 14 anterior, and 8 horizontal afferents. For regular (n=21), intermediate (n=3), and irregular (n=5) firing afferents, the average sensitivities at 0.5 Hz were 0.6[+ or -]0.3 spikesx[s.sup.-1]/degx[s.sup.-1], 1.7[+ or -]0.6 spikesx[s.sup.-1]/degx[s.sup.-1], and 0.8[+ or -]0.4 spikesx[s.sup.-1]/degx[s.sup.-1], respectively. The average phase leads relative to head velocity were 9.4[+ or -]4.4[degrees], 24.0[+ or -]9.3[degrees], and 31 .4[+ or -]5.5[degrees] respectively. Regular firing afferents had response dynamics that consisted of flat gains and constant phase advances across stimulus frequencies. Irregular firing afferents had pronounced gain increases and increased phase advances as stimulus frequency increased. The maximum sensitivity vector for each afferent was calculated from the responses to rotations in different planes. The vectors were plotted in the head-fixed coordinate system, defined by the alignment of the horizontal semicircular canals with an earth horizontal plane, using direction cosines. Other than a small yaw component for vertical canal afferents, the direction vectors tended to be orthogonally oriented to each other. Technical assistance was provided by Bennie Harris, Jesus Loya, and Quinn McHenry. This work was supported in part by NIH EY10851 and NASA NAG2-1204.

10:15 INTERACTION OF HUMAN COMPLEMENT FACTOR H WITH PSPC 

Corunda T. [Pruitt.sup.*] (1), Sandhya Dave (2), Joseph A. Cameron (1), and Larry S. McDaniel (2), (1.) Jackson State University, Jackson, MS 39217, and (2.) University of Mississippi Medical Center, Jackson, MS 39216

Streptococcus pneumoniae can evade the host immune system by a variety of mechanisms. The pneumococcal polysaccharide capsule and various surface proteins can prevent complement mediated opsonophagocytosis. PspC is one of the multi-functional pneumococcal surface proteins capable of eliciting an antibody response m mice. PspC consists of three domains: N-terminal a-helical domain, proline-rich domain, and choline-binding domain. We observed that PspC binds human Factor H (FH), a regulatory protein of the alternative complement pathway. The binding was demonstrated by Western blot analysis. Binding of FH to PspC on viable pneumococci was confirmed by flow cytometry and fluorescence microscopy. FH consists of 20 short consensus repeat (SCR) domains, with each domain containing approximately 60 amino acids. The sites of interaction between FH and PspC have not been previously localized. We used a series of FH truncated mutants to localize the region of FH that binds to PspC. The PspC truncated mutants were used to localize the region of PspC that binds FH. The site of FH that binds to PspC is located within the SCR 6-10 of FH. We have also localized the corresponding binding region on PspC to the first 225 amino acids of the a-helical domain.

10:30 SELECTIVE TOXICITY TO HEPG2 AND RAT PRIMARY LIVER CELLS BY 2,3 BUTANEDIONE

Ibrahim O. [Farah.sup.*] and Ali B. Ishaque, Jackson State University, Jackson, MS 39217

2,3 Butanedione (diacetyl) is the buttery flavor naturally found in daily products, fermented meat and beverages. This chemical is ubiqutous in its production and use. Its uses include the production of diones, dienes and oximes; analytical and pharmaceutical reagents. It is employed as starter in chemical reactions; in modification of proteins; photo initiation in polymers; and is a known preservative and additive for flavor enhancement in food production. Although its antimicrobial activity is well documented in literature, its effects on mammalian cells are not known. The objectives of this study are (1) to evaluate the survival response of mammalian HepG2 and Wistar Rat liver cells to diacetyl and (2) to evaluate its toxicity, inactivation mode and dose response trends. Since these responses were not known, expectations on adverse chronic outcomes are beyond the scope of this study. Cell culture techniques and chemical exposure protocols for concentrations ranging from 490 to 250.000 ppm (serial dilutions ; DMEM-Hyclone) were incorporated. Surviving cells following a 24 hour exposure period were measured using an Accent Fluoroskan at 485/538 nm. HepG2 and rat liver cells showed a toxicity index LC-50 of 530 /[+ or -]35 and 3400/[+ or -]354 ppm respectively. Both cell types showed a dose response to diacetyl with rat liver cells being less sensitive (6 fold increase in LC50). Interesting inactivation trends warranting further studies were seen, however, the study is promising with regards to the selective toxicity of diacetyl.

10:45 DOES NEONATAL METHYL PARATHION (MP) ALTER BRAIN ACETYLCHOLINESTERASE AND WITHDRAWAL FROM MORPHINE DEPENDENCE IN ADULT RATS?

Robin W. [Rockhold.sup.*] (1), A. Lampton (2), J. Lee (3), D. Yokum (4), and He Zhu (1), (1.) University Mississippi Medical Center, Jackson, MS 39216-4505; (2.) Murrah High School, Jackson, MS 39202; (3.) St. Andrew's Episcopal High School, Ridgeland, MS 39157; and (4.) St. Joseph Catholic School, Madison, WI 39110

The effects of repeated administration of MP, an organophosphate insecticide, on brain cholinesterase activity and motor function were investigated in neonatal rat. Pups were treated dermally with 0, 1, 3, or 5 mg/kg of MP daily for 21 days after birth. Cholinesterase activity decreased dose-dependently after MP. Pups exposed to MP, 5 mg/kg, showed signs of intoxication, e.g. slight tremor, and (cortex, hippocampus) cholinesterase activities were 20% of control. However, neither body weight nor locomotor function (open field tests) were reduced. In adult rats, three months after neonatal MP treatment, cholinesterase activities had returned to normal. To test whether neonatal exposure to MP altered the development of opioid dependence, adult rats were made dependent by continuous intracerebroventricular (i.c.v.) infusion of morphine (26 nmol/mI/h) for 3 days. Withdrawal was precipitated after three days of infusion by an opioid antagonist, naloxone (48 nmol/5 ml, i.c.v.). Eight withdrawal behaviors were scored . Although the rats that were treated with 5 mg/kg MP exhibited a higher opioid withdrawal score compared to the control group, the involvement of neonatal cholinergic modulation in opioid dependence still remains to be determined. (Sponsored by the Howard Hughes Medical Institute and RO6CCR419466-01)

11:00 TRANSMISSION GENETICS OF MACHADOJOSEPH DISEASE FAILS TO SUGGEST MALE MEIOSIS MEIOTIC DRIVE

Kim A. Parker, Millsaps College, Jackson, MS 39210

The purpose of this project was to determine the transmission rate from affected males and females of the expanded CAG sequence in the MJD1 gene at 14q32.1 which causes Machado-Joseph disease (MJD), a neurological disease. Previous research projects have suggested a favored transmission from affected fathers to their offspring. Thirteen extensive pedigrees detailing history of affected family members were examined. The number of transmissions from affected males and females were recorded. The offspring of 64 affected individuals (34 male, 30 female) were evaluated. Of 303 offspring (150 male, 153 female) who were evaluated, 137 (62 male, 75 female) were affected by Machado-Joseph disease. The research shows no distortion in transmission ratios that would suggest a meiotic drive in male meiosis. Current observations suggest that there is no favored transmission and the rate that offspring inherit the disease from affected males and affected females operates in accordance with traditional Mendelian ratios.

THURSDAY AFTERNOON

Gulf Hall

1:00 Divisional Poster Session

INFLUENCE OF LEAD ON ENERGY LEVELS AND METAL SEQUESTRATION IN THE BRAIN, LIVER, AND KIDNEY OF ADULT RAT

D. [Jones.sup.*] (1), B. Rajanna (1), J. Butler (1), D. Donald (1), L. Davis (1), S. Rajanna (1), K. Kumar (2), S. Shirisha (2), Neelima (2), and Y.P. Rao (2), (1.) Alcorn State University, Lorman, MS 39096, and (2.) Andhr University, India

Lead (Pb) is a potent toxicant and it disturbs cellular energy metabolism of the brain as well as the liver and the kidney. The objective of this study was to determine the energy levels of different regions of the brain, as well as the liver and the kidney of adult rats exposed to Pb. Lead as lead acetate (500 ppm) was administered through drinking water to male (Wistar strain) rats weighing 125-150 g for 8 weeks and the controls received similar doses of sodium acetate. Rats were sacrificed at intervals of 1, 4, and 8 wks. The whole brain tissue was separated into four regions. Tissues were individually dried at 80[degrees]C for 48 h and then homogenized into dry powder, which was used to estimate total carbohydrates, proteins and lipids. The resulting data was converted into energy levels by using specific constants. Estimation of Pb content was done in the dry tissue powder. The results indicated that Pb decreased energy levels in the liver and kidney as well as in all regions of the brain. The concentrat ion of Pb was higher in all tissues tested. A significant correlation was observed between these two parameters in all tissues. The influence of Pb on energy levels followed a pattern of region specificity and exposure period. The results suggest the Pb interferes in the cellular energy metabolism in different tissues studied. (Supported by NIH/FIC/MIRT #TW00132, NIH/MBRS-SCORE #GM55356)

REGRESSION OF HYPERTROPHY BY CARDIO-SPECIFIC INHIBITOR OF METALLOPROTEINASE IN CHRONIC HEART FAILURE IN MICE

Urseline A. [Hawkins.sup.*] and Suresh C. Tyagi, University of Mississippi Medical Center, Jackson, MS 39216

Left ventricle hypertrophy (LVH) is the hallmark of chronic heart failure (CHF). Previous studies have suggested decreased levels of cardiac inhibitor of metallo-proteinase (CIMP) in heart failure. To test the hypothesis that decreased levels of CIMP lead to LVH, and that increased levels of CIMP ameliorate LVH in CHF, we sought to purify and characterize CIMP from mouse hearts. The heart extracts were prepared, and samples were then loaded onto a preparative continuous gel electrophoresis column. The fractions were collected and analyzed for protein by the SDS-PAGE as well as by the Western blot analysis using a CIMP antibody. Fractions with single bands on the SDS-PAGE, and on the Western blot analysis were identified as being positive for CIMP. To create CHF, an arteriovenous (AV) fistula was created in normal wild type C57BL/J6 mice. The CIMP protein transfer (40 micrograms/day) was performed by a minipump for 4 weeks. The heart weights were measured. The results suggested an increase in heart weights in the AV fistula mice. The heart weight was significantly decreased in AV fistula mice treated with CIMP. These results suggest that CIMP protein transfer decreases heart weight, thereby decreasing hypertrophy in CHF mice.

COMPARISON OF EXTRACTION METHODS AND PROCEDURAL DEVELOPMENTS FOR DRUGS OF ABUSE ANALYSIS IN SERUM USING EMIT[R]

Kirk C. [Eddleman.sup.*], Arthur S. Hume, Patrick B. Kyle, J. Lee Spencer, and Christine M. Purser, University of Mississippi Medical Center, Jackson, MS 39216

A comparison of three different methods using methanol, acetone, and acetonitrile as extracting solvents for drugs of abuse in serum was conducted. The purpose of this study was to determine which of these methodologies is the most effective in the analyses of serum for drugs of abuse. The extracts were analyzed by enzyme-multiplied immunoassay technique (EMIT[R] II Plus, Syva Company, Dade Behring, Inc.). Seven urine assays were used: amphetamine, barbiturate, benzodiazapine, cannabinoids, cocaine, opiate, and phencyclidine (PCP). A control with a known concentration of each drug and a drug free control were prepared and processed through each extraction procedure. The sensitivity of each method was determined by comparing the absorbance differences in the positive and negative controls. For our applications, the acetonitrile method proved to be the most efficient extraction method. The development of cutoff values and concentrations for the controls was then conducted based on the toxic and lethal levels of the selected drugs. One from each drug class was selected at a specific concentration to be used in the positive control for routine analytical testing. Cutoff values for the drugs in the positive control were designated as d-amphetamine-50 ng/ml, butalbital-300 ng/ml, oxazepam-200 ng/ml, carboxytetrahydrocannabinol-30 ng/ml, benzoylecgonine-50 ng/ml, oxycodone-50 ng/ml, and phencyclidine-25 ng/ml.

DRUGS DETECTED BY IMMUNOASSAY DRUGS OF ABUSE SCREENS VS. GAS CHROMATOGRAPHY/MASS SPECTROSCOPY IN SUSPECTED PEDIATRIC INGESTIONS

Patrick B. [Kyle.sup.*], Arthur S. Hume, James L. Spencer, Christine M. Purser, and Kirk C. Eddleman, University of Mississippi Medical Center, Jackson, MS 39216

Rapid and accurate analytical testing can be of great value when determining treatment for pediatric patients suspected of ingesting an unknown chemical. Though the patient's condition may potentially deteriorate into coma or death, clinicians often overlook gas chromatography/mass spectroscopy (GC/MS) as a valuable resource in toxicology testing. This report relates that analysis by GC/MS offers more significant information about the toxicological exposure of the pediatric patient than immunoassay drug screening. In a recent 24-month period, the University of Mississippi Medical Center Analytical Toxicology Laboratory tested 139 urine samples referred for STAT toxicology screening from the hospital's Pediatric Emergency Department. Most patients exhibited symptoms such as ataxia, seizures, and unresponsiveness. Toxicology testing was ordered to confirm or rule out a chemical ingestion. When tested by immunoassay (Dade Behring EMITO II Plus), 17.3% of the samples were positive for a drug of abuse. When analyzed by GC/MS (Thermoquest GCQO), drugs were detected in 88.5% of the samples. Caffeine and theobromine accounted for 21.1% of the positive samples by GC/MS. A total of 64 different pharmaceuticals were identified by GC/MS with an average of 2.21 drugs per sample. In conclusion, analysis by GC/MS offers the clinician a more comprehensive view of exposure patterns of pediatric patients presenting with an unknown chemical ingestion.

THE VALUE OF BENZODIAZEPINE CONFIRMATIONS IN A VETERAN POPULATION

Patrick B. [Kyle.sup.*] and Arthur S. Hume, University of Mississippi Medical Center, Jackson, MS 39216

As the cost of analytical testing continues to rise, many hospitals order drug screen confirmations only as directed by the patient's physician rather than automatically. While this may appear adequate in some clinical settings, confirmatory testing is often required in possible medico-legal situations when the presence of drugs may have serious implications in the workplace or courts of law. In a recent 12-month period, a local Veterans Affairs Medical Center referred 409 urine samples to the UMMC. Analytical Toxicology Laboratory for benzodiazepine confirmations. All samples had screened positive using the Abbot AXSYMO immunoassay analyzer. A second screen was performed with the ROCHEO MIRAO S using Dade Behring EmitO II Plus reagents. Confirmations were performed utilizing a WatersO high pressure liquid chromatograph equipped with an ultraviolet photodiode array detector and a computerized library established in house. Of the 409 samples tested positive by the AxSYMO, 382 (93.4%) generated positive results when tested by the MIRAO S. Benzodiazepines were confirmed in 322(78.7%) of the samples. The AXSYMO generated 87 (21.3%) false positives while the MIRAO S generated 71(17.4%) false positive results respectively. Oxaprozin was identified as the causative agent in 42 of the false positive samples. Finally, discontinuation of benzodiazepine confirmations would result in inclusion of a significant amount of false information in the medical files of some patients, resulting in negative consequences affecting patient treatment, drugs rehabilitation, parole compliance, etc.

CYTOKINE PROMOTER GENE POLYMORPHISM IN ASSOCIATION WITH CYTOKINE EXPRESSION IN PATIENTS WITH HYPERTENSION

Taylor [Butler.sup.*], Scott Rhodes, Vernetta Colman, Maria Oropeza, and D. Olga McDaniel, University of Mississippi Medical Center, Jackson, MS 39216

Hypertension is the number one public health problem in the United States, particularly among African Americans. It can cause a stroke, cardiovascular disease, and kidney failure. Many aging Americans experience elevated blood pressure with age. Overall prevalence of hypertension is 25% and is 35% in African Americans. A number of biological factors contributes to an elevated blood pressure, including growth factors, cytokines and hormones. The effective role of cytokines and growth factors contributing to the induction of hypertension is unknown. It was demonstrated in our recent study that TGF-b1 was hyperexpressed in African-American patients with renal transplant, and majority of these patients had suffered from hypertension. Therefore, the goal was to investigate the possible association between cytokine gene polymorphism and clinical characteristics of hypertension. We have analyzed DNA samples from 160 well-characterized African-American patients with known hypertension and 77 African-American individu als from the normal pool with no evidence of elevated blood pressure. Cytokine gene polymorphism was tested by cytogene protocol using a polymerase chain reaction based sequence specific polymorphism (SSP) detection method. DNA was amplified for identification of TNF-a, TGF-b1, IL-10, IL-6 and IFN-g. A high TGF-bl, T/C G/G genotype was found in 40% of African-American patients with hypertension as compared with 27% of African-American controls, p=0.03. A low IFN-g genotype was increased 67.5% in hypertension group, compared with 57% in non-hypertension group (p = 0.03, RR = 1.18) indicating a new mediator of hypertension. This data demonstrates TGF-b1 and IFN-g genotype polymorphism in concert may contribute to the elevated level of blood pressure in African-American.

AFFERENT INNVERVATION PATTERNS OF THE HORIZONTAL CRISTA AMPULLARIS IN PIGEONS

Asim [Haque.sup.*] and J. David Dickman, Alcorn State University, Alcorn State, MS 39096, and Central Institute for the Deaf, St. Louis, MO 63110

Primary afferents that innervate receptor cells of the semicircular canal neuroepithelium have generally been characterized based upon their regional innervation patterns. The avian labyrinth is of particular interest because of recent evidence showing that both hair cells and their innervating afferents regenerate after death produced by ototoxic insult. In the present study, biotinylated dextran amine (BDA) tracer was injected into the vestibular nuclei of pigeons (Columbia livia) for retrograde transport by canal afferents in order to visualize individual fiber innervation patterns. After post-injection survival, the labyrinth was exposed followed by fixative perfusion, subsequently harvested, reacted in whole mount, embedded in plastic, sectioned, and mounted on glass slides. Serial reconstructions were performed with visual imaging and 3D reconstruction software. In four pigeons, the horizontal canal neuroepithelium was examined and traced. Out of 135 identifiable horizontal semicircular canal afferents, sufficient labeling was present to reconstruct 70 units, including 24 calyces, 25 dimorphic, and 21 bouton units The lateral portion of the neuroepithelium was innervated primarily by bouton type afferents where only type II cells were present. Dimorphic afferents were spread throughout the intermediate and medial portions of the crista. Calyx type afferents were mostly observed in the medial portions of the crista. In the transverse plane of the crista, calyces, boutons, and dimorphs were located throughout the epithelium from the apex to both edges. However, type I cells were concentrated near the apex and type II cells near the peripheral edge. This work was supported by funds from the NIDCD (DC02386).

THE EFFECTS OF TRI-CALCIUM PHOSPHATE (TCP) CERAMICS SINTERED AT DIFFERENT TEMPERATURES ON THE PROLIFERATION OF MRC-5 FIBROBLASTS

Michael [Cox.sup.*], John Ward, Brian Trussell, Hamed Benghuzzi, Michelle Tucci, and James Hughes, University of Mississippi Medical Center, Jackson, MS 39216

Calcium phosphate ceramics have recently been used as dental implants, drug delivery systems, and models for studies on bone remodeling. Although their use has been very successful, problems such as aseptic loosening, coagulation, fibrous tissue formation, and inflammation have been noted. The objective of the study was to investigate the effects of tri-calcium phosphate (TCP) ceramics sintered at different temperatures on the proliferation and cellular alteration of MRC-5 fibroblasts like cells. This was accomplished by light microscopy morphologic analysis, measurements of MDA (a measure of cell damage), total protein, and cell counts. A total of 96 TCP ceramics were divided into 4 groups of 24, with each group being assigned a different sintering temperatures of 800, 1000, 1200, or 1400[degrees]C. Each temperature group was then further divided into 8 ceramics per phase of 24, 48 and 72 hours. All data were analyzed using Sigma Stat software. The data collected from this study provided useful information p ertaining to the optimum manufacturing conditions that produce high proliferation rates with minimal cellular damage. At 72 hours (the phase simulating chronic conditions), cell counts and MDA analysis showed that the cells incubated with the TCP sintered at 1200[degrees]C had the greatest proliferation and least damage when compared to the other groups. Morphological, MDA and protein analysis showed the cells incubated with the 1400[degrees]C TCP capsules to have undergone obvious cellular adaptations or other extreme cellular alterations. Overall, the data collected revealed a marked alteration in cellular function and morphology over the range of sintering temperatures that were tested.

RECOVERY OF RAW 264.7 PHOSPHATIDIC ACID FROM THIN LAYER CHROMATOGRAPHY AFTER STIMULATION WITH LIPOPOLYSACCHARIDE 

Kristie R. [Carter.sup.*], Michelle Tucci, Hamed Benghuzzi, James Hughes, and Rodney Baker, University of Mississippi Medical Center, Jackson, MS 39216

Lipopolysaccharide (LPS) is an endotoxin that has been shown to stimulate the release of inflammatory mediators from RAW 264.7 cells. Ethanol inhibits LPS mediated events as well as changes the profile of the phospholipids on the surface of the cell. The objectives of this experiment were to (1) identify the changes in phosphatidic acid (PA) in RAW 264.7 cells stimulated with LPS or in the presence of LPS and ethanol; and (2) to quantitate the recovery of PA by thin layer chromatography (TLC). RAW 264.7 cells were labeled with [[H.sup.3]]-palmitic acid overnight. The cells were then treated with LPS, ethanol, or both for thirty minutes. Following exposure, phosphatidic acid was isolated by TLC. The tritium-labeled phosphatidic acid band was identified by comparison to a PA standard. The PA region of the plate was scraped and placed in 5 mL of scintillation cocktail. Quantitation of PA was carried out by scintillation counting. The results show addition of2&#61549 g of LPS stimulated RAW 264.7 cells, as eviden ced by a 173% increase in PA within 30 minutes. Also, the addition of 20 mM ethanol prior to stimulation with LPS inhibited the formation of PA by 163% in just 30 minutes. The combination of LPS and ethanol resulted in an 11% increase in PA compared to control cells and a 162% decrease in PA from LPS stimulated cells. The efficiency of recovering the fraction of PA after TLC separation ranged between 10 and 27%. In conclusion, phosphatidic acid is significantly increased in LPS stimulated cells, and TLC separation and extraction allows for sufficient amounts of PA which can be analyzed further by techniques such as mass spectrometry

PROLIFERATION AND MORPHOLOGICAL TRANSFORMATION OF RMK CELLS EXPOSED TO HYDROQUINONE CONTAINING IONOMERS

Veronica [Harvey.sup.*], Hamed Benghuzzi, Michelle Tucci, Zelma Cason, and Aaron Puckett, University of Mississippi Medical Center, Jackson, MS 39216

Recent research in our laboratories have been directed towards the development of ionomeric polymers and monomers for use in biomedical applications such as adhesives, controlled release matrices and tissue scaffolds. The curing reactions are based on free radical chemistry that require a radical scavenger, hydroquinone (HQ) to adjust working and setting times and shelf-life stability. The few studies published on HQ have suggested that high dosages of HQ may stimulate apoptosis as well as an increased cellular leakage, however the effect of HQ on the biocompatability is unknown. Therefore the objectives of this study were to measure the functional capacity, cell proliferation and structural integrity of Rhesus monkey kidney epithelial (RMK) cells exposed to ionomer formulations containing 4 different levels of HQ. A total of 90 tubes of RMK (40,000 cells per tube) cells were divided equally into five equal groups. Group I served as a control and groups II-V were subjected to ionomers containing 0, 500, 1000, and 2000 ppm HQ. Cell numbers, morphology, cellular and supernatant MDA levels, and total protein analysis were performed. The results suggest: (i) All ionomer groups increased cellular proliferation except for the 2000 ppm HQ group; (ii) MDA levels were increased in cells containing 2000 ppm HQ at 24 hours; and 0 ppm at 48 hours whereas the other ionomer treated groups exhibited decreased MDA levels. It may be concluded that HQ concentrations over 1000 ppm may adversely effect biocompatability.

THE ROLE OF DHEA ON THE VIABILITY OF UV RADIATED A549 CELLS

Sandi [Zimmerman.sup.*], Hamed Benghuzzi, Zelma Cason, and Michelle Tucci, University of Mississippi Medical Center, Jackson, MS 39216

Dehydroepiandrosterone (DHEA) is a hormone with antioxidant properties that is endogenously produced in the body, which declines with age. DHEA was shown to protect epithelial cells from direct UV damage. Therefore, the objectives of this study were: (1) to evaluate the effect of cell proliferation and viability after UV exposure, and (2) to determine the effectiveness of DHEA treatment prior to or following UV exposure. A549 cells (n = 96) were divided randomly into 4 equal groups. Control (untreated), UV exposed, DHEA added prior to exposure and DHEA added following exposure. Eight tubes from each group were terminated at 24,48, and 72 hours, and cell counts, protein and MDA levels, as well as, morphology were analyzed. The results revealed exposure to UV light for 30 minutes significantly reduced cell number at 24, 48, and 72 hours. DHEA added prior to or after UV exposure caused a slight increase in cell number over UV exposed cells, but was significantly less than the untreated group at 24-72 hours. DHEA added after UV exposure began to offer protection at 48 hours and by 72 hours the cell numbers began to approach those seen in untreated cells. MDA levels were elevated in all UV exposed groups at 24 hours, and by 72 hours the MDA levels in the DHEA treated groups were not statistically different from the control. In conclusion, the use of antioxidants such as DHEA after UV exposure resulted in minimal cellular damage, suggesting that DHEA is more affective when used as a treatment.

CYTOPATHOLOGICAL EVALUATION OF THE FEMALE REPRODUCTIVE TRACT EXPOSED TO SUSTINED DELIVERY OF DHEA OR DHEA E IN RATS

Tinetta [Robinson.sup.*], Hamed Beughuzzi, Michelle Tucci, and Zelma Cason, University of Mississippi Medical Center, Jackson, MS 39216

The specific objective of this investigation was to investigate the cytopathological changes associated with sustained delivery of DHEA alone or in combination with estrogen (E) using adult intact female rats as a model. A total of nine adult rats allocated for this study (BW 220-250 g). The animals were divided into three equal groups (G1 = control, G2 = DHEA (2-5 ng/ml), DHEA E (10 pg/ml). At the end of 21 days post-treatment the reproductive organs (ovaries, cervix, fallopian tube, uterus and vaginal tissue) were retrieved and subjected to two different extensively used stains (H & E and PAP stains). The results of this study demonstrated that: (1) the use of H & E staining procedure is considered favorable in tissues like ovaries, tubes, and uterus. In contrast, the use of PAP staining is recommended for the cervix and vaginal tissue, (2) no significant difference was observed in ovaries, uterus, and fallopian tubes compared to the control group, (3) treatment with DHEA induced nuclear hyperchromasia of c ervical cells compared to DHEA E and control, (4) the use of DHEA alone or in combination induced an increase in mitotic ability of the vaginal tissue in comparison to the control, and (5) the treatment with DHEA or DHEA E resulted in occasional hyperchromatic nuclear condition compared to the control group.

THE EFFICACY OF TCPL AS E PLUS P REPLACEMENT THERAPY MODEL USING OVARIECTOMIZED RATS AS A MODEL

Zelma [Cason.sup.*] (1) Hamed Benghuzzi (1), Michelle Tucci (1), and Barry [England.sup.*] (2), (1.) University of Mississippi Medical Center, Jackson, MS 39216, and (2.) The University of Michigan Medical Center, Ann Arbor, MI

Tricalcium phosphate lysine (TCPL) delivery system was used to deliver various organic compounds at sustained levels in many different models. The specific aim of this investigation was to utilize TCPL delivery system as a model for estrogen (E) plus progesterone (P) replacement therapy in post-ovariectomized adult rats mimicking a postmenopausal condition. A total of 13 adult female rats were used in this study. The animals were randomly divided into four different groups: groups 1, 2 and 3 were overiectomized (OVX), OVX plus E (20 mg loaded TCPL), and OVX plus E (TCPL, 20 mg) plus P (TCPL, 60 mg) treatment. Group 4 animals (n = 4) served as intact control group. Blood samples were collected biweekly for 33 days. Vaginal smears were taken and screened daily during the entire investigation. The total serum levels of E, P, luteinizing hormone (LH), and follicle stimulating hormone (FSH) were measured by means of radioimmunoassay procedure. Data obtained from this investigation suggest the following: (1) OVX re sulted in an increase in total serum levels of LH and FSH within 2 days post-ovariectomy, (II) TCPL were capable of releasing sustained levels of E (10-40 pg/ml) and P (2.30-3.75 ng/ml) at the end of second day and continued until the [33.sup.rd] day, (III) the sustained levels of E plus P were able to suppress the post ovariectomy rise of LH and FSH to almost undetectable levels, (IV) sustained delivery of E resulted in maturation of vaginal epithelium and the smears exhibited the estrus phase throughout the investigational period (V) E P treatment induced no estrus and the epithelial changes resembled the OVX group.

CYTOPATHOLOGICAL AND BIOCHEMICAL EVALUATION OF A549 CELLS TREATED WITH MINERAL AND GLUCOCORTICOIDS 

Audrey [Cage.sup.*], Hamed Benghuzzi, Michelle Tucci, and Zelma Cason, University of Mississippi Medical Center, Jackson, MS 39216

The synergistic interaction of aldosterone and cortisol on respiratory cells has not been fully investigated. The objectives of this study were: (1) to determine the role of aldosterone and cortisol on the viability of A549 cells and (2) to evaluate the morphology of the cells treated with aldosterone and cortisol. A549 cells (147 tubes) were divided into seven equal groups, (control, vehicle control, aldosterone 0.015 pg, 0.033 pg, and 0.066 pg, cortisol and cortisol aldosterone (0.033 pg) (A C)). Cells were incubated for 24, 48, and 72 hours and analyzed for cell number, morphology and biochemical markers. Neither cortisol nor aldosterone increased cell numbers during the entire experiment. However, addition of A C caused significant decreases in cell number (at 24 and 48 hours) that was not evident at 72 hours of exposure. Cellular protein concentrations were similar for all groups at 24, 48, and 72 hours. Cellular MDA levels were increased in treated groups at 24 hours. Morphological evaluation revealed t he following: (1) treated cells were hyperchromatic with lower nuclear/cytoplasmic (N/C) ratio at 24 & 48-hours, (2) aldosterone treated cells appeared anucleated, vacuolated, with increased N/C ratios 24 & 48-hours, (3) cortisol increased spindle-shapes and cells with foamy cytoplasm, (4) cortisol and A C caused vacuolization, enlarged nuclei and cellular degeneration at 48 & 72 hours. Overall, the data suggest cells have either adapted to their environment or have metabolized the hormones. Future studies should include continuous hormone supplementation and compared with cells given a bolus dose.

THE EFFECTS OF TRICALCIUM PHOSPHATE CERAMICS WITH AMINO ACID BINDERS ON THE PROLIFERATION AND VIABILITY OF MRC-5 CELLS

John [Ward.sup.*], Brian Trussell, Michael Cox, Hamed Benghuzzi, Michelle Tucci, Audrey Tsao, and James Hughes, University of Mississippi Medical Center, Jackson, MS 39216

Ceramic material interacts with surrounding tissue. Therefore, ceramics can serve as scaffolds for tissue growth. The objective of this study was to determine if ceramics formulated with amino acid binders can enhance and support cell growth. A total of 147 tubes containing 1 x [10.sup.5] MRC5 cells were divided into seven equal groups. Group I served as control, Groups II-VI were incubated with ceramics containing amino acid binders (ASP, CYS, HIS, LYS, or SER, respectively) and Group VII served as ceramic without binder. Equal numbers of tubes were terminated at 24, 48, and 72 hours. Cell numbers, morphology and damage were determined at each time point. At 24 hours, cell numbers were increased in experimental groups, and. MDA levels were increased in the control. Cell numbers at 48 hours were increased in all groups with the exception of HYS. HYS treated cells also had higher MDA levels. In addition to cell number, cellular adhesion was also increased in the experimental groups at both 48 and 72 hours. At 72 hours total cell number was increased in CYS and LYS groups. Cells exposed to ceramics with SER as a binder showed decreased cell numbers as well as increased MDA levels at 72 hours. Morphological evaluation of the cells showed amino acid binders with acidic side chains caused an increase in the presence of spindled shaped cells, neutral side chains resulted in cells with elongated appearances, and basic side chains caused the cells to be less elongated. In conclusion, amino acid binders are able to induce the cells to adhere, proliferate and affect their morphological appearance as early as 24 hours.

LOCALIZATION OF IL-6 ON VENTRICULAR TISSUES EXPOSED TO SUSTAINED DELIVERY OF AED, T, AND DHT USING A RAT MODEL

Shontell [Credit1.sup.*] (1), Hamed Benghuzzi, (2) Joseph A. Cameron, (1) and Michelle Tucci, (2) (1.) Jackson State University, Jackson, MS 39204, and (2.) University of Mississippi Medical Center, Jackson, MS 39216

Interleukin-6 has been shown to play a potential role in myocardial injury. This complication can result from normal postreperfusion injury as well as reduced oxygen levels as seen in congestive heart failure. It has been shown in rats that androgen therapy improves coronary blood flow and increases both fractional shortening and peak myocardial oxygen consumption, thereby improving cardiac function. The objectives of this study were: (i) to morphologically (H & E) evaluate the cellular alternations associated with sustained delivery of testosterone (T), dihydrotestosterone (DHT), and androstenedione (AED), and (i) to localize cytokine production mainly interleukin-6 (IL-6) in the ventricular tissues exposed to sustained levels of T (5 ng/ml), DHT (2 ng/ml) and EAD (2 ng/ml). A total of 16 rats were divided into four equal groups. Animals in group I served as control and animals in groups II-IV were implanted with drug delivery systems containing T, DHT, and EAD, respectively. At the end of 90 days post treat ment, the animals were euthanized and the tissues were retrieved and fixed for histological procedures. Representative apical ventricles were sectioned and immunostained for IL-6 producing cells. The results revealed that (i) the exposure of sustained levels of androgenic hormones exhibited myocardial hypertrophic condition compared to the control animals, and (ii) the control animals had a two-fold increase in IL-6 production over T treated animals and approximately fivefold increase over DHT and AED treated animals. In conclusion, IL-6 production is decreased in animals given androgenic steroids and this decrease can be explained by a possible increase in blood flow, resulting in an increase in oxygenation of the myocardium.

BIOCHEMICAL AND STRUCTURAL CHANGES ASSOCIATED WITH SUPPLEMENTATION OF ANTIOXIDANT TO MRC-5 FIBROBLAST CELLS EXPOSED TO UVR

Pamala Fair [Jones.sup.*], Hamed Benghuzzi, and Michelle Tucci, University of Mississippi Medical Center, Jackson, MS 39216

The long-term goal of this study is to provide an effective preventative mode as well as treatment/cure for skin damage mediated by exposure to radiation. The specific aims of this project were: (i) to evaluate the morphological changes of MRC-5 fibroblast cells exposed to ultraviolet radiation (UVR) at various time periods, (ii) to investigate the pathophysiological responses associated with ultraviolet radiation exposure to MRC-5 fibroblast cells, (iii) to evaluate the response(s) of UV exposed MRC-5 fibroblast cells to antioxidant supplementation as a treatment measure, (iv) to evaluate the effects of supplementation of antioxidants to MRC-5 fibroblast cells before exposure to ultraviolet radiation as a preventative measure. A total of 115 vials (100,000 Cells/Vial) of MRC-5 fibroblast cells were used, the MRC-5 fibroblast cells were treated with UVR from a standard light bulb at various time intervals (24, 48, and 72 hours) and in the second phase the cells were treated with vitamin E (0.04 U/ml) to asses s the antioxidants effect. Proliferation rate, biochemical marker changes (total protein, MDA, GTH) and morphological evaluations were performed following standard laboratories protocols. The results of the project revealed that UVR induced significant effect on the proliferation rate of MRC-5 fibroblast cells upon the exposure duration of 45 and 60 minutes. Structural and biochemical changes were noted throughout the experimental phases (24, 48, and 72 hours) among the UVR exposed groups compared to control cells. However, treatment with Vitamin E prior and post exposure provided a protective role by demonstrating lower levels of MDA (P < 0.05) as well as normal structural appearance.

THE GENDER EFFECT ON THE VIABILITY AND PROLIFERATION OF MRC-5 CELLS INCUBATED WITH LIPOPROTEINS

Felicia Magee [Tardy.sup.*], Hamed Benghuzzi, and Michelle Tucci, University of Mississippi Medical Center, Jackson, MS 39216

Increasing evidence reveals that low-density lipoprotein (LDL) and gender are significant risk factors in the development of cardiovascular disease. The specific aims of this investigation include: (1) to evaluate the effect of estrogen (E) and testosterone (T) on the proliferation of MRC-5 cells, (2) to investigate the role of E and T on the viability of MRC-5 cells exposed to physiological and supraphysiological levels of LDL, and (3) to evaluate the morphological changes associated with E, T, and LDL, alone or in combination, on MRC-5 cells. MRC-5 cells exposed to T and LDL resulted in increased proliferation rates and remarkable cellular damage. In contrast, E exposure induced decreased levels of MDA compared to T exposure. Results from this investigation suggest that the development of collagen matrix post cardiovascular necrosis can be attributed to the presence of T. This response could be triggered directly at the fibroblast level or by altering the physiochemical characteristics of LDL.

MORPHOMETRIC ANALYSIS OF CUBOIDAL EPITHELIUM IN THE VENTRAL PROSTATE DURING EARLY DEVELOPMENT OF BENIGN PROSTATIC HYPERPLASIA 

Melanie C. [Pollan.sup.*], Hamed Benghuzzi, Michelle Tucci, and Zelma Cason, University of Mississippi Medical Center, Jackson, MS 39216

This study was designed to provide broader insights into the preventative measures to be considered in interfering with the development of benign prostatic hyperplasia (BPH). To accomplish this, Sprague Dawley rats (16, 250-300 g) were divided into four equal groups. Three treatment groups were each implanted with ceramic drug delivery devices designed to deliver continuous physiologic doses of testosterone (T), dihydrotestosterone (DHT), or androstenedione (AED). Morphometric analysis of epithelial cells lining the tubules of the ventral prostate in these rats gave an indication of pre-clinical manifestation as seen at the cellular level during early development of BPH. Using Image Pro software, the total cell area, nuclear area and cell length were measured. Also counted the number of cells per high power field and calculated the nucleus to cytoplasm ratio (n/c). The results of this study demonstrated that the number of cells counted was not significantly different between the treatment and control groups ( p <0.05), and the control tissues showed a larger cell area and cell length than the three treatment groups. The T and DHT groups showed larger nuclear area than the control. All three treatment groups showed a larger n/c ratio than the control group (p<0.05). In conclusion, the changes occurring at the cellular level of the prostate during early development of BPH due to sustained delivery of androgens are directly related to adaptive responses (hyperplasia and hypertrophy) of the prostatic tissues to injury. This was evidenced by changes in which are primarily seen in the increased nuclear area and increased n/c ratios. These results suggest that a possible genomic stimulation occurred as a response to irritation caused by the delivery of androgens.

SYNERGISTIC ROLE OF VITAMINS ON OF A549 CELLS EXPOSED TO CORTISOL

Letitia [Veals.sup.*], Hamed Benghuzzi, Michelle Tucci, and Zelma Cason, University of Mississippi Medical Center, Jackson, MS 39216

A549 cells are Type II pneumocytes, which can proliferate and be maintained for long-term survival in culture media. The objective of the present study was to determine if antioxidants with cortisol increase viability and metabolic functions of A549 cells. A total of 105 tubes of A549 cells were divided into six equal groups. Group 1 served as control and Groups II-VI were incubated with cortisol, vitamin E, vitamin C, vitamins E C, and vitamin E C cortisol. Seven tubes per group were terminated at 24,48, and 72 hours. Total cell numbers, morphology, and cell damage (MDA level)) was determined. Exposure of A549 cells to physiological levels of cortisol as well as E, C, or E C did not induce any untoward responses as indicated by cell proliferation rate. However, the exposure of cortisol demonstrated a decrease in supernatant MDA levels compared to cortisol or Vitamin C exposure alone. The data suggests that E C treatment plays a major role in protecting membrane integrity (MDA) compared to the use of Vitamin E alone. A possible synergistic effect within the genome could be a possibility and should be fully investigated. Overall conclusion, this investigation suggests that the use of antioxidants such as Vitamin E and C play a major role in minimizing the cellular damage and optimizing functional capacity of A549 cells exposed to physiological levels of cortisol.

EXERCISE TRENDS AT WORK

Michelle L. [Hawkins.sup.*] (1) Macolm Wimbish (2), and Babu P. Patlolla (1), (1.) Alcorn State University, Alcorn State, MS 39096, and (2.) United States Army Corps of Engineers, Vicksburg, MS 39180

A large percentage of America's workforce is getting older. Affectionately known as the baby boomers, they were nutritionally maintained on a high fat, cholesterol, and carbohydrate diet with little regard to exercise. Decades later, emphasis is being placed on the body and its relationship between diet and exercise. In the present study 222 individuals were selected from a work place and are registered online. The activities included were walking, free weights, treadmill, stationary bikes, cross trainers, elliptical riders, ab-doers, and aerobics. Some of the participants selected more than one activity. Questionnaires revealed that those continued for six weeks had decrease in blood pressure, gained muscle tone and high self esteem. In conclusion, as America's workforce gets older, the consequences of poor diet and little exercise will become more evident. It is likely that the health care system will observe more cardiovascular related illnesses, and the consequences.

A QUESTIONNAIRE TO DETERMINE FACTORS ASSOCIATED WITH PLANNING FRUITS AND VEGETABLES IN THE CHILD AND ADULT CARE FOOD PROGRAM: A FOCUS GROUP TECHNIQUE

Charlotte [Oakley.sup.*], Rebecca [Kelly.sup.*], and Sylvia Byrd, Mississippi State University, Mississippi State, MS 39762

The purpose of this research project was to develop a questionnaire that would collect data on multiple factors that have been shown to influence decisions that affect the inclusion of fruits and vegetables on childcare menus. Previous studies demonstrate the value of focus group techniques when conducting qualitative research. Participants were identified by the researchers and the Mississippi Department of Education, Child Nutrition Program office, which administers the Child and Adult Care Food Program (CACFP). The researchers served as the focus group facilitator and recorder. Participants included a Head Start Program director, two school nutrition program directors responsible for feeding pre-school children in the school setting, a representative of the state Department of Education, and a child development expert and a registered dietitian with child care research experiences. Focus group techniques described by Kruger and King were used. The results of an extensive literature search formed the basis of the content discussion. The outcome of the focus group discussion was the development of a comprehensive questionnaire, which explored purchasing and menu planning factors associated with fruits and vegetables. This questionnaire was sent to childcare agencies and centers, participating in the CACEP. Approval of the focus group discussion was obtained from the Internal Review Board, Mississippi State University.

3:30 Divisional Business Meeting