Browsing by Subject "function"
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Item Changes in Mammalian Chromatin Structure as a Function of Protein-Poly(ADP-Ribosyl)ation by Endonuclease Digestion(2004-06-01) Perez-Lamigueiro, Maria A.; Alvarez, Rafael; Das, Hriday K.; Basu, AlakanandaPerez-Lamiguerio, Maria A., Changes in Mammalian Chromatin Structure as a Function of Protein-poly(ADP-ribosyl)ation by Endonuclease Digestion. Master of Science (Biochemistry and Molecular Biology), June 2004. 66 pages, 12 illustrations, Bibliography, 45 titles. Mammalian chromatin was exposed to either Deoxyribonuclease I or Micrococcal Nuclease digestion as a function of time of incubation and enzyme concentration. Endonuclease enzymatic reactions were stopped with EDTA. Samples were run in 1.5% agarose gels and the oligonucleosomal electrophoretic migration patterns compared. Endonuclease experiments were carried out with rat liver chromatin pre-incubated in the presence or absence of 200 μM βNAD+. A solution of 1.0 mM benzamide was used to stop enzymatic modification. The electrophoretic observations demonstrated a faster and increased degradation of chromatin when proteins were poly(ADP-ribosyl)ated prior to digestion. These results support the hypothesis that that the covalent poly(ADP-ribosyl)ation of chromatin proteins, particularly histones, induces a more relaxed structure, rendering chromatin more sensitive to endonuclease digestion.Item Regulation of Myocardial Blood Flow and Function During Exercise in Dogs(1995-06-01) Kim, Song-Jung; Patricia A. Gwirtz; Peter B. Raven; James L. CaffreyIntroduction. Background. Coronary circulation during exercise. Coronary blood flow is regulated primarily by local metabolic mechanisms according to the oxygen and nutrient needs of the heart (2, 4, 19). The local “metabolic signal” involves vasoactive metabolites, such as adenosine, released from myocytes in direct proportion to myocardial work (Figure 1). However, other external factors are superimposed on local regulatory mechanisms and can substantially modulate coronary blood flow. One of these modulatory factors is the sympathetic nervous system. Sympathetic vasoconstriction mediated by α-adrenergic receptors in the coronary circulation has been shown to oppose metabolic vasodilation and limit oxygen supply to the myocardium during physiologic and pathophysiological cardiac stresses, such as exercise and myocardial hypoperfusion (1, 6, 7, 8, 10-14, 17, 18, 21). This limitation on myocardial oxygenation appears to impose a restriction on the increase in regional left ventricular subendocardial contractile function during submaximal exercise (7). In this regard, studies have shown that removing this α1-constrictor tone leads to an increase in coronary blood flow and, as a result, regional contractile function (8). This adrenergic coronary constriction during exercise is mediated by neutrally released norepinephrine, not by circulating catecholamines (8). Endothelial-mediated control of coronary vascular tone. Recent investigations indicate that another factor involved in modulating coronary blood flow is the vascular endothelium. The endothelium exerts an influence on vascular smooth muscle vasomotor tone by releasing an endothelium-derived relaxing factor (EDRF) or nitric oxide (NO), which is derived from the amino acid L-arginine by nitric oxide synthase (5, 22). Synthesized NO diffuses into the underlying vascular smooth muscle to activate cytosolic guanylate cyclase (GC), thereby stimulating the intracellular accumulation of cyclic GMP (cGMP). This is illustrated in Figure 2. NO is released by the stimulation of muscarinic receptors on endothelial cells by acetylcholine, as well as by other agonists or physical stimuli (e.g., shear stress) at the interface between blood and endothelial cell surface (15). During exercise, for example, the work output of the normal heart may increase several-fold by the stimulation of sympathetic nerves to heart. The increased work output of the heart increases myocardial oxygen demand. Consequently, the coronary circulation undergoes vasodilation due to local metabolic mechanisms. The elevation in shear stress caused by increases in coronary blood flow triggers release of NO from the endothelium because of the extremely pulsatile nature of the flow. Therefore, it is likely that during exercise, release of NO by shear stress and by neurohormonal stimuli, concomitant with local release of metabolites, contributes to coronary dilation. These vasodilatory influences counteract a sympathetic α-adrenergic coronary constriction, which limits the increase in coronary blood flow and cardiac performance. Accordingly, coronary vascular smooth muscle tone during exercise is modulated by the endothelium, which responds to the increased shear stress and adrenergic stimulation, which provides the major extrinsic input.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 The Effects of Lifelong Glutathione Deficiency on Functional Decline and Redox Signaling(2018-08) Mock, J. Thomas; Sumien, Nathalie; Forster, Michael J.; Salvatore, Michael; Yang, Shaohua; Zode, Gulab S.Purpose: A recent paradigm shift has implicated redox state as a potential key determinant underlying the aging process. Specifically, a pro-oxidizing shift in the ratio of reduced to oxidized glutathione (key substrate in redox status) is hypothesized to disrupt cellular signaling leading to functional impairments and mortality. Chronic glutathione deficiency is achieved by global knockout of glutamate-cysteine ligase modifier (GCLM), an enzyme subunit at the rate-limiting step in glutathione synthesis. Glutathione levels in GCLM-/- mice are 10-30% of those in GCLM+/+ mice. Our hypothesis stated that diminished glutathione synthesis would be sufficient to produce an accelerated, aging-like pattern effect on function, a shortened lifespan, and negative alterations in redox state. Methods: We characterized GCLM+/+ and GCLM-/- male and female mice with a functional battery (n = 15-23 / sex / age / genotype) measuring motor, cognitive and affective function. We also measured redox state, inflammation, metabolism and autophagy markers in central and peripheral tissues (n = 3-6 / group) at 5, 10, or 20 months of age. Lastly, survivorship and body weights were recorded for all animals (n = 455). Results: Overall, age-related declines in function were observed in all functional tests. In young and adult mice glutathione deficiency did not negatively affect function, rather it decreased anxiety-related behavior, improved coordinated running performance in young females and adult males, and delayed general motor decline in both sexes. In old mice, glutathione deficiency improved balance in males and worsened age-related motordecline in females, yet it had no negative effects on cognition in either sex. Lifespan was also extended in male and female GCLM-/- mice (median and maximum). Lastly, GCLM-/- had reduced liver redox state throughout life but only at 5 months in the young, and had increased inflammatory markers in old mice. Discussion: These data imply that (i) motor and cognitive domains appear to be differentially affected by glutathione deficiency and led to benefits in young/adult GCLM-/- mice, (ii) functional and biochemical outcomes were sexually dimorphic, (iii) glutathione deficiency did not decrease lifespan, but rather extended lifespan, and (iv) redox state was impaired in GCLM-/- mice across the lifespan peripherally, but primarily only at 5 months in central tissues. These data do not support the redox stress hypothesis of aging and require further investigation of the beneficial outcomes associated with chronic glutathione deficiency.Item The vascular aging of the cerebellum(2017-08-01) McElroy, Christopher L.; Sumien, Nathalie; Singh, Meharvan; Cunningham, J. ThomasThe cerebellum has been discovered to have an increased role in our daily lives than was previously recognized. It not controls fine motor skills, posture, and gait, but newer research has shown that it has a role in cognitive thought, memory and emotion, as well as other non-motor specific functions. It is important to know how this part of the brain ages and why. It is well established that during the aging process the cerebellum in some regions decreases in size due to atrophy, but the reasoning for this is not well understood. One possible mechanism that could explain the physical functional deficits contributed to the aging cerebellum is the aging of the vasculature in the cerebellum. We propose that there are changes to the surface area, volume, length, diameter, and branching of the blood vessels between young and aged C57/B6 black mice, specifically in the vermis and Crus I/II areas. These two areas are especially susceptible to the morphological changes caused by aging. To observe these changes traditional fluorescence and a new technique called Clarity will be implemented to evaluate the structural changes of the vessels in the cerebellum, and laser Doppler flowmetry will be used to evaluate the functional changes. Our hypothesis is that aging will cause a decrease in the surface area, volume, length, diameter, branching, and blood flow. What was observed was that there were not any significant changes to any of these parameters.