Browsing by Subject "Medical Microbiology"
Now showing 1 - 14 of 14
- Results Per Page
- Sort Options
Item A 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.Item A Novel sRNA Member of the Carbon Storage Regulatory System of Escherichia Coli(2002-12-01) Weilbacher, Thomas; Jerry SimeckaWeilbacher, Thomas S., A Novel sRNA Member of the Carbon Storage Regulatory System of Escherichi coli. Master of Science (Microbiology & Immunology), December, 2002, 57 pp., 2 tables, 12 illustrations, bibliography, 44 titles. Small untranslated RNAs (sRNAs) perform a variety of important functions in bacterial systems. The 245 nt sRNA of Escherichia coli K-12, CsrC, was uncovered using a genetic screen for genes that regulate glycogen biosynthesis. CsrC RNA binds multiple copies of CsrA, a protein that post-transcriptionally regulates central carbon flux, biofilm formation, and motility in E. coli. CsrC antagonizes the regulatory effects of CsrA, presumably by sequestering this protein. The discovery of CsrC is intriguing, in that a similar sRNA, CsrB, performs essentially the same function. Both of these sRNAs possess similar imperfect repeat sequences (18 in CsrB, 9 in CsrC), primarily localized in the loops of predicted hairpins, which may serve as CsrA binding elements. Transcription of csrC increases as the culture approaches the stationary phase of growth and is activated by CsrA and the response regulator UvrY. Complementation and in vitro transcription-translation experiments reveal that CsrA effects on csrC are mediated indirectly, through UvrY. Because CsrB and CsrC antagonize the activity of CsrA and are dependent on CsrA for their synthesis, a csrB null mutation causes a modest compensatory increase in CsrC levels and vice versa. An updated model for the signaling circuitry of the Csr system is discussed.Item A Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes Required for DNA Damage-Induced Mutagenesis(2008-07-01) Gong, Jinjun; Siede, Wolfram; Sheedlo, Harold; Reeves, RustinA Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes required for DNA Damage-Induced Mutagenesis. Jinjun Gong Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107. Summary. Deoxyribonucleic acid (DNA) damage is common in a cell’s lifetime. DNA can be damaged by endogenous factors such as reactive oxygen species (ROS) or exogenous agents such as ultraviolet (UV) or industrial chemicals. DNA damage will trigger cell responses including cell cycle arrest, transcription activation, DNA repair or apoptosis. In addition to various DNA repair mechanisms including damage reversal, base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end joining, translesion DNA synthesis is an important DNA damage tolerance pathway that can bypass the lesion on template DNA to finish the replication for cell survival but at the risk of potential mutation in the daughter cells. Accumulation of mutation may lead to cancer occurrence. Translesion DNA synthesis components are highly conserved from yeast to humans. Important players in trans-lesion synthesis pathway such as Rev1, Rev3 and Rev7 were first discovered in budding yeast. Saccharomyces cerevisiae. Homologues were found later in human cells. I used the Saccharomyces cerevisiae deletion mutant collection to do a systematic screen to search for novel genes required for DNA damage induced mutagenesis in yeast. After CAN1 forward mutation assay for the systematic screen and reverse mutation assay for further confirmation, two candidate genes SWI6 and DOA4 were detected. Deletion of SWI6 and DOA4 decreases mutagenesis of cells. At the molecular level, Swi6, a transcription cofactor, is involved in mutagenesis by regulating expression of REV7 at the mRNA and protein levels. Rev7 is a regulatory subunit of DNA polymerase zeta, which is essential for DNA damage induced mutagenesis as well as spontaneous mutagenesis. Rev7 is not UV inducible or cell cycle regulated. The regulation of Rev7 at the transcriptional level by Swi6 is essential. Future experimental approaches are planned to address the mechanism by which DOA4 is involved in mutagenesis.Item Approaches to Cloning and Identification of the Ligand for Natural Cytotoxicity Receptor NKp44(2008-07-01) Horton, Nathan C.; Harlan Jones; Stanley Stevens; Raghu KrishnamoorthyHorton, Nathan C., Approaches to Cloning and Identification of the Ligand for the Natural Cytotoxicity Receptor, NKp44. Masters of Science (Microbiology & Immunology), July 2008, 64 pp., 22 illustrations, 37 titles. Natural Killer (NK) cells represent a specialized lymphoid population that mediate innate immune responses against tumor or virally infected cells. NK cell cytotoxicity is regulated by inhibitory and activating receptors. Activating receptors include the Natural Cytotoxicity Receptors (NCRs), 2B4, and NKG2D. The NCRs play a key role in recognition and killing of tumor cells and include the receptors NKp30, NKp46, and NKp44. The ligands for the NCRs are not yet known. NKp44 is of particular interest because it is only expressed on activated NK cells, and is implicated in increased cytotoxicity and HIV infection. To identify and clone the ligand for NKp44, a recombinant fusion protein containing the extracellular domain of NKp44 was constructed and used to identify a cell line, DB, expressing a ligand for NKp44. A directional complimentary DNA (cDNA) library was constructed from this cell line and screened by mammalian expression cloning, resulting in the isolation of several putative cDNA clones of NKp44 ligands.Item Characterization of the Role of PKN in TGF-Beta 1-Mediated Cell Cycle Regulation of Vascular Smooth Muscle Cells(2005-12-01) Su, Chang; Neeraj Agarwal; Glenn Dillon; Robert MalletChang Su, Characterization of the role of PKN in TGF-beta-1 induced cell cycle inhibition in vascular smooth muscle cells. Doctor of Philosophy (Biomedical Sciences), November 2005, 173 pp, 2 tables, 34 illustrations, 225 references. Mature vascular smooth muscle cells (VSMCs) are unique in that they can switch between proliferative and differentiated phenotypes. Aberrant proliferation of VSMC is regarded as a central feature in vascular diseases such as atherosclerosis and restenosis following balloon angioplasty. Transforming growth factor-β1 (TGF-β1) is known to inhibit smooth muscle cell progression; however, the signaling pathway(s) through which this is accomplished is poorly understood. Entry into mitosis in dividing VSMCs is triggered by Cdc2/cyclin B1 complex, which is tightly controlled by phosphatase Cdc25C that dephosphorylates tyrosine-15 and threonine-14 on Cdc2 at onset of mitosis. A serine/threonine protein kinase, PKN, was recently reported to inhibit Cdc25C activity. PKN has been identified as a downstream target for TGF-β1 signaling in VSMCs. Therefore we hypothesize that PKN mediates TGF-β1-delayed cell cycle progression by inhibiting Cdc25C. In this study, TGF-β1 is shown to delay G2/M phase progression timing in PAC-1 VSMCs. This effect is blocked by pretreatment of cells with either HA1077 of Y-27632, two pharmacological inhibitors of PKN, as well as by reduced expression of PKN by RNA interference (RNAi). Oscillation of PKN activity temporally correlates with G2/M phase progression. Co-immunoprecipitation suggests that Cdc25C and PKN physically associate with each other. Immunocytochemistry demonstrate that PKN and Cdc25C co-localize in the nuclei and peri-nuclear region of only dividing (M phase) cells but not in the interphase cells. Additionally, PKN phosphorylates Cdc25C in PAC-1 cell cultures. Finally, TGF-β1-induced delay of Cdc2 activation is abolished by pretreating the cells with Y-27632. These data suggest that PKN inhibits G2/M progression by directly binding to Cdc25C and inhibiting its activity by phosphorylation. In addition to the PKN-Cdc25C signaling pathway, TGF-β1 strongly induces the transcriptional activity of the Smad-dependent enhancer in PAC-1 cells. This effect is attenuated by blocking PKN function by either chemical inhibitors or RNAi. Active forms of MKK3/6 alone are sufficient to increase the Smad enhancer activity, and co-expression of dominant negative MKK3/6 decreases TGF-β1-induced activation of the Smad enhancer. Lastly, the Smad reporter activity induced by TGF-β1 is also significantly attenuated by SB203580, a highly specific pharmacological inhibitor for p38 MAPK. These data demonstrate a novel mechanism of PKN-MKK3/6-p38 MAPK cascade to cross talk with the Smad pathway in PAC-1 VSMCs. Taken together, findings presented in this dissertation identify components of important intracellular signaling pathways through which TGF-β1 activates PKN to inhibit proliferation and promote differentiation of SMCs. Augmenting PKN-Cdc25C-Cdc2 signaling may provide a potential therapeutic approach to counter abnormal VSMC proliferation, prevent the clinical consequences of atherosclerosis and improve outcomes after angioplasty.Item Diverse Immune Responses Mediated by Beta-Adrenergic and Corticotropin-Releasing Hormone Receptors in a Model of Pneumococcal sepsis(2010-08-01) Kim, Byung-Jin; Harlan JonesNeuroendocrine stimulation can impact disease states by regulating immune function. The purpose of our studies was to define the functional role of stress-induced neuroendocrine factors, catecholamines and corticotropin-releasing hormone (CRH) on immune responses involved in the pathogenesis of Streptococcus pneumoniae (S. pneumoniae) infection implementing both in vitro and in vivo methodology. Dendritic cells play a pivotal role in antigen presentation and cytokine production, influencing both innate and adaptive immunity. Initial studies examined the potential immunomodulatory effect of epinephrine and CRH on DC cytokine production in response to the bacterial pathogenic ligand, lipopolysaccharide (LPS). In addition, the ability of DC to dictate CD4+ T cell activation as a consequence of CRH or epinephrine pre-treatment was examined using an in vitro co-culture system. Epinephrine and CRH pre-treatment resulted in a preferential increase in IL-23 and IL-10 cytokine production. In contrast, IL-12p70 was significantly attenuated in response to epinephrine and CRH pre-treatment. Preferences in IL-23 and IL-10 cytokine production by DC pre-treated with epinephrine and CRH corresponded with an increase in IL-4 and IL-17A, but not IFN-y cytokine production by CD4+ T cells. These results suggest that exposure to stress-derived epinephrine/CRH dictates dendritic cells to generate a dominant Th2/Th17 phenotype in the context of subsequent exposure to a pathogen. Our second study examined the functional properties of IL-23 during pulmonary S. pneumoniae infection. IL-23 plays a crucial role in establishing host defenses against extracellular pathogens. Further investigation is still required to define the impact of IL-23 on acute pulmonary S. pneumoniae infection. Utilizing IL-23p19 genetic deficient mice, we determined bacterial load, cytokine production and the contribution of neutrophils against S. pneumoniae infection using monoclonal antibody-mediated systemic neutrophil depletion. The absence of IL-23 induced a higher bacterial load in lung and blood as compared to IL-23 competent counterparts. In the absence of IL-23, production of proinflammatory cytokines such as IL-6, IL-12p70 as well as IL-17A and IFN- were dampened as compared to wild type mice. In addition, neutrophil distribution was also altered in IL-23-deficient mice, suggesting impaired neutrophil recruitment into lung. Interestingly, neutrophil depletion did not impact bacterial load in lung and blood in both IL-23 competent and deficient mice. These findings, suggest a novel role of IL-23 in pulmonary S. pneumoniae infection, potentially independent of neutrophil function. We next examined the possible impact of CRH and catecholamines as regulators of immune function against acute bacterial infection in response to stress. Utilizing a murine model of acute pulmonary S. pneumoniae infection and restraint stress, we selectively blocked CRH receptors (CRHR1 and CRHR2) as well as the 2 adrenergic receptor prior to restraint stress followed by intranasal pulmonary S. pneumoniae infection. Antagonist administration did not impact restraint stress-induced physiological responses as compared to restraint stressed mice, which did not receive receptor antagonists. However, following S. pneumoniae infection, physiological changes including weight and temperature were altered in response to administration of selective CRH receptor and β2 adrenergic receptor antagonists. Survival rate, bacterial load and cytokine production corresponded with physiological differences observed in response to selective CRH receptor and 2 adrenergic receptor antagonists. Importantly, preferential differences in bacterial colonization and survival corresponded with distinct differences in inflammatory cytokine production and immune cell distribution along pulmonary airways. In particular, opposing effects in IL-17A and neutrophil accumulation was found among mice administered the CRHR1 versus the CRHR2 antagonists. Together, these findings indicate that activation of each receptor can influence immune responses against S. pneumoniae infection. Thus, our findings provide further understanding of how stress-derived neuroendocrine factors directly impact immune responses related to immunopathology and immunoprotection.Item Human Hair Shaft Volume and Mitochondrial DNA Recovery(2007-08-01) Kreikemeier, Melissa A.; Arthur Eisenberg; Joseph Warren; John PlanzKreikemeier, Melissa A., Human Hair Shaft Volume and Mitochondrial DNA Recovery. Master’s of Science (Biomedical Science, Emphasis in Forensic Genetics) August, 2007, 54 pp., 6 tables, 26 figures, References, 45 titles. The goal of this study was to determine if there was a relationship between hair volume and amount of amplified mtDNA for all head hair samples, as well as among the three major ethnic groups, different hair colors, and different donors. This relationship could be used to determine how much of a hair sample is needed for extraction so that enough mtDNA PCR product is obtained, while preserving forensic hair samples and avoiding unnecessary consumption of evidence. Amplification success rates were also calculated for each of these categories to determine if the findings coincided with previously published literature.Item Interleukin-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.Item Mechanisms of Cell Surface Trafficking and Potential Functions of Extracellular Annexin A2(2010-12-01) Valapala, Mallika; Jamboor VishwanathaAnnexin A2 (AnxA2) belongs to a family of Ca2+-dependent phospholipid binding proteins. AnxA2 is abundantly expressed in the cytosol of many different cell types and is translocated to the outer surface of the plasma membrane in response to elevated concentrations of intracellular Ca2+. It is known that the binding of Ca2+ to the C-terminal Ca2+-binding domains is essential for the recruitment of AnxA2 to the inner leaflet of the plasma membrane. However, the mechanism by which AnxA2 is translocated from the inner leaflet of the plasma membrane to the outer leaflet remains unknown. Since, AnxA2 does not possess a signal sequence that is essential for the classical ER-Golgi secretion; we believe that the protein follows a non-classical pathway of protein secretion. Here, we show that Ca2+- induced translocation of AnxA2 to the cell surface is a multi-step pathway that involves association of AnxA2 with specific domains of the plasma membrane called lipid rafts and its recruitment to the intracellular membranes of the endosomal pathway followed by extracellular secretion by association with the secretory vesicles called exosomes. In our studies, we also investigated the role of AnxA2 in inducing neovascular responses. We have shown that angiogenic insults like hypoxia upregulate the expression of AnxA2. AnxA2-induced angiogenic responses were identified to be regulated by the N-terminus which is important for the binding of several proteolytic components including, tissue plasminogen activator (tPA), plasminogen, procathepsin B and tenascin C. Targeting the Nterminus of AnxA2 with a competitive hexapeptide that prevents the binding of proteolytic components inhibited the AnxA2-mediated neovascular responses. In summary, our data suggests that AnxA2 is transported to the cell surface following an unconventional pathway of protein secretion and extracellular AnxA2 is an active proteolytic receptor that can have potential roles in pathological conditions associated with excessive extracellular proteolytic events.Item Pharmacological Assessment of Novel Phenylacetamide as a Sigma 1 Receptor Ligand(2014-05-01) Malik, Maninder; Robert LuedtkeThe symptoms of psychosis have been categorized as positive, negative and cognitive. Traditionally drug discovery in psychiatric disorders has focused on positive symptoms of the disease. However, cognitive impairment is equally prevalent and represents a major impediment to the recovery of patients. Hence, research on the drug discovery and development that can improve overall quality of life of patients with neuropsychiatric conditions is important. The main aim of this project was to evaluate a selective and potent sigma 1 receptor phenylacetamide (LS-1-137) as a potential pharmacotherapeutic agent for treating neuropsychiatric disorders and associated cognitive impairment. The sigma 1 receptor is an endoplasmic reticulum (ER) resident protein located on the interface of ER and mitochondria. The sigma 1 receptor is a 25 KDa protein that shares no amino acid sequence homology with any other known mammalian proteins. The research being done on these novel receptors suggests that rather than being a classical receptor-signaling unit sigma 1 receptors act as a molecular chaperone. Several studies suggest sigma 1 receptor ligands modulate abnormal neurotransmission that contributes to the pathogenesis of several CNS disorders. In recent studies by our laboratory, it was found that our novel sigma 1 receptor ligand, LS-1-137 (developed by our collaborators Mach and colleagues) exhibit neuroprotection both in vitro and in vivo. In this project we further characterized LS-1-137 as a potential treatment option for cognitive and neuropsychiatric disorders. Our fundamental hypothesis is that sigma 1 receptor selective compounds represent a potential neuroprotective pharmacotherapy for treating psychosis and cognitive deficits associated with neuropsychiatric disorders. LS-1-137 was evaluated in a 2, 5-dimethoxy-4-iodoamphetamine (DOI) induced head twitch response (HTR) model and a scopolamine-induced cognition impairment model. Our findings suggest that LS-1-137 attenuates the DOI-induced HTR and alleviates scopolamine-induced impairment in learning. Our in vitro data suggest that LS-1-137 is an agonist at sigma 1 receptors and triggers the release of BDNF from rat astrocytes. Furthermore, rotarod and swim test studies indicated that unlike currently prescribed neuroleptics, LS-1-137 does not compromise the agility or muscular coordination of animals. Therefore, in this dissertation we have assessed a novel pharmacotherapeutic agent that may treat the psychosis and cognitive dysfunction associated with neuropsychiatric disorders.Item The Impact of the Mycoplasma pulmonis MALP-2 Homologue on Disease Progression(2008-04-01) Spear, Marcia G.; Simecka, Jerry W.; Hodge, Lisa M.; Mathew, Porunelloor A.Spear, Marcia. The Impact of Mycoplasma pulmonis MALP-2 Homologue on Disease Progression. Master of Science (Biomedical Sciences), April 2008. 64 pp., 3 tables, 8 illustrations. Using Mycoplasma pulmonis, this project looked at a possible critical component in mycoplasma disease, the MALP-2 homologue lipoprotein. Studies demonstrated other lipoproteins besides the MALP-2 homologue were critical for in vivo disease progression and in vitro macrophage IL-6, IL-12, and TNF-α cytokine production. This trend was also seen human endothelial kidney (HEK) cells transfected with toll-like receptor 1 (TLR2) and the heterodimer TLR2/6. An increase in IL-8 cytokine production seen in all stimulated HEK cell lines, indicating the lipoproteins involved in cell interactions are TLR2 mediated. This project suggests the M. pulmonis MALP-2 homologue is not the main lipoprotein involved in disease progression and cell interactions, indicating the MALP-2 homologue may not be an ideal target for vaccines or antibiotics.Item The Neuroprotective Efficacy of Antioxidants Against In Vitro Models of Oxidative Stress and Their Theoretical Application Via Intravitreal Injection Encapsulated in Nanoparticles(2010-05-01) Ondricek, Amber J.; Jamboor VishwanathaThe purpose of this study was to explore the possibility of utilizing antioxidants to mitigate oxidative stress induced apoptosis related to neurodegenerative diseases such as glaucoma. Our hypothesis is that in diseases related to an imbalanced redox status, whatever the primary cause may be, the loss of function may be prevented by antioxidants at the level of alleviating oxidative burden and preventing apoptotic signaling events. Application of these antioxidants to the site of injury can be improved using nanoparticle delivery methods. We have done work to characterize a model of mitochondrial associated oxidative stress induced cell death and obtained neuroprotective profiles on a group of antioxidants using this model. We have found that estrogens and phytoestrogens, as well as thiol containing antioxidants, function well as neuroprotectants in our in vitro model. Nanoparticle delivery of these models is a promising intervention and we therefore did work to optimize the characteristics of encapsulating one of these antioxidants, N-acetyl cysteine, in Poly(lactic-coglycolic acid) nanoparticles, which can be localized to the retina. Intravitreal injection of these particles is the preferred delivery route to retinal cells and has not been fully explored. We provide evidence to suggest that the intravitreal injection of nanoparticles is not detrimental to an animal’s vision. Taken together, the results of our experiments suggest that antioxidants remain a promising intervention in diseases related to mitochondrial associated oxidative stress, and that these drugs, when encapsulated in nanoparticles, can be delivered to the retina via intravitreal injection without deleterious side effects.Item The Phosphorylation of Endogenous Substrates by Calcium/Calmodulin-Dependent Protein Kinase II in Pancreatic β-Cells(1998-06-01) Krueger, Kimberly A.; Richard Easom; Rafael Alvarez; S. Dan DimitrijevichKrueger, Kimberly A., The Phosphorylation of Endogenous Substrates by Calcium/Calmodulin-dependent Protein Kinase II in Pancreatic β-cells. Doctor of Philosophy (Biomedical Sciences), June, 1998, 165 pp., 35 illustrations, references, 259 titles. Increasing evidence supports a physiological role for calcium/calmodulin-dependent protein kinase II (CaM kinase II) in the secretion of insulin from pancreatic β-cell. While it has been previously demonstrated that CaM kinase II is activated by glucose in isolated rat islets implicating this enzyme in the secretion process, its cellular targets are unidentified. Potential candidates would likely exhibit strong binding to the enzyme, an association with the cytoskeleton, or an involvement in the secretion process. Based on these criteria, the following study represents an evaluation, in situ, of two proteins to function as substrates for CaM kinase II. Microtubule-associated protein, MAP-2 is one of the best substrates of CaM kinase II in vitro thought to be involved in secretion process. Synapsin I phosphorylation in the neuron by CaM kinase II is essential for neurotransmitter release. Unique to this study, both proteins were determined to be expressed in clonal mouse β-cells (βTC3) and primary rat islet β-cells. By immunoprecipation, in situ phosphorylation of MAP-2 and synapsin I was induced in permeabilized βTC3 cells within a calcium range shown to activate endogenous CaM kinase II under identical conditions. Two-dimensional tryptic phosphopeptide mapping of both proteins revealed that sites phosphorylated by CaM kinase II in vitro, while distinct from sites phosphorylated by protein kinase A in vitro, were largely homologous to those sites phosphorylated in situ upon incubation of the βTC3 cells with increased free calcium. Immunofluorescence verified expression of both proteins in βTC3 cells and pancreatic slices, however, synapsin I exhibited little co-localization with insulin containing dense core granules as demonstrated by immunogold electron microscopy. These data provide evidence that MAP-2 and synapsin I are phosphorylated by CaM kinase II in the pancreatic β-cell in situ. While the data suggest that synapsin I may not be involved in insulin secretion, an association with other known microvesicles of the β-cell, similarly secreted, may be possible. The phosphorylation of these CaM kinase II substrates may reveal an important intermediate step in the mediation of the glucose response in the pancreatic β-cell.Item Vagotonic Effects of Enkephalin Are Not Mediated by Sympatholytic Mechanisms(2005-05-01) Barlow, Matthew A.; James L. Caffrey; H. Fred Downey; Glenn DillonBarlow, Matthew A., Vagotonic Effects of Enkephalin are not Mediated by Sympatholytic Mechanisms. Master of Science (Biomedical Sciences), May, 2005, 50pp. 1 table, 10 illustrations, bibliography. This study examined the hypothesis that the vagotonic and sympatholytic effects of cardiac enkephalins are independently mediated by different receptors. In study one, the heart rate response to increasing doses of the δ-receptor opioid, MEAP was determined during nerve stimulation. MEAP was administered by microdialysis into the interstitium of the canine sinoatrial node during sympathetic (right ansa subclavia) and parasympathetic (right cervical vagus) stimulation. This protocol was conducted to illustrate that the vagotonic effect of MEAP was independent of any simultaneous sympatholytic activity that might be intrinsic to MEAP. The right cardiac sympathetic nerves were isolated as they exited the stellate ganglion and were stimulated at frequencies selected to produce an intermediate increase in heart rate (HR) of approximately 35 bpm. The cervical vagi were ligated and the right vagus nerve was stimulated at frequencies related to produce a two-step decline in heart rate of approximately 25 and 50 bpm. Dose response relationships were constructed by recording the change in heart rate during nerve stimulation as the dose of MEAP was increased in 5-min steps from 0.05 pmoles/min to 1500 pmoles/min. A significant increase in vagal transmission was observed during the administration of δ-agonist, MEAP at 0.5 pmoles/min as evident by a greater decline in heart rate. The sympathetically mediated tachycardia was unaltered at this or any other dose of MEAP. In study two, a similar dose response relationship was constructed with the κ-opioid receptor agonist, U-50488H to illustrate an independent sympatholytic effect and to verify its κ-receptor character. U-50488H gradually suppressed the sympathetic tachycardia with a significant effect obtained at the highest dose (1500 pmoles/min). U-50488H had no effect on vagally mediated decreases in heart rate. Surprisingly the sympatholytic effect of U-50488H was not reversed by withdrawal of the agonist or by addition of the κ-antagonist, norBNI. Study three was conducted to determine whether the sympatholytic effect to U-50488H could be prevented by co-administration of norBNI. NorBNI blocked the sympatholytic effect of the U-50488H throughout 90 min of exposure. When norBNI was discontinued after 90 min and U-50488H was continued alone, its sympathetic effect reappeared within 30 min. Collectively these observations support the hypothesis that the vagotonic influence of MEAP was independent of sympathetic transmission and sympathetic transmission was unaltered by MEAP. Furthermore the observed sympatholytic effect of U-50488H was mediated independently by κ-receptors. The sympatholytic effect of sustained κ-receptor stimulation appears to evolve gradually into a functional state not easily reversed.