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ItemRat Naphthalene Cataract Studies: Mechanisms and Prevention(1994-06-01) Xu, Guo-Tong; Thomas YorioXu, Guo-Tong, Rat Naphthalene Cataract Studies: Mechanisms and Prevention. Doctor of Philosophy (Biomedical Sciences/Pharmacology), June, 1994, 134 pp., 16 tables, 34 figures, references, 153 titles. The mechanism of naphthalene-induced cataract in rats and the preventive action of AL01576 (an aldose reductase inhibitor, ARI) were studied in both in vivo and in vitro systems. In the in vivo studies, cataracts were induced in five strains of rats (2 pigmented, 3 albino) by naphthalene feeding (1g/kg/day). The cataractous changes occurred in 1 week as watercleft and spoke-like opacities which merged to form a shell-like opacity in the deep cortex by 3 weeks. Semi-quantitation of the opacities with an arbitrary six-score grading system showed little difference in the cataract development between the pigmented and albino strains. Major biochemical changes observed were a decrease of 20%-30% in GSH by one week of feeding, the appearance of disulfide cross-linking of lens proteins by 3 weeks, and a more than ten fold increase in the content of protein-GSH mixed disulfide. Neither damage to lens membrane functions as measured by 3H-choline or 86Rb uptake or loss of Na+/K+-ATPase activity was detected AL01576 (10 mg/kg/day) completely prevented the naphthalene-induced lens changes in both pigmented and albino rats. These results indicate that pigmentation is not required for induction of naphthalene cataract in rats and suggest that tyrosinase action on naphthalene metabolites (such as 1- or 2- naphthol) is not involved in this cataract formation. The in vitro “naphthalene cataract” was established by exposing rat lens to each of 5 potential naphthalene metabolites in organ culture system (in modified TC-199 medium) for 48 hrs. When naphthalene dihydrodiol was used, both the morphological and biochemical changes in the lens were very similar to those observed in lenses of naphthalene-fed rats, and AL01576 completely blocked these in vitro changes as it did in vivo. Other naphthalene metabolites (1,2-dihydroxynaphthalne, 1-naphthol, 2-naphthol and 1,2-naphthoquinone) caused changes which were different from those induced by naphthalene in vivo and one of them was prevented by AL01576. Therefore, naphthalene in vivo and none of them was prevented by AL01576. Therefore, naphthalene dihydrodiol is the key naphthalene metabolite which reaches the lens via blood and aqueous humor and causes cataract when it is metabolized to 1,2-naphthoquinone. This mechanism is further supported by the detection of naphthalene dihydrodiol in the lens and aqueous humor of naphthalene-fed rats. Examples of various classes of ARI (AL01576, AL04114, Sorbinil and Tolrestat) were compared for their effects on the formation of naphthalene cataract and a dual cataract induced with simultaneous feeding of galactose and naph-thalene. Both AL01576 and AL04114 (spirohydantoin derivatives) completely prevented the changes in the lenses of naphthalene-fed rats. However, Sorbinil (another spirohydantoin ARI) demonstrated a much weaker efficacy in this model and the carboxylic acid ARI, Tolrestat, showed no efficacy at all. In the dual cataract, Tolrestat prevented galactose cataract formation and reduced the lens dulcitol accumulation but showed no protection against the shell-like opacity caused by naphthalene. On the other hand, AL01576 protected the lens from the cataractogenic action of both compounds. These results rule out the involvement of aldose reductase in naphthalene cataract formation. Furthermore, AL04114 (not a cytochrome P-450 inhibitor) showed a similar efficacy as AL01576 (a inhibitor of cytochrome) in naphthalene cataract prevention. Therefore, the inhibition of cytochrome P-450 may not be involved in the prevention of this cataract. Based on these findings and the fact that AL01576 prevents the changes induced by naphthalene dihydrodiol (ND) but not 1,2-naphthoquinone (NQ), a new mechanism for rat naphthalene cataract formation is proposed: naphthalene is converted by cytochrome P-450 to ND, which reaches the eye via the blood and penetrates into the lens. By the action of dihydroxynaphthalene which autoxidizes to form NQ and H2O2 production and thus causes cataract. AL01576 and AL04114 inhibit DDD activity, block NQ and H2O2 production and thus prevent the cataract formation. ItemThe Influence of Aging and Glycation on Protein-Thiol Mixed Disulfides in the Eye Lens(1994-06-01) Dickerson Jr., Jaime E.; Walter J. McConathy; Thomas Yorio; Marjorie LouJaime E. Dickerson, Jr., The Influence of Aging and Glycation on Protein-Thiol Mixed Disulfides in the Eye Lens. Doctor of Philosophy (Biomedical Sciences), June, 1994, 163 pp., 9 tables, 28 illustrations, bibliography, 116 titles. The human lens is continually growing. As new cells are formed they differentiate into fiber cells which have no organelles, no protein synthesis or turnover. Lens protein aging involves formation of very large aggregations and insoluble complexes. These are held together through disulfide linkages. Reduced gluthathione (GSH) is present in high concentrations. The oxidized form, (GSSG), (5% of the total) can form mixed disulfides with proteins. This can destabilize the protein conformation. Accumulation of mixed disulfides may increase the potential for further modification. The participation of a PSSG (protein/gluthathione mixed disulfide) in the formation of a protein-protein disulfide becomes increasingly likely. The purpose of this work is to document PSSG and protein-cysteine mixed disulfide (PSSC) accumulation in human lenses (through eight decades), and to identify a third mixed disulfide discovered in this research. The free thiol molecules GSH and cysteine were also quantitated for normal and cataractous lenses. Glycation may alter conformation similar to mixed disulfides and potentiate mixed or protein-protein disulfide formation. This model was evaluated two ways. First, purified alpha crystalline was incubated with ascorbate and conformational changes were evaluated with CD spectroscopy. Second, rat lenses were cultured under high sugar conditions to determine if the resulting glycation influenced the level of mixed disulfides. Conversely, the effect of prior mixed disulfide formation on the extent of glycation in another purified crystalline, gamma, was evaluated. The results indicate: GSH declines in the lens with age, cysteine exists in the lens albeit at relatively low levels, PSSG shows a triphasic pattern of accumulation, PSSC accumulated linearly with increasing age, the existence of a third mixed disulfide species, gamma glutamylcysteine mixed disulfide, detected in old or cataractous lenses, has been confirmed, glycation by ascorbic acid alters α- crystalline secondary structure, the influence of glycation is minimal on mixed disulfide formation, mixed disulfide formation affects glycation of gamma crystalline. ItemA Study of Some Aspects of the Role of Mast Cells in Experimental Autoimmune Uveitis(1994-06-01) Lee, Carol Hamberlin; Edward Orr; Robert Gracy; Laura S. LangLee, Carol Hamberlin, A Study of Some Aspects of the Role of Mast Cells in Experimental Autoimmune Uveitis. Doctor of Philosophy (Biomedical Sciences), June 1994, 141 pp., 6 tables, 29 illustrations, bibliography, 115 titles. Choroidal mast cells have been implicated in experimental autoimmune uveitis (EAU), an ocular inflammatory disease induced by S-antigen (Sag). Activation of ocular mast cells in Lewis rats was evaluated by determining changes in numbers of mast cells, levels of histamine, and wet weights of ocular tissues. A decrease in choroidal mast cells was confirmed statistically, and limbal mast cells were found to be activated earlier than choroidal mast cells. The ocular histamine distribution was altered during EAU, decreasing in the anterior eye, and increasing in the posterior eye. Retinal histamine levels increased when EAU symptoms occurred, but decreased while the disease was still intense. Levels of histamine methyltransferase, which degrades histamine, increased significiantly in retinal tissue when histamine levels fell. Signficant weight increases indicated edema, which can result from mast cell mediator action. Leflunomide, an immunomodulating drug that is known to affect mast cells in vitro, prevented induction of EAU. Leflunomide also suppressed changes in the mast cell-related parameters, histamine levels and wet weights. Mechanisms for activation of ocular mast cells in EAU were investigated. Results suggest that mast cell activation does not occur through mast cell surface IgE-antigen crosslinking. The adjuvant used, complete Freund’s adjuvant, is not conducive to IgE production. Histamine releasing factors, HRFs, are produced by various immune system cellular components. Preliminary efforts did not demonstrate HRF activity. Mast cell numbers, histamine levels, and wet weights were also evaluated in a milder form of EAU induced by M-peptide (Mpep), a peptide fragment of Sag. Mpep/EAU produces few disease symptoms in the anterior eye, but destroys the same retinal area as Sag/EAU—photoreceptor cells and their outer segments. Inflammation is less intense, restricted primarily to the target area. Mast cell numbers did not change, but histamine levels and wet weights changed significantly, suggesting that mast cells are also involved in Mpep/EAU. Overall, the results of this study add to evidence that mast cells are involved in pathogenesis of EAU. The results also point to topics of further investigation into the role of mast cells in EAU and in normal function in ocular tissues. ItemFunctional Heterogeneity in Canine Coronary Resistance Arteries(1994-06-01) Parker, James Bruce; Peter B. Raven; Patricia A. Gwirtz; James CaffreyParker, James B., Functional Heterogeneity in Canine Coronary Resistance Arteries. Doctor of Philosophy (Biomedical Sciences), June, 1994, 89 pages, 21 illustrations, bibliography, 82 titles. Two thirds of the coronary vascular resistance resides in the smallest arteries and investigators have hypothesized that they may respond differently to endogenous vasoactive substances. The arterial responses to norepinephrine, acetylcholine, and adenosine were evaluated in large ([greater than] 700 μm, n=24), intermediate (400 600 μm, n=24), and small arteries (μm, n=24). Maximal vessel lumen diameter (Dmax) was determined in CA++ free medium. A reference diameter (84 ± 4.3% of Dmax) was established by re-equilibration in medium containing 2.0 mM Ca++. Arterial maximal responses as a percentage of Dmax to norepinephrine, acetylcholine, and adenosine are given in table 1: Table 1; Large % of Dmax; Inter. % of Dmax; Small % of Dmax; Norepinephrine; 41 ± 2.3; 50 ± 4.2; 83 ± 2.4; Acetylcholine; 96 ± 2.7; 88 ± 3.9; 78 ± 1.9; Adenosine; 71 ± 1.8; 81 ± 4.2; 96 ± 1.4. The sensitivity of canine coronary arteries to norepinephrine, acetylcholine, and adenosine in terms of ED50’s are given in table 2: Table 2; Agonists; Large ED50 μM; Inter. ED50 μM; Small ED50 μM; Norepinephrine; 0.037 ± 0.002; 0.078 ± 0.004; no response; acetylcholine; 0.028 ± 0.003; 0.087 ± 0.005; 0.309 ± 0.03; Adenosine; 0.295 ± 0.002; 0.095 ± 0.004; 0.035 ± 0.03. These data indicate that canine arterial responses to the native agonists norepinephrine, acetylcholine, and adenosine are heterogeneous and that neural control predominates in the larger “transport” arteries while local control predominates in the smaller “distributive” arteries. Responses of small and intermediate isolated canine coronary arteries (lumen diameter 147±42μm, and 531±37μm respectively) to norepinephrine were evaluated after pharmacological or mechanical interruption of endothelial relaxing activity. Following with the nitric oxide synthase inhibitor N-Nitro-L-Arginine Methylester (L-NAME) 10^-5 M the small and intermediate vessels spontaneously constricted to 73±4.1% of Dmax indicating a significant basal release of nitric oxide. After L-NAME or endothelial disruption graded additions of norepinephrine now reduced the vessel diameter in previously unresponsive small arteries. These data suggest that the weak and equivocal response of coronary resistance arteries to norepinephrine results from the competitive dilatory influence of endothelial derived nitric oxide production and not to the absence of norepinephrine receptors. ItemInterleukin-8: Baculovirus Expression and the Receptor Signal Transduction Pathway(1994-06-01) Kang, Xiaoqiang; Stephen R. Grant; Rafael Alvarez; Paula SumstomXiaoqiang, Kang., Interleukin-8: Baculovirus Expression and the Receptor Signal Transduction Pathway. Doctor of Philosophy (Biomedical Sciences), June, 1994, 150 pp., 4 tables, 36 illustrations, bibliography, 212 titles. The cDNA for human interleukin-8 (IL8) was subcloned from a bacterial source into the eukaryotic baculoviral vector expression system. Recombinant human IL-8 (rhIL-8) was synthesized and secreted from SF9 cells following infection of a recombinant virus harboring the full-length IL-8 structural gene. Recombinant human interleukin-8 was purified ([greater than] 600 fold) to homogeneity using preparative HPLC. The rhIL-8 preparation retained all of the physical, immunological, and biochemical properties of the natural product (monocyte-derived IL-8). Baculovirus vector expression coupled to preparative HPLC proved to be a very efficient method for large-scale recombinant interleukin production. Biochemical mechanisms that mediate IL-8 receptor-stimulated activities are poorly understood. In this study, I have explored the intracellular mechanism(s) induced by IL-8 in differentiated HL-60 cells. IL-8 induced a rapid and transient activation of phospholipase A2 in differentiated HL-60 cells. A consequence of phospholipase A2 activation was the release of arachidonic acid and the generation of lysophospholipids from membrane phospholipids. The IL-8 stimulated-arachidonic acid release was pertussis toxin and phospholipase A2 inhibitor sensitive, and protein kinase C independent. In contrast to another neutrophil chemotactic factor, fMLP, IL-8 did not stimulate the activation of phospholipase C and phospholipase D. When comparing the phosphorylation events induced by IL-8 and fMLP, I found that these two chemotactic factors triggered different protein phosphorylation profiles. Tyrosine phosphorylation of proteins was not detected following IL-8 stimulation in HL-60 cells. However, IL-8 stimulated the rapid autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaM kinase II). These results strongly suggest that the IL-8 receptor is closely coupled to the activation of PLA2 and that CaM kinase II is an integral component of IL-8 receptor signal pathway. ItemAnatomical and Biochemical Characterization of the Porcine Spinal Arachnoid(1994-06-01) Taylor, Martin T.; James Turner; Thomas Yorio; Robert WordingerTaylor, Martin T., Anatomical and Biochemical Characterization of the Porcine Spinal Arachnoid. Doctor of Philosophy (Biomedical Sciences), June, 1994. Mast cell products modulate many biologic barrier systems. In the central nervous system (CNS) two such barriers are the blood-brain barrier (B-B-B) and its analogous cerebrospinal fluid-blood barrier (CSF-B-B). Published research has demonstrated that mast cell products increase the permeability of the BBB, but no comparable research has been described for the CSF-B-B. The main goal of this dissertation was to begin to assess the role of mast cell products on the chief component of the CSF-B-B, the arachnoid mater. Specifically, the hypothesis that mast cell products modulate arachnoid function through receptor mediated second messenger system regulation was postulated. Initially, the light and electron microscopic features of the porcine spinal meninges generally and the arachnoid mater specifically were characterized and found to be similar to those of other species and CNS regions. In addition, mast cells were found to be present in the meninges where their product could affect the arachnoid mater. To study the effects of the selected mast products on the arachnoid mater, arachnoid cells were isolated and cultured. Morphologic, immunohistochemical, and physiological studies confirmed the cultured cells were arachnid cells and that they were capable of developing attributes of a barrier membrane in vitro, (e.g. tight junctions, increased transcellular resistance). The effects of mast cell products on arachnoid cells were then assessed biochemically. Arachnoid cells were found to produce cyclic adenosine monophosphate (cAMP) in response to forskolin and prostaglandin D2 (PGD2). Histamine inhibited both forskolin and PGD2 stimulated production of cAMP. Additionally, arachnoid cells produced inositol phosphates (IP) in response to carbachol and histamine via muscarinic and H1-histamine receptors respectively. Since histamine and PGD2 are produced and released by activated mast cells, and since cAMP and IP levels are known to modulate cellular barrier systems, it is concluded that meningeal mast cells and their products may regulate or modulate permeability of the CSF-B-B. An understanding of the specific biochemical actions of mast cell products on the arachnoid may ultimately aid in the understanding of many physiologic and pathologic processes involving the arachnoid such as hydrocephalus, subarachnoid and subdural hemorrhages, cerebral edema, meningitis, and meningiomas. ItemStudies 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. ItemHemorrhagic Hypotension Alters Circulating and Myocardial Enkephalins and Catecholamines(1994-11-01) Mateo, Zaira; James Caffrey; Leslie Napier; Darice YoshishigeMateo, Zaira, Hemorrhagic hypotension alters circulating and myocardial enkephalins and catecholamines. Master of Science (Biomedical Sciences). November, 1994. A variety of plasma and intrinsic cardiac enkephalins were extracted, chromatographed and assayed under control conditions and during two hours of hemorrhagic hypotension. The animals were anesthetized, instrumented and sufficient blood was withdrawn as required to reduce mean arterial pressure and maintain it at 40 mmHg. Central venous blood samples were obtained 15 minutes before and at 30 minute intervals during the experiment. Arterial blood gases remained stable throughout the experiment while pH declined from above 7.4 to near 7.1. Heart rate rose gradually by 100 bpm. Plasma catcholamines were unchanged during two hour time-controls. Plasma norepinephrine and epinephrine increased by 6 and 100 fold respectively, during the first hour of hypotension and remained high through the second hour. All eight enkephalin immunoreactivities monitored were unchanged during the time-controls. Plasma met-enkephalin (ME) and Peptide-F both gradually increased by 70-100% during the hypotension. Plasma Met-enkephalin-Arg-Phe (MEAP) and Peptide-B concentrations increased 4-5 fold during the same interval. Proenkephalin and other large enkephalin containing peptids though present, were unchanged during hypotension. Myocardial norepinephrine was preferentially concentrated about 3:1 in the atria. Both atrial and ventricular concentrations were reduced by one third or more following two hours of hypotension. Proenkephalin and peptide-B accounted for 75% of the intrinsic enkephalins and their ventricular concentrations were 3 to 4 times atrial concentrations in the same hearts. Intrinsic cardiac MEAP concentrations were 15-25 times higher than comparable ME concentrations in the same myocardial regions. Hypotension produced a significant increase in Peptide-B and proenkephalin compared to controls. The increase was consistent throughout the heart, thus maintaining the preferential concentration in the ventricles. Myocardial ME, MEAP and Peptide-F were largely unchanged in hypotensives compared to time-controls. The data demonstrate the preferential processing and retention of MEAP rather than ME-immunoreactive enkephalins in heart tissue. The data also indicate the responsiveness of MEAP-ir to changes in the circulatory environment and their subsequent appearance in plasma during hemorrhagic hypotension. Prior data suggests that intrinsic cardiac enkephalins may actively regulate either vagal control of the heart or sympathetic control of vasomotor tone. ItemAge Related Changes in Rabbit Cornea: Permeability and Membrane Properties(1994-12-01) Ke, Tai-Lee; Abbot F. Clark; Robert W. Gracy; Walter J. McConathyKe, Tai-Lee, Age Related Changes in Rabbit Cornea: Permeability and Membrane Properties. Doctor of Philosophy (Biochemistry), December, 1994, 139 pp., 26 tables, 13 illustrations, bibliography, 117 titles. This investigation was designed to characterize age-related changes in corneal function and biochemical structure. The specific aims were to: 1) systematically assess changes in permeability to compounds of different molecular weights and lipophilicities, 2) examine differences in tissue binding by utilizing a theoretical transport model, and 3) evaluate the biochemical changes in lipid composition and distribution. Experiments to compare young (six weeks) versus old (three to four years) rabbit corneal permeability were carried out utilizing an in vitro diffusion model. Changes in corneal transmembrane resistance, permeability to various compounds, and metabolic capability were examined by various analytical techniques. In addition, a theoretical penetration model which took into account stromal binding was studied. Corneal lipid composition and distribution were assessed by HPLC and GC. in corneal transmembrane resistance, permeability to various compounds, and metabolic capability were examined by various analytical techniques. In addition, a theoretical penetration model which took into account stromal binding was studied. Corneal lipid composition and distribution were assessed by HPLC and GC. Permeabilities of selected compounds with different physicochemical properties were evaluated in young and old intact and denuded (wounded) rabbit corneas. With age, the membrane permeability significantly decreased in parallel with an increase in transmembrane resistance. Age-related changes in activities of esterase and phosphatase were also found. For some compounds, the aged corneas exhibited longer lag times in penetration studies. This suggested that the binding constant in the cornea from older animals was higher than in young animals. Maximum binding capacity from theoretical model calculations correlated well with experimental results in the young corneal stroma but correlation was less rigorous for old corneal stroma. Age-related changes in lipid composition and distribution in corneas were observed and provide indirect evidence for a decrease in membrane fluidity (decrease in the ratio of phosphatidylcholine/sphingomyelin) in the aged cornea. Results indicate that the aging process in the cornea is associated with changes in biochemical structural matrix including membrane lipid composition and physical properties such as fluidity (microviscosity). Functional correlations include changes in: 1) transmembrane resistance, 2) membrane permeability, 3) enzymatic activities (esterase and phosphatase), and 4) binding properties of the cornea. A possible mechanism for understanding and developing an intervention for age-related changes in the cornea is postulated. ItemThe Effects of Elevated Glucose Upon Na+/K+-ATPase in Bovine Retinal Pigment Epithelial Cells(1994-12-01) Crider, Julie Y.; Thomas Yorio; John Lane; Edward OrrCrider, Julie Y., The Effects of Elevated Glucose Upon Na+/K+-ATPase in Bovine Retinal Pigment Epithelial Cells. Doctor of Philosophy (Biomedical Sciences, Pharmacology), December, 1994, 154 pp., 14 tables, 31 illustrations, bibliography, 288 titles. Bovine retinal pigment epithelial (RPE) cells were cultured under 1, 4.5 and 10 g/l glucose conditions in order to characterize the effects of hyperglycemia upon Na+/K+-ATPase. Functional activity of Na+/K+-ATPase was measured as ouabain-sensitive Rb+ uptake. 3H ouabain was used to assess binding characteristics of Na+/K+-ATPase. The major contributors to rubidium (mRb+) uptake activity were the ouabain-sensitive Na+/K+-ATpase and a bumetanide-sensitive NA+/K+/Cl- cotransporter. Dose response curves for ouabain and bumetanide produced IC50 values for 86Rb+ uptake of 60-100 nM and 120 nM, respectively. At elevated glucose concentrations, the aldose reductase inhibitor (ARI) AL-1576 stimulated 86Rb+ uptake upon chronic treatment. A sensitive new nonradioactive Rb+ uptake assay was developed which utilized suppressed conductivity detection and provided several advantages over the radioactive method. The average ouabain IC50 value was confirmed to be 100nM and was not significantly affected by elevated glucose concentrations. The bumetanide sensitive component was responsible for approximately 30% of Rb+ uptake at all glucose concentrations. Potassium efflux out of the cells was observed that was sensitive to the Na+/K+/Cl- cotransport inhibitor bumetanide. Elevated glucose appeared to increase Rb+ transport through potassium channels was also reduced Rb+ uptake indicating a decrease in Na+/K+-ATPase activity. Bovine RPE cells exposed to both high glucose and AL-1576 for one month showed mild stimulation of Rb+ uptake compared to the activity in high glucose alone. Ouabain and strophanthidin inhibition of 3H ouabain binding, in bovine RPE cells, appeared to be unaffected by hyperglycemia. The average IC50 values for these compounds were 5.02 x 10^-8 M, respectively. The results of this study indicate that Na+/K+-ATPase activity in bovine RPE is decreased by hyperglycemic state, and can be stimulated by treatment with an aldose reductase inhibitor administered from the onset of the hyperglycemic insult. ItemDiscriminative and Negative Reinforcing Properties of the Periaqueductal Gray and the Medial Hypothalamus(1994-12-01) Jung, Marianna E.; Michael W. Emmett-Oglesby; Thomas YorioMarianna Eunsun, Jung, Discriminative and Negative Reinforcing Properties of Electrical brain stimulation of the Periaqueductal Gray and the Medial Hypothalamus. Master of Science [Biomedical Sciences, (Pharmacology)], December, 1994, 123 pp., 24 figures, references, 137 titles. Electrical brain stimulation (EBS) of the periaqueductal gray (PAG) and the medial hypothalamus (MH) is known to serve as a discriminative and a negative reinforcing stimulus (NRS). Using a two-lever food reinforced discrimination paradigm and a switch-off paradigm, the present study investigated the effects of anxiolytic drugs and an anxiogenic drug on these stimulus effects. A prototypic anxiogenic, pentylenetetrazole (PTZ) potentiated both discriminative stimulus and NRS effects, whereas the full benzodiazepine (BZD) agonist diazepam (DZP), the partial BZD agonist abecarnil (ABC) and 5-HT1A agonist buspirone (BUS, chronic regimen) attenuated a NRS effect. A BZD antagonist, flumazenil (FLU) blocked the effects of DZP and ABC on the NRS effects. DZP failed to attenuate the discriminative stimulus effect. Thus, present study extended the use of a switch-off paradigm to detect novel anxiolytic ABC (putative) and BUX as well as an anxiogenic PTZ. In addition, under the condition used in this study, the use of NRS in a switch-off paradigm more reliably detected both anxiolytic drugs and an anxiogenic drug than the use of discriminative stimulus in a two-lever food reinforced paradigm. ItemConformational Properties of Circulating and Recombinant Forms of Human Plasma Lecithin Cholesterol ACYL Transferase(1995-06-01) Sundarrajan, Geetha; Walter McConathySundarrajan, Geetha, Conformational properties of circulating and recombinant forms of human plasma LCAT Master of Science (Biomedical Sciences), June, 1995, 69pp., 3 tables, 18 figures, 47 references. The relationship between enzymatic activity and conformational properties of the circulating and recombinant forms of human plasma LCAT were examined in the native and denatured states. The two denaturing agents used in this study were guanidine hydrochloride and heat. These studies led to the following conclusions: (1) Although the alpha helical content of desialylated recombinant LCAT (d-LCAT) is comparable to that of the other two forms of the enzyme (p-LCAT and r-LCAT), the desialylation of LCAT is associated with an increase in the beta sheet and a decrease in beta turn content. (2) The presence of sialic acids, in addition, seems to influence the local environments of aromatic amino acid residues. (3) From the denaturation and renaturation studies with guanidine hydrochloride and heat, it appears that the N-glycan structures of p-PCAT and r-LCAT may contribute differentially to the conformational stability of the enzyme. (4) The alpha helical structure of LCAT may not be involved in maintaining the active conformation of the enzyme. ItemRegulation 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. ItemEffects of the Pleiotropic Gene csrA on Glycogen Metabolism in Escherichia Coli(1995-06-01) Yang, Honghui; Ming-Chi Wu; Wayne NicholsonYang, Honghui, Effects of the pleiotropic gene csrA on glycogen metabolism in Escherichia coli. Master of Science (Biomedical Sciences), June, 1995, 78 pp., 3 tables, 17 illustrations, bibliography, 59 titles. The csrA gene negatively regulates the expression of four genes glgB, glgC, glgA and glgS involved in glycogen synthesis. It also negatively regulates glgY, which encodes the enzyme glycogen phoshorylase involved in glycogen degradation, but no effect was observed on the glycogen debranching enzyme in this pathway. In addition, csrA exhibits a positive effect on the glycolytic enzyme triosephosphate isomerase. No significant effects were observed on the expression of two genes (zwf & gnd) participating in the pentose phosphate pathway. In vitro expression of glgB, glgC and glgA was specifically inhibited by cell extracts containing the csrA gene product (CsrA). This study provides evidence that csrA encodes an important regulator of intermediary carbon metabolism in Escherichia coli. ItemA Calcium-Dependent Nuclear Signaling Pathway Transcriptionally Silences Atrial Natriuretic Factor Gene Expression(1995-08-01) Zeng, Hong; Stephen R. Grant; Walter McConathy; Richard EasomZeng, Hong, A Calcium-Dependent Nuclear Signaling Pathway Transcriptionally Silences Atrial Natriuretic Factor Gene Expression. Master of Science (Biomedical Science), August, 1995, 85 pp., 2 tables, 20 illustrations, bibliography, 90 titles. A cultured myocardial cell model was used to examine a potential role of calcium-dependent protein kinases and phosphatases in regulating the induction of the atrial natriuretic factor (ANF) gene mediated through adrenoreceptor signaling. In primary culture, rat neonate cardiomyocytes supplemented with phenylephrine (PE) following transfection (24 h) with a full length ANF promoter-reporter construct, showed elevated levels of promoter activity when compared to transfected cardiomyocytes cultured in the absence of PE. Prazosin, a dedicated α1-antagonist, completely blocked the transcriptional induction mediated through PE stimulation. Two different calcium mobilizing agents, BAY K8644 and gramicidin D, significantly reduced PE-stimulated ANF promoter activity. The over-expression of co-transfected exogenous CaM kinase II isoforms resulted in transcriptional silencing of PE-induced promoter activity for cardiac ANF. Transfection of a constitutively active, mutant form of the calcium-dependent phosphatase 2B, calcineurin, gene also transcriptionally silenced ANF gene expression. Exposure of PE-induced cardiomyocytes to either FK-506-treated cells in the absence of PE exposure suggesting that transcriptional silencing may be mediated through a transcriptional repression mechanism. Taken together, these results suggest that the activation of a Ca2+-dependent nuclear signaling pathway mediated through either CaM kinase II or calcineurin leads to complete transcriptional silencing of the embryonic ANF gene expression. ItemMechanism of Gramicidin D-Induced Insulin Secretion From BTC3 Cells(1995-08-01) Dibas, Adnan I.Dibas, Adnan I., Mechanism of Gramicidin D-Induced Insulin Secretion From BTC3 cells. Doctor of Philosophy (Biomedical Sciences), August, 1995, 190 pp., 5 tables, 38 illustrations, bibliography, 265 titles. Gramicidin D, a sodium ionophore, was discovered to be a potent insulin secretagogue in the B-cell line BTC3 cells. Gramicidin D (1 uM) induced a 3.28-fold increase in insulin release relative to control, and when studied in a dynamic cell-perifusion system, was biphasic. Insulin secretion was accompanied by effects of gramicidin D to increase intracellular concentrations of Na+([Na+]i) and Ca2+ ([Ca2+)i) in BTC3 cells as determined by dynamic single-cell video imaging techniques, gramicidin D had no effect on cellular pH. The mechanism of gramicidin D-induced increase in [Ca2+ and suggested to be mediated by a combination of membrane depolarization-induced activation of voltage-sensitive Ca2+ channels and the activation of a Na+/Ca2+ exchanger in the reverse mode. Gramicidin D-induced increase in [Ca2+]I in the first phase correlated temporally with a profound (5.56-fold) activation of multifunctional Ca2+/calmodulin-dependent protein kinase II. While these observations are consistent with the involvement of this enzyme in gramicidin D-induced insulin secretion, further observations suggested that the kinase may play only a modulatory role in insulin secretion. A similar activation of myosin light chain kinase was not detected. In contrast to BTC3 cells, gramicidin D failed to induce insulin secretion from pancreatic islets. BTC3 cells and pancreatic islets exhibited distinct responses to ouabain, an inhibitor of the Na+/K+ ATPase, with respect to [Ca2+]I and insulin secretion suggesting that different mechanisms controlling Na+ homeostasis exist in these B-cell preparations. Furthermore, Na+/K+ ATPase activity in BTC3 cell membranes was found to be approximately fifty percent that of primary B-cells. Gramicidin D was identified as a secretagogue in BTC3 cells with a novel mechanism of action. The ability of this ionophore to induce insulin secretion from these cells and not primary B-cells is thought to be a function of different mechanisms of Na+ homeostasis and documents a functional difference in this insulinoma cell line. ItemRole of Adenosine in Acute Hibernation of Guinea-Pig Myocardium(1995-08-01) Gao, Zhi-Ping; H. Fred Downey; James L. Caffrey; Patricia A. GwirtzGao, Zhi-Ping, Role of Adenosine in Acute Hibernation of Guinea-Pig Myocardium Doctor of Philosophy (Biomedical Sciences), August, 1995; 111 pp; 3 tables; 15 figures, bibliography, 158 titles. Myocardial hibernation is a state of depressed contractile function and energy demand during chronic ischemia. When coronary flow is restored, depressed contractile function can partially or completely recover to the pre-ischemic level, and ischemic injury of the myocardium in not evident. This project tested the hypothesis that endogenous adenosine mediates hibernation in guinea-pig myocardium. Isolated working guinea-pig hearts, perfused with glucose fortified Krebs-Henseleit buffer, were subjected to global low-flow ischemia. Left ventricular performance and cytosolic energy level were assessed. Lactate and purine nucleotides were measured in venous effluent. Heart were perfused with [U-14C]glucose to investigate the role of adenosine on glucose metabolism in myocardium. Left ventricular function in untreated hearts decreased by 80% and remained stable during ischemia, and completely recovered upon reperfusion. Neither adenosine receptor blockade with 8-p-sulfophenyl theophylline (8-SPT; 20 μM) nor ecto 5’-nucleotidase inhibitor αβ-methylene adenosine 5’-diphosphonate (AOPCP; 50μM) affected left ventricular function either ischemia or during reperfusion. Cytosolic energy level fell by 67% at 10 min ischemia in untreated hearts, but subsequently recovered to the pre-ischemic level despite continued ischemia. Adenosine receptor blockade increased cytosolic energy level at 10 min ischemia relative to untreated hearts, but blunted the subsequent rebound of phosphorylation potential. Moreover, 8-SPT doubled ischemic lactate release. Adenosine receptor blockade also increased glucose uptake during pre-ischemia and hypoperfusion, but did not stimulate glucose oxidation. Crossover plots of glycolytic intermediates revealed that phosphofructokinase, a key rate-controlling step in glycolysis, was activated by adenosine receptor blockade in both pre-ischemic and hibernating myocardium. We conclude that 1) activation of adenosine receptors results in recovery of cytosolic energy level of moderately ischemic working myocardium, but this energetic recover is not solely responsible for post-ischemic contractile recovery; 2) endogenous adenosine attenuates anaerobic glycolysis during myocardial hibernation by blunting phosphofructokinase activity. ItemCross-Tolerance Between the Discriminative Stimulus Properties of Ethanol, Diazepam and Pentobarbital(1995-12-01) Lytle, Douglas A.; Michael Forster; Glenn Dillon; Thomas YorioLytle, Douglas A., Cross-Tolerance Between the Discriminative Stimulus Properties of Ethanol, Diazepam and Pentobarbital. Doctor of Philosophy (Biomedical Sciences), December, 1995, 132 pp., 8 tables, 19 figures, bibliography, 176 titles. Ethanol, benzodiazepine agonists and barbiturates all facilitate GABA-mediated CT flux. The present experiments tested the hypothesis that, because these agents share this common action, tolerance to discriminative stimulus properties of one of these drugs would result in cross-tolerance to the others. Rats were trained to detect either ethanol (EtOH; 1.0 g/kg), the benzodiazepine diazepam, (DZP; 5.6 mg/kg), or the barbiturate pentobarbital (PB; 10.0 mg/kg) from vehicle using a two-lever choice procedure where food was available under a fixed-ration ten schedule of reinforcement. Subsequently, dose-effect curves for EtOH (0.1-1.78 g/kg), DZP (0.56-17.8 mg/kg), or PB (1.0-17.8 mg/kg) were tested before and after chronic administration of EtOH 96.0 g/kg/12hrs for seven days), DZP (20.0 mg/kg/8hrs for seven days), or PB (32.0 mg/kg/8hrs for seven days). The chronic administration of EtOH conferred tolerance to itself in all cases and cross-tolerance to DZP and PB in subjects trained to detect EtOH, but did not confer cross-tolerance to these agents in their respective discriminations. The chronic administration of DZP conferred tolerance to itself substituting for DZP. Although tolerance developed to DZP substituting for PB after treating animals with chronic DZP, this regimen on DZP did not confer tolerance to itself substituting for EtOH. This regimen of DZP failed to confer significant cross-tolerance to either EtOH or PB under any conditions. The chronic administration of PB conferred tolerance to itself substituting for PB. Although tolerance developed to PB substituting for DZP after treating animals with chronic PB, this regimen of PB did not confer tolerance to itself substituting for EtOH. This regimen of PB failed to confer significant cross-tolerance to either EtOH or DZP under any conditions. In summary, EtOH was found to confer cross-tolerance to DZP and PB only in animals trained to detect EtOH. The chronic administrations of DZP and PB failed to confer tolerance to themselves substituting for EtOH. These results are parsimonious with the heterogeneous nature of the GABA receptor. Finally, tolerance to either DZP or PB does not result in cross-tolerance to the discriminative stimulus properties of the other drug. These results suggest that the mechanisms mediating tolerance to BZs and barbiturates are not linked. ItemAdenosine Receptor Blockade Increases Lactate and Purine Release But Does Not Affect Functional Recovery in Isolated Rabbit Myocardium(1995-12-01) Wang, Sheng; H. Fred Downey; Miao-Xiang He; Robert T. MalletWang, Sheng, Adenosine Receptor Blockade Increases Lactate and Purine Release but does not Affect Functional Recovery in Isolated Rabbit Myocardium Master of Science (Biomedical Sciences), December 1995; 67 pp; 3 tables; 8 figures; bibliography, 121 titles. This study tests the hypothesis that endogenous adenosine mediates recovery of cardiac function in ischemia/reperfused rabbit hearts. Isolated isovolumic rabbit hearts perfused at constant pressure was subjected to mild ischemia (perfusion pressure 50 cm H2) or moderate ischemia (perfusion pressure 30 cm H2O) for 90 min followed by 60 min of reperfusion. In treated hearts, infusion of 100 μM 8-p-sulfophenyl theophylline (SPT) was initiated 20 min before ischemia and maintained throughout the experiment. Adenosine receptor blockade did not affect left ventricular function assessed from pressure-heart rate product (PRP). Lactate release increased to 152 ± 24% of baseline during mild ischemia and 259 ± 26% of baseline during moderate ischemia in untreated hearts. Lactate release was markedly elevated at baseline, ischemia and reperfusion by SPT treatment (p [less than] 0.05 compared to untreated). Purine nucleoside release was 4.1 ±0.7 nmol · min-1 · g-1 in SPT treated group and 1.8 ± 0.24 nmol · min-1 · g-1 in untreated group during moderate ischemia (P [less than] 0.05). Myocardial efficiency was significantly lower in the SPT treated hearts (240 ± 11 mmHg · g=1 · μl-1 O2) compared to untreated hearts (300 ± 22 mmHg · g-1 · μl-1 O2) during reperfusion after moderate ischemia. In conclusion, adenosine receptor blockade stimulates glycolysis in normoxic and ischemic myocardium, but does not affect post-ischemic functional recovery. ItemIsotope Partitioning and Initial Velocity Studies with 6-Phosphofructo-1-kinase from Ascaris suum(1996-05-01) Gibson, Grant E.; Robert EasomGibson, Grant E., Isotope Partitioning and Initial Velocity Studies with 6-Phosphofructo-1-kinase from Ascaris suum. Doctor of Philosophy (Biomedical Sciences), May, 1996, 91 pages, 2 tables, 18 figures, 2 schemes, 1 reaction, 2 mechanisms, 1 diagram, bibliography, 61 titles. The natives Ascaris suum 6-phosphofructo-1-kinase (nPFK) and a chemically modified form (dPFK) which is desensitized to allosteric behavior have been studied using isotope partitioning and initial velocity techniques to determine the kinetic mechanism as well as the effects of fructose 2,6-biphosphate (F26P2) and Mg2+ on the mechanism. At 8 mM Mg2+, complete trapping (P*max≈100%) of E:MgATP* complex as fructose 1-(32P), 6-biphosphate for both enzyme forms is consistent with the previously proposed steady-state ordered mechanism ((Rao, G.S.J., Harris, B.G., and Cook, P.F. (1987) J. Biol. Chem. 262, 14074-14079) with MgATP binding before fructose 6-phosphate (F6P). A saturating amount of F26P2 causes no change in the trapping parameters for nPFK but causes a decrease in both P*max and K’F6P for dPFK. The partial trapping of E:MgATP* in the presence of F26P2 for dPFK at high MG2+ suggests that the activator changes the kinetic mechanism from an ordered to a random binding of substrates. Initiial velocity studies at 8 mM Mg2+ confirm the change in mechanism. Uncompetitive inhibition by arabinose 5-phosphate (Ara5P), a dead-end inhibitory analog of F6P, versus MgATP for nPFK in the absence and presence of F26P2 is consistent with an ordered mechanism with MgATP adding to enzyme prior to F6P. An uncompetitive pattern is also obtained with dPFK for Ara5P versus MgATP in the absence of F26P2, but the pattern becomes noncompetitive in the presence of F26P2, consistent with a change to a random mechanism. No trapping of the dPFK: (14C)F6P complex could be detected 8mM Mg2+, indicating either that dPFK:14C-F6P complex does not form or that the off-rate for F6P from enzyme is much faster than the net rate constant for formation of the first product, FBP. Initial velocity data indicate that a second Mg2+ ion in addition to the one bound in MgATP is an essential activator of Ascaris suum PFK which decreases the off-rate for MgATP. Kact for Mg2+ is estimated to be 0.47±0.08mM. Isotope partitioning data at 0.1 mM Mg2+ indicate that dPFK is able to trap only 20% of the E:MgATP* both in the presence and absence of F26P2, consistent with a faster off-rate for MgATP at low Mg2+ than at high Mg2+. Partial trapping of MgATP* at low Mg2+ again suggests a random binding of substrates. Noncompetitive Ara5P inhibition versus MgATP at low Mg2+ confirms the random mechanism. An active site role both in binding MgATP and in facilitating catalysis is proposed for the second Mg2+. Furthermore, calculations from the isotope partitioning and initial velocity data as well as changes that are seen in the circular dichroic spectra for both nPFK and dPFK indicate that an enzyme structural isomerization occurs upon binding mgATP.