Co-Chair: Tina Martin, University of Mississippi Medical Center
Co-Vice Chair: Mary Tan, Holmes Community College
Co-Vice Chair: Edwin Swiatlo, University of Mississippi Medical Center
Bost Auditorium North
Session I: Oncology Research
8:00 Opening Remarks
8:15 PHENOXAZINES INHIBIT AKT AND INDUCE APOPTOSIS IN CANCER CELLS
K. N. Thimmaiah (1*), H Simmons (1), A Rinaldy (1), L Sylvester (1), P Grisham (1), and Peter J. Houghton (2), (1) Northwest Mississippi Community College, Southaven, Mississippi 38671 and (2) St Jude Children's Research Hospital, Memphis, Tennessee, 38105
Phenoxazines shut down the activation of Akt/mTOR/p70S6/S6 kinase pathway and induce apoptosis to a significant extent in rhabdomyosarcoma cells. Within the series, compounds having 3 carbon alkyl bridge were less potent than compounds with 4 carbon chain length. We proposed that by increasing the alkyl chain length to (-[CH.sub.2])[.sub.5] or (-[CH.sub.2])[.sub.6] at [N.sup.10]-position, the potency will be increased to a significant extent. Towards this goal, thirteen new derivatives have been synthesized and the parent 2-chlorophenoxazine was reacted with chlorobromoalkanes which resulted in [N.sup.10]-(chloroalkyl)-derivatives. Nucleophilic substitution reactions of these compounds with various secondary amines resulted in the desired products which were investigated to determine whether they would inhibit the phosphorylation of Akt. Serum starved cells were exposed to 100 nM-2000 nM phenoxazine derivatives before stimulating with IGF-I (10 ng/mL) for 10 min. Akt or Erk-1/2 phosphorylation was detected. Indeed, an increase in the expression of phosphorylated form of Akt at Ser 473 or Erk-1/2 was observed in response to IGF-I stimulation. These compounds at 100 nM inhibited the phosphorylation of Akt without affecting the phosphorylation of Erk-1/2. The potency of these compounds has been increased by 10-fold, and additional experiments are in progress (Supported by NIH grant CA 115404-01 to KNT).
8:30 COMPARISON OF POTENTIAL CHEMOTHERAPEUTIC AGENTS, 5-FLUORURACIL, GREEN TEA, AND THYMOQUINONE ON COLON CANCER CELLS
Anne A. Norwood*, Mary Tan, Marilyn May, Michelle Tucci, and Hamed Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216
Antioxidants have been found to be quite successful in deterring certain disease processes for years, especially cancer. Antioxidants protect the body by neutralizing the free radicals and donating one of their own electrons, thus ending the scavenger reaction. Epigallocatechin-3-gallate (EGCG), the most abundant catechin found in green tea, is a valuable scavenger of reactive oxygen species in vitro as well as in vivo. Thymoquinone (TQ), a major active component of black seed (Nigella sativa), is also known for its powerful scavenger abilities as an inhibitor of oxidative stress and has been utilized in the Middle East for centuries for healing properties. These two potent antioxidants when compared to the chemotherapeutic drug of choice, 5-fluorouracil (5-FU), have demonstrated incredible chemotherapeutic responses to the SW-626 cell line. The objective of this study was to evaluate and compare the effects of SW-626 colon cancer cells after a 24, 48, and 72 hour incubation periods with low, medium, and high doses of EGCG, TQ, and 5-FU. Cell viability, cell number, cellular morphology, and cellular metabolism were compared for the control and treatment groups. The results of this study evidenced a similar significant decrease in cell number as early as 24 hours in the groups treated with TQ and EGCG compared to 5-FU. Increases in cellular damage were evident after 24, 48, and 72 hours and in all treated groups compared with the control. Reduced cell numbers in the treated groups suggests the possibility that TQ and EGCG may have similar chemotherapeutic effects on cancer cells as 5-FU.
8:45 POTENTIAL EFFECT OF A-ESTRADIOL IN HUMAN JURKAT T-CELLS
Michael Johnson (1*), Danielle Wells (1), Erika Brown (2), Clement Yedjou (2), and Joseph A. Cameron (2), (1) Hinds Community College, Raymond, MS 39154 and (2) Jackson State Universtiy, Jackson, MS 39216
a-estradiol is the most potent estrogen of a group of endogenous estrogen steroids, which includes estrone and estriol. This steroid hormone is the most potent natural estrogen, produced mainly by the ovary, placenta, and in smaller amounts by the adrenal cortex, and the male testes. Although a-estradiol protects the renal and cardiovascular systems, the mechanisms involved remain unclear. In this research, we performed both MTT assay and trypan blue exclusion test to evaluate the effect of a-estradiol in HL-60 and Jurkat T-cells; and to compare the sensitivity of these two cells types. The results from both MTT assay and trypan blue exclusion test demonstrated that low, physiological levels of a-estradiol induce cellular proliferation in Jurkat T-cells. At higher dose of exposure (16 uM), a-estradiol decreases the viability of Jurkat T-cells compared to the control cells. Similar trend was obtained with the trypan blue exclusion test using the hemacytometer to count the cells manually. In summary, the results of the present study demonstrate that physiological levels of a-estradiol induce cell growth, and cellular proliferation of HL-60 and Jurkat T-cells.
9:00 COMPARISON OF GENE EXPRESSION IN MALIGNANT GASTRIC AND ENDOMETRIAL TUMORS
Sireesha Chinthaparthi Reddy (1*), Margot Kaelbling (2), Warren May (2), John Cleary (2), (1) Mississippi College, Clinton, MS 39058 and (2) University of Mississippi Medical Center, Jackson, MS 39216
We aimed to (1) identify genes that are differentially expressed in gastric adenocarcinoma, the second most common cause of cancer-related death worldwide, versus uninvolved tissue of the same patients and (2) assess if genes that are differentially expressed in gastric tumors were similarly expressed in endometrial tumors that were analyzed previously (MK). Total RNA was isolated from six tissue panels. RNAs from tumor and uninvolved tissue were separately reverse-transcribed and simultaneously hybridized onto a glass microarray spotted with 19,008 human ESTs. Detection was with Cy3[TM] (tumor) and Cy5[TM] (uninvolved tissue). Scanned arrays were analyzed with QuantArray[R] software. Statistical analysis involved correction of unequal dye intensity, bas[e.sub.2] transformation, LOESS normalization, SAM-type analysis, and volcano plots. Spots lacking gene information were eliminated. Seventeen genes were upregulated and 63 were downregulated in at least three of the six analyses. Two upregulated genes (CASP4 and ARL6IP) are at loci reported by other investigators to have gained chromosomal material in gastric tumors while six downregulated genes (SYNPO2, COX7C, SNAPC3, CDH5, DHX33, and CNN1) had chromosomal loss. Genes that were differentially regulated in both our gastric and endometrial tumor studies were CASP4 and ARL6IP (upregulated) and SYNPO2 (downregulated). Supported by NIH grant RR106476 from the MFGN INBRE Program of the National Center for Research Resources and the Cooperative Human Tissue Network.
9:15 PHARMACOPHORE MODEL FOR ANTIMALARIAL FARNESYLTRANSFERASE INHIBITION
Prasanna Sivaprakasam*, Aihua Xie, and Robert J. Doerksen, University of Mississippi, University, MS 38677
A very promising anticancer and antimalarial target is the heterodimeric zinc-containing protein farnesyltransferase (FT), which is one of the key enzymes in post-translational modification of proteins by prenylation, an important mechanism of cellular regulation. In order to help understand the balance of forces responsible for protein-ligand interaction, a useful approach is to prepare a pharmacophore model which summarizes the key interaction elements from amongst a group of diverse ligands. We selected representative antimalarial FT inhibitors including a potent benzophenone derivative and tetrahydroquinolines plus the known antimalarials chloroquine and artemisinin and prepared pharmacophore models using Catalyst software. One hydrogen bond acceptor and two hydrophobic features were found to be essential for in vitro growth inhibition of Dd2 strain of Plasmodium falciparum (Pf). We also developed a 3D pharmacophore model exclusively for benzophenones that are reported to be FT as well as Pfinhibitors. Several pharmacophore models either for FT inhibition or antimalarial activity exist in literature but to our knowledge this is the first report on a pharmacophore model for antimalarial FT inhibition. Our results showed four important pharmacophoric elements for antimalarial FT inhibitory activity of these benzophenones: one ring aromatic, one hydrophobic and two hydrogen bond accepting features. We will use these models for identifying novel lead antimalarial FT inhibitors using virtual screening.
9:45 Session II: Posters
THE EFFECTS OF H2O2 ON CELL VIABILITY, CELLULAR MORPHOLOGY AND CELLULAR MEMBRANE DAMAGE USING TYPE II PNEUMOCYTES
Alicia Martin (1*), Michelle Tucci (2), Hamed Benghuzzi (2), and Joseph A. Cameron (3), (1) Hinds Community College, Raymond, MS 39154, (2) University of Mississippi Medical Center, Jackson, MS 39216 and (3) Jackson State University, Jackson, MS 39217
Oxygen supplementation at supraphysiologic levels is necessary in patients with respiratory failure, especially in those with acute lung injury. Oxygen in these settings is a life-preserving supportive measure until the initial pathologic process that elicited the respiratory disease subsides. It has been suggested that the toxic effect of oxygen is mediated by increased reactive oxygen intermediates, such as superoxide anion ([O.sub.2.sup.-]), hydrogen peroxide (H2[O.sub.2]), and hydroxyl radical (O[H.sup.-]). In this study, administration of low, medium and high doses of hydrogen peroxide to mimic oxygen overload or hyperoxia was investigated. In this study, increasing concentrations of [H.sub.2][O.sub.2] predisposed cells to lipid peroxidation, cellular damage, and increased membrane phosphatidylserine a sign of cellular apoptosis. Injury to the respiratory cells resulted in distortion of the alveolar architecture such as morphologic changes characterized by cell flattening/stretching, hyperchromasia, and cellular death. Increasing concentrations resulted in reduced cell numbers with time as well as increased levels of MDA and annexin V staining. Low-level administration of [H.sub.2][O.sub.2] did not induce alteration in cell numbers or cellular damage. The data indicates that type II pneumocytes have cellular machinery capable of detoxifying [H.sub.2][O.sub.2] below 80 FM, and as the dose reaches 220 FM cellular toxicity results and cells undergo cell death by apoptosis as evidenced by an increase in Annexin V staining.
THE EFFECTS OF SUSTAINED DELIVERY OF IGF-1 ON KIDNEY AND TESTICULAR TISSUES USING ADULT MALE RATS AS A MODEL
Phatia Wells (1*), Michelle Tucci (2), and Hamed Benghuzzi (2), and Joseph A. Cameron (3), (1) Hinds Community College, Raymond, MS 39154, (2) University of Mississippi Medical Center, and (3) Jackson State University, Jackson, MS 39217
It's common knowledge that growth hormone (GH) and insulin-like growth factor-1 (IGF-1) are some of the agents used by bodybuilders, athletes. These agents are banned from being used by athletes in most international sport federations, including the International Olympic Committee (IOC). Since 1990, the use of these agents has increased tremendously because of their potential use to produce gains in lean body mass and strength with concurrent loses in fat mass. Supplement companies, sensing the public demand for GH and IGF-1, have released all kinds of supplements that allegedly elevate these anabolic hormones. IGF-1 has been shown in vitro to produce changes in kidney epithelial cells and it is known that GH and IGF-1 play major roles in the development of tissues such as pancreas, kidney and testes. This study evaluated the effects of sustained administration of 5ng/day IGF-1 for 4 weeks on body weights, organ weights and morphology of vital and reproductive organs. Body weights over the 4-week period did not differ from control or sham operated rats. Decreased testicular and kidney weights were observed in the IGF-1 treated rats when compared with control and sham operated rats. Epididymal and seminal vesicle weights were increased at 4 weeks in animals treated with IGF-1. Morphological evaluations revealed structural changes in the proximal tubules of the kidney with increased area and length when compared to control and sham operated animals. In addition to changes within the kidney, there was an increase in the area of the seminiferous tubules as well as marked reduction in sperm by four weeks. The data suggests that doses of 5ng/day IGF-1 does play a significant role in kidney function and can alter reproductive tissues when given over long periods of time.
PASSIVE IMMUNIZATION WITH PNEUMOLYSIN ANTISERUM AS TREATMENT FOR STREPTOCOCCUS PNEUMONIAE KERATITIS
Sherrina Robinson*, Sherrina N. Dixon, Melissa Sanders, Kathryn S. Monds, Armando R. Caballero, Larry S. McDaniel, and Mary E. Marquart, University of Mississippi Medical Center, Jackson, MS 39216
Streptococcus pneumoniae (pneumococcus) is one of the top three causes of bacterial keratitis. Though pneumococcus can be treated with antibiotics, alternatives are necessary due to the increase in antibiotic resistance. Pneumolysin is a pneumococcal cytotoxin that stimulates the host inflammatory response and forms pores in host cell membranes. Antibiotics are prescribed to treat pneumococcal infections, however, pneumolysin remains and continues to damage the eye. Immunization methods to prevent or treat pneumococcal ocular infections could be beneficial in providing protection against the bacteria. The aim of this study was to determine if passive immunization with antiserum to pneumolysin could protect rabbit corneas from the damage associated with pneumococcal keratitis. Two forms of pneumolysin were used for immunization, heat-inactivated pneumolysin and non-heat-inactivated pneumolysin with a single amino acid change that renders it non-cytolytic. High titer antiserum to each immunogen was produced in rabbits by three monthly subcutaneous injections of each immunogen. Control serum was produced by three monthly subcutaneous injections of PBS. The corneas of new rabbits were injected with [10.sup.5] colony-forming units of S. pneumoniae strain WU2, and then these rabbits were immediately injected intravenously with control serum, antiserum to each of the immunogens. Clinical examination of the corneas was performed at 24, 36, and 48 hours post-infection, and the rabbits were sacrificed. The clinical scores of the corneas from rabbits that received passive immunization with either antiserum to the heat-inactivated toxin or antiserum to the cytolytic-negative toxoid were significantly lower than the scores from rabbits that received control serum. The bacterial colony-forming units recovered from all of the corneas harvested after sacrifice were not significantly different between experimental groups and control groups. These data show that passive immunization with antiserum to pneumolysin is an effective means to treat S. pneumoniae keratitis. (Supported in part by the Howard Hughes Medical Institute)
INFLUENCE OF TERMINALIA CHEBULA ON PHARMACODYNAMIC AND PHARMOCOKINETICS OF GLICLAZIDE IN NORMAL AND DIABETIC RATS
Clareice Stewart (1*), S. Satyanarayana (2), J Rajasekhar (2), T Sailaja (2), Bettaiya Rajanna (1), and Sharada Rajanna (1), (1) Alcorn State, Lorman, MS 39096 and (2) Visakhapatnam, India
It is a fact that countries like India and China with large populations use drugs from oriental medicine along with allopathic drugs. We are also aware of the fact that optimal blood sugar control is needed in diabetes. Diabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycemia caused by inadequate secretion of the hormone insulin, an inadequate response of target cells to insulin, or a combination of these factors. Albino rats (Wistar strain) of either sex were divided into 3 groups of 6 each and fasted for 18h prior to the experiment. Water was provided to the animals ad libitum. During the experiment they were withdrawn from food and water. Animals in Group-I, II and III were treated with gliclazide with doses equal to pre-determined Therapeutic Dose (TD), 1/2TD & sub TD respectively. Following these treatments, animals in group I, II and III were treated with Terminalia Chebula (TC) at 30 mg/kg, 100mg/kg and 300 mg/kg respectively with a washout period of one week between the treatments. Then, animals in group II were selected and treated with the combination of TC 100 mg/kg + gliclazide 1/2 TD after a washout period of one week. Diabetes was induced by alloxan monohydrate 100 -150 mg / kg body weight i.p. A group of 6 diabetic rats were treated with TC 100mg/kg, gliclazide 1/2 TD and the combination with a washout period of one week between the treatments. Blood samples were collected from retro-orbital plexus at 0, 1, 2, 3, 4, 6, 8, 10 and 12 h. and were analyzed for blood glucose by GOD-POD method using Semi Auto analyzer (Screen Master 3000). Gliclazide levels were estimated by HPLC. The results suggested that Gliclazide & TC produced hypoglycemia dose dependently in rats. Bi-phasic Peak effects were observed in gliclazide and terminalia chebula. TC produced hypoglycemia when given alone and enhanced the effect of gliclazide in normal and diabetic rats. The pharmacokinetics of gliclazide was not altered in the presence of TC. The results of this study indicate that TC enhances the effects of gliclazide, which is of pharmacodynamic in nature and varies with species. Hence, we plan to conduct further studies in humans to establish the clinical significance of the interaction.
SYNTHESIS AND CHARACTERIZATION OF DIORGANOTIN(IV) COMPLEX OF 2-ACETYLPYRAZINE N(4) -3-THIOSEMICARBAZONE
Tramarea Adams (1*), Lugile Sitole (2), Ramaiyer Venkatraman (2), and Joseph A. Cameron (2), (1) Hinds Community College, Raymond, MS 39154 and (2) Jackson State University, Jackson, MS 39216
Thiosemicarbazones exhibit a broad spectrum of pharmacological properties, including antibacterial, antiviral, antifungal, antimalarial, and antineoplastic activities. In solution, thiosemicarbazone molecules can exist in thione--thiol tautomeric form. The unique property of thiosemicarbazone is not only the presence of many electron donors centers in the structure but also the bonding characteristics. As a ligand, thiosemicarbazones are well known to behave as chelating agents towards a wide range of metallic ions forming structurally different complexes. In many instances, thiosemicarbazones act as a bidentate ligand and bind to the metals through the sulfur and hydrazinic nitrogen atom. Further, metal complexes often display enhanced activities when compared with the parent compound. Thiosemicarbazones can easily be modified by variation of the parent aldehyde or ketone used for their synthesis, and reactivity studies of thiosemicarbazones and their metal complexes. Among the metal ions that complex with thiosemicarbazones organotin compounds are of current interest due to their industrial, agricultural and biological applications. Relatively few studies on tin compounds were reported in the literature. In this research, we synthesized 2-acetylpyrazine N(4)-ethyl-3-thiosemicarbzone, [Hapetsc], by using 2-acetylpyrazine and 4-ethyl-3-thiosemicarbazide in a 1:1 molar ratio in ethanol. The tin(IV) complex of the ligand, [Sn Ph2Cl2(Hapetsc)2], was synthesized by using diphenyltin(IV) dichloride and 2-acetylpyrazine N(4)-ethyl 3-thiosemicarbazone in a 1:2 mole ratio in ethanol. The synthesized compounds were characterized by X-ray diffraction, and IR spectroscopy in the solid state. Electrical conductance, UV/Vis and by multinuclear (1H, 13C) NMR spectroscopic studies were carried out in DMSO. The ligand revealed to be monomeric with orthorhombic crystal system and Pnama space group with Z configuration. The crystal structures of the tin complex revealed it to be a monomeric six-co-ordinate complex with monoclinic crystal system and P21/c space group. Each of the two apetsc molecules functions as a monodentate ligand, co-ordinating to the Sn atom through the pyrazine ring nitrogen atom and conferring a distorted-octahedral geometry upon tin.
SURVIVAL RESPONSES OF A549 AND MRC-5 CELLS TO D-GLUCOSE AND D-FRUCTOSE DIPHOSPHATE EXPOSURE
Ashley Stowers (1*), Ashley Blackwell (2), Clement Yedjou (2), Ibrahim O. Farah (2), and Joseph A. Cameron (2), (1) Hinds Community College, Raymond, MS 39154 and (2) Jackson State University, Jackson, MS 39216
Many studies showed that tumor growth and abnormal cell survival is associated with a number of metabolic abnormalities. Glucose metabolism is known to be deranged as frequently revealed by an impaired oral glucose tolerance test on patients. Protein metabolism was also found to be deranged in cancer patients as revealed by changes in plasma amino acid profile and evidenced by an increased plasma free tryptophan levels in cancer patients and depressed lipoprotein lipase activity, depressed, resulting in hypertriglyceridemia. The above findings seem to relate to or pose as a consequence of a shift to non-oxidative metabolic pathways. The role of carbohydrate metabolism in cancer was not clearly established in the literature. Therefore the purpose of this study was to evaluate differential metabolic toxicity/enhancement as well as survival responses of A549 (lung cancer) and MRC-5 (normal lung fibroblast) cells exposed to different concentrations for different times of D-glucose and D-Fructose diphosphate (FDP), in growth media. Tests were conducted using phase contrast micro scanning, a vital Mito-PT staining for survival/death indices as well as digitizing of cellular morphology assessments. Results showed that, both sugars have a range of different effects on cell survival. The effects seen on these cells could be attributed to either an increase in the pro-oxidant activity leading to increased ROS or the increase in internal pH (lactic acid) due to glycolytic metabolism that would differentially target cancer cells. Future studies will focus on the mechanisms of death due to the differential manipulation of non-oxidative metabolism and glucose transport mechanisms in cancer prevention /control.
AGE-RELATED DIFFERENCES IN DETOXICATION AND ACUTE TOXICITY LEVELS OF THREE ORGANOPHOSPHATE COMPOUNDS IN RATS
Edward Meek*, Valerie C. Beasley, Russell L. Carr, Howard W. Chambers, Jennifer Wagner, and Janice E. Chambers, Center for Environmental Health Sciences, Mississippi State University, Mississippi State, MS 39762
The organophosphorus (OP) insecticides which cause acetylcholinesterase (AChE) inhibition, display greater acute toxicity in juveniles than in adults. Active metabolites (oxons) of three of these insecticides, chlorpyrifos, methyl parathion, and parathion, were used to investigate esterase-mediated detoxication. The roles of carboxylesterases (CbxE) in stoichiometric detoxication and A-esterases (paraoxonases) in catalytic detoxication were investigated in liver and serum from adult (70 days of age) and juvenile (1 and 12 days of age) rats using spectrophotometric and HPLC methods. Detoxication ability of all 3 compounds by both esterases increased with age. Chlorpyrifos-oxon was detoxified to the greatest extent (both CbxE and A-esterases) followed by paraoxon (CbxE only), and methyl paraoxon the least (neither esterase). Rats of the same 3 ages were exposed IP to each of the oxons to determine the dosages required to inhibit brain AChE at 3 levels (80-90%, 40-50%, and 10-20%). The dosages producing the same level of AChE inhibition in adults and juveniles were most different for chlorpyrifos-oxon and least different for methyl paraoxon. These results support the concept that the greater detoxication of organophosphates in adults is an important factor in the greater tolerance of OP's by adults, and that the extent of esterase-mediated detoxication among compounds is critical in determining toxicity levels. (Supported by NIH R01 ES11287).
SEX DIFFERENCES IN ANGIOTENSIN-II INDUCED HYPERTENSION
Rachel Lockhart (1*), Julio Sartori-Valinotti (2), and Jane F. Reckelhoff (2), (1) Murrah High School,Jackson, MS 39202 and (2) University of Mississippi Medical Center, Jackson, MS, 39216
Angiotensin (Ang) II is capable of producing hypertension when given chronically. Sex hormones modulate the renin-angiotensin system (RAS). Whether there is a sexual dimorphism in the response to chronic Ang II when the endogenous RAS is blocked has not been determined. Objectives: 1) Determine whether there are sex differences in Ang-II induced hypertension in the presence of endogenous RAS blockade; and 2) Determine whether sex differences in the pressor responses to Ang II are exacerbated by high salt diet. Methods: Age matched (12 weeks old) male and female rats were assigned to receive either Ang II (150 ng/kg/min SC) or vehicle for 3 weeks. To block the endogenous RAS, enalapril was given in the drinking water. The rats were kept on regular diet (1% NaCl) during the first week of Ang II infusion, and thereafter, were challenged to 4% NaCl diet. Blood pressure (BP) was recorded by telemetry. Results: We found no sex differences in Ang-II induced hypertension when rats were fed a regular diet. On high sodium diet, males exhibited higher BP than did females. Conclusions: When the endogenous renin-angiotensin system is blocked and rats are kept on a normal salt diet, Ang II increases BP to a similar extent in males and females. In contrast, when placed on a high salt diet, males exhibit higher BP than females during the second week of high salt intake. We speculate that testosterone in males promotes proximal tubular reabsorption of sodium and water, leading to increased BP compared to females. (Supported in part by the Howard Hughes Medical Institute)
INFLUENCE OF GENDER, ETHNIC, AND SOCIOECONOMIC FACTORS ON PATIENTS WITH EPILEPSY
Manju Pande (1*), Veronica Scott (1*), Ruby Mason (1), Ravi Pande (2), and Parminder J.S. Vig (3), (1) Mississippi Valley State University, Itta Bena, MS 38941, (2) Neurology Clinic, Greenwood Leflore Hospital, Greenwood, MS 38930, and (3) University of Mississippi Medical Center, Jackson, MS 39216
Epilepsy a chronic condition of the nervous system, often leads to substantial disability and thus has a major socioeconomic impact. The present study was done to evaluate prevalence in different ethnic groups and gender susceptibility to the development of epilepsy in patient population in delta. The information was obtained form Greenwood neurology clinic. This clinic has a large patient population from all surrounding areas. The data was collected for 117 patients visiting over the last two years. First looking at the gender differences, there was a slightly higher occurrence in females (56%) than males (44%). However, there was no difference in the two ethnic groups whites (34) versus blacks (31). In contrast, a marked difference was observed in male ethnic groups (36bl versus 15wh), which resulted in overall difference in the two ethnic groups (58% in black compared to 42% in whites). Evaluation of the patients age showed a uniform distribution between the age groups of 18-45 and 45 and over, but interestingly the number of female patients increased in the age group of 45 and above. It will be interesting to find out if hormone or stress has any relationship with the higher incidence of seizures in females in the older age group. In order to evaluate the socioeconomic impact of epilepsy on the region, we found only 17% of the total patients had insurance while 83% of the patient population was mainly on medicare or medicaid. It is difficult to say if socioeconomic stress in these patients, was a contributing factor to the already existing epileptic seizures.
THE OZONE LAYER--EARTH'S NATURAL SUNSCREEN
Amin Haque, Alcorn State University, Lorman, MS 39096
The ozone layer is a concentration of ozone (O3) located in the stratosphere about 30 km above the surface of the Earth. It is considered the Earth's natural sunscreen because it absorbs most of the harmful ultraviolet (UV) radiation coming from the sun. O3 is produced due to the photochemical reaction of oxygen molecules (O2) in the stratosphere. Each particle of UV radiation, called a photon, has about 5.2 eV energy, which is enough to break O2 into two atoms of oxygen (O) or produce free hydroxyl radicals from water (H2O). Thus, an ozone layer is formed when a large number of O2 in the stratosphere absorb UV radiation from the sun and dissociate into two atoms of oxygen. The oxygen atoms combine with O2 to form O3. While O3 are being formed, some of them are absorbing UV radiation of higher energy, which causes them to be broken down to form O and O2. A dynamic equilibrium is established, maintaining a fairly constant concentration of ozone in the stratosphere. The concentration varies with season and latitude but averages 10 parts per million. Although it is low, this concentration of ozone is sufficient to block 95 % to 99 % of the sun's dangerous UV radiation. Until recently, ozone was created at least as quickly as it was destroyed. A substantial reduction in the amount of ozone in the ozone layer could threaten all life on Earth.
THE EFFECTS OF CORTISOL ADMINISTRATION ON PHOSPHOLIPID PROFILES, CELLULAR VIABILITY, AND CELLULAR MORPHOLOGY USING TYPE II PNEUMOCYTES
Destiny Foster (1*), Michelle Tucci (2), Hamed Benghuzzi (2), and Joseph A. Cameron (3), (1) Hinds Community College, Raymond, MS, 39154, (2) University of Mississippi Medical Center, Jackson, MS 39216, and (3) Jackson State University, Jackson, MS 39217
Neonates exhibit a high risk of developing acute and/or chronic lung disorder, often associated with surfactant deficiency. Alveolar type II cells synthesize dipalmitoyl phosphatidylcholine (DPC) as the main surfactant phospholipid. Cortisol stimulates the production of surfactant and is used to induce surfactant in premature infants. The objective of our study was to administer various concentrations of cortisol to A549 and MVLU1 mixture of cells and determine the level of cellular viability, cellular damage, cellular morphology, and degree of surfactant production at 24, 48 and 72 hours of incubation. The results of our study show increased cell numbers in the high dose treatment without increases in cell damage or alterations in cellular morphology. Analysis of the cellular lipids revealed an increase in the production of dipalmitoyl phosphatidylcholine in treated cells. The increase in DPC was in a time and dose dependent manner with the largest increase in the 72-hour treatment period. Overall, the results show that physiological and supraphysiological doses of cortisol induce surfactant production in type II pneumocytes without evidence of cell loss and destruction.
THE ROLE OF CAV 1.3 IN MECHANOTRANSDUCTION
Cheryl Bell (1*), Jiliang Li (2), David Burr (2), and Joseph A. Cameron (1), (1)Jackson State Univerisity, Jackson, MS 39216 and (2) Indiana University School of Medicine, Indianapolis, IN 46202
The L-type voltage sensitive calcium channels (L-VSCCs) have been shown to be involved in bone mechanotransduction in vivo. Our previous studies have shown that blockers of VSCCs suppress the load-induced bone formation in vivo, suggesting that L-VSCCs play an important role in bone response to mechanical loading. In the present study, we investigated the in vivo effect of the Cav 1.3 subunit on bone formation induced by mechanical loading. The right forearms of sixteen, twenty-week old mice were mechanically loaded for three consecutive days, while the left forearms served as nonloaded controls. The fluorochrome bone labels calcein (30 mg/kg) and alizarin (50 mg/kg) were injected two and six days, respectively, after the last loading session. The ulnas were processed for bone histomorphometry and calculations were done to measure mineralizing surface (MS/bone surface [BS]), mineral apposition rate (MAR), and bone formation rate (BFR/BS) of the periosteal surface for both the loaded and nonloaded ulnas. We report a positive bone response to mechanical loading in both WT and KO for the parameters MS/BS, MAR, and BFR/BS; however there were no significant differences, in reference to genotype, between the loaded and non-loaded ulnas. Similar results were found when comparing male versus female WT and KO. This study shows that loss of the Cav 1.3 subunit of the L-VSCCs in vivo does not suppress the mechanically induced response to bone formation that has been previously shown. Our data suggests that the L-type calcium channel does not affect the mechanically induced bone adaptation in vivo.
INDUCTION OF VESTIBULAR DAMAGE IN THE RATS VIA INTRA-TYMPANIC OR TRANS-BULLAR APPLICATIONS OF AMINOGLYCOSIDES
Jay Culpepper*, Rob Cannon, Jadrien Young,James Randall Jordan, and Hong Zhu, University of Mississippi Medical Center, Jackson, MS 39216
The loss of primary vestibular afferent signals by destruction of one labyrinth causes characteristic ocular, postural, and autonomic symptoms. However, a remission of a large part of these symptoms occurs within a few days. This phenomenon is called vestibular compensation. To study the neural mechanism of vestibular compensation, it is important to develop an effective protocol to induce unilateral peripheral vestibular damage in animals. Aminoglycoside antibiotics are toxic to the sensory hair cells of the inner ear. Some aminoglycosides such as gentamicin and streptomycin have selective toxicity for vestibular hair cells. In the present study, intra-typanic or trans-bullar applications of aminoglycosides were performed in rats to induce unilateral vestibular damage. An elevated body rotation test was used to assess the abnormal rotation behavior induced by unilateral vestibular damage. Of the seven rats that received single or repeated intra-tympanic injection of gentamicin (150mg/ml), only one rat showed abnormal rotational behavior. The second method involved a placement of frozen streptomycin pellets (2 mg) into the mastoid bulla. The two rats that underwent the trans-bullar approach displayed significant asymmetrical behavioral changes following recovery from the surgery, including head tilt, spontaneous turning behavior and high score of the elevated body rotation test. The severe spontaneous turning behavior disappeared 3 days after the surgery. The score of the elevated body rotation test improved 2 weeks after the surgery. These results suggest that the trans-bullar approach produce more rapid and reliable unilateral vestibular damage compared to the intra-tympanic injection of aminoglycosides in rats.
IMPACT OF ALF-1 AND IL-18 GENE POLYMORPHISMS ON REJECTION EPISODES AND CORONARY VASCULOPATHY AFTER CARDIAC TRANSPLANTATION
Yolanda Jackson (1*), Olga McDaniel (2), Charles Moore (2), Andy Barker (2), Tammy Thomas (2), Rae Calcote (2), and Michael Mitchell (2), (1) Tougaloo College, Tougaloo, Mississippi 39174 and (2) University of Mississippi Medical Center, Jackson, Mississippi 39216
Development of coronary vasculopathy (CV) plays a major role in determining the clinical condition of cardiac allograft. Inflammatory factor-1 (AIF-1) and interleukin-18 (IL-18) was previously shown to be over-expressed in CV patients with severe rejection episodes. The role of gene polymorphism in the promoter regions of Alf-1 and IL-18 in association with allograft rejection was investigated to explore their prognostic marker capability. Using SNP assay, DNA samples of 113 patients and 76 controls (African American and Caucasian) were tested. The various levels of CV (grade I-III) and rejection scores 0-3A/3B were compared. Fisher's exact test analysis was significant; p<0.05. The C [right arrow] ?T AIF-1 mutation, position -932, produced three genotypes; IL-18 mutations, position -607 and -137, produced six genotypes. Globally, frequency distributions of genotypes were not significant between patients and controls when clinical conditions were excluded for both IL-18 and AIF-1 genes. Majority of patients with rejection score >3A carry AIF-1 C allele (88.2%) as compared with T allele (11.8%); p<0.008. These data provide evidence that IL-18 with AIF-1 association might predict clinical outcomes of cardiac allograft and with direct correlation between expression levels of AIF-1 and IL-18 in association with the clinical outcome may prove a useful prognostic marker in management of cardiac allograft rejections.
INTERFERON-B ANTI-VIRAL THERAPY INDUCED TYPE II DIABETES
Victoria Beck (1*), Katherine S. Toomey (2), Andrew B. Strawbridge (2), Joseph A. Cameron (1), Ibrahim O. Farah (1), James P. Walsh (2), and Janice S. Blum (2), (1) Jackson State University, Jackson, MS 39217 and (2) Indiana University School of Medicine, Indianapolis, IN 46202
Studies have shown that extensive interferon treatment can trigger autoimmune disorders such as thyroiditis, systemic lupus erythematosus and diabetes. In all cases, patients exhibited no preexisting autoimmunity, suggesting that the induction of auto-reactive antibodies (Ab) was a result of the immunosuppressive therapy. In this study, we investigated the molecular events which may have lead to the development of insulin resistance in a 55 year-old African American male diagnosed with insulin-dependent type II diabetes after a 35 week treatment with polyethylene glycol (PEG) interferon/ribavirin therapy for hepatitis C virus. Patient serum and sera from healthy human male donors were tested for the presence of Abs reactive against human insulin receptor (hIR). Insulin receptor-specific Abs were monitored by immunoprecipitation and Western blotting lysates from Chinese Hamster Ovary (CHO) cells lacking or expressing the human receptor (the latter CHO/IR membrane fragments). While patient serum was not reactive with membrane fraction of CHO/IR cells on Western blots, the hIR from CHO/IR, the serum immunoprecipitated the hIR from CHO/IR membrane fragments such that IR was detected with commercial Abs insulin receptor-alpha and insulin receptor-beta subunits. We are currently, in the process of testing whether these Abs are capable of disrupting IR function in vitro and our results indicated that the patient has developed antibodies against his insulin receptors.
NEUTRALIZATION OF STREPTOCOCCUS PNEUMONIAE PNEUMOLYSIN BY ANTI-PNEUMOLYSIN IMMUNOGLOBULIN G
Melissa Sanders*, Sherrina N. Dixon, Larry S. McDaniel, and Mary E. Marquart, University of Mississippi Medical Center, Jackson, MS 39216
Pneumolysin is a cytolysin produced by Streptococcus pneumoniae that binds to cholesterol in host cell membranes and forms pores. These pores disrupt the cell membranes, leading to host cell lysis. In this study, antiserum to pneumolysin was produced in New Zealand white rabbits. This serum possessed a high anti-pneumolysin IgG titer of 819,200 as determined by ELISA. Control serum was also produced, generating a negligible anti-pneumolysin IgG titer of 1600. IgG was purified from both the antiserum and the control serum using Protein A-Sepharose, and was tested for its capacity to neutralize the ability of pneumolysin to lyse erythrocytes. IgG from the antiserum, but not from the control serum, was able to protect rabbit erythrocytes from lysis by pneumolysin. IgM from the antiserum was also tested, and did not produce high ELISA titers against pneumolysin or neutralize pneumolysin. These findings suggest that the host response to S. pneumoniae pneumolysin is predominated by IgG.
CHARACTERIZATION OF PEDIATRIC PNEUMOCOCCAL OTITIS MEDIA ISOLATES
Chinwendu Onwubiko*, Courtney Shires, Lisa R. Quin, Edwin Swiatlo, and Larry S. McDaniel, University of Mississippi Medical Center, Jackson MS 39216
Streptococcus pneumoniae is the main cause of otitis media infections in children. A vaccine based on capsule types is currently used in order to prevent this and other pneumococcal diseases. However, the efficacy of the current vaccine against otitis media was not as high as anticipated, so there is need to examine other possibilities. To further our understanding of pneumococcal otitis media, we examined 29 isolates obtained from children 5 years old or younger throughout Mississippi in the pre-vaccine era (1999-2000). The isolates were characterized based on pneumococcal surface protein A (PspA) family typing, capsular typing, antibiotic susceptibility, and DNA fingerprinting. We found that most of the strains studied were either vaccine- or vaccine-related serotypes and all belonged to either PspA family type 1 or 2. Over 65% of the strains were resistant to penicillin, with only 5 strains showing susceptibility to all of the antibiotics tested. Our study has shown that these strains have many genetic differences, except when it comes to PspA family type. These results indicate that development of a PspA-based vaccine could aid in the future prevention of otitis media.
IDENTIFICATION OF RRP2-CONTROLLED MAMMALIAN INFECTION ASSOCIATED PROTEINS IN BORRELIA BURGDORFERI
Kristen Hosey (1*), Joseph A, Cameron (1), and Frank Yang (2), (1) Jackson State University, Jackson, MS 39217 and (2) Indiana University School of Medicine, Indianapolis, IN 46202
Lyme disease is the most commonly reported arthropod-borne illness in the United States and Europe. The infection is caused by the tick-borne spirochete, Borrelia burgdorferi, resulting in a multisystem, multistage, inflammatory illness. Despite its medical importance, very little is known about the virulence determinants of B. burgdorferi. In this regard, we recently identified a genetic regulatory network that is critical for B. burgdorferi infection in mammalian hosts. This network constitutes a bacterial two-component response regulator, Rrp2, and a novel cascade of the alternate sigma factors, RpoN and RpoS. This regulatory network modulates expression of numerous B. burgdorferi genes and has emerged as a central regulatory pathway for B. burgdorferi pathogenesis. Through microarray and sequencing analyses, we have identified five prospective target genes in Borrelia burgdorferi, bb0681, bb0844, bba05, bba07, and bbb09, upon which Rrp2 controls their transpcription. In this study, we have chosen three of those genes, bb0844, bba05, and bba07, to generate recombinant proteins and, subsequently, the respective antibodies to determine their regulation at the protein level.
EFFECTS OF CHLORPYRIFOS AND METHYL PARATHION ON CHOLINESTERASE AND MUSCARINIC ACETYLCHOLINE RECEPTOR IN YOUNG RAT BRAINS
Shirley X. Guo-Ross*, Russell L. Carr, Edward Meek, and Janice E. Chambers, Mississippi State University, Mississippi State, MS 39762
Organophosphorus (OP) insecticides are widely used in both the US and worldwide. The neurotoxic effects of these compounds in developing animal are necessary to characterize in order to protect human health. In this study, rats were orally gavaged with either chlorpyrifos (CPS) or methyl parathion (MPS) starting at postnatal day (PND) 1 until PND4 or PND8 with low, medium, and high dosages of CPS or MPS. Cholinesterase (ChE) activity and muscarinic acetylcholine receptor (mAChR) binding were investigated in three brain regions (anterior to optic chiasm, posterior to optic chiasm excluding medulla/pons and cerebellum, and medulla/pons) at PND4 or PND8 following treatment with either CPS or MPS. Radioactive ligands were used to measure the maximal binding of the M1, M2/M4, M3-subtype and total mAChRs. Overall, in the anterior and posterior forebrain, the levels of all mAChRs nearly doubled from PND4 to PND8. However, in the medulla/pons, M1/M3-subtype densities were low and remained same, while M2/M4- and total mAChR levels increased a relatively small amount from PND4 to PND8. Dose-dependent effects of CPS and MPS on ChE activity and mAChR binding were more evident in rats at PND8 than at PND4. These results demonstrate that OPs exert adverse effects on rat cholinergic development in an age- and region-dependent manner. (Supported by NIH R01 ES 10386).
SURVIVAL RESPONSES OF A549 AND MRC-5 CELLS TO SODIUM ASCORBATE AND SODIUM BICARBONATE EXPOSURE
Bradley Adams (1*), Ashley Blackwell (2), Clement Yedjou (2), Ibrahim O. Farah (2), and Joseph A. Cameron (2), (1) Hinds Community College, Raymond, MS 39154 and (2) Jackson State University, Jackson, MS 39217
Tumor growth and abnormal cell survival was shown to be associated with a number of cellular metabolic abnormalities revealed by changes in plasma amino acid profiles in patients with breast, lung, colon, stomach, and other cancers from various origins. Glucose is the major energy source in cancer cells where it utilizes aerobic/anaerobic glycolysis with the resultant lactic acid formation. In contrast to normal cells, cancer cells may lose the ability to utilize aerobic respiration due to either defective mitochondria or hypoxia within the tumor microenvironments. The role of energetic modulations and use of glycolytic inhibitors on cancer / normal cell survival were not clearly established in the literature. Therefore, the purpose of this study was to evaluate two natural and potential glycolytic inhibitors namely, Sodium ascorbate and sodium bicarbonate on growing A549 and MRC-5 cell lines. The two cell types (cancer and normal) were exposed for various times to different concentrations of the chemicals in growth media. Exposed cells were tested with phase contrast micro scanning, a Mito-PT K it, and cell digitizing to determine their differential influence on cell survival. Sodium bicarbonate and sodium ascorbate both showed various levels of modulation within the two cell lines. These studies show the potential for exploiting cellular metabolic differences in cancer control. Supported in part by JSU-Center for University Scholars and NIH-RISE.
THE ROLE OF VISUAL CUES IN THE LINEAR ACCELERATION-INDUCED BLOOD PRESSURE CHANGES IN RATS
Bowen Zhou (1*) and Hong Zhu (2), (1) Jackson Academy, Jackson, MS 39236 and (2) University of Mississippi Medical Center, Jackson, MS 39216
Vestibular function is not only essential for posture and balance, but also is important for maintaining stable blood presseure. Otolith is the part of the vestibular system that senses linear motion and head orientation with respect to gravity and it has been suggested to play a primary role in the vestibular-cardiovascular reflex. Our previous studies show that pure linear acceleration, which selectively activates otolith system, induces characteristic cardiovascular responses in rats. Since linear head motions not only activate the vestibular system, but also activate the visual system by generating retinal slips, in the present study, we investigated whether visual cues play a role in the linear acceleration-induced blood pressure responses. Conscious Sprague-Dawley rats were stabilized on a linear sled through a surgically implanted head holder. Blood pressure was measured via a chronically implanted abdominal aortic catheter. The linear motion consisted of an acceleration phase of 200ms (3m/s2) followed by a deceleration phase of 200ms (3m/s2). The linear motion-induced blood pressure changes were monitored with visual cues present (light on) or visual cue absent (light off). Our data show that there was no significant difference in the linear motion-induced blood pressure change when visual information was available (light on condition) and when the visual cues were absent (light off condition). The results suggest that visual information may not play an important role in the linear acceleration-evoked autonomic responses.
CHRONIC BLOCKADE OF VEGFR-2 REVEALS NEGATIVE FEEDBACK REGULATION OF VEGF IN EXERCISING MUSCLES OF MICE
Janelle S. Pryor* and Thomas H. Adair, University of Mississippi Medical Center, Jackson, MS 39216
Exercise conditioning causes VEGF-mediated angiogenesis in skeletal muscle. Previous studies indicate that VEGF mRNA increases initially (days 1-7) in response to exercise-induced hypoxia, and then returns to nearly normal levels after 14-28 days when capillarity has increased and normoxic conditions have been achieved. This temporal relation between muscle capillarity and VEGF expression suggests that VEGF production may be subject to negative feedback regulation. To test this hypothesis, we used a VEGFR-2 inhibitor, PTK787 (Novartis), to prevent angiogenesis in skeletal muscle during exercise conditioning, i.e., to "open" the negative feedback loop. Male C57BL/6J mice were dosed with either PTK787 (50 mg/kg/day) or vehicle, and run on a motorized rodent treadmill for 1 hr/day (18 m/min, 10[degrees] incline); age-matched cage-confined mice dosed with PTK787 or vehicle served as additional control groups. mRNA expression was evaluated from gastrocnemius muscles collected at days 1, 4, 7 and 14. VEGF and VEGFR-1 expression were significantly higher in PTK787-exercise mice compared to cage-confined mice at all time points, whereas both factors returned to near normal levels in the vehicle-exercise mice by day 14. Because VEGF and VEGFR-1 are known to be upregulated by hypoxia, the results suggest that blocking the actions of VEGF prevents adaptation of skeletal muscles to exercise conditioning, and thus support our primary hypothesis that VEGF is subject to negative feedback regulation. NHLBI (HL-51971)
Session III: Nursing Education
11:30 GENERATIONAL AGE DIFFERENCES IMPACT THE COLLEGE CLASSROOM
Lisa Haynie, Tina Martin, Jill White, Anne A. Norwood, and Jean T. Walker, University of Mississippi School of Nursing, Jackson, MS 39216
A generational age transformation is occurring across the country in higher education classrooms. Generation Y is coming to higher education in record numbers and merging with significant numbers of Generation X. Boomers remain a significant force as faculty and the combination of the generations in the classroom is yielding interesting results. Educators need to prepare for the different values and expectations of students from Generation X and the newly emerging Generation Y in the educational environment. This quantitative, descriptive research begins to examine these differences and the potential impact for the college classroom of the future.
11:45 PREDICTORS OF SUCCESS AND ATTRITION RATES IN AN ASSOCIATE DEGREE NURSING PROGRAM IN MISSISSIPPI
Joyce Vaughn, Mary Tan*, Cindi Eads, Merilyn Long, Alice Austin, Becky Daniels, Mary McNair, and Pam McCollum, Holmes Community College, Ridgeland, MS 39157
Prediction of variables for academic success in associate degree nursing programs has intrigued nursing educators for decades. Compounding this issue is the urgency for producing more than one million registered nurses by the year 2010. The identification of these variables would enable nursing admissions committees to formulate relevant admission criteria, identify and develop programs for failing students as well as implement advisory and academic support programs to increase the probability of passing NCLEX-RN. This study examined both academic and non-academic variables. Study variables included ACT composite scores, ACT sub-math scores, ACT sub-English scores, ACT sub-reading scores, successful passage of NCLEX-RN scores, age of students, race, gender, prior nursing school failures, GPA and NUR 2119 and NUR 2123 test scores. Although many of these variables have been explored in past studies the majority of the studies have examined baccalaureate of science nursing (BSN) populations. Currently there is a lack of studies of associate degree nursing program populations. The purpose of this study was to examine previous study variables identified in the literature and determine if these variables have relevance or predictability of success or failure of associate degree nursing (ADN) students on NCLEX-RN passage. Data were obtained from a convenience sample of thirty-five, May 2005 graduates from an urban Mississippi public community college ADN program. Results from the study indicated that ACT composite and sub-scores scores had significant relationships with NCLEX-RN passage rates (p<0.05).
Bost Auditorium North
Session IV: Drug Delivery Symposium
1:15 EFFECTS OF SUSTAINED DELIVERY OF EPIGALLOCATECHIN--3--GALLATE, THYMOQUINONE, AND TANNIC ACID ON LNCAP CELLS
La'Toya Ross Richards*, Pamala Jones, Hamed Benghuzzi, and Michelle Tucci, University of Mississippi Medical Center, Jackson, MS 39216
Antioxidants are substances that play an essential role in protecting cells from damage caused by unstable free radicals, which are implicated in the development of cancer. A review of research on nutritional supplements and cancer risk has demonstrated that antioxidants have been reported to prevent prostate cancer development. High consumptions of antioxidants have been hypothesized to contribute to a reduced incidence of prostate cancer. The aim of this study was to utilize the drug delivery system to deliver antioxidants (epigallocatechin-3-gallate (EGCG), thymoquinone, and tannic acid) in a sustained manner and characterize the behavior and response of LNCaP prostate cancer cells after 24, 48 and 72 hours of treatments. Following treatment periods, cell number, cell damage and PSA levels were determined. Groups treated with EGCG + TCP had the largest reduction in cell number and the largest increase in membrane damage for the duration of the experiment. Cells treated with TA +TCP also showed significant damage to the cells membrane in comparison to the control group. Evaluation of prostate specific antigen (PSA) levels in the EGCG + TCP, TQ + TCP, and TA + TCP groups demonstrated significant decreases in the levels after 24, 48, and 72 hours of incubation (p < 0.001). The results of this study indicate that sustained delivery of antioxidant results indicate in addition to preventing cancer formation, they also have the ability to cause a substantial loss of cancer cells with altered functional abilities. These findings may enhance the healthcare industry and yield safer and effective treatments for prostate cancer.
1:45 THE EFFECTS OF SUSTAINED ADMINISTRATION OF GROWTH FACTORS ON TRAUMATIZED DISC USING THE ADULT MALE RAT AS A MODEL
Scott Wingerter*, James Woodall, Jr., Laura Franklin, Joel Davis, Michelle Tucci, Ashraf Ragab, and Hamed Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216
The current modalities of treating symptomatic degenerative disc disease are either conservative non-surgical or surgical modalities. However, none of these modalities is a true cure for the degenerative process. Ideally, the best treatment would be preventing the progression of degeneration. The goal of our research is to identify factors that may slow or stop the degenerative process. A rat degenerative disc model induced by piercing the center of the disc was implemented. In this phase of the study, it was hypothesized that continuous sustained release of transforming growth factor (TGF) would reverse the loss of cellularity associated with degenerating discs. A total of twelve rats were divided into three equal groups. Group I served as control and groups II and III were subjected to a surgery where a 21 -gauge needle was used to pierce the L4/L5 disc posteriorly. Animals in group III received TGF over a four week period via a tricalcium phosphate sustained delivery device. After 4 weeks the animals were sacrificed and the traumatized discs were removed. Sections of 10mM were taken and stained with hematoxylin and eosin and evaluated using light microscopy techniques. Using Image Pro software the area of the transition zone was calculated and the number of chondrocyte nuclei per area was determined. The results show that after four weeks, animals in group II (trauma only) showed evidence of disc degeneration with the largest decrease in cell number anterior to the site of trauma. Treatment with TGF resulted in chondrocyte numbers similar to control in posterolateral views of the disc, while lateral views and views of the site directly opposite the trauma (anterior) had approximately 45% less chondrocytes per area than the control; however, the chondrocyte numbers in the anterior views were twice as many as seen in the discs retrieved from trauma only animals.
2:15 COMPARATIVE EFFECTS OF CORTISOL ON KIDNEY TUBULE EPITHELIAL CELL USING CELL CULTURE AND ANIMAL STUDIES
Stacy Hull Vance*, Hamed Benghuzzi, and Michelle Tucci, University of Mississippi Medical Center, Jackson, MS 39216
Various studies have investigated the effects of kidney epithelial cells to various agents but there are few studies that compare an in vitro to an in vivo environment. The specific objectives of this study were to investigate the effects of cortisol (C) on the proliferation and viability kidney epithelial cells (KEC) and compare the finding with cortisol administered via drug delivery for 30 days using adult rats as a model. In phase I, a total of 30 tubes plated with KEC were divided into three equal groups. Cells were treated with either supraphysiological or physiological doses of cortisol and compared with untreated control cells. In phase II of the experiment, animals were divided into three equal groups and were given a TCPL drug delivery system containing supraphysiological levels of cortisol, SHAM or left untreated for 30 days. The animals were euthanized and their kidneys were compared using histological techniques. In Phase I: Epithelial damage was evident 24 after receiving a supraphysiological dose of cortisol, with increased hydrophic effects and alterations in cellular metabolism. In phase II, after four weeks of treatment, kidney tissues of animals exposed to sustained release of cortisol resulted in a significant reduction of glomerular area, with evidence of KEC damage. The overall histological and cytological data suggest that test compounds tested in tissue culture provide a correlation as to how they will perform in vivo. Testing in compounds in vitro will allow the investigator to establish the [IC.sub.50]'s of the test compounds as well as calculate the number of animals needed to reach statistical power.
Session V: Health Research
3:00 BLOOD PRESSURE IS NOT SALT SENSITIVE IN OBESE DOGS
Shardale McAfee and Jeff Henegar, Murrah High School, Jackson, MS, 39202 and University of Mississippi Medical Center, Jackson, MS, 39216
Blood pressure can change based on salt intake. This is referred to as salt sensitivity. Some rat models exhibit large increases in blood pressure when fed a high-salt (HS) diet. Obese Zucker rats have a significant increase in blood pressure when fed a high salt diet and thus are salt sensitive. However, it is unclear if the increase in blood pressure is due to obesity in these rats. Also, it is unclear if obesity salt sensitivity is specific for rats or is true for other species more similar to humans. Therefore, the present study tested the hypothesis that blood pressure in obese dogs is salt sensitive. The obese dog model mimics what happens in human obesity with regards to cardiovascular, endocrine, and metabolic parameters. To test our hypothesis, six obese dogs were given normal salt (NS, 85mEq/day), then low salt (LS, 7mEq/day), and then high salt (300mEq/day) for 7 days each, intravenously. Blood pressure and sodium excretion were measured. Neither LS diet nor HS levels significantly altered blood pressure in obese dogs (98 [+ or -] 4 LS and 104 [+ or -]5 HS versus 97 [+ or -] 4 NS, p=0.65 LS vs. NS, p=0.35 HS vs.NS). Sodium excretion was significantly less in LS (12.2 [+ or -]1.3 mEq/day) and significantly higher in HS (278 [+ or -] 6.8 mEq/day) compared to NS (86.2 [+ or -] 3.8 mEq/day). Thus, the kidneys were able to excrete enough sodium and fluid and prevent further increases in blood pressure. These data suggest that blood pressure is not salt sensitive in dogs. (Supported in part by the Howard Hughes Medical Institute)
3:15 TB DIAGNOSTIC USING EPITOPE PEPTIDE COUPLED WITH SPACER TECHNOLOGY IN AN ENHANCED ELISPAC DETECTON SYSTEM
A Rinaldy (1,3,4,*) K. N. Thimmaiah (1,2), H Simmons (1), L Sylvester (1), P Grisham (1), J Henry (3), K Gregorius (3), R Ilyas (4) and B Setiawan (4), (1) Northwest Mississippi Community College, Southaven, MS 38671, (2) St. Jude Children's Cancer Research Hospital, Memphis, TN 38105, (3) ImmunoCon Diagnostic, Cordova, TN 38016 and (4) Innogene Kalbiotech, Kalbe Farma, Singapore 139951
Rapid and sensitive diagnosis for infection by Mycobacterium tuberculosis is necessary in order to combat the spread of this ranking one infectious disease globally. To achieve this objective we focused on the membrane bound specific antigenic molecule isolated and purified from enriched membrane fractions of the virulent Erdman strain of Mycobacterium tuberculosis. Three epitope peptide domains with the highest hydophylicity were identified followed by its application in ELISA in combination with Tresyl-Activated-Dextran spacer molecule. The result is a minimal steric-hindrance configuration of these epitope peptides allowing the highly sensitive capturing of patient's specific IgG. Due to the nature of Mycobacterium tuberculosis as well as the mode of infection of this disease, the P3 prototype in this ELISPAC was assessed using 7 groups of serum samples. Specificity, Sensitivity, Positive and Negative Predictive Value were determined of the overlapping values primarily between group I ([TB.sup.+] [AFB.sup.+]) and group VI ([TB.sup.-]/healthy). Sensitivity and positive predictive value was 88% whereas specificity and negative predictive value was 68%. (Supported by Kalbe Farma Research Foundation, Singapore, to AR).
3:30 THE EFFECT OF ELLAGIC ACID ADMINISTERED INTRAPERITONEALLY AND ORALLY IN TRYPANOSOMA LEWISI INFECTED RATS
Joan Dickerson*, Emily Fortenberry*, Carey Klotz*, Marlena Mattingly*, Zack Owens*, and Aubrey Voorbrood*, Belhaven College, Jackson, MS 39202
Trypanosoma lewisi, a non-pathogenic form of the protozoan parasite that causes African sleeping sickness, was used in an experiment to examine the effects of ellagic acid on parasitemia. Recent trypanosome research has focused on the use of anti-carcinogenic drugs to reduce parasitemia due to their antiproliferative effects on cells. Ellagic acid, a phenolic compound used to treat malignancy and found in many fruits, was administered to rats both orally and intraperitoneally to examine its antiproliferative and antioxidant effects. Rats were separated into four treatment groups: injection low dose, injection high dose, dietary low dose, and dietary high dose. Adult trypanosome counts in the injection high dose group were found to be significantly lower than adult counts in the control. It was also observed that in treatment groups immature forms of the parasite remained at a fairly constant level, while adult forms appeared to decrease in number. This indicates that ellagic acid may inhibit the maturation of immature forms. However, it was not clear if the treatment had apoptotic effects on the parasite. Further studies are required to indicate the specific pathway through which ellagic acid prevents maturation. Ellagic acid could prove to be a treatment for trypanosomiasis that is cost-effective and readily available in underdeveloped countries.
3:45 OZONE DEPLETION AND ITS POTENTIAL EFFECTS
Amin Haque, Alcorn State University, Lorman, MS 39096
The chlorofluorocarbon (CFC) gases released in the atmosphere might seriously damage the ozone layer in the stratosphere. The major CFCs are CFC13 and CF2C12. The CFC molecules are broken apart by UV radiation, with the release of reactive chlorine radical atoms, which react with ozone molecules (O3) and convert O3 to O2 in a repeating cycle. It has been estimated that a single Cl radical may destroy as many as 100,000 ozone molecules. Other chemicals that damage the ozone layer include halons used in fire extinguishers, and chloroform used as a solvent in industrial processes. As methyl bromide and halons are broken apart, they release bromine atoms, which are 40 times more destructive to ozone molecules than chlorine atoms. Satellite images have revealed a "hole" in the ozone layer over the South Pole since 1985. The largest hole, 2.9 x 107 km2, was recorded on September 10, 2000. The second largest ozone hole area, 2.7 x 107 km2, was recorded on Sept. 11, 2003. Ozone levels over northern Europe, Russia, and Canada during the winter and spring of 1992 have been 12% below the seasonal average. The chlorine monoxide radical chain reaction is now thought to account for approximately 80 % of the ozone loss in the stratosphere. At present, relatively little is known or understood about the possible consequences of enhanced ultraviolet (UV) radiation levels in our environment. However, scientists anticipate and warn that depletion of ozone layer may have many significant effects on our health and the environment.
Bost Auditorium North
8:30 Health Fair
10:30 Poster Session
TISSUE TRANSGLUTAMINASE DEPENDENT AND INDEPENDENT INTERACTION OF CALCIUM BINDING PROTEIN CALBINDIN D28K WITH ATAXIN-1
La'Mont Sutton*, Parminder J.S. Vig, Jinrong Wei, Michael D. Hebert, and S.H. Subramony, University of Mississippi Medical Center, Jackson, Ms 39216
Spinocerebellar ataxia-1 (SCA1) is caused by the expansion of a polyglutamine repeat within the disease protein, ataxin-1. In normal neuronal tissues, ataxin-1 localizes to the nucleus in a diffuse fashion. However, in the affected neurons ataxin-1 precipitates as large intranuclear aggregates. These aggregates may protect neurons from mutant protein and/or trigger neuronal degeneration by encouraging recruitment of other essential proteins. Our previous studies have shown that calcium binding proteins, especially calbindin D28k (CaB) and parvalbumin have a role in SCA1 pathogenesis. In addition, we demonstrated that CaB is recruited to ataxin-1 aggregates in Purkinje cells of SCAI mice. Since our recent findings suggest that tissue transglutaminase 2 (TG2) may be involved in cross-linking and aggregation of ataxin-1, the present study was initiated to determine if TG2 has any role in CaB-ataxin-1 interaction. The guinea pig TG2 covalently cross-linked purified rat brain CaB. Time dependent progressive increase in aggregation produced large multimers, which stayed on top of the gel. To determine if CaB interacts with ataxin-1, we studied the effects of exogenous TG2 on HeLa cell lysates expressing GFP and GFP tagged ataxin-1 with normal and expanded polyglutamine repeats (Q2, Q30, and Q82) in the presence and absence of exogenously added CaB. The reaction products were analyzed by Western blots using anti-polyglutamine, CaB, or GFP antibodies. CaB crosslinked preferentially with Q82 ataxin-1. The present data indicate that CaB may be a TG2 substrate. Further, mutant ataxin-1 recruits CaB and TG2 stabilizes this complex by covalently cross-linking these interacting proteins, supporting the argument that ataxin-1 aggregates may be toxic to neurons. (Supported in part by the Howard Hughes Medical Institute)
METABOLIC EFFECTS OF FRUCTOSE 1,6-BISPHOSPHATE IN NORMOXIC AND HYPOXIC STATES OF MG63 OSTEOSARCOMA CELLS
Ameze Adah (1*), Hamed Benghuzzi (2), Michelle Tucci (2), Derrick Huang (1), Laura Franklin (2), and Felix Adah (2), (1) University of Mississippi, University, MS 38677 and (2) University of Mississippi Medical Center, Jackson MS 39216
Glycolysis is a very important process which contains very intricate steps that play a role in cellular performance and viability. Fructose 1,6-bisphosphate (FBP) is a glycolytic intermediate that has proven to improve cellular conditions under hypoxic and ischemic conditions. Osteoblasts are key regulators of skeletal matrix synthesis and degradation. Thus, considering FBP's positive effects on ameliorating hypoxia-induced injuries, the objective of this study was to determine its effects and comparative effects on osteoblast cells under normoxic and hypoxic states. MG63 osteoblast-like cells were cultured in 24-well culture plates and treated with high, medium and low dosages of FBP at 24, 48, and 72 hours. At the end of each time period, cellular number, damage by a malondialdehyde assay (MDA), and glutathione levels were evaluated. There was a significant increase in cell number for the low level of FBP in normoxia at 48 hours (p <0.05). For the cells in hypoxia, there was a significant decrease in cell number for the medium level at 48 hours (p <0.05). At 48 hours there was a significant decrease in cell damage through MDA measurement for the cells in normoxia and hypoxia when compared to the control. Cellular damage was not evident in the supernatant in either oxygen condition for the duration of the study. A significant decrease in glutathione levels was also noted for the cells in hypoxia. Cellular morphology included multiple nucleoli, vacuolated cytoplasm, abnormal cells, and web-like cytoplasm. The results indicate that FBP does protect bone cells exposed to hypoxic injuries, and while doing so, ameliorating the states of the cells in shock.
ANALYSIS OF MULTIPLE MEDWATCH AND AdDEERS FORMS OF CLINICAL RESEARCH TRIALS AND ADVERSE EVENT OUTCOMES OF STUDY DRUGS AND DETERMINATION OF RELATIONSHIP TO STUDY DRUG
Rachelle Cooley (1*), Andy Patel (1), Ednita Street (1), Maggie Clarkson (2), and Mitch Harrala (2), (1) Mississippi Gulf Coast Community College, Gautier, MS 39533, National Aeronautics and Space Administration, Stennis Space Center, MS 39522, (2) Gulf Coast Research Laboratory, Ocean Springs, MS 39566, and Singing River Hospital Cancer Research Center, Pascagoula, MS 39581
The purpose of this project was to analyze different MedWatch and AdDeers forms from clinical research trials and summarize them on an internal report form for the Singing River Hospital's (SRHS) Cancer Center Office of Clinical Research (OCR). The SRHS Cancer Center OCR is required to submit Adverse Event (AE) reports received from study sponsors to the local Internal Review Board (IRB) within a specified time frame in accordance with the Food and Drug Administration (FDA). Each study sponsor provides the OCR investigators with adverse events using the standard version of the MedWatch form. The AdDeers AE reports contain information on all grade three, four, and five toxicities experienced by patients receiving the sponsor's study drugs. The form requests specific information regarding the patient, study, drug regimen, incident/event and outcome. The investigator makes a determination of whether the event is related to the study drug or not. The study sponsor reviews the AdDeers form submitted by the investigator and also makes a determination as to relation of the event with study drug. The AE reporting process supports efforts by the FDA to notify individual investigators and the approving IRB's of expected as well as unexpected toxicities involved with use of the drug. The SRHS IRB's Standard Operating Procedures (SOP) require that all study sponsor generated AE reports be summarized on an internal report form. Once the report is reviewed by the local investigator for input and approval, the summary report is then submitted to the SRHS IRB for final review and approval. This notification to the local IRB insures patient safety on a local, national, and international level. The final disposition of all adverse events experienced by patients receiving a specific drug is included in the sponsor's application to the FDA for approval of the agent for commercial/retail use. The potential toxicities listed in drug inserts provided by the pharmaceutical company are a compilation of these adverse events.
COMPARISON OF TWO DIFFERENT FIXATION TECHNIQUES FOR A SEGMENTAL DEFECT IN A RAT FEMUR MODEL
Scott Wingerter*, Graham Calvert, Michelle Tucci, Audrey Tsao, George Russell, and Hamed Benghuzzi, University of Mississippi Medical Center, Jackson, MS, 39216
Studies have attempted to identify the osteogenic effects of bone morphogenetic proteins using a rat femur model, which commonly involves the creation of a critical size defect followed by internal fixation of the femur. Among the most familiar fixation methods are either plating or intramedullary placement of a Kirschner wire (K-wire). There are advantages and disadvantages to each method; however, this study attempts to identify the best method by exploring the histological effects of each technique. The experiment involved two groups with no added treatment: Group P (plate fixation method) and Group K (K-wire fixation method). The animals were allowed a four week interval for the femurs to heal, and proximal, distal, and two midshaft cuts were examined under high-power microscopy after the fixation apparatus was removed. Group K exhibited a peculiar fibrotic healing pattern that followed the shaft of the then vacated K-wire and there was minimal new viable bone formation. Group P, however, exhibited a more natural ingrowth of newly formed bone that began at the proximal and distal cuts and proceeded centrally into the core of the defect. Due to the fibrotic tissue in Group K, this study shows that the model is insufficient due to the micromotion created and, therefore, supports plating of critical defects as the fixation method of choice due to the creation of a stable healing environment.
EXPERIMENTAL RESEARCH FOR DEVELOPING A NOVEL BANDAGE ADHESIVE THAT DEGENERATES UNDER WHITE LIGHT
Marshall Y. Bartlett* and Paige Phillips, University of Southern Mississippi, Hattiesburg, MS 39406
The loss of stickiness and tackiness in polymers Poly(Styrene-Butadiene-Styrene) (SBS) and Poly(Styrene-Isoprene-Styrene) (SIS) containing varying amounts ofC6o was observed after exposing the adhesive to an intense white light for varying time increments. After diluting the polymer in toluene, C60 was added to the solution to maximize cross-linking under white light. The more cross-linking occurs in a solution, the more stickiness and tackiness it loses. The gel fraction method was used to determine what percentage of C60 caused the most cross-linking in the solution. A dynamic mechanical analysis instrument was also used to measure the T 9 of the solution. As little .20 percent weight of C6o in SBS caused significant cross-linking and loss of stickiness/tackiness. The longer the system was exposed to white light, the more it cross-linked, but significant cross-linkage did occur within five minutes. Adding C60 also caused a great (30- 40[degrees]F) rise in the T g. With these results, it was determined that a novel bandage adhesive can be developed using photo-responsive polymers and fullerenes that crosslink and produce a non-sticky, non-tacky system.
Bost Auditorium North
1:15 Poster Session
HISTOPATHOPHYSIOLOGICAL COMPARISON OF MALE AND FEMALE HEART TISSUE FOLLOWING SUSTAINED RELEASE OF SUPRAPHYSIOLOGICAL LEVELS OF CORTICOSTEROID
Kenneth Powell*, Hamed Benghuzzi, and Michelle Tucci, University of Mississippi Medical Center, Jackson, MS 39216
Chronic stress may result in abnormal laboratory findings including urine free cortisol, and elevated late-night salivary cortisol levels. There is also data that supports the concept that stress-related increases in cortisol may be associated with known physiological abnormalities such as hypertension, insulin resistance, dyslipidemia, and higher incidence of cardiovascular alterations. The onset of adverse cardiovascular events differs between males and females, and it generally reported that stress and hormone status are responsible for these differences. The aim of this study was to investigate the heart tissue of adult male and female animals following twenty-eight days of sustained supraphysiological corticosterone levels. Animals were divided into four equal groups containing four animals per group. Male animals in group I and female animals in group III served as control. Male animals in group II and female animals in group IV were surgically implanted with a TCP delivery device containing 50 mg corticosteroid. Twenty-eight days post surgery, the heart were collected and processed for histological evaluations. Histomorphometric measurements including total area, bundle lengths within the ventricles and the length of the apex were recorded. No differences in the wet weights were recorded in the male and female hearts when compared to the control. The length of the apex was increased following exposure to corticosterone in both male and female animals. The results also indicate an increase in the length of the bundles on the left side of the heart in the male animals exposed to corticosterone when compared to the male control heart tissues. No differences were seen in the female animals in the bundle lengths when compared with female control animals. The data overall suggest the possibility of structural differences in the heart that are gender related and suggest hyperatrophy.
IDENTIFICATION AND APPLICATION OF EFFECTIVE EARLY LITERACY PRACTICES FOR DEAF CHILDREN
Kimberly Barnes, University of Southern Mississippi, Hattiesburg, MS 39406
Literacy is the ability of an individual to write, speak, compute, and solve problems at levels of proficiency necessary to function in various roles. Historically the literacy levels of students who are deaf have been very low. Approximately 20 percent of deaf students graduate from high school at or below second grade reading levels. The written expression of these students is also very low when compared to their hearing peers. Promoting the literacy development of students who are deaf is a highly valued objective. The instructional practices, methods, and strategies should reflect the special needs of the hearing impaired. Thus this study identifies the best early literacy practices and assesses their implication with deaf children.
ARTERIAL STIFFNESS IN ADOLESCENTS EITH TYPE II DIABETES AT RISK FOR CARDIOVASCULAR DISEASE (CVD)
Ervin Coburn (1*), Bruce Alpert (2), Rachel Beecham (1), (1) Mississippi Valley State University, Itta Bena, MS 38941 and (2) University of Tennessee Health Science Center, Memphis, TN 38163
In western societies, atherosclerotic cardiovascular disease is the leading cause death in adult population. One complication of obesity that is associated with cardiovascular disease (CVD) includes Type II diabetes. Most children with type II diabetes are overweight or obese. In fact, studies prove that 85 percent of type II diabetics are considered overweight or obese. This study wanted to prove that type 2 diabetic adolescents' pulse wave velocity (PWV) was faster than that of healthy adolescents. The PWV appears to be the strongest predictor of cardiovascular mortality by measuring arterial stiffness. The method of the study was to recruit blacks, whites, and hispanics that were type 2 diabetics. We gave questionnaires to screen for personal health and family health history, recorded BMI, and measured PWV with Colin Medical Instrument VP-1000 Vascular Profiling System by placing BP cuffs on all four extremities, ECG on both wrist, and a phonocardiographic microphone over the precordium. The results were inconclusive and the goal that was set was not reached by the end of the study. Full statistical analysis are yet forthcoming and we are hoping that the hypothesis stand true. The study should reveal whether data collected are significant. The study is hope to also prove that non-invasively measurements of PWV is an excellent screening tool for both diabetes and CVD and that it will help design investigations on whether reducing BMI in obese patients slows PWV.
EXAMINING TRAUMA-INDUCED CLINICAL COMPLICATIONS IN THE CONTEXT OF SOCIAL AND HEALTH DISPARITIES
Brandon Newsome (12*), Xinchun Zhou (2), James Hamilton (2), Jake Olivier (2), and Olga McDaniel (2), (1) Murrah High School, Jackson, MS 39202 and (2) University of Mississippi Medical Center, Jackson, MS, 39216
Traumatic injuries often become a life altering and threatening event. The outcome however, depends on the strength of the individual's immune response to the injury. In posttraumatic injuries, the systemic inflammatory response syndrome (SIRS) often leads to clinical complications such as sepsis, multiple organ failure and death. An individual's genetic make up and social and health disparities are influential factors in the overall outcome. Our previous studies demonstrated the presence of multiple gene markers associated with sepsis at posttraumatic injuries. Here, we have tested the hypothesis that existing social and health disparities might have impact on post injury clinical outcome. Two hundred thirty African American (AFAM) and Caucasians (CAU) patients were studied. Health disparity data (HDD) including diabetes, hypertension, and social habits (smoking, drinking and drug abuse) as well as patient's education and the employment status were assessed. Overall, presence of sepsis and SIRS were inversely associated with age group in AFAM patients. There was a 2-fold increase in the presence of sepsis in AFAM patients >35 compared with <35. Frequency of sepsis was the same in both AFAM and CAU at age >35, but was increased 1.8-fold in CAU patients as compared with AFAM at age <35, p<0.006. Thus, factors beyond HDD influence posttraumatic clinical outcome.(Supported in part by the Howard Hughes Medical Institute and Project EXPORT-Jackson Heart Study
THE EFFECTS OF SYNTHETIC BONE GRAFTING MATERIALS ON THE VIABILITY AND MORPHOLOGY OF MG-63 OSTEOBLAST LIKE CELLS
Mark Barron*, James Woodall, Jr., Michelle Tucci, and Hamed Benghuzzi, University of Mississippi Medical Center, Jackson, MS 39216
Bone grafting has become a routine addition to fixation of many types of complex fracture patterns. Several synthetic bone grafting materials are currently used. The objective of this study was to evaluate the effect that these synthetic materials have on the body's capability to produce bone. The three synthetic grafting materials tested were tricalcium phosphate, zinc calcium phosphate, and pro-Osteon. The MG-63 cell line that was used is a human osteoblast like cell. The cells were tested for compatibility and resorbability. Measurements of calcium and alkaline phosphatase were taken at regular intervals over a 15 day period. Histological analysis was performed at the end of 15 days to evaluate cellular activity, osteocalcin level, and Collagen I. Our findings suggest that the cells show similar levels of osteogenic activity in all groups. However, the pro-Osteon treated cells resulted in bone cell formation only on the interior of the graft. The ceramic capsules resulted in formation of bone cell layers throughout the entire grafting material. All materials seem to be osteoconducive. However, the different patterns of bone formation within the materials will determine which is better suited as a bone grafting agent.
MONITORING FLEA CONTROL INSECTICIDE (CHLORPYRIFOS, TETRACHLORVINPHOS, AND PERMETHRIN) EXPOSURE LEVELS FROM THE FUR OF TREATED DOGS
Michael Davis*, Mississippi State University; Mark Russak, Mississippi State University, Mississippi State, MS 39762; John W. Tyler, Mississippi State University, Mississippi State, MS 39762; J. Scott Boone, Mississippi State University, Mississippi State, MS 39762; Matthew K. Ross, Mississippi State University, Mississippi State, MS 39762; Janice E. Chambers, Mississippi State University, Mississippi State, MS 39762
Pet owners have relied heavily upon both organophosphorus (OP) and pyrethroid insecticides for control of fleas and other pests of pets, thus leading to potential insecticide exposures in children. In the present study, pet dogs were treated with over-the-counter flea collars containing the OP insecticides chlorpyrifos (CP) or tetrachlorvinphos (TCVP), or a spot treatment with permethrin (PER), a pyrethroid insecticide. Transferable insecticide residues were quantified on cotton gloves used to pet the dogs, and on cotton t-shirts worn by a child. First morning urine samples were also obtained from adults and children for metabolite quantification. Transferable residues were highest near the neck of the dogs and were lowest in areas most distant from the neck for all of the insecticides. Three weeks after application, the average amounts of CP and TCVP transferred from fur of the neck (treated area) to a glove were 447 [+ or -] 57 and 15,788 [+ or -] 2194 [micro]g/glove, respectively, and the average amounts of CP and TCVP transferred from the fur of the back to a glove were 8 [+ or -] 16 and 82 [+ or -] 32 [micro]g/glove, respectively. The average amount of PER transferred to a glove from the fur of the neck (treated area) 4 hours post application was 24,521 [+ or -] 7,382 [micro]g/glove while the average amount transferred at 20 days post application was 509 [+ or -] 274 [micro]g/glove. T-shirts worn by the child on the day following application showed levels in ng/g shirt of 134 [+ or -] 41, 1692 [+ or -] 657, and 8297 [+ or -] 3421 for CP, TCVP, and PER, respectively. There were no significant differences between adults and children in the levels of urinary metabolites of CP; however, children typically had somewhat higher urinary levels of metabolites than adults. (Supported by EPA R828017)
SEASONAL VARIATION IN ANTI-CCV SERUM ACTIVITY IN CHANNEL CATFISH: IMPLICATION FOR BROOD-STOCK SCREENING
Yonas Habte (1*), and N. Chatakondi (2), Larry A. Hanson (1), (1) Mississippi State University, Mississippi 39762 and (2) Eagle Aquaculture, Pike Road, Alabama 36064
Channel catfish virus (CCV) is the etiological agent of channel catfish disease (CCVD), a fatal hemorrhagic viremia of Ictalurus punctatus fry. Using diagnostic real time PCR, we found up to 90% prevalence of latent CCV infection in sac-fry produced from carrier broodstocks; indicating high level of vertical transmission. Control of CCVD would therefore necessitate culling of carrier broodstocks. Serum neutralization index (SNI) assay and PCR have been suggested to be useful methods for broodstock screening. However, the effectiveness of these techniques have not been adequately evaluated. Screening of broodstocks by PCR and SNI assay in May 2004 and subsequent rearing of the fry lead to an outbreak of CCVD in 11.1% of CCV negative tanks. Re-evaluation of the broodstocks in October by SNI assay, and comparison to the May data demonstrated significantly higher titers. The results clearly indicate a better detection of CCV specific antibodies in October. In May, 4.2% of the broodstocks had SNI within the "CCV positive" range; compared to 40% in October. The same trend was observed in 2005. This suggests that virus expression is occurring in the summer in adult fish and implies that screening of broodstocks by CCV specific serology should be conducted in the fall.
BUTORPHANOL-INDUCED NEURONAL ACTIVATION WITHIN THE PARAVENTRICULAR NUCLEUS (PVN) OF THE HYPOTHALAMUS AND RESULTING ACTIVATION OF THE HYPOTHALAMIC-PITUITARY-ADRENAL (HPA) AXIS
George Howell (1*), Melissa Parker (1), Ryan Veach (1), and Deandria Magee (2), Rob Rockhold (1), (1) University of Mississippi Medical Center, Jackson, MS 39216 and (2) Tougaloo College, Tougaloo, MS 39174
The present study seeks to determine the in vivo dose-response relationship between intravenous butorphanol administration and neuronal activation within the PVN and activation of the HPA axis. In addition to dose-response relationships, the role of the kappa opioid receptor (KOR) subtype in butorphanol-induced neuronal activity within the PVN was explored. To determine the dose-effect relationship between butorphanol and PVN neuronal activity, butorphanol (0.1, 1.0, or 10.0 mg/kg) was administered and neuronal activity assessed by c-Fos immunoreactivity. Administration of butorphanol resulted in significant, dose-related increases in the number of c-Fos immunoreactive cells, percentage of c-Fos immunopositive cell area, and plasma corticosterone levels compared to vehicle, c-Fos expression within the PVN was positively correlated with increases in plasma corticosterone. The role of the KOR in butorphanol-induced PVN neuronal activation was explored by intracerebroventricular administration of nor-binaltorphimine (nor-BNI), prior to butorphanol (10 mg/kg) administration. Nor-BNI (20 [micro]g) pretreatment did not reduce either butorphanol-induced c-Fos expression within the PVN or plasma corticosterone increases. However, nor-BNI (35 [micro]g) pretreatment significantly reduced butorphanol-induced c-Fos expression within the PVN. Our results indicate acute administration of butorphanol elicits dose-related increases in neuronal activity within the PVN which is associated with activation of the HPA axis. Butorphanol-induced neuronal activity within the PVN is mediated by the KOR in the central nervous system.
SELECTIVE HUMAN MELANIN-CONCENTRATING HORMONE RECEPTOR 1 AND MELANIN-CONCENTRATING HORMONE RECEPTOR 2 ANTAGONISTS FOR THE TREATMENT OF METABOLIC DISEASES
Joshua Swan*, Alexander Quesenberry*, Mitchell Avery, and Ziaeddin Shariat-Madar, University of Mississippi, University, MS 38677
Melanin-concentrating hormone (MCH) is an appetite regulating neuropeptide highly expressed in the lateral hypothalamic area and the zona incerta. MCH regulates energy balance and feeding behavior as well as the modulation of cardiovascular and metabolic processes. These physiological functions are mediated by melanin-concentrating hormone receptor 1 (MCHR1) and melanin-concentrating hormone receptor 2 (MCHR2) in the G protein-coupled receptor family. Our goal here was to screen our in-house synthesized compounds to discover potent and selective inhibitors of human MCHR1 and MCHR2 receptors. Chinese hamster ovary (CHO) cells transfected with MCHR1 or MCHR2 receptor will be incubated with [125.sub.I]-MCH or biotinylated MCH in the absence or presence of synthesized compounds to determine and evaluate for MCHR1 and MCHR2 antagonist properties. The binding of [125.sub.I]-MCH to CHO cells stably expressing MCHR1 or MCHR2 will be determined. The pharmacological and physiological effects of MCH at MCHR1 and MCHR2 will be assessed by intracellular signaling and radioligand binding assays. In sum, we anticipate that this information may lead to the development of improved medications for cardiovascular and metabolic disorders in which these receptors have been compromised.
DEVELOPMENT OF A MODEL FOR ALCOHOLIC LIVER DISEASE
Veronica Levison (1) Rodney C. Baker (2) Keila Brown (3), Stanley V. Smith (2), Christine A. Purser (2), Tina G. Smith (2), Stanley V. Smith (2), and Michelle Tucci (2), (1) Alcorn State University, Alcorn State, Mississippi, 39096, (2) The University of Mississippi Medical Center, Jackson, MS 39216, and (3) Tougaloo College, Tougaloo, MS 39174
Introduction: Development of alcoholic liver disease in animal models has proved to be difficult, primarily because most experimental animals do not readily drink alcohol (ethanol). Purpose: Establish conditions under which zebrafish could be treated with ethanol continuously, and find methods suitable for histological examination of zebrafish liver morphology. Procedure: Test fish were placed in a 50 mM ethanol solution, controls were placed in a similar environment 22[degrees] C or 26[degrees] C with exception of ethanol in the aquarium water. Water samples were taken daily for ethanol measurements. The behavior of the fish was monitored and water samples taken for ethanol determination. Fish were sacrificed at various time points and used to develop histology methods. Results: Both treated and control fish held in water at 22[degrees]C stopped eating. The swimming and schooling behavior of zebrafish was not significantly altered by one week of treatment at 22[degrees]C. The fish held at 26[degrees]C exhibited normal feeding and swimming behavior. The control and treatment groups were not significantly different for approximately the first 30 days of treatment. After 45 days of treatment the fish started swimming in a more random manner and did not display normal schooling behavior. Conclusion: This study demonstrated that zebrafish can be treated with 50 mM ethanol for up to 2 months without extensive morbidity. After extended treatment ethanol treated fish consumed food slower than controls, and the normal schooling behavior is disrupted. Methods normally used to prepare histological specimens must be modified to obtain good quality zebrafish histology specimens.
INTRAOPERATIVE EVALUTION OF FEMORAL HEAD VASCULARITY DURING OPEN REDUCTION AND INTERNAL FIXATION OF ACETABULAR FRACTURES
James Woodall, Jr. (1), Barnaby Dedmond (2), Scott Wingerter (1), Robert Reddix (2), Ben Jackson (2), Steven Flores (2), Brenda Kulp (2), and Lawrence X. Webb (2), (1) University of Mississippi Medical Center, Jackson, MS 39216 and (2) Wake Forest University Baptist Medical Center, Winston-Salem, NC 27157
This study was designed to investigate the link between the amount of bleeding observed intra-operatively when the femoral head was drilled and the subsequent progression to avascular necrosis. This technique could be used diagnostically to establish a protocol for patients that are at increased risk of progression to avascular necrosis. This would give the surgeon an opportunity to initiate a more vigilant post-operative follow up plan with serial MRI and physical exams, or even an early intervention. Diagnosis of avascular necrosis at an early stage before collapse of the femoral head would give the surgeon a chance to perform core decompression or free fibular graft at that time. A retrospective review of all patients undergoing internal fixation of acetabular fracture from August 1996 to April 2005 was performed. The surgeon used a 3.5 mm Kirschner wire to drill a hole off of the weight bearing axis of the femoral head of each patient. The results were dictated in the operative note (did not bleed, bled poorly, bled freely). All patients who had greater than twelve months of follow up were included, resulting in seventy-one patients with an average follow up of 32.5 months. The results of this review showed that 2 of the 9 patients (22.2%) that "did not bleed" progressed to avascular necrosis. Three of the thirteen patients (23%) that "bled poorly" progressed to avascular necrosis, and 5 of the 49 patients (10%) that bled freely progressed to avascular necrosis. These results indicate that patients with femoral heads that "did not bleed" or "bled poorly" were more than twice as likely to progress to avascular necrosis as patients that "bled freely". These preliminary findings are impressive, and warrant a prospective randomized control trial to determine is intervention is beneficial at the time of primary surgery.
COMPARISON OF WATER SOLUBLE AND LIPID SOLUBLE ANTIOXIDANTS AS SCAVENGERS IN A HYPOXANTHINE-XANTHINE-OXIDASE AND LIPID RADICAL GENERATING SYSTEMS
Omonuwa Adah (*1), Michelle Tucci (2), and Hamed Benghuzzi (2), (1) Mississippi College, Clinton, MS 39058 and (2) The University of Mississippi Medical Center, Jackson, MS 39216
The aim of the study was to investigate the reactive oxygen species (ROS) production in the hypoxanthine-xanthine-oxidase (HX-XO) and lipid free radical (LOO-) systems by using various concentrations of ROS scavengers, such as vitamin E, vitamin C, green tea, thymoquinone, and Tannic Acid to determine the concentrations of radical scavengers that are effective against intracellular (HX-OX) and cellular membrane radical (LOO-) damage. Various doses of antioxidants were evaluated at 1, 4 and 24 hours two different radical generating systems. In the lipid radical generating system we hypothesized that vitamin E and thymoquinone would protect against radical formation in a dose dependent manner and for a longer period of time. In our assay system, vitamin E and thymoquinone were more effective in squelching radicals at 1 and 4 hours, but were not able to sustain the protection at 24 hours. The water soluble compounds, vitamin C, green tea, and tannic acid were more effective in squelching radical formation after 24 hours when compared with control and lipid soluble compounds. The HX-XO pathway has been implicated as an important route in the oxidative injury to tissues, and is a source of oxygen free radicals. In our HX-XO assay system we found that green tea was most efficient at squelching radicals followed by thymoquinone and vitamin E in both a dose and time dependent manner. Tannic acid was not effective in eliminating radical formation after 4 hours and vitamic C was only effective at the initial time point. This information is important for the development of antioxidant regimes for use in the clinical setting.
HISTOLOGICAL AND RADIOGRAPHIC COMPARISON OF ALLOGRAFT SUBSTITUTES USING A CONTINUOUS DELIVERY MODEL IN SEGMENTAL DEFECTS
Tyler Marks *, Scott Wingerter, James Woodall, Jr., Laura Franklin, Michelle Tucci, George Russell, Rameesh Patel, and Hamed Benghuzzi, University of Mississippi Medical Center, Jackson, MS, 39216
Demineralized bone matrix (DBM) has been shown to possess osteoinductive capability and one of the specific bone morphogenetic proteins (BMPs) found within DBM that has been attributed with this osteoinductive ability is osteogenic protein-1 (OP-1). The specific aims of this study were (1) to compare the treatment of segmental bone defects with OP-1 and DBM in a rat femur model and (2) to determine the effects of the two treatments given at high and low doses via sustained release drug delivery. Animals in Group 1 acted as the control and Group 2 had a created segmental defect with plating and placement of a calcined tricalcium phosphate lysine (TCPL) capsule containing antibiotic (sham). Group 3 and 4 animals had a created segmental defect and received a TCPL carrier containing antibiotic along with DBM or OP-1, respectively. After 2 and 4 weeks post-implantation, 5 animals in each group were sacrificed before the retrieval of the bone. The femora were analyzed radiographically and histologically for bone growth. Analysis of the gross specimens showed considerable bone regeneration at low and high doses for both DBM and OP-1 when compared to the shams. At low levels bone regeneration between DBM and OP-1 was very similar. However, at high doses, OP-1 was shown to cause bone overgrowth with a greater curvature and an increased thickness of the distal and proximal ends of the femur. The stained slides showed the defects treated with DBM and OP-1 to be bridged with lamellar and woven bone that was continuous with the original bone. Histologically, the experimental femurs demonstrated natural remodeling processes with new osteons and angiogenesis.
COMPARISON BETWEEN THE EFFECTS OF MEVINOLIN, ALENDRONATE, AND PARATHYROID HORMONE ON MG63 OSTEOBLAST-LIKE CELLS
Laura Franklin*, Joyce Belcher, Hamed Benghuzzi, and Michelle Tucci, University of Mississippi Medical Center, Jackson, MS 39216
Increasing osteoblast activity in an anabolic fashion may offer an ideal therapeutic treatment for various orthopedic complications including osteoporosis. The purpose of this study was to evaluate the effect of mevinolin, a clinical statin drug, and alendronate, a bisphosphonate, on osteoblast function (MG63 Cell Line) and compare its mode of action with the conventionally utilized parathyroid hormone (PTH). MG63 cells were treated with different concentrations (control, low (100nM), medium (1uM), and high (10uM)) of mevinolin, alendronate, or parathyroid hormone. The cells were incubated for 24, 48, and 72 hours at 37[degrees]C in a 95% air and 5% CO2 environmental chamber. Data obtained in this study revealed that: (I) there were significant decreases in cell number after 24 hours upon the exposure of medium and high doses of mevinolin, but cells rebounded back toward control after 48 hours and were similar in number at 72 hours, (II) there were increases in cell number upon the exposure of medium doses of alendronate for 24 and 48 hours, but a decrease in cell number occur during 72 hours for low, medium, and high dose levels of alendronate, (III) there were no significant changes in calcium activity observed for mevinolin and alendronate. However alkaline phosphatase activity for alendronate showed lower levels compared to mevinolin and the greatest increase in activity occurred during 24 and 48 hours with the low and medium doses. Mevinolin showed no significant changes in alkaline phosphatase activity throughout the study. The concentrations (100nM and 10uM) of mevinolin and alendronate used did not trigger the differentiation process of the cells throughout the experimental phases. This observation led us to suggest that the reason for such an outcome could be attributed to the lack of a response in calcium production or alkaline phosphatase activity.
ANALYSIS OF PESTICIDE EXPOSURE AND RISK OF BREAST CANCER MORTALITY IN MISSISSIPPI
Elgenaid Hamadain (1*), Ibrahim O. Farah (2), and Mohamed H. Abdalla (2), (1) University of Mississippi Medical Center, Jackson, MS 39216 and (2) Jackson State University, Jackson, MS 39217
Breast Cancer is the most common form of cancer among women in the United States. Established risk factors include advancing age, early menarche, late menopause, positive first relative, late age at first birth and socioeconomic status. According to the American Cancer Society, breast cancer case estimates were at 2,480 with 460 deaths for the year 2004 in Mississippi State. Mississippi State has a combination of risk factors making it suitable for studying the pathways of breast cancer etiology. The purpose of this study was to analyze the role of pesticide exposure as a risk factor for breast cancer mortality in Mississippi women. Data for this study consisted of secondary analyses of the Mississippi age-adjusted breast cancer mortality aggregated by two periods (1970-1994 & 1996-1999) and total number of acres of planted crops (as a proxy for pesticide exposure) for 1997-2001. Descriptive statistics, Spearman and Pearson correlations and geospatial analysis (GIS) by State Economic Area (SEA) were used for the analysis. Significant correlation between pesticide exposure and breast cancer mortality was detected in several SEAs. The total number of acres planted was positively associated with female cancer mortality and this association differed by race. Significant linear associations were found between level of pesticide exposure (acres planted) and breast cancer mortality rate in Mississippi women per SEA for both periods of study (1970-1994 and 1996-1999). We conclude that there were moderate statistically significant correlations between number of acres planted, type of crops and mortality rate of breast cancer in Mississippi women. Our findings may well relate to the pesticide bioaccumulation hypothesis
ASSOCIATION OF MRI-DEFINED SUBCLINICAL CEREBROVASCULAR DISEASE AND TRANSIENT ISCHEMIC ATTACK-STROKE SYMPTOMS: THE ARIC STUDY
Kenneth Butler*, Thomas H. Mosley, Jr., and Alan D. Penman, University of Mississippi Medical Center, Jackson, MS 39216
The purpose of this study was to examine the association between prevalent subclinical cerebral abnormalities identified by MRI and self-reported stroke and transient ischemic attack (TIA) symptoms in a large population-based cohort. During the third ARIC clinic visit, 1,934 of 2,821 cohort members aged 55 years and older at the Forsyth County, NC and Jackson, MS field centers were eligible for cerebral MRI examination, in addition to regular clinic examinations. MR images were evaluated for the presence and location of infarcts > 3mm in size and white matter hyperintensities (WMHs), and ventricular and sulcal size. TIA/stroke symptoms were assessed using a standardized questionnaire, and an algorithm was used to classify TIA/stroke symptoms as positive or negative. After excluding those with missing data or prevalent stroke, data were available for analysis on 1,278 persons. Infarct-like lesions were found in 145 persons (11%), including 111 with subcortical (lacunar) infarcts and 34 with cortical infarcts. WMHs were found in 150 (12%) study participants. High ventricular size was found in 211 (17%) participants, and high sulcal size was observed in 374 (29%). A history of TIA/stroke symptoms was reported by 112 (9%) persons. The adjusted prevalence odds ratio (OR) of having TIA/stroke symptoms was 1.9 (95%CI, 1.2-3.2) for persons with any infarct, increasing to 4.7 (95%CI, 1.3-16.4) in those with 3 or more infarcts. The adjusted prevalence ORs for persons with WMHs, high ventricular size, and high sulcal size were 1.7 (95%CI, 1.0-2.9), 1.3 (95%CI, 0.8-2.2), and 0.9 (95%CI, 0.6-1.5), respectively. Persons with both infarct and WMHs had an adjusted prevalence OR of 3.0 (95%CI, 1.3-6.5) for having TIA/stroke symptoms. In this population based middle-aged and young elderly cohort free of prevalent stroke at baseline, subclinical infarcts and WMHs detected by MRI are not always asymptomatic. These common changes in brain morphology are not necessarily benign.
THE EFFECTS OF SUSTAINED DELIVERY OF CORTICOSTEROIDS ON THE ADRENAL GLAND OF FEMALE RATS
Tamika Taylor (1*), Vanessa Price (1), Steven Farris (2), Hamed Benghuzzi (1), and Michelle Tucci (1), (1) University of Mississippi Medical Center, Jackson, MS 39216 and (2) Belhaven College, Jackson, MS 39216
The role of stress hormones on the structural and functional capacity of multiple organs has not been well defined. The objective of this investigation was to morphometrically evaluate the effect of sustained delivery of corticosteroids for periods of 28 days to adult female rats. Twelve animals were divided equally into three groups. Animals in Group I were implanted with TCP delivery capsules containing supraphysiological levels of corticosterone (50 mg). Animals in group II were implanted with an empty TCP capsule and served as operated and drug delivery device control (SHAM). Animals in group III were not implanted with TCP devices and served as control. Surgical techniques were performed following standard lab protocols. Body weights were collected weekly, and at the end of 28 days the adrenal glands were harvested, fixed, embedded, sectioned and stained with hematoxylin and eosin. The 5mm sections were subjected to qualitative and quantitative evaluations using Image Pro Digital Analysis System. Data revealed no differences in body weights or vital and reproductive organ wet weights after 28 days of treatment when compared to sham and control animals. Histopathological evaluation of the tissue suggests atrophy in the zona glomerulosa in animals treated with corticosterone. The area most affected is responsible for the secretion of mineralocorticoids. The data suggests the interaction of corticosteroid with mineralcorticoid receptors with the adrenal gland as well as suggest adrenal suppression by cortisol.
DOES YOUR IMMUNE SYSTEM HAVE STIPES? INVESTIGATING THE SIMILARITIES AND DIFFERENCES BETWEEN THE IMMUNE SYSTEM OF MAN AND ZEBRAFISH
Claudia Hohn* and Lora Petrie-Hanson, Mississippi State University, Mississippi State, MS 39762
Zebrafish share many orthologous genes with mouse and man. Studies have identified several regions of the zebrafish and human genome that encode the same (or similar) genes. Definitive blood cell lineages in zebrafish show a high degree of morphological similarity to mammalian counterparts. Gene expression studies and functional studies have indicated the general mechanisms of hematopoietic development and effector cell functions are likewise conserved. The similarities of these features and developments in the cell biology and functional genomics of zebrafish are making this model increasingly more attractive for immunobiological investigations. But the differences may also further our understanding of immunology. Zebrafish possess at least one unusually large family of putative immune genes, novel immune-type receptor genes (NITRs), for which a corresponding mammalian ortholog is not evident. Although the precise functions of NITRs are not yet known, it is likely that their membrane disposition and signaling are equivalent to other activating/inhibitory leukocyte regulatory receptors, like the mammalian natural killer (NK) cell receptors. NITRs could be evolutionary precursors of NK cell receptors, enhancing our understanding of mammalian innate immunity. Specific pathogen free (SPF) zebrafish are propagated and reared in the Mississippi State University College of Veterinary Medicine's fish hatchery. Mutant zebrafish lines are utilized to study innate immunity and the pathogenesis of channel catfish diseases. Wild-type zebrafish are available to researchers upon request.
THE EFFECTS OF FRUCTOSE-1, 6-BISPHOSPHATE ON CELLULAR ATP LEVEL AT REDUCED OXYGEN LEVELS
Derrick Huang (*1), Ameze Adah (1), Michelle Tucci (2), and Hamed Benghuzzi (2), (1) University of Mississippi, University, MS 38677 and (2) University of Mississippi Medical Center, Jackson, MS 39216
Background and significance: Fructose-1, 6-bisphos-phate (FDP) is a glycolytic intermediate that has been used to protect tissues in various hypoxic and ischemic conditions. Under ischemic conditions where ATP levels are much lower than normal, bypassing the initial steps of glycolysis offers protection by allowing the cell to produce ATP without expending energy. Hypothesis: Exogenously added fructose-1, 6-bisphosphate to MRC-5 fibroblast cells under ischemic conditions will maintain cellular ATP levels and ultimately allow cells to proliferate in a similar manner to untreated cells maintained at ambient air. Objectives: (1) To provide increasing concentrations of fructose-1, 6-bisphosphate for 24, 48, and 72 hours under ischemic and ambient conditions and compare growth characteristics of cells under the similar conditions, and (2) To evaluate the cellular ATP levels at each time period following ischemia. Results: Under ambient conditions, FDP increased cell umber in a dose dependent fashion at 24 hours. Cell number was similar at 48 and72 hours. Cellular glutathione levels were decreased in all treatment groups as early as 24 hours, and MDA was increased in the medium and high dose treatment group for the duration of the study. Under ischemic conditions, FDP reduced cell numbers by 50% in the medium and high dose group at 24 hours. Cell numbers were not different at 48 and 72 hours of treatment. Cellular glutathione levels were not significantly different from control. Cellular MDA levels were increased in the medium and high dose levels. Cellular ATP levels were maintained in the fructose 1,6-bisphoshate treated cells in a dose dependent manner. Conclusions: Overall FDP was able to protect cellular glutathione levels and ATP levels under ischemic conditions. Increasing concentrations of FDP regardless of oxygen concentration resulted in increased evidence of cellular damage.
THE EFFECT OF DAKIN'S SOLUTION ON COLLAGEN AND CELL MIGRATION
Roshada Bozeman (1), Ryan Claire Propst (2), and Annette B. Wysocki (2), (1) Tougaloo College, Tougaloo, MS 39174 and 2University of Mississippi Medical Center, Jackson, MS 39216.
Open chronic skin wounds are colonized with bacteria that can lead to subsequent infection. The use of Dakin's solution, a topical antiseptic, on chronic open skin wounds remains controversial in clinical care because of its reported damaging effects. However, the effect of Dakin's solution on collagen and cell migration remains open to further investigation. Here we tested four different formulations of Dakin's solution, 0.5% (full strength), 0.25%(half-strength), 0.125% (quarter strength) and 0.0125% (diluted strength), to determine if it degrades collagen or impairs cell migration that may lead to delayed wound healing. To do this we first added varying amounts of Dakin's solution to acid solubilized type I collagen, with and without serum, at two different time points that were then mixed. Subsequently we used 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) to determine the extent of degradation using colorimetric detection with Coomassie stain to visualize the collagen. Our results indicate that the 0.0125% Dakin's solution resulted in little or no collagen degradation compared to higher concentrations, where collagen was either completely or partially degraded. Likewise, cell migration was completely inhibited using the 0.5% Dakin's solution compared to the 0.0125% solution where cells were still able to migrate. Furthermore, we noted that serum had a protective effect for both collagen degradation and cell migration and that time exposure was a factor in our collagen degradation experiments. Thus, we conclude that there is a dilution, serum, and time effect that can be used to attenuate the effect of Dakin's solution on collagen degradation and cell migration. (Supported by a fellowship from the Mississippi Functional Genomics Network).
ANALYSIS OF THE 16S-23S INTERGENIC SPACER REGION OF FLAVOBACTERIUM COLUMNARE
Lorelei Ford (1*), Brian Scheffler (2), Esteban Soto (1), Mark Lawrence (1), and Larry A. Hanson (1), (1) Mississippi State University, Mississippi State, MS 39762 and (2) United States Department of Agriculture, Stoneville, MS 38776
The 16S, 23S, and 5S ribosomal RNA (rRNA) genes are highly conserved sequences in bacteria. For this reason, rRNA genes are often used for phylogenetic classification. On the other hand, the regions between the structural sequences, known as intergenic spacer regions (ITS) are under less evolutionary pressure to be conserved. Because they are not as highly conserved, they can be used to differentiate strains of the same bacterial species. The purpose of this study was to evaluate the 16S-23S ITS of Flavobacterium columnare, an important pathogen of catfish, by comparing of the sequences from 70 isolates. We developed two PCR assays that amplify overlapping regions of one large previously identified ITS. The primers targeted the 16s sequence and isoleucine tRNA and the 23s sequence and alanine tRNA. The PCR products were cloned and sequenced. We also targeted pulse field gel electophertically-separated, I-CeuI restriction fragments from the ATCC type strain. We found that the genome of this species harbors at least 2 intergenic spacer regions that are very similar and contain the same tRNA encoding sequences. This suggests that earlier studies that used the ITS for distinguishing between strains of Flavobacterium columnare may be comparing sequences from different structural RNA operons and thus have misleading data.
THE EFFECTS OF DEFEROXAMINE ON THE SURVIVAL OF FREE FLAPS USING THE ADULT MALE RAT AS A MODEL
Nicholas Longstreet (1*), Sheila Lindley (2), Hamed Benghuzzi (2), Michelle Tucci (2), Feng Zhang (2), Steven Longstreet (2), and Michael Angel (2), (1) College of William and Mary, Williamsburg, VA 23187 and (2) University of Mississippi Medical Center, Jackson, MS 39216
The ability to transfer tissue based on microvascular anastomoses has created a revolution in reconstructive surgery. Patients whose injuries and tissue defects had been considered unsalvageable can now be rescued by the appropriate free-tissue transfer. However, secondary ischemia is a serious problem for the survival of a free flap after transfer. In order to improve the flaps tolerance to secondary ischemia, deferoxamine, a powerful iron chelator and free radical scavenger was administered following three hours of secondary ischemia. Sprague Dawley rat were used as a model for the study. Epigastric island flap was isolated and raised in the right groin area. Primary ischemia was induced for one hour. Twenty four hours later, a secondary ischemia by venous occlusion was performed for three hours. The flaps were clinically evaluated at five days and tissues were collected after fourteen days, fixed, embedded, sectioned, and stained using rigorous staining methods to qualify the health of the flap. The flaps were stained with H & E to characterize the cells present within the tissue. Additional immunostains methods using antibodies against T-cells (to indicate immune response and rejection of transplant tissue), presence of fibrosis, and inflammatory cells were also evaluated. The findings showed administration of deferoxamine showed a trend in the both the reduction of CD 40 which indicates fibrosis and CD 28 which is indicates lack of flap rejection compared with the saline control. The levels of inflammatory cells were not different between the two groups. Overall, the findings of this study were intriguing and suggest that additional experiments on length of secondary ischemia need to be performed to further characterize the importance of reactive oxygen species on flap survival.
THE EFFECTS OF MULTIPLE RADIATION EXPOSURE TO MRC-5 FIBROBLAST CELLS IN CULTURE
Pamala Jones (2*), LaToya Richards (1), Hamed Benghuzzi (1), and Michelle Tucci (1), University of Mississippi Medical Center, Jackson, MS 39216 and (2) University of Southern Mississippi, Hattiesburg, MS 39406
The primary mechanism of biological damage to macromolecules from ionizing radiation is an indirect interaction that begins with the radiolysis of water. The event is a cascade of chemical transformations that result in the formation of free radicals. Free radicals are highly reactive particles that can indirectly harm DNA and cause cellular damage. The aim of this study was to assess cell proliferation and structural integrity of the fibroblast by evaluating the MRC-5 cells morphologically after multiple exposures to ionizing radiation. Following multiple exposures (2, 3, or 4 times) with a single dose of X-radiation (10Gy), the cells were harvested at 24, 48 and 72 hours. An interesting finding occurred with 3(X) and 4(X) exposure to a dose of 10 Gy. With 3(X) exposure, the cell number decreased after 24,48 and 72 hours. However, after 4(X) exposure, the cell number increased after all durations. Data analysis revealed that there was a statistically significant difference in the mean values between the treatment groups and the control (p=<0.001) for all durations. The cells exposed to 2(X) the radiation dose, demonstrated swelling, pleomorphism, and the nucleoli which were not as prominent as the groups exposed 3(X) and 4(X) to a dose of 10Gy. After 72 hours the group receiving 2(X) radiation dose revealed a characteristic owl eye nucleoli, usually seen after radiation exposure. After 3(X) and 4(X) the cells showed hydropic swelling and pleomorphism with multiple nucleoli present; however, after 72 hours, the cells receiving 4(X) the radiation dose of 10 Gy showed more cells that were spindle shaped with significantly less damage than 24 and 48 hours. These findings showed significant effects caused by free radicals generated from X-radiation.
IMMUNIZATION WITH PSPA INCORPORATED INTO A POLY(ETHYLENE OXIDE) MATRIX ELICITS PROTECTIVE IMMUNITY AGAINST STREPTOCOCCUS PNEUMONIAE
Quincy Moore (1*), Lashundra Johnson (1), Michael Repka (2), and Larry S. McDaniel (2), (1) University of Mississippi Medical Center, Jackson, MS 39216 and (2) University of Mississippi, University, MS 38677
Vaccine delivery is an important issue especially if the delivery system can augment the immune response and does not require an injection. Novel vaccine delivery systems will likely lead to broader acceptance of vaccines. We used our pneumococcal immunization/ challenge model to examine the ability of a poly(ethylene oxide) matrix incorporated with PspA to deliver the antigen and modulate a protective immune response. The matrix containing PspA was implanted subcutaneously in CBA/N mice for direct comparison to subcutaneous injection of the antigen. Mice were boosted with a second implant at two weeks. One week later, sera were collected from the mice, and the mice were challenged with a lethal dose of Streptococcus pneumoniae. All of the mice that receive PspA in the matrix survived pneumococcal challenge and had serum-specific anti-PspA antibody levels statistically higher than mice receiving PspA alone (p<0.005). We also demonstrated in western blot analysis that the immune serum was reactive to PspA of several pneumococcal strains. As a proof of concept, our data indicate that PspA can be incorporated into the matrix and the matrix has the potential to be a novel vaccine delivery system.
2:15 Divisional Business Meeting
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|Publication:||Journal of the Mississippi Academy of Sciences|
|Date:||Jan 1, 2007|
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