HEALTH SCIENCES.

Chair: Zelma Cason, University of Mississippi Medical Center 

Vicechair: Aaron D. Puckett, University of Mississippi Medical Center

THURSDAY MORNING

Gulf Hall

8:30 Introduction

Zelma Cason, University of Mississippi Medical Center, Jackson, MS 39216

8:40 CYTOPATHOLOGICAL ANALYSIS OF THE TISSUE-IMPLANT REACTION FOLLOWING SURGICAL IMPLANTATION OF UHMW POLYETHYLENE INTO ADULT MALE RATS

Kenneth Butler [*], A.D. Puckett, Hamed Benghuzzi, and Audrey Tsao, University of Mississippi Medical Center, Jackson, MS 39216

It is well demonstrated in the literature that polyethylene materials used in orthopedic and dental applications are highly compatible and non-immunogenic with host tissues. However, recent studies in our laboratory have demonstrated the need for further study of these devices in vivo to further elucidate the mechanisms involved in biocompatibility. The purpose of this investigation was to study the tissue-implant response resulting from the implantation of ultra-high molecular weight polyethylene (PE) at 90 days post-implantation. In this investigation, implants of PE coated with RGE, RGD, Poly-L-Lysine, and Saline were implanted into 15 adult male rats subcutaneously (S/C) and intraperitoneally (I/P). At 90 days post-implantation, the animals were euthanized and the PE devices, the fibrous tissue, and vital organs were harvested. Evaluation of routinely stained sections (5 [micro]m, Hematoxylin & Eosin) of the fibrous tissue capsule surrounding the PE revealed the following: 1) all the devices had fibrous connective tissue capsules of slightly varying degrees of thickness present at the time of sacrifice, 2) the thickness of the fibrous tissue capsule was dependent on the site of implantation and type of coating, 3) the thickness of the fibrous tissue capsules around the subcutaneous devices differed markedly than those retrieved from the peritoneal cavity, and 4) there were statistically significant differences (ANOVA, p[less than]0.05) in the numbers and types of cellular components with respect to implantation site and type of coating. The number of macrophages, neutrophils, fibroblasts, degree of vascularity, and thickness of the fibrous tissue matrix was found to be statistically different between the subcutaneously implanted PE groups. The presence of collagen was found to be statistically different when comparing the S/C and lIP fibrous tissue surrounding PE.

8:55 DETECTION OF REMOTE EXOGENOUS HER2/NEU ANTIBODIES IN RAT SALIVA

W.R. McReynolds [*], J.T. Brinkley , Hamed Benghuzzi, Michelle Tucci, X. Dai, L.G. Bigler, and Charles F. Streckfus, University of Mississippi Medical Center, Jackson, MS

In recent years, there has been an increased interest in saliva as a diagnostic tool. Diagnosis of many diseases, including gastric cancer and immunodeficiency, has been made using saliva. The purpose of this study was to determine whether Her2/neu antibody, made in response to certain carcinomas of the breast, could be detected in saliva when experimentally placed at a remote site. This study was conducted using two male HSD rats, each weighing between 300 g and 500 g. One experimental animal received 200 microliters, and the other, 500 microliters, of encapsulated c-erbB-2 monoclonal antibody (Signet, Dedham, MA) intraperitoneally. Prior to capsule placement, baseline serum and saliva samples were taken. Samples were also taken 20, 68, 140, 188, 308 and 356 hours post-placement. Saliva flow was induced by administration of ophthalmic pilocarpine prior to sampling. All samples were kept at -20 degrees Celsius. Antibody detection was performed using the ELISA technique. The subject receiving the higher antib ody concentration showed a markedly greater saliva level of the antibody than the other (peak 24.158 hnu/ml vs. 7.95 hnu/ml at 308 hours post-injection). These results seem to indicate that Her2/neu antibody saliva levels mqy serve as a useful, non-invasive method in the early detection of some breast cancers. Supported by NIH/NIDCR grant IR55DE/OD12414.

9:10 THE PATHOPHYSIOLOGICAL RESPONSES ASSOCIATED WITH SUSTAINED DELIVERY OF DHEA AND DIOSGENIN IN OVARIECT-OMIZED RATS

Aaron Scott [*], Kent Kihyet Higdon, Hamed Benghuzzi, Michelle Tucci, Zelma Cason, Barry England, James L. Hughes, and Audrey Tsao, University of Mississippi Medical Center, Jackson, MS 39216

Numerous studies have shown that dehydroepiandrosterone (DHEA) exhibits a host of benefits at the vital and reproductive organ levels. However, the use of naturally occurring DHEA is hindered by its inability to survive the first metabolic process of the liver. One possible alternative means that deserves consideration is the administration of DHEA's precursor, namely, Diosgenin (DO). The specific objectives of this investigation are: 1) to deliver DHEA and DG at sustained levels by Tri-Calcium Phosphate Lysine (TCPL) drug delivery systems using ovariectomized (OVX) adult rats as a model, and 2) to evaluate the biochemical and histopathological changes associated with the sustained delivery system. A total of 14 adult female rats were used, and further divided into 4 groups. Group 1 animals served as an intact control group while each rat in Groups 2, 3, and 4 were ovariectomized (sham, n=3), OVX + DHEA, OVX + DG, respectively. Surgical aseptic techniques were employed according to standard procedures. Malio ndialdehyde (MDA) and hormonal levels were measured from serum, collected semi-weekly, during the entire investigation (for 33 days). A routine of H & E staining conducted for histopathological evaluation on the reproductive and vital organs. Pap Smears were collected daily for 33 days to ensure the endometrial changes associated with DHEA and DG treatment. Results of this investigation demonstrated: 1) OVX resulted in a decrease in total body weight, and the use of DHEA and DG returned the body weight to normal levels compared to intact control, 2) TCPL delivered DHEA and DG at a sustained level during the 33 day phase, 3) the ease of MDA levels is as follows: DG [greater than] DHEA = OVX[greater than] control, 4) no significant changes were observed in total wet weights, as well as, the morphology of the spleen, kidney, adrenal, heart, liver, and lung tissues, 5) OVX resulted in an atrophy and non-keratinization trend in the reproductive tissues, and sustained delivery of DHEA and DG showed no remarkable ch ange in these tissues, 6) the use of sustained delivery of DHEA and DG resulted in a significantly higher weights of uteri compared to the OVX group. In conclusion, this study provided more information regarding the interrelationship between DHEA and DG, and the physiological responses encountered when they are administered continuously using adult OVX rats as a model.

9:25 PHYSICAL PROPERTIES COMPARISONS FOR NEW APACKABLE@ DENTAL RESTORATIVES

W.B. Jenkins [*] and A.D. Puckett, University of Mississippi Medical Center, Jackson, MS 39216

The objective of this study was to determine the polymerization shrinkage and coefficient of thermal expansion (CTE) for the recently introduced packable composites, Surefil (Dentsply/Caulk, Milford DE) and Prodigy (Kerr Sybron, Orange, CA.).The polymerization shrinkage was measured from the change in composite density produced by light curing a 0.3 gram disc having a diameter of approximately 15mm and a thickness of[less than]2 mm. The mass of the cured sample was also measured in air and water. The formula, (1-specific gravity of uncured composite / specific gravity of cured composite [H.sub.2]O)[*]100, was used to calculate shrinkage. Additional samples were prepared for Thermal Mechanical Analysis (TMA) to determine the CTE of the composites. The CTE was measured using a Mettler TMA at a rate of 2[degrees]C/minute over the temperature range of 30[degrees]C 50[degrees]C. The CTE for Prodigy was 43.55 ppm/K and Surefil=s CTE was 38.15 ppm/K. The polymerization shrinkage determined at 22[degrees]C was 2.23% and 1.72% for Prodigy and Surefil respectfully. These new restorative materials may behave more like amalgam during placement but their shrinkage and expansion properties are not significantly different than conventional hybrid composites.

9:40 INTERVENTIONAL MRI GUIDED STEREO-TACTIC ASPIRATION OF ACUTE/SUBACUTE INTRACEREBRAL HEMATOMAS

Donnie Tyler, University of Mississippi Medical Center, Jackson, MS 39216

Surgical Interventions for hypertensive intracerebral hematomas are still controversial. Many believe only hyperacute intervention is of any real utility. The majority of present interventions require a formal craniotomy with standard neurosurgical techniques. There are, though, a few reports on CT guided stereotactic aspiration of these hematomas with favorable results. We report often patients treated with frameless fiduciless stereotactic means using an intraoperative MRI scanner (GE 0.5T Signa SP). These patients were initially diagnosed with hypertensive ICH and operated on within 1 to 34 days post hemorrhage. The actual operating time averaged less than 120 minutes, including intraoperative imaging. Clot volumes ranges from 2.5-75 cc with a mean of 31 cc. There were two thalamic hematomas and eight basal ganglial hematomas. Three patients had intraventricular hematoma extension and all three as well as an additional patient required extra ventricular drainage.! However, no patients required permanent p ost hemorrhage ventriculo-peritoneal shunt. Aspiration was successful in all cases to 70-90% of clot removal; two cases utilized intrahematoma t-PA infusion with subsequent 80-90% clot removal. There were no complications or rehemorrhages. All patients showed some form of improvement that included either improved blood pressure control, speech or cognitive abilities. We conclude that using an intraoperative MRI scanner to perform frameless, fiduciless stereotactic aspiration of acute/subacute ICH is a safe and potentially effective means of treating intracerebral hematomas.

9:55 THE EFFECTS OF CIRCADIAN RHYTHM ON SALIVARY C-ERBB-2 LEVELS IN HEALTHY INDIVIDUALS

A.N. McArthur [*], W.J.. Cox, X. Dai, L.G. Bigler, T.M. Dellinger, and Charles F. Streckfus, University of Mississippi Medical Center, Jackson, MS 39216

C-erbB-2, commonly known as Her2/neu, is a prognostic marker used in the diagnosis of malignant tumors in breast cancer patients. The purpose of this study is to valuate the effects of circadian rhythm on the protein cerbB-2 salivary secretions in both males and females. CerbB-2 protein levels in saliva were determined using enzyme-linked immunosorbent assay in two separate groups. The two groups consisted of 10 healthy males with the ages ranging from 22 to 45 (mean 26.3) and 10 healthy females with the ages ranging from 19 to 62 (mean 37.4). Saliva specimens were collected 3 times daily for 5 consecutive days. Each specimen was collected during a 5 minute stimulation period. Using the subjects' baseline values as a control, serial assessments were compared to the control using a one way analysis of variance. The data indicated that there were no statistical differences for salivary c-erbB-2 levels throughout the circadian cycle. The data also indicated that gender had no effect on the salivary c-erbB-2 lev els (mean of males 15.06 hnu/mL; mean of females 15.03 hnu/mL). This study suggest that the secretion of oncogene c-erbB-2 is not affected by circadian rhythm.

10:10 Break

10:25 A RETROSPECTIVE STUDY OF ORAL DISORDERS AMONG PATIENTS WITH HYPOTHYROIDISM IN A DENTAL SCHOOL PATIENT POPULATION

P. Steven Arnold [*], Horace E. Combs, Jason S. Grissom, Brian C. Talley, Sigurds 0. Krolls, J. Perry McGinnis, Jr., and Charles F. Streckfus, University of Mississippi Medical Center, Jackson, MS 39216

One thousand and twelve patients comprising a lower to middle income class were evaluated for dental treatment in 1999 at the UMC School of Dentistry. The cohort with hypothyroidism was comprised of46 individuals or 5% of the total patient population and comprised of 5 males (11%) and 32 females (70%) Caucasians and 1 male (2%) and 8 female (17%) African-Americans. Results of oral examinations revealed that 50% of the cohort had evidence of periodontal disease. Seventeen percent of those exhibiting hypothyroidism had class V carious lesions and 24% had class 11 lesions. Of those patients with hypothyroidism, 27% had missing teeth, 28% had loose teeth and 30% open contacts. Of the total non-hypothyroidism population studied, 51% presented with evidence of periodontal disease. In addition, the control group exhibited 15% class V carious lesions and 30% presented with class 11 lesions. The control group presented with 29% missing teeth, 23% loose teeth and 24% open contacts. This study suggests that caries and periodontal disease in a hypothyroid patient are no more prevalent than in a patient presenting without a hypothyroid disorder.

10:40 DEVELOPMENT OF A NOVEL SUSTAINED DELIVERY SYSTEM FOR ANTI-CANCER

AGENT IN TISSUE CULTURE SETTING

Kent Kihyet Higdon [*], Aaron Scott, Hamed Benghuzzi, Michelle Tucci, and James L. Hughes, University of Mississippi Medical Center, Jackson, MS 39216

The specific objectives of this investigation were: (1) to develop a ceramic delivery system for the purpose of releasing sustained levels of IP6 + I in a tissue culture setting; and (2) to assess the proliferation and viability of HTB 122 Intraductal Breast Cancer (IBC) cells exposed to sustained levels of IP6 + I compared to conventional means of drug delivery. The Tricalcium Phosphate - Lysine (TCPL) capsules were fabricated according to standard laboratory procedure. A total of 4O mg of IP6 + I was loaded into each capsule (n=12) and sterilized by gas sterilization (24 hours). The experimental design consisted of four groups: Groups 1-4 were consisted of media + cells (50,000 cells / well); media + cells + 10 [micro]g (IP6 + I) daily; media + cells + sham capsule; of media + cells + TCPL loaded with IP6 + I, respectively. At the end of 24, 48, and 72 hours, the cells were terminated, and the supernatant was removed for biochemical analysis. Total cell counts, protein content, and MDA levels were conducte d by following standard laboratory protocols. Data obtained from this investigation suggest the following: (1) TCPL delivery systems can be utilized in the tissue culture setting to deliver IP6 + I in a sustained manner for 72 hours; (2) the use of IP6 + Tat the physiological dose did not induce any significant change in the biochemical marker (MDA); and (3) regardless of route of administration, treatment with 1P6 + I resulted in a decrease in total protein content compared to the sham capsules and control groups.

10:55 ADVANCES IN CURING OF DENTAL RESTORATIVE MATERIALS 

S.B. Roberts [*], A.D. Puckett, C.C. Inman, and J.G. Fitchie, University of Mississippi Medical Center, Jackson, MS 39216

The purpose of this study was compare the degree of conversion and mechanical properties obtained for a hybrid composite cured by a plasma-arc light curing unit on two available settings to a conventional halogen light curing unit. The hybrid composite APH (Caulk Dentsply, Milford, NH) was cured by three methods: A) Apollo 95E plasma-arc light for 3 seconds B) Apollo 95E on "step-cure" mode (2 seconds at 50% full power, followed by 4 seconds at full power) C) Demetron Optilux 401 light for 40 seconds. Five standard diametral tensile specimens (d 6 mm and t = 3 mm) were prepared for each experimental group using a stainless steel mold and glass cover slips. Immediately after curing the hardness measurements for both top and bottom surfaces were recorded using a Shore D durometer (Pacific Transducers Corp, Los Angeles, CA). After 1 hour at room temperature, diametral tensile strengths (DTS) were determined using a Sintech 21/G mechanical testing system at a load rate of 1 cm/min. Another series of samples were prepared, cured, and stored in DI [H.sub.2]O for 24 hr @ 37[degrees]C before repeating the mechanical analysis. A series of samples were also cured, crushed and extracted with chloroform to evaluate the residual monomer using gel permeation chromatography (GPC). The results from this study suggest that the three curing methods are not equal.

11:10 PHYSICIAN TECHNOLOGY PREFERENCES: EVOLUTION DURING TRAINING AND TECHNOLOGIC CHANGE

Kendall P. Brown [*], and W.B. Lushbaugh, University of Mississippi Medical Center, Jackson, MS 39216

Given the expected convergence of local area network (LAN) technology and hand-held computers within hospital practice, a survey of 4th year medical students was performed, to evaluate how attitudes and preferences towards this evolving technology have been formed, and what subsets of users practicing physicians might be divided into. The results further elucidate the gap between physician preferences and the process by which default technologies become standards. For example, the institutional process by which technologies are chosen and become standards are opaque relative to the clear process evidenced by these survey results, even in this first approximation of the physician preference-determining process. Portable handheld devices were preferred. This cohort chose optimal device ergonomics. A majority of respondents desired to delegate responsibility for maintenance of interface standards, as well as software and hardware maintenance. Wide bandwidth was preferred, by [greater than] implication, given a desire for real-time access to network databases. Transmission range and data security issues were not fully resolved, and warrant further investigation. Respondents chose a stable interface, and a high level of inter-device data transfer transparency. LAN designs are consistent with this need, and further research needs to be conducted to verify which LAN software schemes are most adaptable to physician tool designs.

11:25 USING PROCRIT TO MINIMIZE ANEMIA SIDE-EFFECTS OF CHEMOTHERAPY

Renetta King [1][*], Richard Mansour [2] and Howard Wold [2], (1.) University of Southern Mississippi, Hattiesburg, MS 39406-5043 and (2.) Freedman Clinic of Internal Medicine, Alexandria, LA 71303

In addition to killing cancer cells, chemotherapy also destroys erythrocytes, thereby causing anemia. Anemia results when there is not enough oxygen released in the cells or when levels of hemoglobin are low. Normally the hormone, Erythropoietin would stimulate the production of red blood cells, but during chemotherapy the levels of Erythropoietin are not high enough to recover such a great loss of erythroctyes. Procrit or epoetin alpha is a replacement for human Erythropoietin. Cancer patients usually receive an injection of Procrit three times a week, a dose that is based on the regimen of Procrit administration for renal failure. However, a 16 week study in 500 clinics found that Procrit could be administered once a week to cancer patients and produce the same results as compared to three doses a week. Overall, there were a 3.0 g/dL mean increase in hemoglobin levels as well as improved patient-reported quality of life parameters. These results have been published and presented to FDA for approval of once weekly dosing of Procrit.

11:40 THE EFFECT OF THE PHYTOCHEMICALS FISETIN AND LUTEOLIN ON MCF-7 BREAST CANCER CELL GROWTH

William J. Steelman [*], Kedra L. Martin, and Nancy Eddy Hopkins, Millsaps College, Jackson, MS 39210

Recent research and media attention has focused on the estrogenic and the cancer chemopreventative effects of phytochemical isoflavanoids such as genistein and daidzein in soybased foods as well as the cancer chemopreventative effects of the flavanoids such as catechin and epicatechin found in tea. Women such as those in Thailand and Japan who eat diets rich in plant foods have a decreased risk of breast cancer. Many other flavanoids and isoflavanoids are found in plant foods. Using a modified E-screen (Wang, et al., Carcinogenesis 17:271-275, 1996), we have evaluated the effects two phytochemicals, fisetin and luteolin, on MCF-7 cell growth of in the presence and absence of 17-[beta] estradiol (E2). These tetrahydroxyflavone isomers produced similar growth effects in this system. Both fisetin and luteolin increased cell growth in a dose depend manner in the absence of E2 but inhibited cell growth in a dose depended manner in the presence of E2. Studies are underway to evaluate the effects of other phytochem icals with similiar structures and to elucidate the mechanism of these effects.

THURSDAY AFTERNOON

Gulf Hall

1:30 DEVELOPMENT OF A CACO-2 COLON CANCER CELL ASSAY TO EVALUATE CHEMOPREVENTATIVE PHYTOCHEMICALS

Alexa N. Ivancac [*], Kedra L. Martin, and Nancy Eddy Hopkins, Millsaps College, Jackson, MS 39210

The Caco-2 colon cancer cell line is well characterized and is a useful model for many tissue culture models. This line will undergo early differentiation and expresses many markers and receptors in culture that are expressed in normal colon epithelium. Recently, these cells were reported to express estrogen, receptor in serum-free media. Phytochemicals such as the catechins found in green tea and flavanoids and isoflavanoids found in soybean foods have been shown to be chemopreventative in several colon cancer animal models. These same chemicals have been shown to decrease the risk of breast cancer. In order to study these phytochemicals, we have developed an assay using 2.5% dextran-charcoal stripped serum to evaluate the effect of phytochemicals on estrogen -induced Caco-2 cell growth. We have found that 1 [micro]M 17-[beta] estradiol will give a 2- to 2.5-fold increase in cell growth. Studies are underway to determine the effects of phytochemicals in this system.

1:45 PSPA FAMILY TYPING OF CLINICAL ISOLATES OF STREPTOCOCCUS PNEUMONIAE

LaTonya B. Washington [*], LaShandra Johnston, and Larry S. McDaniel, University of Mississippi Medical Center, Jackson, MS 39216

Streptococcus pneumoniae is an important human pathogen worldwide for which a more effective vaccine is needed. There has been considerable interest in pneumococcal protein antigens as potential vaccine components. One of the most well characterized pneumococcal proteins is pneumococcal surface protein A (PspA). PspA has been shown to play a role in pneumococcal virulence and can elicit protective immune responses against pneumococcal infection in a mouse model. Despite being a serologically variable protein, PspA is able to elicit cross-protection against different variants. Sequence analysis has defined 7 clades that can be placed into 4 PspA families. Rabbit antiserum is available that reacts specifically with family I and family 2 PspA. Most clinical isolates have been typed using the rabbit antiserum as family 1 or 2. We examined 167 pneumococcal isolates collected over a four year period from the University of Mississippi Medical Center Clinical Laboratory. We found that 81/167 (48.5%) were family 1, 7 6/167(45.5%) were family 2,2/167(1.2%) were both, and 8/167(4.8%) were neither. Our results are in agreement with those reported for pneumococcal clinical isolates from other geographic locations. If PspA is to be used as a pneumococcal vaccine, it is important to identify, and continue to monitor, the relatedness of pneumococcal clinical isolates.

2:00 EFFECT OF EPHEDRINE ANALOGS AND ISOMERS ON HUMAN a2a- AND a2b- ADRENOCEPTORS EXPRESSED IN CHINESE HAMSTER OVARY CELLS

Yolande Davis [1][*] Shilpa G. Lalchandani [2], and Dennis Feller [2], (1.) Mississippi University for Women, Columbus, MS 39701 and (2.) University of Mississippi, Oxford, MS 38655

Ephedrine and its alkaloids are used for the treatment of asthma, nasal congestion and obesity. Ephedrine, with two chiral centers exist as four isomers which exhibit direct and indirect affects on both a- and b-adrenergic receptors (AR). Our main goal was to study the binding affinity and selectivity of ephedrine isomers and its analogs on human a2a- and a2b-adrenergic receptor subtypes expressed in Chinese hamster ovary cells (CHO). Radioligand binding assay was performed on racemic ephedrine, its hydroxyl and methyl substituents and also on four isomers of ephedrine. The binding data showed that the addition of 4-hydroxry and N-methyl substituents reduced the binding affinity of ephedrine on the alpha-adrenergic subtypes. Also among the isomers the 1R, 2S and the iR, 2R ephedrine isomers showed greater binding than the 1S, 2R and the 1S, 2S isomers, indicating the importance of the 1R hydroxyl substituent for alpha-adrenoceptor binding.

2:15 DETERMINATIN OF TAMOXIFEN AND ITS METACOLITES IN FETAL TISSUE USING CAPILLARY ELECTROPHORESIS (CE) CHROMATOGRAPHY

K.L. Alford [1][*], Henry Outlaw [1], P.J. Schupp [2] , J.M. Sanders [2], and M. L. Cunningham [2], (1.) Delta State University, Cleveland, MS 38733 and 2Laboratory of Pharmacology and Chemistry, NISHS, Research Triangle Park, NC 27709

Tamoxifen (TAM), an antiestrogen, has been approved for use by women with increased risk of developing hormone-dependent breast cancer. However, little data exist describing potential TAM-induced fetal toxicity to women of child-bearing age. Transplacental exposure of TAM has been linked to reproductive tract toxicity in female offspring of CD-1 mice. In support of the National Toxicology Program's characterization of reproductive and developmental effects of AM, this work describes a CE-based analytical technique for the detection of TAM and two of its major metabolites, N-desmethyltamoxifen (DMT), and 4hydroxytamoxifen (4-HT) in mouse fetal tissue. The present technique was developed and validated following 98:2 hexane/isoamyl alcohol extraction of TAM, DMT, and 4HT from spiked fetal tissue homogenates. Optimum resolution of TAM, DMT, and 4-HT was obtained on a 57cm x 50 [micro]m capillary using a nonaqueous buffer system of 1:1 methanol/acetonitrile containing 50 mM ammonium acetate and 1.5% acetic acid. 4-Dimethylaminopyridine was used as an internal standard. Temperature and voltage were optimized at 40[degrees]C and 15 kV, respectively. This CE-based analytical technique was used to analyze for the presence of TAM, DMT, and 4-HT in TAM-treated CD-1 mice. All three compounds were detected at 2, 4, 8, and 12 hr post-dosing in fetal tissue collected at gestation day 16 from mice receiving a single dose of 100 mg TAM/hg. Results of the present study demonstrate transplacental transport of TAM derived material to fetuses of TAM-treated dams. Further, this work presents a sensitive new methods for the detection of TAM and two major metabolites in fetal rodent tissues.

2:30 TRANSFECTION STUDIES WITH A CANDIDATE HUMAN IMIDAZOLINE RECEPTOR cDNA

Michael Chen*, Mary Elise Lutrick, He Zhu, James Baldwin, Victor Stuckey, and John Piletz, University of Mississippi Medical Center, Jackson, MS 39216

Imidazoline receptors (IR) are involved in blood pressure and stress responses from brain nuclei. We have previously cloned a candidate [IR.sub.1] cDNA by screening from a human hippocampal cDNA expression library with two IR-selective antisera (J. Autonomic Nervous System, 72, 98-110, 1998; DNA and Cell Biology, submitted). That cDNA, designated imidazoline receptor antisera-selected (iras-1) cDNA, contains 5,131 bp and is predicted to encode a 167 kDal protein. Transfection of iras-1 cDNA into CHO (Chinese hamster ovary) cells results in a three-fold increase in the [B.sub.max] of[[[blank].sup.125]I]-p-iodoclonidine for [I.sub.1] sites; with a high-affinity component revealed only in transfected CHO cells for the [I.sub.1] ligands, moxonidine and rilmenidine. In PC-12 (phaeochromocytoma) cells, over 80 stably-transfected subclones were screened both by Western blotting and PCR, and six of these showed at least a two-fold increase in [I.sub.1] [B.sub.max] values. Transfected CHO and PC-12 cells showed a 167 kDal band as well as smaller bands ([sim]85 kDal) on Western blots. On the other hand, transient trasfections into COS-7 and Sf9 cells failed to result in an increase in [I.sub.1] binding sites, even though there was an abundance of the [sim]167 kDal protein made. To determine if there is any possible interaction between [I.sub.1] sites and [alpha.sub.2A] adrenergic sites, CHO cells stably transfected with the human [alpha.sub.2A] adrenergic receptor cDNA were transiently transfected with iras-1. These cells produced both [alpha.sub.2A] and [I.sub.1] receptors (immunologically) with the appropriate binding sites observed, and the results provide support for studies aimed at characterizing the pharmacological and functional interactions between alpha2-adrenergic and [I.sub.1] receptors. Supported by NIMH grant MH49248-06 and grants from Eli Lilly and Solvay Pharmaceutical Companies.

2:45 Break

3:00 Divisional Poster Session

GROWTH HORMONE (GH) AFFECTS A-549 LUNG CELLS INDIRECTLY THROUGH IGF-l

Robert Triplett*, Bobby Thomas, Joseph A. Cameron, Leon Anderson, Michelle Tucci, and Hamed Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216 and Jackson State University, Jackson, MS 39216

In reviewing the literature, lung cell proliferation and differentiation is controlled by a complex interaction of endocrine and autocrine growth factors, mechanical forces and developmental regulators. In the present study, we have examined the regulation of GH and IGF-1 in A549 cells to gain more insights into the role of growth factors might have in neoplastic lung growth. The specific objectives of this investigation were: (I) to study the effect that IGF-1 and GH might have directly or indirectly on the proliferation of A549 cells in culture, and (ii) to evaluate the role of IGF-1 and GH on the cellular membrane integrity (MDA levels) of A-549 cells. A549 cells were grown m 15-ml glass tubes in Ham's F-12 medium. The cells were then incubated with IGF-1 (26 pg/ml) or GH (48 pg/ml) for either 24 or 48 hours. At the end of each phase, the media was removed and saved in a 1.5 ml microcentrifuge tube for MDA analysis. The cells were washed twice with 5 ml of phosphate buffered saline. The cells were detache d from the side of the tube, and collected into a volume of I ml of phosphate buffered saline for cell counts and protein analysis. At the time of termination, the cells were evaluated morphologically, trypsinized, and cell numbers were calculated using a hemacytometer. Results obtained from this investigation suggest that: (I) IGF-l stimulated the A549 at concentration of 26 pg/[micro]1 at both 24 and 48 hours without causing cellular damage, (II) GH at concentration of 48 pg/[micro]1 was unable to stimulate cell proliferation at either 24 or 48 hrs, and (III) supernatant MDA levels were increased in the growth hormone treated cells in comparison to the IGF- 1 and media treated cells. Overall conclusion obtained from this study reveals that GH regulates the proliferation of A549 cells indirectly through IGF-1.

INFLUENCE OF LEAD ON NITRIC OXIDE IN THE LIVER AND KIDNEY OF NEONATAL RATS

Regina L. Bell, S.L.N. Reddy, Sharonda Swilley, Vanessa Hinton*, S. Rajanna, and B. Rajanna, Alcorn State University, Alcorn State, MS 39096

Nitric Oxide (NO) is implicated in the control of short and long term regulation of arterial pressures in the liver and kidney. We have earlier reported hepatic and renal toxicity of lead (Pb). The purpose of this study is to determine the effects of Pb on the production of NO measured as nitrate and nitrite (NOx) in the liver and kidney of the rats. Pregnant Sprague Dawley rats in their 5th day of gestation were orally treated with 0.1% and 0.2% PbAcetate. Treatments of mothers were continued until 21 post natal days (PND). The pups were sacrificed at 5, 10, 15, 20, and 25 PND. The liver and kidney were excised. The NOx levels were estimated by the chemiluminescent method using Sievers 280 Nitric Oxide Analyzer. 0.1% and 0.2% PbAcetate significantly reduced NOx levels in both the liver and kidney at PND 5,10,15, and 20. At PND 25, NOx reductions by PbAcetate at both doses were not significant. These results suggest that Pb play a significant role in reducing the NO production in the liver and kidney of neon atal rats. These studies have been extended to include PND 35, 40, and 60. Supported by NIH/MBRS #GM 55356).

TRANSFER OF IMMUNITY TO TRYPANOSOMA LEWISI FROM INFECTED MOTHERS TO THE OFF-SPRING IN RATS

Kristi McGillivray [*], Daila Morgan, John Rentrop, and Kristi Tucker, Belhaven College, Jackson, MS 39202

Trypanosoma lewisi is a cosmopolitan blood parasite of rats, which is non-pathogenic in humans. Another species causes African sleeping sickness in humans and is the fifth leading cause of death from parasites in the world. It is known that humans receive immunity from the mother's milk through breast-feeding. It has been shown that suckling rats can also receive immunity from their mother. In this study a group of mothers (I) were inoculated with T. lewisi before giving birth to determine if they will develop immunity to the parasite and transfer that immunity to their offspring. Another attempt was made to determine if immunity was passed trans-placentally. A cross fostering method was used: the I mothers gave birth and then their offspring were switched to nurse from a non-infected (NI) mother. This would determine if immunity was passed trans-placentally. The offspring from the NI mother nursed from an I mother, which would determine if immunity to the mother's milk was the result of infection prior to g iving birth. It was observed that the levels of trypanosomes in the offspring that nursed from the I mother were almost zero. This demonstrated that the immunity passed in the milk was sufficient to halt the infection. Immunity to the trypanosomes passing trans-placentally resulted in an infection lower than the control group. Another aspect of this study was an in vitro use of antibodies derived from rat's milk against live trypanosomes. The results were inconclusive after twenty minutes because none of the trypanosomes were killed.

CYTOHISTOLOGIC CORRELATION IN PATIENTS WITH CLINICAL SYMPTOMS OF POSTMENOPAUSAL BLEEDING

Stephanie D. Webster [*], Zelma Cason, and Hamed Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216

Today, the life expectancy for women is longer; therefore, many will likely experience the postmenopausal period (termination of fertility and menstrual bleeding). Uterine bleeding after this period could be attributed to several pathologic conditions. The specific objective of this project was to evaluate the cytohistologic findings in women with postmenopausal bleeding (PMB) and to determine the presence of any significant pathologic lesions. Cytohisto logic correlations from 66 patients attained in 1993 from the University of Mississippi Medical Center were evaluated. The population evaluated were divided into three groups: (control group 1) dysfunctional uterine bleeding (DUB), (control group 2) postmenopausal (PMP), and (test group 3) the group of women with postmenopausal bleeding. The DUB and PMP age-matched controls (n=12, mean age 51 [+ or -] 5 and 57 [+ or -] 5 years) were randomly selected, and correlated with the actual group being tested (54 PMB, mean age 57 years). The distribution among the 54 PMB women evaluated were 69% (37/54) black, and 31% (17/54) white. The DUB and PMP control groups consisted of 50% (6/12) black and 50% (6/12) white, respectively. Histopathological confirmation (62/66-94%) revealed 47/66 as negative, 5/66 as endometrial hyperplasia and 10/66 as squamous cell carcinoma or adenocarcinoma. A significant lesion with endometrial pathology was found in 23% of the patients. These findings suggest that the majority of women in t his study with clinical symptoms of postmenopausal bleeding were negative for malignancies. While these results lean more towards a normal cytologic evaluation, postmenopausal bleeding should not be taken lightly. Post menopausal bleeding could represent signs of more serious lesions such as squamous cell carcinoma or endometrial adenocarcinoma if not detected and managed early.

THE EFFECT OF TCPL DENSITY ON THE RELEASE PROFILES OF HYDROPHOBIC AND HYDROPHILIC COMPOUNDS

Krashod Byrd [*] and Hamed Benghuzzi, Jackson State University, Jackson, MS 39216 and University of Mississippi Medical Center, Jackson, MS 39216

Several studies have shown that Tri-Calcium Phosphate Lysine Drug Delivery Systems (TCPL) are capable of releasing various organic compounds at a sustained manner. The fabrication of these devices is a very complex and the manipulation of one of various procedures can alter the release profile. The compression of the TCPL material into the capsules is an instrumental factor when studying the release profile. The objectives of this investigation were: 1) to study the interrelationship between the release profiles of devices that had been cold pressed to a density of 1.53 [+ or -] 0.15 (Low density, TCPL-LD) and 1.98 [+ or -] 0.7 gm/[cm.sup.3] (High density, TCPL-HD), and 2) to evaluate the role of the change in density that might have on the release of hydrophobic and hydrophilic compounds in an in vitro environment. A total of 16 serum bottles were used and later subdivided into four groups (n =4). Groups I and II contained TCPL-HD and TCPL-LD capsules each loaded with 20 mg of progesterone (P). Groups III & IV contained TCPL-HD and TCPL-LD capsules that were loaded with 20 mg of Bovine Serum Albumin (BSA). Each serum bottle in groups III and IV was filled with a 100 ml of the experimental buffer (phosphate buffer saline, pH 7.4) and 10 [micro]1 of sodium azide (antibacterial agent). Serum bottles in groups I and II were filled with a buffer of 50% alcohol (wt/vol). Samples were withdrawn at various time intervals (168 hours) and analyzed by using standard spectrophotometer techniques and Jandel Sigmastat Statistical program. Results of this investigation suggest that: 1) TCPL-HD and TCPL-LD devices were capable of releasing P and BSA at a sustained levels, 2) regardless of the loaded drugs, the release profiles from TCPL-LD devices was found to be significantly higher (p [less than]0.05) than the release profiles of P and BSA from TCPL-HD, and 3) physiochemical characteristics of the drug to be delivered are instrumental in regulating the rate of release and duration or availability of an effective and safe dos e (BSA [greater than] P).

BIOMECHANICAL EVALUATION OF SUTURE BONE ANCHORS: IMPROVING ORTHOPEDIC SURGICAL REPAIR OF TENDONS TOM OFF BONE

Philip Eichhorn [1][*], Douglas Parsell [2], and Thorn Tarquinio [2],

(1.) Jackson Preparatory School and (2.) University of Mississippi Medical Center, Jackson, MS 39216

Bone anchors are tiny metal screws with a central hole in the outer end for connecting sutures from tom off tendons or ligaments being reattached to bone (heel, shoulder) in orthopaedic surgery. Repair strength is critical. This study evaluates failures of suture-anchor combinations as a function of anchor design and orientation during use. Four commercially available bone anchors were evaluated using heavy suture (#2 Ethibond). Sutures, n = 232, (looped through the eyelets of anchors inserted into high-density polyethylene, UHMWPE, that simulates bone) were pulled at three angles of orientation using four different anchors of each type, pulled five times in each orientation. Using a computerized screw-type load frame, pull was applied rapidly to simulate a patient's traumatic fall. Two of four brands showed significant variation among samples. Also, suture failure strength varied significantly as a function of pull angle. Sutures in Fastin and SuperQuick anchors at 90[degrees] and 90[degrees]/90[degrees] br oke easily. Suture strength in the Mainstay anchor highly depended on eyelet orientation: weak at 90[degrees] but strongest of all tested at 90[degrees]/90[degrees]. Suture in the Peba anchor had good strength at both orientations. Significant differences were shown among these bone anchor types. Most sutures are used at 90[degrees] and 90[degrees]/90[degrees] in anchors in surgery. Therefore, the Peba anchor and, particularly, the Mainstay used at 90[degrees]/90[degrees] have the greatest repair strength. Application of these results can improve orthopedic surgical repairs needing bone anchors. Future work towards more orientationally robust bone anchor designs is warranted.

CYTOMORPHOLOGICAL ASSESSMENT OF BENIGN AND MALIGNANT DENSE HYPERCHROMATIC GROUPS IN CERVICOVAGINAL SMEARS

Marlene Harris [*], Zelma Cason, and Hamed Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216

Dense hyperchromatic cell groups are considered one common diagnostic problem in cytopathological evaluations. This morphological feature represented by cluster of cells with scant cytoplasm and dark nuclei. Cytologic evaluations may result in high false-positive or false negative rates in cervicovaginal smears, due to overcalling benign groups as malignant and malignant groups as benign. The key is to differentiate among the dense groups and to appropriately classify the lesions based on strict morphologic criteria. The objective of this study was to evaluate the cytomorphology of benign and malignant dense hyperchromatic groups in cervicovaginal smears at the University of Mississippi Medical Center. Six types of dense hyperchromatic groups were selected (sixty cervicovaginal smears) to represent all of the entities. The cases were divided into; group 1, atrophic pattern (n=10), group 2, endocervical cells (n=10), group 3, endometrial cells (n=10), group 4, high-grade squamous lesions (HSIL) (n=l0), group 5, invasive squamous cell carcinoma (n=10), and group 6, endometrial adenocarcinoma (n=10). Light microscopy was used to evaluate the background, arrangement, and chromatin pattern. ImagePro [R] computer software (x40 magnification) was used to measure and evaluate the nuclear area, ratio, density, and shape. Data obtained from this investigation suggest the followings: (I) there were no significant difference can be observed in the total areas among all groups, (ii) nuclear density of the cells have shown to be higher in HSIL, invasive squamous and adenocarcinoma cases, and (iii) evaluation of cellular shape in groups 1, 2, 3, 4, and 5 demonstrated round to oval, predominate round, oval to irregular, round to oval, round to irregular, and round to oval, respectively. In conclusion, cytomorphometric analysis can be utilized as an ideal diagnostic tool in differentiating between the ambiguous or suspicious groups of dense hyperchromatic cells. Ultimately, this diagnostic tool can minimize the high false-positi ve and false-negative rates resulting in better cytologic evaluations and patient prognosis.

NEUROTOXIC EFFECTS OF LEAD ON NITRIC OXIDE IN THE DEVELOPING RAT BRAIN

Regina L. Bell [*], Sharonda Swilley, S.L.N. Reddy, S. Rajanna, and B. Rajanna, Alcorn State University, Alcorn State, MS 39096

Nitric Oxide (NO) is a free radical gas synthesized by Nitric Oxide Synthase (NOS) during the conversion of Larginine to L-citruline. Lead (Pb) is an environmental toxicant that affects neuronal functioning. NO, a neuronal messenger, has a short half-life and converts immediately to nitrite and nitrate. The present study is designed to determine the effects of Pb on NO production in four regions of the developing rat brain: the cerebellum, the hippocampus, the frontal cortex, and the brain stem. Pregnant Sprague Dawley rats in their 5th day of gestation were treated with 0.1 % PbAcetate and 0.2% PbAcetate, and control groups received 0.1% NaAcetate. All treatments were administered through deionized, distilled drinking water. Treatments were continued during pregnancy and until 21 postnatal days (PND). The pups were sacrificed at PND 5, 10, 15, 20, 25, 30, 45, and 60. Whole brain tissues were excised and separated into the cerebellum, hippocampus, frontal cortex and brain stem. The nitrite and nitrate (NOx) were measured by the chemiluminescent method using Sievers Nitric Oxide Analyzer (280). In the cerebellum, at PND 25, 0.1% and 0.2% Pb reduced NOx levels by 428% and 328%, respectively. In the hippocampus, both doses of Pb reduced NOx levels at all PND except PND 15. In the brain stem, there were significant reductions in NOx at PND 20 and 25. Both doses of Pb reduced NOx in the frontal cortex at PND 10, 15, 20, and 25. These results suggest that Pb alter NO production in the rat brain leading to neuronal dysfunction. These studies have been extended to include rat brain tissues of PND 30, 45, and 60. (Supported by NIH/NIGMS/MBRS #GM 55356).

THE ROLE OF GLUCOCORTICOIDS ON HUMAN PULMONARY ADENOCARCINOMA (A549 CELLS) IN CULTURE

Kimberly Cornelius [*], LaShunda Thompson, Hamed Benghuzzi, Michelle Tucci, and James L. Hughes, University of Mississippi Medical Center, Jackson, MS 39216

A549 cell line, a transformed cultured cell line derived from a human pulmonary adenocarcinoma. They are distinguished from the cell lines by having morphologic and biochemical features of the pulmonary alveolar type II cells. Steroid hormones have been used to treat several pulmonary medical complications. The mechanism of action of such steroids is still unclear. The specific objective of this investigation is to study the effect of various concentrations of cortisol that might have on the viability and proliferation rate of A549 cells in culture. The cells were incubated in the presence of 0, 1, or 20 [micro]g/ml cortisol, and the cell number, morphology and cell damaged (MDA) were measured at 24, 48 and 96 hours after cortisol administration. Data obtained from this study demonstrated that the protein content of the cells at 24 hours showed a significant increase (p [less than]0.05) in both the 1 and 20 [micro]g/ml cortisol treated cells. No significant change in protein content at 48 and 96 hours phases between experimental and control groups. Cortisol treatment did not induce any significant changes in cell number, viability and markers of cell membrane at all three phases. In contrast, morphological evaluation showed, regardless of the dose, that there were an increase in cell volume with condensed chromatin in cortisol treated cells. This phenomenon could possibly explain the increase observed in cell protein content. A549 cells act as pulmonary alveolar type II cells, which are capable of secreting surfactant. Corticosteroids have been used in neonates to reduce the incidence of respiratory distress syndrome, and the increase in cell volume may indicate cellular activation and increased surfactant secretions. The results of this study suggest that cortisol at concentrations of I and 20 [micro]g/ml are capable of increasing cellular protein concentration after 24 hours without causing membrane damage (MDA levels) or changes in cell viability (morphology).

RESTORATION OF THE ENDOMETERIAL FUNCTION BY MEANS OF SUSTAINED DELIVERY OF ES AND ES + P IN OVARIECTOMIZED RATS

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

Previous studies conducted in our laboratories have documented that tricalcium phosphate lysine (TCPL) delivery system can be utilized to deliver estrogen (ES) and combination of estrogen with progesterone (ES + P) at a sustained levels for long duration in intact rats. The specific aim of this investigation was an attempt to reverse the endometrial changes resulted from post-ovariectomy by exogenously delivering sustained levels of ES and ES + P. 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 ovariectomized (OVX), OVX plus ES (20 mg loaded TCPL), and OVX plus E (TCPL, 20mg) plus P (TCPL, 60mg) treatment. Group 4 animals served as intact control group. Blood samples were collected biweekly for 33 days. Vaginal smears were taken and screened daily during the entire investigation. Histopathological evaluations were conducted on all reproductive as well as vital organs (H & E). Data obtained from this investigation su ggest the following: (I) OVX resulted in an increase in total body weight, however ES and ES plus P maintained the body weights at prior ovariectomy level, (II) sustained delivery of ES resulted in maturation of vaginal epithelium and the smears exhibited the estrus at the end of 72 hours post implantation and continued this trend for the duration of the study, (III) ES + P treatment induced no estrus and the epithelial changes resembled the OVX group, and (IV) ES and ES + P treatment resulted in a significant different (p [less than] 0.05) in MDA levels compared to OVX and intact control groups. Results of this investigation conclude that sustained delivery of ES and ES + P by TCPL can be utilized to maintain the normal function of the reproductive organs and could serve as an efficient and safe therapy in estrogen deficient patients.

EFFECTS OF THE POLYCHLORINATED BIPHENYL (AROCLOR-1254) ON PRIMARY RABBIT KIDNEY (PRK) CELLS

Annette Davis [*], Michelle Tucci, James L. Hughes, Hamed Benghuzzi, and Leon Anderson, University of Mississippi Medical Center, Jackson, MS 39216

The cytotoxicity of a commercial PCB mixture, Aroclor-1254, was assessed on cultured kidney epithelial cells. Studies using this cell line can be useful to determine the effect of Aroclor-1254 on kidney function such as filtration, excretion, and absorption processes. In PCB exposed and unexposed cells, the following biochemical parameters were measured: cell viability, protein synthesis, and lipid peroxidation. The results show that Aroclor-1254 at concentrations exceeding 0.1mM (but not at lower concentrations) causes irreversible damage to cultured kidney epithelial cells. In cells exposed to concentrations of 10 [micro]g of PCBs, there was decreased in cell number at 24 and 48 hour phases compared to untreated and cells treated with lower doses of Aroclor-1254. Cellular protein concentration confirmed the results of cell number. The decrease in both cell number and protein concentration at both 24 and 48 indicated either cell death due to severe injury or interference with cell ability to divide in cultu re. In order to determine the integrity of the cell, the levels of MDA were determined at both time points. The results obtained from the MDA assay indicate no significant differences in the levels between the treatment groups and vehicle control, suggesting disruption of the cell's ability to divide rather than an indicator of cell death. However, cell morphology evaluation indicated changes in cell structure at concentrations greater than 0.1 [micro]g/[micro]1 as compared to cells treated with media or vehicle alone. In conclusion, Aroclor-1254 at concentrations of 1 and 10 [micro]g/[micro]1 caused disruption of the cells ability to divide possibly by causing changes within the cellular DNA resulting in the cell being held within [G.sub.1] of the cell cycle. Cells exposed to concentrations greater than 0.1 [micro]g/ml showed alterations in cell morphology as compared to untreated cells.

THE EFFECT OF ALDOSTERONE AND CORTISOL ON THE VIABILITY AND PROLIFERATION OF RHESUS MONKEY KIDNEY EPITHELIAL CELLS IN CULTURE

Mi Jung [*], Hamed Benghuzzi, Michelle Tucci, and Zelma Cason, University of Mississippi Medical Center, Jackson, MS 39216

Primary cultures of Rhesus Monkey renal tubular epithelial cells were used to study the effect of two adrenal hormones, aldosterone and cortisol, on cell proliferation and viability as well as cell morphology at 24, 48 and 72 hours after steroid hormone treatments. Proliferation was studied by comparing cells grown in serum; with cells grown in the presence of either 1mg or 20mg cortisol or aldosterone for 24, 48 or 72 hours in culture. In addition to cell counts cellular protein and MDA levels were determined at each phase. Proliferation was assessed by determining cell number (population doublings), and the results show that cells treated with cortisol caused a slight increase in cell number at both 24 and 48 hours, where as cells treated with low dose aldosterone had slight effects on cell doubling. Supernatant levels of MDA in cortisol treated cells were significantly higher than cells treated with aldosterone or media alone. At 72 hours the cells treated with both the low and high dose of either cortiso l or aldosterone showed the highest levels of supernatant MDA. Morphological evaluation of aldosterone treated cells revealed changes in size and shape as early as 24 hours after treatment. The changes observed included vacuolation, irregular spindle shapes, lack of cell aggregates and occasional pyknosis. Cortisol treatment caused the appearance of vesicular nuclei, columnar shape cells, shriveling, and predominant pyknosis. The results from this experiment show that the proximal tubule epithelial cells retain a number of functional characteristics and that they represent an excellent model for studies of normal and abnormal biology of the renal proximal tubule epithelium.

THE EFFECTS OF AMYLOID [beta]-PROTEINS ON PROSTAGLANDIN E2 PRODUCTION IN RAT MICROGLIA Shuang Chen [*] and March D. Ard, University of Mississippi Medical Center, Jackson, MS 39216

The hallmarks of Alzheimer's disease include senile plaques that contain amyloid [beta]-proteins (A[beta]), neuritic tangles, loss of neurons and gliosis which involves microglia and astrocytes. Microglia are immune cells of the central nervous system. Many activated microglia are found near the senile plaques. In this study, we investigated the regulation of prostaglandin E2 (PGE2) production by A[[beta].sub.1-40] and A[[beta].sub.1-42] in adult rst microglia after exposure to bacterial lipopolysaccha ride (LPS) (a classical inducer of microglia). Rat microglia were cultured from cortex of adult rat brain and were used within seven days of culture. We tested the effects of both A[[beta].sub.1-40] and A[[beta].sub.1-42] on PGE2 production in rat microglia with or without exposure to LPS. PGE2 was assayed by commercial enzyme-linked immunoassay kit. The results showed that both A[[beta].sub.1-40] and A[[beta].sub.1-42] alone didn't increase the production of PGE2 compared with the controls. LPS significantly increased PGE2 production. Interestingly, we found that A[[beta].sub.1-40] increased PGE2 production induced by LPS, whereas A[[beta].sub.1-42] decreased the PGE2 production induced by LPS. We also tested expression of cyclooxygenase-2 (prostaglandin synthase) which rises rapidly in response to inflammatory stimuli. In each condition the level of expression of cyclooxygenase-2 was consistent with the production of PGE2. The results suggest that A[[beta].sub.1-40]and A[[beta].sub.1-42] have opposite effects on the production of PGE2 induced by LPS.

THE EFFECTS OF GROWTH HORMONE AND INSULIN-LIKE GROWTH FACTOR ON THE PROLIFERATION RATES OF RAW 264.7 MACROPHAGES

Jayson R. Smith [*], Michelle Tucci, James L. Hughes, and Hamed Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216

It is well documented that growth hormone (GH) functions to regulate both cell growth and cell number and is considered the master hormone because it affects almost every cell of the body. Growth hormone stimulates the liver to secrete insulin-like growth factor, which is also capable of binding insulin as well as insulin-like binding receptors on the cell surface. It is possible that GH cellular effects are mediated by insulin-like growth factor (IGF) rather than GH itself. In this study, RAW f264.7 cells were challenged with high dose GH (48 ng4[micro]1), low dose GH (4.8 ng/[micro]1), high dose IGF-1 (26 mg/[micro]1) or low doses IGF-1 (6.3 mg/[micro]1) for 24, 48, 72, and 96 hours. Cell number, cell protein, concentration, cell damage, and cellular morphology were measured at each time point and compared to untreated RAW 264.7 cells. The results show significant increases in cell number for cells treated with low doses of GH and IGF-1 at 24 hour phase. Cell proliferative effects were also observed at 48 hours in IGF-1 treated cells. Cellular damage (MDA levels) was not statistically significant for any treated group for the entire duration of the experiment. Most notable differences were observed in cellular morphology for both IGF-1 and GH treated cells. IGF-1 resulted in condensation of the nuclear material as early as 24 hours after treatment. In conclusion: (1) RAW 264.7 responded to both IGF-1 and GH equally (viability and proliferation), and (2) morphological changes were observed in all cells treated with both hormones compared to control group. This study indicate that GH hormone could induce its effect directly or indirectly through IGF-1.

INHIBITION OF NITRIC OXIDE POTENTIATES GLUCOSE-HYPERTENSION IN RATS

Christie R. Claxton [1][*], Michael W. Brands [2], and Joseph A. Cameron [1], [1] Jackson State University, Jackson, MS 39217 and [2] University of Mississippi Medical Center, Jackson, MS 39216

Endothelial dysfunction, has been proposed to contribute to vasoconstriction or hypertension in many conditions characterized by hyperinsulinemia or hyperglycemia. However, there is little known about the role of the endothelium before dysfunction develops. This study tested whether nitric oxide production, before endothelial dysfunction develops, plays an important role in counteracting the hypertensive response to chronic glucose infusion. Glucose was infused (18.6 mg /kg/min, iv.) for 7 days in 8 normal rats (G) and in 9 rats with a chronic background iv. infusion of L-NAME at 10 mg/kg/min (G+L). Mean arterial pressure (MAP), measured 24 hr/day, increased an average of approximately 11 mmHg in the G rats. L-NAME treatment increased MAP an average of 28[+ or -]2 mm Hg in the G+L rats, and glucose infusion raised MAP more than 30 nun Hg above that, averaging 155[+ or -]8 mm Hg by day 6. In addition, heart rate increased from an average of 389[+ or -]8 bpm to 441[+ or -]16 bpm by day 6, while there was no si gnificant change in the G rats. Glomerular filtration rate decreased significantly with L-NAME treatment and decreased in both groups by day 3 of glucose infusion, reaching lower levels in the G+L rats. These results show that NO is required to minimize the increase in MAP during glucose infusion, and suggest renal and neural mechanisms may be important in mediating that effect.

THE EFFECTS OF INOSITOL HEXAPHOSPHATE ON THE INFLAMMATORY RESPONSE IN TRANSFORMED RAW 264.7 MACROPHAGES

Mara Johnson [*], Michelle Tucci, James L. Hughes, Zelma Cason, and Hamed Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216

Inositol hexaphosphate (IP-6) has received much attention for its role in interfering with tumor progression and slowing the metastasis of neoplastic cells. However, there is little information regarding the antioxidant properties of IP-6 or its ability to enhance the natural disease resistance of the body. The specific objectives of this experiment were to investigate the effects of IP-6 that might have on the proliferation and viability of RAW 264.7 transformed macrophages and to morphologically and biochemically investigate the role of IP-6 as a free radical scavenger. Transformed RAW macrophages were obtained from the American Type Culture Collection (Rockville, MD) and maintained in sterile media (RPMI) supplemented with 10% fetal bovine serum and 1% antibiotics and antimycotics. The cells were plated on to 24 well plates at a density of 1x[10.sup.6] cells/well. The cells were divided into five groups of four wells per group per phase (24, 48, and 72 hours). Cells in Group I were treated with media alon e and served as controls. Cells in Group II were treated with lipopoly-saccharide (LPS) only. Cells in Groups III, IV, and V cells were treated with 1000 ugh of IP-6 + LPS, 500 [micro]gh of IP-6 + LPS, and 100 [micro]gh of IP-6 + LPS, respectively. Cell number, as well as, histopathology, MDA, and protein was determined at the end of 24, 48, and 72 hours. Data obtained from this investigation revealed that the rate of cell proliferation was totally dependent on the dose of IP-6. At 24 and 48 hours and upon the exposure of high dose of IP-6 the mitotic ability of the cells was higher (p[less than]0.05) than the rate at 72 hour phase. Morphological evaluation of cells at all three phases revealed that there were no significant changes in the architecture of cells upon the exposure of IP-6 compared to the control group. The results of this study suggest that IP-6 may have had an excitatory effect on the inflammatory cell secretions and this phenomenon was found to be dose dependent.

HISTOPATHOLOGICAL EVALUATION OF KIDNEY TISSUES EXPOSED TO SUSTAINED DELIVERY OF STEROIDS IN OVARIECTOMIZED ADULT FEMALE RATS

John Bullock [*], Hamed Benghuzzi, and Michelle Tucci, University of Mississippi Medical Center, Jackson, MS 39216

Kidney tissue has been used extensively to investigate the mechanisms of steroid action. The specific objective of this investigation was to histopathologically evaluate the kidney cortical changes associated with sustained delivery of dehydroepiandrosterone (DHEA), diosgenin (DG), or estrogen (E) using ovariectomized rats. A total of 15 rats were divided randomly into five groups of 3 rats per group. Animals in groups II-V were ovariectomized (OVX), and animals in groups III-V were implanted with a single tricalcium phosphate lysine drug delivery device (TCPL) loaded with DHEA (40 mg), DG (40 mg), or E (20 mg), respectively. Animals in Group I served as unimplanted intact control. At the end of 17 days post implantation, the animals were sacrificed and the kidneys were harvested, processed, embedded, sectioned and screened for cellular alterations. Digital image analysis (glomeruli areas, and diameter) as well as light microscopy (glomeruli number) were employed to screen the cross sections of kidney tissue s. Data obtained from this study show significant decreases in glomeruli area in OVX animals treated with estrogen (p[less than]0.05) compared with either control groups or other treatment groups. DHEA and its precursor, DG, had no effect on glomeruli area, number or length when compared by analysis of variance to the control intact or OVX control groups. The length of glomeruli in OVX estrogen treated animals was not different from any treatment or control group. Estrogen treatment also caused a significant decrease in body weight in the animals compared with the other groups (p[less than]0.05), and in kidney weights between the estrogen treated animals and DG treated animals at p[less than]0.05. Overall conclusion: sustained delivery of DHEA or DG resulted in a maintenance of the kidney function in OVX animals while E treatment resulted in severe nephrotic hypotrophy conditions.

SCREENING ANTISENSE OLIGONUCLEOTIDES FOR THE DOWNREGULAITION OF THE EXPRESSION OF THE EQULIBRATIVE NBMPR-SENSITIVE NUCLEOSIDE TRANSPORTER (ES)

James C. Elledge [1] Alicia D. Andrews [1][*], and John K. Buolamwini[1],[2], (1.) NIH NHLBI STTMS Cardiovascular Biomedical Research Program and (2.) University of Mississippi, University, MS 38677

In view of the fact that heart disease is the leading cause of death among Americans and the world population, there is an urgent need for innovative therapeutic intervention approaches. Nucleoside transport inhibition has been demonstrated to be an effective means of enhancing the cardioprotective effects of adenosine, and is being investigated as a viable therapeutic approach in ischemic heart disease and stroke. This study was initiated to discover antisense oligonucleotides (ODNs) as potential downregulators of the expression of the NBMPR-sensitive equlibrative nucleoside transporter (es). In this regard, thirteen (13) ODNs were screened as inhibitors of the es transporter protein synthesis by a flow cytometry method using CEM cells and a liposomal delivery system. The results suggest that four of the thirteen antisense ODNs tested, namely, ES1 (5'-ATGGTGATGGTGTTCTCGGT-3'), ES-4 (5'CCGTCCCTCGACTCTG - 3'), ES-S (5'TCCTTCTGTCCATCCTTT-3') and ES-11 (5'CCAATAGCCACAGCAGGA-3') may be used to down-regulate the cellular expression of the es transporter.

THE EFFECT OF DHEA AND AED ON THE VIABILITY OF RAW 264.7 MACROPHAGES

Tyklie Necole Coleman [*], Hamed Benghuzzi, Michelle Tucci, and James L. Hugh]es, University of Mississippi Medical Center, Jackson, MS 39216

The effect of adrenal hormones namely dehydroepiandrosterone (DHEA, 10-50 [micro]g/day) and androstenedione (AED, 10-50 [micro]g/day), on RAW 264.7 macrophage survival at 24,48 and 72 hours after lipopoly-saccharide (LPS, 2 mg/ml) exposure was investigated in an in vitro environment. RAW cells were obtained from American Type Culture Collection and standard laboratory protocols were followed in cells plating ([10.sup.6] cells/well), phase terminating, morphological evaluation, and biochemical marker analysis. From physiologic to supraphysiologic doses of DHEA and AED at 24 hours caused increased levels of cellular proteins and cell number without causing any significant (p[less than]0.05) change in cellular membrane integrity (Maliondialdehyde, MDA) or viability (morphology). At 48 and 72 hours, cells treated with either AED or DHEA did not sustain the increased cellular proliferation as observed at 24 hours and did not significantly differ (P [less than] 0.05) in cellular protein content. RAW 264.7 cells tr eated with LPS for 30 minutes prior to AED or DHEA exposure caused slight decrease in cell number and cell protein content. The decrease in both cell number and cell protein content were not attributed to increased cell damage or decreases in cell viability due to the fact that the cellular MDA levels were not statistically higher than the control values (p[less than]0.05). Dot.blot analysis of pro-inflammatory cytokine (TNFa) production after LPS treatment was suppressed by DHEA while AED had a minor influence on the responses. These data imply that LPS mediated activation of RAW 264.7 cells can be inhibited by the addition of pharmacological doses of adrenal hormones such as DHEA.

THE ROLE OF THE ROUTE OF ADMINISTRATION OF POLY-CHLORINATED BIPHENYLS (PCBs) ON THE REPRODUCTIVE AND VITAL ORGANS OF ADULT FEMALE RATS

Kevin Blake [*], Hamed Benghuzzi, Zelma Cason, and Michelle Tucci, University of Mississippi Medical Center, Jackson, MS 39216

Studies have shown that high doses of Polychlorinated biphenyls (PCBs) given by conventional methods (orally or injections) have adverse effects on the reproductive and vital organs of adult female rats. However, there has not been documentation as to the effects of PCBs on adult female rats by means of a sustained delivery system. The specific objectives of this study were: (1) to investigate the effects of sustained delivery (TCPL ceramics) of PCB versus conventional mode of administration (injection) on the reproductive and vital organs of the adult female rat, (2) to evaluate the role that PCB that might have on the estrus events of adult female rats upon the exposure by sustained delivery (TCPL ceramics) and conventional mode (injection), and (3) to histopathologically evaluate the effect that PCB might have on the ovarian and accessory organs upon the sustained delivery for 21 days. A total of 10 adult female rats (BW 270-300 gm) were randomly divided into three groups. Group 1 (n=3) served as the contr ol, group 2 (n=4) was injected intramuscularly every other day with Aroclor 1254 (0.1 cc), and each rat in group 3 (n=3) was implanted with TCPL capsules (5 mg of 2, 3, 3', 4, 5-Pentachlorobiphenyl). Aseptic surgical techniques were performed throughout the experiment. Blood (1 cc) was collected biweekly for biochemical analysis, and body weights were recorded as well. Pap smears were taken daily at approximately the same time for 25 days, and two slides were made for each pap taken (1 pap stain, 1 Diff Quik). At the end of 2l days post-implantation, all control and experimental animals were sacrificed following standard lab procedures (overdose of Halothane). The reproductive and vital organs were collected, weighed, fixed, embedded, sectioned, and stained (H&E) for histological evaluations. Data obtained from this investigation suggest the following: (1) TCPL devices were able to deliver PCB at sustained levels for 21 days, (2) regardless of the route of PCB administration no significant change was observed in total body weight compared to the controls, (3) conventional administration of PCB resulted in a remarkable changes in the fallopian tubes compared to control and sustained delivery implanted animals, (4) there were no obvious change was observed in the phases of estrus cycles upon the exposure of PCB, and (4) histopathological evaluation of spleen, kidneys, heart, adrenals, ovaries, uterus, and cervix tissues exposed to PCB did not reveal any significant changes compared to the intact group.

THE EFFECT OF POLYCHLORINATED BIPHENYL ON RAW 264.7 CELLS

Daphanie Denise Webster [*], Michelle Tucci, Zelma Cason, and Hamed Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216

Polychlorinated biphenyls (PCBs) refer to a group of chlorinated hydrocarbons that have been used widely in industry since the early 1900's. Among their physical properties, PCBs are thermally stable, non-reactive, have low water solubility and low electrical conductance. These features made PCBs the perfect choice for use in flame-retardants, electrical insulators, lubricants and liquid seals. However, these characteristics have also allowed the very stable PCBs to resist degradation treatments and accumulate in the environment. The effects of PCBs exposure, at low doses ranging between 10-100 mg PCB (Arclor-1254), on the viability of RAW 264.7 macrophage cells after 24 and 48 hours of incubation. Furthermore, several studies have indicated that PCBs exposure resulted in pathophysiological conditions at the reproductive level, this study was designed to determine the interrelationship between PCBs and estrogen (E, 10 mg) and its role in the viability of RAW cells. Macrophages were cultured and plated in 24 w ell-plates according to standard protocols. The wells were divided into eight groups (n=4 wells/group, 1 x [10.sup.6]cells/well). The cells in groups 1-3 were treated with vehicle (serum- control for estrogen, DMSO-control for PCB, and media alone, respectively). Cells in groups 4 and 5 were treated with low (10 mg) and high (100 mg) doses of PCB. Cells in group 6 were treated with estrogen, and cells in groups 7 and 8 were treated with low dose PCB + estrogen and high dose PCB + estrogen, respectively. Cell viability and damage (Malinodialdehyde, MDA level) were determined after 24 and 48 hours as well as cell morphology. The results of this study showed that low and high doses of PCB depressed cell number by 52%. Estrogen treatment caused no effect on cell number in comparison to cells treated with serum alone. Cell number in response to estrogen and low and high doses of PCB decreased cell number by 50%. Similar results were also observed at 48 hour time phase. Morphological evaluation of the cells reveale d healthy spindle shaped multinucleated cells in the control group. PCB exposure induced morphological changes includes: cells became small, round, and increased evidence of cellular injury and debris. Estrogen treatment did not show changes from the control group. However estrogen and PCB treatments caused the cells to become round, tightly compact nuclei with evidence of cell fragmentation. The results of this investigation showed that exposure to either 10 or 100mg of PCB had detrimental effects on the RAW 264.7 macrophage cells as early as 24 hours. Combination treatment with estrogen didn't provide any protective measures to the viability of RAW cells exposed to PCBs.

SEARCH FOR NATURAL PRODUCT INHIBITORS OF SECRETED ASPARTIC PROTEASE FROM CANDIDA ALBICANS

Keyana Mitchell [*], A. Clark, M. Jacobs, and A. Nimrod, University of Mississippi, University, MS 38677

The yeast Candida albicans invades the tissues of the body via the SAP (secreted aspartic protease) enzymes. The proteolytic activity of the SAP enzymes lyses cell membranes, allowing the yeast to cause infection. Therefore, the SAP enzymes are important virulence factors and reasonable targets for drug therapy in the prevention and treatment of yeast-borne diseases. The SAP assay screens natural products for inhibition of the SAP enzyme using a fluorescent substrate. The substrate is specific for aspartic proteases and the assay is rapid, amenable to high throughput, and can be done in a 96-well plate. Due to the large number of natural product extracts available to the National Center for Natural Products Research (NCNPR), an elimination strategy is used in the assay. Samples of the natural product extracts are first tested at 200 mg/mL. The samples that are active (showing[greater than]80% inhibition of SAP) are then tested at a lower concentration of 10 mg/mL. The samples that continue to show inhibitory activity are then tested in a secondary assay at 50, 10, and 2 mg/mL to determine the IC50, the concentration that results in 50 % inhibition of the SAP. These samples are then prioritized according to their IC50's and undergo bioassay directed fractionation done by the chemists at the NCNPR. The samples are fractionated until a pure compound is obtained that shows appreciable SAP inhibitory activity. As of September 20, 1999, 3,256 plant extracts have been tested in the SAP assay. Approximately 5% of these extracts were tested in the secondary assay based on their inhibitory activity. 122 of the extracts (approximately 3.7% of the plant extracts tested in the primary assay) had 1C50's of less than 50 mg/mL. Two pure compounds have been isolated from plant extracts of Miconia myriantha which have IC50's in the range of 7-10 mg/mL. A second assay is being developed to determine the specificity of the samples that inhibit SAP. Renin and pepsin are being explored as possible aspartic protease enzymes for this a ssay. The isolated pure compounds that specifically inhibit SAP activity are potential sources for drugs to treat infections by Candida albicans.

THE EFFECTS OF CORTISOL ON THE VIABILITY AND PROLIFERATION OF HEP-2 CELLS

Melissa P. Daniel [*], Michelle Tucci, and Hamed Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216

It is well known that bronchial epithelial cells are able to convert cortisol and synthetic cortisols such as hydrocortisone to inactive metabolite cortisone via the enzyme 11 beta-hydroxsteroid dehydrogenase. The effects of cortisol, however, have not been investigated in the human larynx carcinoma cell line, Hep-2. Because the enzyme is expressed in human airway epithelial cells as well as other types of epithelial cells, we hypothesize that cortisol effects are limited due to the rapid metabolism to cortisone, which is then unable to bind to the glucocorticoid receptors. In this study, Hep-2 cells were treated with various doses of cortisol (0, 1, 5, 10, 20 and 50 mg/dl) for 24, 48 and 72 hours. After 24,48 and 72 hours of treatment the cells were harvested and cell numbers were determined by hemacytometer counting. In addition to cell number, cell damage (Maliondialdehyde, MDA levels), cellular protein content, and cellular morphological characteristics were determined at each phase. The data obtained fr om this investigation demonstrated the following: (1) Cell counts: Cell number was suppressed at all doses of cortisol tested at the initial time points until the cells were able to metabolize the cortisol to the inactive cortisone. All treatments showed statistical differences at p[less than]0.001 using the Kurskal Wallis one way ANOVA on ranks. (2) MDA: The exposure to the highest cortisol concentration (50 mg/dl) showed evidence of increased MDA levels after 24 hours, and by 48 hours there was a statistically significant increase in MDA levels in cells treated with either 20 and 50 mg/dl cortisol. (3). Protein: Cortisol caused a statistically significant increase in the median values among the treatment groups for protein concentration at all time points investigated. (4) Morphology: Morphological differences existed among the cortisol treated cells compared with cells treated with media alone. In conclusion, the investigation provides significant information regarding the interrelationship between the ste roid hormone cortisol and the viability and proliferation of human larynx carcinoma cells in culture.

THE CHARACTERIZATION OF ACETYLCHOLINESTERASE INHIBITION BY ALDICARB IN JAPANESE MEDAKA AT HIGH SALINITY

Sonja Grisle [*], Daniel Schenk, and Abir El-Alfy, University of Mississippi, University, MS 38677

Aldicarb is a carbamate pesticide used on crops in the Southeastern United States to kill insects and nematodes on crops. These crops include cotton, sugar cane, peanuts, potatoes, sugar beets, and other ornamental plants. The corps are often grown adjoining estuaries, which undergo large fluctuations in salinity. Aldicarb inhibits cholinesterase enzymes. This results in a build up of acetylcholine. This buildup, cholinergic overload, causes over stimulation at the neuromuscular synaptic joint leading to convulsions and death. Salinity has been shown to enhance toxicity of thioether pesticides, terbufos and aldicarb, in euryhaline fish (Hemmer et al., Schlenk & El-Alfy, 1998). Results from recent studies show that female Japanese medaka, Oryzias latipes, are more sensitive to aldicarb at high salinity in comparison to male Japanese medaka. The reason for this is currently unknown. In this study the inhibition of acetylcholinesterase (AChE) is analyzed in euryhaline fish, Japanese medaka, exposed to aldicarb at high salinity. Time course characterization AChE inhibition by different aldicarb zones (LC 20, LC 50, and LC 90) in Japanese Medaka at different salinity regimens (1.5, 12, and 20 ppths) is presented. To examine the developmental differences, the effect of salinity on toxicity of aldicarb in Japanese medaka hatchlings is also presented.

LEVELS OF HYDROGEN PEROXIDE IN TISSUES RETRIEVED FROM PATIENTS WITH FAILING IMPLANT DEVICES MAY PLAY AN ACTIVE ROLE IN CYTOKINE PRODUCTION

Michelle Tucci [*], Rodney Baker, Hamed Benghuzzi, Audrey Tsao, and James L. Hughes, University of Mississippi Medical Center, Jackson, MS 39216

Late aseptic loosening of total joint implants continues to be a common cause of implant failure. However, the pathophysiology of implant loosening remains controversial as to which factors at the interface between tissue and implant play a crucial role in implant failure. The most prominent feature of the foreign body membrane obtained from patients undergoing revision hip surgery in the presence of lymphocytes, histiocytes, giant cells, and immature collagen formation. Biochemical and immunochemical analysis of the tissues obtained from implant failure patients revealed increased levels of pro-inflammatory cytokines as well as increased levels of hydrogen peroxide and decreased catalase activity. Macrophage activation by cytokine (TNFa) or lipopolysaccharide (LPS) is mediated via translocation of NFkB from the cytosol to the nucleus and appears to be dependent upon phospholipase D (PLD) activation. Increasing concentrations of hydrogen peroxide also caused increases in macrophage release of pro-inflammator y cytokine (IL-1). This response may also be mediated via the PLD pathway. In tissues of patients with failing primary joint implants over production of hydrogen peroxide in response to wear debris stimuli, may activate NFkB and initiate cytokine production.

PHYSIOLOGICAL RESPONSES ASSOCIATED WITH SUSTAINED DELIVERY OF T, DHT, AND AED N MALE RATS

Keith Stokes [*], Hamed Benghuzzi [1], and Joseph A. Cameron [2], (1.) University of Mississippi Medical Center, Jackson, MS 39216 and (2.) Jackson State University, Jackson, MS 39204

The factors responsible for the marked gender differences in risk of coronary disease and atherosclerosis remain controversial. Some clinicians and experimental evidence supports a protective effect of endogenous estrogen, however, the epidemiological data do not support this conclusion. The role of endogenous androgens on atherosclerosis progression has received little attention over the years. The specific objectives of this study are: 1) to deliver testosterone (T), dihydrotestosterone (DHT), or androstenedione (AED) at sustained levels by tricalcium phosphate lysine (TCPL) ceramic delivery devices, and 2) to evaluate lactate dehydrogenase activity (LDH), total cholesterol, thiobarbituric reactive species (TBARS) and histopathological changes associated with excess androgens delivery in adult intact male rats. A total of sixteen male rats were randomly divided into four groups of four rats per group. Rats in group I served as intact controls, and animals in groups II-IV were implanted with a single TCPL d elivery device containing T (40 mg), DHT (40 mg), or AED (40 mg), respectively. Animals in all groups were weighted and blood was drawn biweekly for cholesterol, LDH and TBAR analysis. At the end of ninety days post-implantation, the animals were sacrificed and the organs removed, processed embedded, sectioned and screened for morphological changes. The results of this study suggest that: 1) sustained delivery of androgens caused a statistically significant increase (p[less than]0.05) in the prostate and seminal vesicle weights, while the epididymis weights remained similar to control intact animals, 2) decreases in adrenal gland weight was detected in animals treated with androgens compared to control animals, 3) kidney weights increased in all androgen treated animals compared to control, 4) no differences were observed in heart spleen or body weights between the groups, 5) TBAR analysis, as well as, LDH activity were not different between androgen treated animals and control animals, and 6) total cholester ol was significantly reduced in T and DHT treated groups compared with control and AED treated animals. The decrease in total cholesterol and reduction in adrenal gland weight may suggest impairment of cholesterol synthesis. In conclusion, this study provides information on the effects of sustained release of DHT, T and AED on the reproductive organs, vital organs, as well as physiological parameters (TBARS and total cholesterol) of atherosclerotic plaque formation in adult male rats.

MAPPING OF MONOAMINERGIC RECEPTORS IN HUMAN AMYGDALA 

Jane E. Schenck [*], Violetta Klimek, and Gregory A. Ordway, University of Mississippi Medical Center, Jackson, MS 39216

The amygdala is a key structure in the brain's integration of emotions and stress. The amygdala receives dense noradrenergic and dopaminergic projections from the locus coeruleus and from the ventral tegmental area, respectively. These two catechalominergic neuronal systems are critically involved in the pathophysiology of depression. Therefore, the amygdala is an important region for the study of neurochemical abnormalities in depression that involves norepinephrine and dopamine. The purpose of this study was to create a "neurochemical" map of the distribution of dopaminergic and noradrenergic receptors and transporters in this very complex brain structure. Many of the amygdaloid nuclei can be distinguished based on differences in size, packing density, and staining intensity of their constituent neurons m Nissl preparation. These properties, together with the histochemical localization of an enzyme (acetylcholinesterase) marker, were used to create anatomical templates for measuring the density of certain receptors using quantitative autoradiography at multiple levels in several discrete regions of the human amygdala. Our study demonstrated that dopamine D2/3 receptors and noradrenergic [[alpha].sub.2]-adrenoceptors were predominantly localized in the central and in the baso-lateral nuclei of the amygdala. However, D1 and [beta]-adrenoceptros were more evenly distributed among areas of the amygaloid complex. Both transporters occurred in a low density throughout the amygdala. These neurochemical maps of amygdala are necessary for revealing individual variability and for assessing changes in noradrenergic and dopaminergic proteins in depression.

FRIDAY MORNING

Gulf Hall

8:30 INTRAVAGINAL ANTIMICROBIALS PREVENT TRICHOMONIASIS IN MICE

W.B. Lushbaugh [1][*], A. Paxton [1], P. Shah [1], A. Banga [2], J. Jaynes [3], J. Cleary [1,4] and R. Finley [1], (1.) University of Mississippi Medical Center, Jackson, MS 39216; (2.) Mercer University, Atlanta, GA 30341; (3.) Demeter, Inc., Philadelphia, PA 15221; and (4.) University of Mississippi, University, MS 38677

A chemical prophylactic that protects against acquisition of sexually transmitted infections (STI) without disrupting the vaginal environment is needed. The efficacy of the novel peptide antibiotic (Peptidyl [MIMs.sup.tm] D2A21) to prevent acquisition of Trichomonas vaginal is (Tv) was tested in mice. Trichomonas infections in estrogenized Lactobacilus acidophilus pre-infected mice were sporadic so we examined several other Lactobacillus species as pre-infections to modify the mouse vaginal flora. Tv infections of estrogenized mice pre-infected with L. vaginalis or L. rhamnosus (Lr) were more prevalent and persistent than those in mice pre-infected with L. cripatus or L. acidophilus. The efficacy of intravaginal microbicides to interfere with Tv acquisition was tested in estrogenized, Lr pre-infected mice. The mice were intravaginally pre-treated with placebo gel, 0.5% or 2% Peptidyl [MIMs.sup.tm] D2A21 gel, or 500 mg/mL metronidazole gel just prior to Tv challenge. Pretreatment with 2% D2A21 or metronidazol e gels significantly (P[less than] 0.001) prevented Tv infection. None of 30 metronidazole-treated-mice and three of 30 2%-D2A21-treated-mice became infected while about half of control mice became infected. These studies suggest that intravaginal microbicides incorporating Peptidyl [MIMs.sup.tm] D2A21 with activity against a variety of STI may be practical for prevention of STI in humans.

8:45 LOW DOSE IONIZING RADIATION EFFECTS ON BIOLOGICAL SYSTEMS

Amin Haque, Alcorn State University, Alcorn State, MS 39096

A cell contains many elements and compounds including about 80 % water ([H.sub.2]O). Absorption of ionizing radiation (alpha, beta, gamma, neutron, and x-rays) by the cells causes ionization, and excitation of atoms and molecules, and dissociation of covalent bonds in molecules. Within an extremely short period ([sim][10.sup.-10] seconds) large numbers of ions and free radicals are produced which are highly chemically reactive. The problem in the biological systems is the production of hydrogen peroxide ([H.sub.2][O.sub.2]), which is a poison to the cells. In addition, [H.sub.2][O.sub.2] readily forms a peroxide radical, which then attacks other bio-organic molecules to form. a relatively stable organic peroxide. This process may prove fatal to the cell if the original organic molecule happens to be a basic molecule such as a key enzyme. The change in the nature of the basic molecule effectively removes it from the cell. The process of conversion of the original basic molecules causes serious damage to the c ells. The rate at which energy is transferred per unit length or mass determines how effective a given radiation is at injuning the cells. So far no law exists which would predict exactly the kind and amount of damage produced in a given cell following an irradiation. Electron-Spin-Resonance Spectroscopy is used to study the spectra of the free radicals. The amplitude of the resonance signal is proportional to the number of free radicals which in turn should be proportional to the radiation absorbed dose.

9:00 SYNERGISTIC ENHANCEMENT OF COLLAGENOUS PROTEIN SYNTHESIS BY NIFEDIPINE AND TUMOR NECROSIS FACTOR-[alpha]

Roger B. Johnson, University of Mississippi Medical Center, Jackson, MS 39216

Gingival overgrowth commonly occurs coincident to therapy with calcium channel blockers. The biologic mechanism for this condition is unknown; anecdotal evidence suggests that gingivitis may contribute to development of the overgrowth. This study tests the hypothesis that collagenous protein synthesis is synergistically enhanced by nifedipine (N) and the pro-inflammatory cytokine, tumor necrosis factor-[alpha] (TNF-[alpha]). Fibroblasts were isolated from biopsies of normal gingiva and cells separated into two groups. Group I were exposed to media containing 0, 5, 50, or 500 ng/mL TNF-[alpha], or [10.sup.-7] MN for 7 days; group 2 were exposed to those concentrations of TNF-[alpha] + [10.sup.-7] M N. 3-Hproline was added to the medium for the final 24. hours. Cells and matrix were harvested and radioactivity determined by liquid scintillation analysis. Means (dprn/[10.sup.4] cells) were compared by factorial ANOVA and Scheffe comparisons. Collagenous protein synthesis was significantly reduced by a concentra tions of TNF-[alpha] and was significantly enhanced by [10.sup.-7] M nifedipine and all concentrations of TNF-[alpha] + [l0.sup.-7] M nifedipine. Thus, patients may be more susceptible to gingival overgrowth during nifedipine therapy, due to the synergism between TNF-[alpha] and nifedipine on collagenous protein synthesis by gingival fibroblasts. Supported by the University of Mississippi School of Dentistry.

9:15 EVAPORATIVE LASER LIGHT SCATTERING DETECTOR FOR GPC ANALYSIS OF POLY (LID, L-LACTIDE) POLYMERS

C.C. Inman [*], S.B. Roberts, and A.D. Puckett, University of Mississippi Medical Center, Jackson, MS 39216

The GPC system consisted of a Rheodyne injector (Rheodyne Inc., Cotati, California) with 200 [micro]m injection loop, LDC Milton Roy Constametric 3000 pump, LDC Milton Roy refractoMonitor IV refractive index detector, and a column set consisting of five styragel GPC columns. The column set contained three Shodex (Showa Denko, Tokyo, Japan 8 mm x 250 mm) columns (A-803/S, A-804/S and A-805/S) and two Waters Corporation (Milford, Delaware 7.8 mm x 300 mm) [10.sup.5] A and [10.sup.6] A columns. All solutions were filtered through a 0.45 [micro]m Teflon filter before injection onto the column set. A microcomputer and strip chart recorder collected the output from the detector. The LDC Milton Roy PC GPC software was used for data collection and analysis. The GPC system was calibrated using narrow distribution polystyrene standards obtained from PolySciences. Seven polystyrene standards ranging from 7.8-x[10.sup.6] g/mole to 2.0 x 104 g/mole were dissolved in HPLC grade chloroform at 1-2 wt. % and chromatographed in triplicate to establish a calibration curve. A plot of molecular weight versus elution volume was constructed and fit to a 5th degree polynomial. This calibration was then used to calculate the weight average ([M.sub.w]), number average ([M.sub.n]), and polydispersity index ([M.sub.w]/[M.sub.n]) for the 70:30 poly (L/D, L-lactide) samples from retention volume data collected for each sample.

9:30 A CASE STUDY FOR BUILDING AGENCY SUPPORT FOR COMMUNITY-BASED HEALTH ROJECTS: THE DELTA FUTURES PROJECT

M. Williams [1][*], D. Lamar [2], A. Arrington [3], and Reid Jones [2], (1.) Delta Hills Public Health District, Greenwood, MS 38939; (2.) Delta State University, Cleveland, MS 38733; and (3.) Mississippi Valley State University, Itta Bena, MS 38941

Rural health care agencies are usually small, understaffed, under-resourced, and on the wrong side of the 'digital divide.' Analyses of data from the Mississippi State Department of Health demonstrated that per capita health care concerns were generally more severe in rural districts. Building collaborative relationships was difficult in these circumstances. The report described the Delta Futures Project, an innovative 'peer educator' approach to perinatal health risk management. Guidelines were presented for developing strong collaboratives among largely rural agencies. Strong collaborations were still needed with major health care research and education centers.

9:45 Break

10:00 INFLUENCE OF DIACETYL AND HEAT ON CELLULAR LEAKAGE AND DEATH OF STAPHYLOCOCCUS AUREUS AND SALMONELLA TYPHIMURIUM

I.O. Farah [*] and S.R. Tatini, Jackson State University, Jackson, MS 39217 and University of Minnesota, St. Paul, MN 55108

Influence of diacetyl on cellular leakage and/or death of Staphylococcus aureus (S aureus; FRI- 100) and Salmonella typhimurium (S. typhimurium) in the presence and absence of 30% sucrose and NZ-amine broth at 37,45 and 55[degrees]C was studied using Microbiological and Spectrophotometric techniques. Diacetyl exists naturally in starter distillate and is usually added to enhance the flavor at the end of the fermentation process. Our objective was to examine its use before the onset of fermentation towards the safety of fermented products. Results showed significant difference (p[less than]0.05) between control and diacetyl treatments; average mean difference was [greater than]5 log colony forming units per ml (CFU/ml) within 1 hour (h). Interesting trends were also observed when differences with respect to absorbance ratios, temperature and control were normalized; multiple peaks in relation to incubation times. Results also showed that diacetyl (0.1%) caused more cellular leakage and death of S. aureus at 4 5[degrees]C as compared to 37[degrees]C, and that cellular leakage per se was not directly related to death. Diacetyl, however, caused rapid death of S. aureus and S. typhimurium in the presence of 30% sucrose and NZ-amine broth at 55[degrees]C; [greater than]7 log CFU/ml death within 1 and 2 h respectively. We also conducted a study with commercial starter distillate (7%) and proved its ability to control both organisms under the same test conditions within lh at 55[degrees]C. It is concluded that diacetyl is a potential candidate for the control of foodborne pathogens especially under low water activity conditions such as those encountered during meat fermentation.

10:15 SEALING POTENTIAL OF NEW ONE BOTTLE DENTAL ADHESIVES

J.G. Fitchie [*], G.W. Cobb, A.D. Puckett, S.M. Phillips, and W.B. Jenkins, University of Mississippi Medical Center, Jackson, MS 39216

The purpose of this study was to evaluate in vitro microleakage behavior of several one bottle adhesives and a conventional primer/adhesive system utilizing the universal hybrid composite Amelogen (Ultradent Products, Inc.). Fifty class V cavities were prepared at the facial C.E.J. of bovine incisor teeth to simulate cervical abrasion/erosion lesions. The five groups of ten teeth were treated with the enamel and dentin total etch technique (15 second application phosphoric acid) then restored following manufacturer's directions with the appropriate adhesive: GlumaOneBond (GLU1), SnapBond (SNPB), PQ1 (PQ1), ProBond (ProB), Prime&BondNT (PBNT), and bulk filled with Amelogen universal composite and light cured for sixty seconds. Microleakage was evaluated separately at the enamel (incisal) and dentin (gingival) margins using the following scale: 0 (no leakage) to 4 (gross leakage). Results were analyzed with the Mann-Whitney Test. Mean dentin microleakage values gave the following ranking: PQ1 (1.4) [less than] ProB (2.2) [less than] SNPB (3.1) [less than] PBNT (3.36) [less than] GLU1 (3.7). There were no significant differences in the leakage behavior between any of the groups at the enamel margin. PQ1 exhibited statistically less microleakage at the gingival (dentin) margins than any of the one bottle adhesives, however, the difference between PQ1 and the conventional primer/adhesive ProBond was not significant.

10:30 A CASE STUDY IN BUILDING GRASS ROOTS SUPPORT FOR COMMUNITY BASED HEALTH PROJECTS: THE SAFE AT HOME PROJECT

S. French [1][*], J. McCrory [2], C. Ekanem [3], and Reid Jones [4], (1.) Delta Health Partners, Marks, MS 38646; (2.) Delta State University, Cleveland, MS 38733; and (3.) Mississippi Valley State University, Itta Bena, MS 38941

Federally funded health care programs often neglect the crucial aspect of developing "grass roots" support in the local community. This is particularly important for rural communities, which are distant from major hospitals, research centers, and medical specialists. The Safe at Home Project has been repeatedly funded to provide creative solutions for health problems such as SIDS, teen pregnancy, and low birth weight. Over the years, Safe at Home has built effective and extensive collaborative relationships with health care professionals, Head Start, schools, local government, civic clubs, businesses, and local media. Project personnel are presently expanding existing relationships with regional universities. The presentation provided a program report on the project as well as a case study in effective community support building.

10:45 HEALTH RISKS FROM LOW-DOSE IONIZING RADIATION EXPOSURES

Amin Haque, Alcorn State University, Alcorn State, MS 39096

The average person in the United States is exposed to an effective dose equivalent of approximately 360 mrem per year ftom natural and technological radiation sources. The nature and severity of effects depend on: nature of radiation, absorbed dose, dose rate, dose distribution, age of the object, general health, and previous exposures. The rate at which energy is transferred per unit length determines how effective a given ionizing radiation is at injuring the cells. The toes of biological effects seen in humans from exposures to high doses (100,000 millirem or more) of radiation are: SOMATIC (Prompt and Delayed), TEROTOGENIC, and GENETIC. If individuals cumulative radiation dose is 1000 mrem, the chances of eventually developing cancer during their life may increase by 25%. For low doses, extrapolations are made from the results at high doses and high dose rates, which may not be appropriate. No law exists which would predict exactly the kind and magnitude of damage produced in a given cell following an ir radiation. There is no theory to predict what really happens at the low-dose rates. According to NIH, no biological effects are expected at low dose of less than 10 mrem per year. Extensive literature indicates that minute doses of ionizing radiation actually benefit animal growth and development, fecundity, health and longevity. The mechanism of radiation hormesis is not understood. It is very important to determine a threshold value of radiation below which no damage is caused to the tissues.

11:00 FORMULATION AND STABILITY OF NAL TREXONE SUSPENSION FOR REDUCINC EPIDURAL OPIOED SIDE EFFECTS

Brian K. Tsang, Ahmed S. Abdel-Aziz [*], and John H. Eichhorn, University of Mississippi Medical Center, Jackson, MS 39216

Naltrexone is a pure opioid antagonist that unlike naloxone is highly effective orally. Recently, naltrexone, 6 mg, has been shown to reduce the side effects of epidural morphine without reversal of its analgesic effect. However, naltrexone is only available in 50 mg tablets. This high dose of naltrexone is designed for use in detoxifying addicts. Naltrexone suspensions at concentration of 1 mg/ml were prepared. Our goal is to produce 6 mg doses for use in reducing epidural opioid side effects. The naltrexone suspensions were then tested for stability at room temperature over five consecutive days. High performance liquid chromatography was used for quantifying the concentration of naltrexone samples. There were no detected difference in physical characteristics (color, clarity, and crystal formation) or, pH values of naltrexone suspensions over the course of the five days duration of the stability study. In addition, there was no statistical difference in mean concentration of naltrexone suspensions (1 mg/m l). In conclusion, this study proves that formulation of naltrexone suspensions prepared from the 50 mg naltrexone tablets can be kept in room temperature for five days without a loss of the active ingredient. This easy formulation of naltrexone 6 mg can then become available for wider use in reducing the side effects of epidural opioids.

11:15 Divisional Business Meeting and Awards Presentations.