Browsing by Subject "Medical Biochemistry"
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Item Gracy, Robert, PhD.(1994-01-26) Gracy, Robert; Hailey, BlakeDr. Gracy, Associate Dean of Basic Sciences and Research, recounts the process of bringing TCOM and North Texas together. Interviewed by Blake Hailey, January 26, 1994Item Leucine-Enkephalin and Sympathetic Control of Heart Rate(2001-12-01) Stanfill, Amber; Caffrey, James L.; Downey, H. Fred; Shi, XiangrongStanfill, Amber A., Leucine-enkephalin and Sympathetic Control of Heart Rate. Master of Science (Biomedical Sciences), December, 2001, 51 pp., 1 table, 4 figures, references, 48 titles. The following study examined the role of leucine-enkephalin in the sympathetic regulation of the cardiac pacemaker. Leucine-enkephalin (0.3 mM) was administered, by microdialysis into the interstitium of the sinoatrial node in 10 mongrel dogs in conjunction with either sympathetic nerve stimulation or infused norepinephrine. In study one, the right cardiac sympathetic nerves were isolated as they exit the stellate ganglion and stimulated to produce graded (low, 20-30; high 40-50 bpm) increases in heart rate. Once stimulation frequencies were determined, leucine-enkephalin (0.3mM) was added to the dialysis inflow and perfused at 5: 1/min thereafter. The sympathetic stimulations were repeated after 5 and 20 min exposure to leucine-enkephalin. The resulting increases in heart rate during sympathetic stimulation were attenuated at both low (18.2 ±1.3 to 11.4 ±1.4 bpm) and high (45 ±1.5 to 22.8 ±1.5 bpm) frequency stimulation. The degree of inhibition was nearly identical after 20 minutes exposure providing no evidence for a progressively evolving response and for desensitization. Vagal function was also evaluated at 5 and 20 min by stimulating the right cervical vagus at 1 and 3 Hz. Leucine-enkephalin reduced the vagal bradycardia approximately 50% at both time intervals. The administration of the delta-selective opioid antagonist, naltrindole, restored only one third of the sympathetically medicated tachycardia. The same dose of naltrindole completely reversed the coincident vagolytic of leucine-enkephalin. These observations suggested that the sympatholytic effect was either non-opioid or mediated by a different opioid receptor subtype. Study two was conducted to determine if the sympatholytic effect was prejunctional and post-junctional in character. Norepinephrine was added to the dialysis inflow into the SA node in a concentration (6-9 μM) sufficient to produce an intermediate increase in heart rate. The average increase in heart rate was 35.2 ±1.8 bpm. Leucine-enkephalin was then combined with norepinephrine and sympathetic and parasympathetic responses were recorded at 5-min intervals for 20 minutes. The tachycardia mediated by added norepinephrine was unaltered by leucine-enkephalin or the subsequent addition of naltrindole. At the same time intervals, vagal control of heart rate was reduced by more than 50% and then completely restored by naltrindole. When combined with observations in study one, the data support the conclusion that the local nodal sympatholytic effect of leucine-enkephalin was the result of a reduction in the effective interstitial concentration of norepinephrine and not the result of a post-junctional interaction between leucine-enkephalin and norepinephrine.Item Setting Us Up to Fail: Pulmonary Dendritic Cells Promote Immunopathology during Mycoplasma Respiratory Disease(2010-08-01) Dobbs, Nicole A.; Jerry SimeckaThe purpose of these studies was to define the contributions of T helper 2 cells and dendritic cells toward the development of immunopathology during mycoplasma respiratory disease. IFN-γ+ CD8+ T cells, IFN-γ+ Th1 cells and IL-13+ Th2 cells developed over the course of mycoplasma infection. By day 14 post-infection, the results demonstrated a significant and preferential increase of an IL-13+ Th2 cell sub-population in the LRNs. Additional studies using STAT4-/- animals, which have a Th2 polarized environment, demonstrated no difference in disease compared to the wild-type animals. Absence of STAT6, which strongly contributes to a Th1 polarized environment, conveyed significantly more protection from mycoplasma disease in immunized mice compared to STAT4-/- and WT mice. By day 14 post-infection, all mice had significantly more IL-13+ Th2 cells than IFN-γ+ Th1 in the LRN compared to STAT6-/- immunized mice, thus suggesting that the reduction in the IL-13+ Th2 population leads to protection, while an increase in Th2 is pathogenic. Additional studies demonstrated that pulmonary dendritic cells support the mycoplasma-specific CD4+ and CD8+ T cell activation when stimulated with mycoplasma antigen. Knowing that T cells and DCs have an intimate relationship during mycoplasma disease, sub-classes of cytokine differentiated BMDCs were created to attempt to skew to the protective arm of immunity against mycoplasma disease. However, in vivo adoptive transfer studies demonstrated antigen pulsed DCs accelerated and exacerbated the pathological effects of mycoplasma disease. The exacerbation was antigen-specific and lymphocyte dependent. Mice that received antigen pulsed DCs demonstrated a significant increase in IL-13+ Th2 cell sub-population in the LRNs with a similar trend found in the lungs prior to infection. The same exacerbation was seen when antigen pulsed pulmonary DCs were adoptively transferred into mice, but not with antigen pulsed splenic DCs. Prior to infection, mice that received antigen-pulsed pulmonary DC, not splenic DC, had a significant increase in a IL-13+ Th2 population in the LRNs. Taken collectively, these studies demonstrate two key players in the development of the detrimental response against mycoplasma disease. This knowledge will assist in the development of targeted vaccines that will promote protection over pathology.Item Studies of Protein F1 (GAP-43) Expression and Function in Spinal Neuronal Cultures(1994-08-01) El-Badawy, Hassan M.E. Azzazy; Ming-Chi Wu; Guenter W. Gross; Scott NortonEl-Badawy, Hassan M. E. Azzazy, Studies of Protein F1 (GAP-43) Expression and Function in Spinal Neuronal Cultures. Doctor of Philosophy (Biochemistry and Molecular Biology), August 1994, 167 pp., 32 illustrations, References, 194 titles. Protein F1 (GAP-43, B-50, neuromodulin) is a membrane-bound phosphoprotein that has been studied mainly in neurons and is implicated in synaptic plasticity, axonal growth and regeneration, and neurotransmitter release. In this study, a 21 amino acid polypeptide that corresponds to the C-terminus sequence of protein F1 and contains a potential PKC phosphorylation sequence (SXR) was synthesized. The synthetic peptide was phosphorylated by rat PKC in a concentration-dependent manner suggesting that this site in the intact protein may be phosphorylated by PKC in vivo. Polyclonal antibodies against the peptide were produced in a rabbit and used to: (i) recognize native non-phosphorylated protein F1 purified from rat brain, (ii) immunoprecipitate phosphorylated protein F1, and (iii) stain the cell bodies and neuritis of cultured neurons. Electron microscopic studies revealed intracellular protein F1 immunoreactivity but no specific subcellular association of the gold label could be demonstrated. The antibodies were also used to compare protein F1 levels during the development of spinal neurons in culture and in vivo. The highest levels of protein F1 were detected by ELISA, at 2 days in culture. These results are in accordance with previous reports that correlate high expression of protein F1 to neurite outgrowth. In vivo, however, protein F1 reached maximal level at one day after parturition. Two approaches were utilized to investigate the potential physiological functions of protein F1 in spinal neurons networks. First, interaction of positively charged, rhodamine-labeled liposomes with spinal neurons was characterized by fluorescence microscopy and electrophysiological recording. Uniform, non-toxic, and preferential interaction of liposomes with spinal neurons over glia was established. These liposomes were used to deliver anti-protein F1 antibodies into spinal neurons but did not affect neurite formation by these cells. Second, antisense oligodeoxynucleotides internalized into spinal neurons in order to interfere with protein F1 expression had no effect on the development of these cells in culture. Data from this study suggest that Ser-210 at the C-terminus of protein F1 may be a substrate for PKC phosphorylation in vivo. Antibodies raised against F1 peptide revealed protein F1 immunoreactivity that outlined cell bodies and neuritis of cultured spinal neurons. Positively charged liposomes were characterized as a potential delivery system for macromolecules into spinal neurons. Protein F1 levels were shown to be developmentally regulated in mouse spinal neurons in culture and in vivo. Finally, the use of antisense oligodeoxynucleotides against protein F1 mRNA revealed that protein F1 may not be essential for neurite outgrowth of mouse spinal neurons in culture.Item Studies on solvent induced fluorescence properties of styryl dye, LDS 798, to develop in vitro and in situ assay techniques(2011-05-01) Sarkar, Pabak; Gryczynski, ZygmuntThe styryl group of dyes has been used in cellular studies for over 50 years because of their solvatochromic and/ or electrochromic properties. Here we report characterization of solubility and solvatochromic properties of a near infra-red amiophenylstyrylquinolinum dye, styryl 11 or LDS 798. We have extended our studies to small unilamellar vesicles, lipid based nanoparticles and live cells. Our cellular studies show that LDS 798 preferentially localizes in mitochondria and its fluorescence lifetime changes with change in mitochondrial membrane potential. We have used this change in lifetime as an early marker for mitochondria-dependent apoptosis. We also found that solvatochromic properties of this dye, used in tandem with fluorescence correlation spectroscopy, can be used to efficiently determine the diffusion coefficient and hence the size of the submicron lipid based particles. This dye has the potential to provide essential information about liposomal structures and mitochondrial potential changes in vitro and in situ respectively.Item Synergy 2011: Annual Research Report(2011-01-01)Item Targeted Nanoparticles for the Treatment of Neuroblastoma(2011-05-01) Pratap, Suraj; Lacko, Andras G.Neuroblastoma (NB) is one of the most frequently diagnosed tumors in infants and children. However, the mechanism by which it is initiated and subsequently develops on the molecular and cellular level is yet to be fully elucidated. Its wide spectrum of clinical presentation has baffled physicians and biomedical scientists alike. The variant called high risk neuroblastoma (HRNB) is extremely resistant to the currently available drug regimes. Despite the recent advances in anti-cancer agents and the use of multi-modality therapy for the treatment of HRNB the morbidity and mortality in this group of patients continues to remain high. The purpose of our project was to find novel alternative therapeutic approaches by encapsulating known anti-NB agents in a lipoprotein based formulation to achieve selected, targeted delivery of these drugs to HRNB tumors. We wanted to enhance the therapeutic efficacy of these drugs that have shown encouraging results in pre-clinical trials but have so far exhibited an adverse pharmacokinetic profile precluding their systemic application. Our laboratory has been working for the last several years on a novel drug delivery platform by encapsulating drugs into the core of high density lipoprotein (HDL) type nano-particles. Using this strategy, we encapsulated all-trans retinoic acid (ATRA), fenretinide (FR) and valrubicin into reconstituted HDL (rHDL) nanoparticles and subsequently evaluated some of their physical and chemical properties and their anti-NB potential. Further, we tested the efficiency of an apolipoprotein mimetic peptide called 5-A peptide as a component of rHDL particles and compared its efficiency with apolipoprotein A-1 (Apo-A1). The 5-A peptide offers numerous advantages over the Apo-A-1 both in terms of cost of production as well as manufacturing time. After successfully encapsulating the drugs, we characterized them and tested their cytotoxic potential on various cancerous cell lines. We also conducted cell uptake studies to test our hypothesis of tissue targeting and selective uptake of rHDL nano-particles mediated by the scavenger receptor type B1 (SR-B1). We conducted a pilot study on nude mice in which we administered rHDL containing fluorescent dye intravenously in mice xenografted with NB tumors and took subsequent images to track its distribution in the body. Our results demonstrate that it is possible to encapsulate ATRA, FR and valrubicin into rHDL preparations with a predictable efficiency; these nano-particles show a dose dependent cytotoxic effect on NB cell lines. We anticipate that the results of our studies will facilitate the application of liposomal nano-particles and these novel drugs in the treatment of HRNB in the future.Item The Role of Estrogen and Estrogen Analogues in Friedreich’s Ataxia Cytoprotection(2014-05-01) Richardson, Timothy E.; Simpkins, James W.Friedreich’s ataxia (FRDA) is the most common form of inherited ataxia in the world, affecting roughly 1:50,000 people in the United States. It is inherited in an autosomal recessive manner due to a GAA trinucleotide repeat expansion in the first intron of the FXN gene on chromosome 9q13-21, causing gene silencing and a functional absence of the mitochondrial-localizing protein frataxin. The frataxin protein is responsible for the assembly of iron-sulfur centers in mitochondrial proteins, including the electron transport chain complex I-III, heme synthesis, as well as removing iron from around the mitochondria, preventing the formation of reactive oxygen species (ROS). The loss of FXN function causes an accumulation of mitochondrial iron and ROS, as well as impaired function of Fe-S centers in mitochondrial proteins, leading to mitochondrial damage and a decrease in activity of mitochondrial complexes I-III. The damaged mitochondria are unable to match ATP production to the cell’s energy requirements, resulting in cell death. High energy use cell types, such as neurons and cardiac myocytes, depend almost entirely on oxidative phosphorylation, leaving them especially vulnerable to the mitochondrial damage caused, and it is for this reason that these tissues are the most severely affected by the pathogenesis of FRDA. Cellular models of Friedreich’s Ataxia have employed L-buthionine (S,R)-sulfoximine (BSO), a chemotherapeutic agent which blocks the rate limiting step of de novo glutathione (GSH) synthesis, catalyzed by gamma-glutamylcysteine synthetase. Studies have shown that donor fibroblasts from Friedreich’s Ataxia patients are extremely susceptible to this BSO induced oxidative stress, while fibroblasts from healthy patients are not, due the presence of functional frataxin to absorb the increased load of cellular ROS when GSH is inhibited. Currently, there are few effective treatment modalities for FRDA. Historically, treatment has been focused on palliative care: patient counseling, genetic counseling for prospective parents, speech therapy, physical therapy, wheelchair and other ambulatory device use, propranolol for tremors, dantrolene sodium for muscle spasms and symptomatic treatment for heart disease and diabetes. Recently, antioxidant and mitochondria specific iron chelation therapy have both been proposed as possible therapies to treat the root cause of FRDA. Iron chelation therapy works by a similar principal, removing the iron from around the mitochondria, preventing the formation of free radicals and preventing the associated mitochondrial damage. The neuroprotective effects of 17β-estradiol (E2) have been clearly documented for more than a decade in a variety of disease states involving mitochondrial disruption, but the exact mechanism of action is currently poorly understood. Although the neuroprotective effects of estrogens have never been tested in an FRDA model and FRDA shows no gender-bias in incidence, some epidemiologic studies of FRDA have shown a better prognosis in female patients. Since there is a simple genetic test to determine the presence of FRDA in the children of silent FRDA carriers, it is possible to determine the presence of Friedreich’s ataxia in newborns, years before the cardio- and neurodegeneration and clinical symptoms begin, a time window during which nonfeminizing estrogens and other antioxidants could potentially be clinically useful. Estrogens are putative candidate drugs to provide a neuroprotective effect in Friedreich’s ataxia. The ability of phenolic estrogens to protect against the oxidative damage of ROS, coupled with the possibility that they maintain the integrity of the oxidative phosphorylation process makes them ideal for the treatment of the underlying cellular dysfunction, not just the symptoms of FRDA. This study will determine if E2 and estrogen-like compounds can protect human FRDA fibroblasts from oxidative insults in vitro. In addition, we will attempt to determine the exact mechanism by which E2 acts and investigate the possibility of any synergistic effects with other compounds proposed as putative treatments for FRDA.