• Androgenic maintenance of rat penile erection

      Reily, Christopher M.; School of Graduate Studies (1997-06)
    • Angiotensin II Regulation of Aldosterone Synthase

      Nogueira, Edson da F.; Department of Physiology (2009-07)
      Angiotensin II (Ang II) is the major physiological regulator of aldosterone production acting acutely to stimulate aldosterone biosynthesis and chronically to increase the capacity of the adrenals to produce aldosterone. Aldosterone is principally synthesized in the zona glomerulosa of the adrenal by a series of enzymatic reactions leading to the conversion of cholesterol to aldosterone. The major goal of our study was to define the Ang II-induced mechanisms regulating the expression of aldosterone synthase (CYP11B2) in adrenocortical cells. We approached the analysis of the protein synthesis-dependent regulation of this enzyme by defining, through microarray and real time PCR analysis, the transcription factors that are rapidly induced by Ang II incubation of adrenocortical cell models from three species (human, bovine, and rat). The gene list generated by this comparison included: ATF3, BTG2, NR4A1, NR4A2, NR4A3, EGR1, FOS, FOSB, and JUNB. Importantly, pretreatment of H295R cells with cycloheximide had no effect on Ang II induction of these genes, suggesting that they are direct targets of Ang II signaling. Co-transfection studies, used to investigate the role of these transcription factors in the regulation of CYP11B2, determined that out of the nine transcription factors listed above, only the NGFI-B family members (NGFI-B, NURR1, and NOR1) increased expression of CYP11B2. The importance of NGFI-B in the regulation of CYP11B2 was confirmed by the decrease in CYP11B2 expression in the presence of a dominant-negative (DN)- NGFI-B. A pharmacological approach used to characterize the Ang II pathways regulating transcription of NGFI-B family genes suggested that Ang II binding to the AT1R increases activity of protein kinase C (PKC), Ca -dependent calmodulin kinases (CaMK), and SRC kinase (SRC), which act to regulate the expression of the family of NGFI-B genes as well as CYP11B2. In the current study we also analyzed protein synthesis-independent mechanisms regulating CYP11B2 expression. We studied the role of the ATF/CREB family of transcription factors (ATF1, ATF2, CREB, and CREM), which may bind the cAMP response element (CRE) in the promoter region of the CYP11B2 gene. Importantly, analysis of these transcription factors in the human H295R adrenocortical cell line revealed very low expression of CREB in comparison to the other CRE-binding proteins herein studied. We investigated Ang II-induced phosphorylation of these transcription factors, their binding to the promoter region of CYP11B2, and their effect on CYP11B2 expression. Ang II time-dependently induced phosphorylation of ATF1, ATF2, and CREM in H295R cells. The association of these transcription factors with the CYP11B2 promoter region was induced by Ang II and K+. Transfection of siRNA for ATF1, ATF2, and CREM significantly reduced CYP11B2 expression in Ang II-stimulated conditions. Expression of NURR-1 alone or with constitutively active ATF1, ATF2, CREB, and CREM increased the promoter activity of CYP11B2 in H295R cells. In summary, Ang II rapidly induces expression of newly synthesized transcription factors as well as the phosphorylation of transcription factors already present in the adrenocortical cell. These events are followed by increased CYP11B2 expression and, therefore, represent important mechanisms to increase the adrenal capacity to produce aldosterone.
    • Angiotensin II Signaling Mechanisms Involved in the Elevation of Arginase Activity/Expression and Vascular Dysfunction

      Shatanawi, Alia; Department of Pharmacology and Toxicology (2011-11)
      Vascular endothelial dysfunction is a major cause of morbidity and mortality in patients with cardiovascular diseases such as hypertension, atherosclerosis and diabetes. Nitric oxide (NO) produced by endothelial nitric oxide synthase (NOS) is needed for normal vascular function. During hypertension, diabetes or atherosclerosis, elevated levels of arginase can compete with NOS for available L-arginine thus reducing vascular NO production. Elevated angiotensin II (Ang II) is a key participant of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. In this study we explored the signaling pathway leading to increased arginase expression/activity in responses to Ang II in bovine aortic endothelial cells (BAEC). Treatment of BAEC with Ang II (10-7 M, 24 hrs) caused a 40±6% increase in arginase activity. This was accompanied by 30±8% decrease in NO production. Our studies indicate involvement of the RhoA/ROCK-p38 mitogen activated protein kinase (MAPK) in Ang II-induced arginase upregulation and reduced NO production, as inhibitors of ROCK or p38 MAPK prevented the Ang II-induced increase in arginase activity. Our studies in mice also show involvement of p38 MAPK in Ang II-induced vascular dysfunction associated with elevated arginase activity and expression. Ang II (42 μg/kg/h) caused impaired EC-dependent vasorelaxation in mouse aorta (55±7% vs. 75±8% for control). This impairment was prevented by treatment with p38 inhibitor SB203580 (5 μg/kg/day). Ang II also caused a 6.2 fold increase in vascular arginase activity/expression that was completely prevented by p38 MAPK inhibition. Additionally, treatment of BAEC with Ang II causes phosphorylation of activating transcription factor-2 (ATF-2) and enhancement of the binding of ATF-2 to arginase promotor through an AP-1 site as evident from electrophoretic mobility shift assay experiments. Transfection of BAEC with ATF-2 siRNA prevents Ang II-induced increases in arginase activity/expression and maintains NO production. These results indicate that ATF-2 is necessary for enhanced expression of arginase by Ang II. Collectively, our results indicate that Ang II increases endothelial arginase activity/expression through a RhoA/ROCK-p38 MAPK-ATF-2 pathway leading to reduced NO production and endothelial dysfunction. Targeting these signaling steps might be therapeutic points for preventing vascular endothelial dysfunction associated with elevated arginase activity/expression.
    • Angiotensin II signaling mechanisms involved in the elevation of arginase activity/expression and vascular dysfunction

      Shatanawi, Alia; School of Graduate Studies (2011-11)
      Vascular endothelial dysfunction is a major cause of morbidity and mortality in · patients with cardiovascular diseases such as hypertension, atherosclerosis and diabetes. Nitric oxide (NO) produced by endothelial nitric oxide synthase (NOS) is· needed for norm.al vascular function. During hypertension, diabetes or -atherosclerosis, elevated levels of arginase can compete with NOS for available L-arginine thus reducing vascular NO production. Elevated angiotensin II (Ang II) is a key participant of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. In this· study we explored the signaling pathway leading to increased arginase expression/activity in responses to Ang II.in bovine aortic endothelial cells (BAEC). Treatment of BAEC with Ang II (10-7 _M, 24 hrs) caused a 40±6% increase in arginase activity. This was accompanied by 30±8% decrease in NO production. Our studies indicate involvement of the RhoA/ROCK-p38 mitogen activated protein kinase (MAPK) in Ang 11.,induced arginase upregulation and reduced NO production, as inhibitors of ROCK or p38 MAPK prevented the Ang II-induced increase in arginase activity. Our studies in mice also show involvement of p38 MAPK in Ang II-induced vascular dysfunction associated with elevated arginase activity and expression. Ang 11 (42 · μg/kg/h) caused impaired EC-dependeht vasorelaxation in mouse . aorta (55±7~ vs. -75±8% for control}. This ·impairment was prevented by treatment with p38 inhibitor S8203580. (5 μg/kg/day). Ang II also caused a 6.2 fold increase in vascular arginase activity/expression that was complete_ly prevented by p38 MAPK inhibition. Additionally, treatment of BAEC with Ang II causes phosphorylation of activating transcription factor-2 (ATF-2) and -enhancement of the binding of ATF-2 to arginase promoter through an AP-1 site . as evident from electrophoretic mobility shift assay experiments. Transfection of BAEC with ATF-2 siRNA prevents Ang II-induced increases in arginase activity/expression and maintains NO production. These results indicate that. ATF-2 is necessary for enhanced expression of arginase by Ang II. Collectively, our results indicate that Ang 11 increases endothelial arginase activity/expression through a RhoA/ROCK-p38 MAPK-ATF-2 pathway leading to reduced NO production and endothelial dysfunction. Targeting these signaling steps might be therapeutic points for preventing vascular endothelial dysfunction_ associated with · elevated arginase activity/expression.
    • Angiotensin II-Induced Protein Kinase D Activation and Regulation of Aldosterone Production

      Olala, Lawrence O.; Department of Pathology (2013-02)
      Dysregulated aldosterone production leading to hypertension and its associated complications, such as congestive heart failure, cardiac fibrosis and renal failure, are important public health concerns with a huge impact on the economy and patient quality of life. Thus, there is a high level of interest in the development of medical interventions and lifestyle changes to reduce the incidence of hypertension. Stimulation of the adrenal zona glomerulosa with angiotensin II (AngII), potassium (K+) or adrenocorticotropic hormone (ACTH), increases aldosterone production, to result in increased sodium and water retention. We have recently shown a role for the serine/threonine protein kinase D (PKD) in the regulation of acute aldosterone synthesis upon AngII stimulation. In this study, using both molecular and pharmacological approaches, we demonstrate that Src family kinases and protein kinase C (PKC) activate PKD to increase aldosterone production in bovine adrenal glomerulosa cells. We have also shown that PKD positively regulates expression of steroidogenic acute regulatory (StAR) protein, a protein required for cholesterol transport into the mitochondria, and aldosterone synthesis. PKD plays this role, in part, through activating members of the activating transcription factor (ATF)/cAMP response element (CRE-) binding protein (CREB) family of leucine zipper transcription factors. Therefore, we hypothesize that PKC and Src family kinase-mediated PKD activation in response to AngII increases the phosphorylation and activation of ATF-2 and CREB, which bind the StAR proximal promoter thereby resulting in induction of StAR expression and stimulation of steroidogenesis.
    • Anterograde and Retrograde Regulation of Neuromuscular Junction Formation and Aging

      Zhao, Kai; Department of Neuroscience and Regenerative Medicine (2018-11-29)
      The neuromuscular junction (NMJ) is a chemical synapse that facilitates the neuronal control of muscle contraction. Proper NMJ formation and maintenance require both anterograde and retrograde signaling. In this study, on one hand, we characterized the role of Yes-associated protein (Yap) in the formation of neuromuscular junction (NMJ). In HSA-Yap-/- mice where Yap was mutated specifically in muscle cells, AChR clusters were smaller and distributed in a broader region in the middle of muscle fibers. In addition, HSA-Yap-/- mice also exhibited remarkable presynaptic deficits including less nerve coverage of the endplates, reduced mEPP frequency and increased paired-pulse facilitation, indicating structural and functional defects. Moreover, muscle Yap mutation prevented reinnervation of denervated muscle fibers and the phenotypes were related to compromised β-catenin signaling. Both NMJ formation and regeneration deficits of HSA-Yap-/- mice were ameliorated by inhibiting β-catenin degradation, further corroborating a role of β-catenin as a downstream molecule of Yap to regulate NMJ formation and regeneration. On the other hand, we showed that Lrp4, a receptor for agrin and critical for NMJ formation and maintenance, was reduced at the protein level in aged mice, which was associated with decreased MuSK tyrosine phosphorylation, suggesting compromised agrin-Lrp4-MuSK signaling in aged muscles. Transgenic expression of Lrp4 in muscles alleviated AChR fragmentation and denervation and improved neuromuscular transmission in aged mice. Lrp4 ubiquitination was augmented in aged muscles, suggesting increased Lrp4 degradation as a mechanism for the reduced protein level. We also found that sarcoglycan alpha (SGα) interacted with Lrp4 and delayed Lrp4 degradation in co-transfected HEK293 cells. AAV9-mediated expression of SGα in muscles mitigated Lrp4 degradation and NMJ decline in aged mice. These observations support a model where compromised agrin-Lrp4-MuSK signaling serves as a pathological mechanism of age-related NMJ decline and identify a novel function of SGα in stabilizing Lrp4 for NMJ maintenance in aged mice.
    • ANTI-INFLAMMATORY ROLE OF 17β-ESTRADIOL IN THE BRAIN

      Thakkar, Roshni Dinesh; Department of Neuroscience and Regenerative Medicine (2017)
      17β-estradiol (E2) is a well-known neuroprotective hormone, but its role in regulation of neuroinflammation is less understood. In the current study, we examined whether E2, acting via PELP1, can exert anti-inflammatory effects in the ovariectomized rat and mouse hippocampus to regulate NLRP3 inflammasome activation, cytokine production and microglial M1/M2 phenotype after global cerebral ischemia (GCI). The results showed that activation of the NLRP3 inflammasome pathway and expression of its downstream products, cleaved caspase-1, and IL-1β, are temporally increased in the hippocampus after GCI, with peak levels observed at 6-7 days. E2 robustly inhibited NLRP3 inflammasome pathway activation, caspase-1 and pro-inflammatory cytokine production, as well as gliosis after GCI at gene as well as protein levels. Moreover, E2 also profoundly suppressed the pro-inflammatory M1 microglial phenotype, while increasing the anti-inflammatory M2 microglial phenotype after GCI. Intriguingly, the ability of E2 to exert all of these anti-inflammatory effects was lost in PELP1 forebrain-specific knockout mice. These robust effects of E2 may be mediated directly upon microglia, as we found that E2 suppressed the M1 while enhancing the M2 microglia phenotype in LPS-activated BV2 microglia cells. Furthermore, E2 treatment also prevented the neurotoxic effects of BV2 microglia cells upon hippocampal HT-22 neurons, suggesting a novel E2-mediated neuroprotective effect via regulation of microglia activation and phenotype. Mechanistically, E2 strongly suppressed expression and activation of the transcription factor NF-κB in BV2 microglia cells, which is known to be a critical regulator of both microglia pro-inflammatory effects and M1/M2 microglia phenotype. Additional studies revealed that NF-κB inhibition also prevents the cytotoxic effects of BV2 microglia cells upon hippocampal HT-22 neurons. Collectively, our study suggests a novel E2-mediated neuroprotective effect via regulation of inflammasome and microglia activation and promotion of the M2 “anti-inflammatory” phenotype in the brain. KEY WORDS: Estrogen, global cerebral ischemia, NLRP3 inflammasome, microglia phenotype, cytokines, neuroprotection.
    • Antitumor Activity of 3-Phenylacetylamino-2, 6-Piperidinedione and Its Computer Modeled Analogs

      Copeland, John A. III; Department of Physiology and Endocrinology (1992-06)
      N/A
    • APPLICATIONS OF MACHINE LEARNING TO GENOMICS: STUDIES IN TYPE 1 DIABETES AND CANCER

      Tran, Paul; Center for Biotechnology and Genomic Medicine (Augusta University, 2020-04)
      Introduction: A major aim of modern medicine is to translate basic genomics findings using machine learning and other data analysis methods into clinical tests for improving patient care. Herein, I applied machine learning methods to publicly available genetic and genomic data to address three clinical problems in cancer and type 1 diabetes (T1D) research. Project 1: Cancer classification mostly depends on the anatomic pathology workforce; hence, diagnosis is slow, stepwise, and prone to errors and systemic bias. Using a transcriptome-based cancer classification method, I reconciled the 18% disagreement rate between histology and mutation-based classifier for brain cancer. Project 2: I applied the same transcriptome-based classification method to lung adenocarcinoma and identified 3 novel subgroups comprising ~30% of lung adenocarcinoma. Project 3: The estimated genetic heritability of T1D is up to 80%. Identifying those most genetically susceptible to T1D can lead to reduction of the number of islet autoimmunity cases and the number diabetic ketoacidosis episodes. I developed a genetic risk prediction model using neural networks which performs better than currently published methods. I applied model interpretation methods to the neural network and identified important genetic drivers for characterizing T1D molecular subgroups. Conclusion: These projects are small steps in translating genomic medicine projects to clinical applications but represent a future with more objective and automated tools to aid in clinical decision making.
    • Arteriolar Responsiveness In Adrenal Crisis In the Dog

      Brown, Faith Kipp; Department of Physiology (1955)
    • Artificial Chromosome Transgenesis Reveals Long-Distance Negative Regulation of ragl in Zebrafish

      Jessen, Jason R.; Department of Biochemistry and Molecular Biology (1999-11)
      Despite the essential roles played by the recombination activating genes (ragl and rag2) during V(D)J recombination, the mechanisms that restrict their expression to lymphoid cells are undefined. Using a novel approach to achieve artificial chromosome transgenesis in zebrafish, we demonstrate that distal regulatory elements are critical to suppress ragl expression in inappropriate tissues. In contrast to smaller reporter gene constructs, 125 and 75 kb artificial chromosomes containing the zebrafish rag genomic locus directed GFP expression in a pattern reflective of endogenous rag 1. Mapping experiments identified a positive element 5' of ragl that enhances GFP expression in both lymphoid and non-lymphoid tissues and a negative element 5' of ra g l that specifically suppresses GFP expression in the skeletal muscle. Our transgenic zebrafish also express GFP in olfactory neurons which we show represent an authentic ra g l expression site in zebrafish.
    • Arylamidase of human liver : purification and catalytic properties

      Little, Gwynne Hamilton; Department of Cell and Molecular Biology (1970-04)
    • Assessment of Renal Ischemia Reperfusion Induced Injury in Male and Female Rats

      Crislip, Gene Ryan; Department of Physiology (2017)
      Acute kidney injury (AKI) is a clinical problem often induced by ischemia reperfusion (IR). Males are reported to have worse outcomes following IR compared to females based on measurements of blood urea nitrogen and creatinine. However, these markers are produced at different levels depending on body mass. The goal of Aims 1 and 2 was to do a complete assessment of the impact of sex on IR to establish a model that displays a sex difference. We measured multiple markers, including inulin clearance which is the gold standard of determining renal function. We determined there is no sex difference in response to IR after 24 hours. However, males had impaired renal function, higher vascular congestion and tubular injury than females 7 days following IR. A consequence of vascular congestion and tubular injury is fluid leakage into interstitial space, which increases renal volume. The goal of Aim 3 was to determine if ultrasound could be used as a tool to detect progressive changes in regional kidney volume following IR. To do this, we compared renal volume measurements with stereological assessment and examined the use of renal volume as an injury marker following IR. We verified the use of ultrasound to monitor renal volume after IR and the changes in volume correlated with the extent of medullary injury. Limiting vascular congestion improves recovery following IR. Pericytes are contractile cells that line the vessels in the renal medulla that are prone to congestion following IR. The goal of Aim 4 was to determine the role of renal pericytes following IR. To do this, we decreased pericytes in rats before IR to determine if this effected injury. We found that lower pericyte density was associated with greater vascular congestion following IR, additionally, males lose more pericytes than females. From these studies, we concluded that there was no sex difference in IR induced injury after 24 hours, however, following 7 days males had poorer recovery than females. We hypothesize that this poorer recovery is attributed to less pericytes in males following IR resulting in the inability to reduce vascular congestion compared to females.
    • Assessment of the immune status of dialysis of patients following donor specific blood transfusions

      Kapp III, William Karl; Department of Cell and Molecular Biology (1985-06)
    • Attempts to infect poikilothermic animals with the dimorphic fungus, blastomyces dermatitidi

      Kangelos, Marilyn; Department of Cell and Molecular Biology (1965-05)
    • Attenuating the Interaction Between Delta Protein Kinase C and the "d" Subunit of FIFo ATp Synthase Protects Against Cardiac Ischemia/Repferusion injury

      Walker, Matthew; Deparment of Pharmacology and Toxicology (6/3/2016)
      Cardiac ischemia / reperfusion (IR) injury most often results from the thrombotic blockade of the coronary arteries and is the most frequent cause of death in humans. Despite the significant role energy deprivation plays in cardiac IR injury, few studies have targeted the IR-induced impairment of the mitochondrial F1Fo ATP synthase. We have previously demonstrated delta protein kinase C (δPKC) involvement in cardiac myocyte energy deprivation via its interaction with the “d” subunit of F1Fo ATP synthase (dF1Fo) and have developed a peptide inhibitor [NH2YGRKKRQRRMLATRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK- COOH] of this interaction. It targets to the mitochondrial matrix / inner membrane. The inhibitor peptide contains a FLAG epitope which allowed confirmation of its uptake into cardiac mitochondria. Our early studies in neonatal cardiac myocytes (NCMs) led us to the hypothesis that PKC inhibits ATP production in vivo via an interaction with dF1Fo to exacerbate cardiac IR injury. To directly test our hypothesis, we first utilized the Langendorff isolated heart model to show that PKC co-immunoprecipitates (co-IPs) with antisera to dF1Fo in myocardium exposed to simulated IR injury. Administration of the inhibitor peptide to the isolated rat hearts prior to cardiac IR attenuated the co-IP of 􀁇PKC with dF1Fo, improved recovery of contractility, diminished levels of tissue t-carbonyls and 4-hydroxy-2-nonenal (HNE), and reduced myocardial infarct size (as assessed by 2, 3, 5 triphenyltetrazolium chloride (TTC) staining) following simulated IR exposures. Additionally, this peptide enhanced ATP levels 2.1 fold, improved ADP-stimulated mitochondrial respiration, and attenuated Ca++-induced mitochondrial swelling in ischemic myocardium. We next evaluated the inhibitor peptide in an in situ rat coronary ligation model for its ability to protect live rats from cardiac IR injury. A 10 min coronary ligation increased the PKC-dF1Fo co-IP in the region at risk (RAR) by 5-fold which was attenuated by 71% with intravenous infusion of the inhibitor peptide. This response correlated with an enhancement of ATP levels, a 2-fold reduction in oxidative stress markers, improvement in systolic cardiac function, and a reduction in TTC monitored myocardial infarct size in the RAR. These results support further development of this peptide as a first-in-class-translational therapeutic for the treatment of cardiac IR injury.
    • The Autoimmune Regulator (Aire) Confers Immunosuppressive Properties to Dendritic Cells

      Eisenman, Daniel; Center for Biotechnology and Genomic Medicine (2007-05)
      The Autoimmune regulator (Aire) is a transcription factor that controls expression of self antigens by thymic epithelium and it plays a critical role in the deletion of autoreactive thymocytes and prevention of autoimmunity. Recent studies have reported Aire expression in dendritic cells (DC) located in spleen and lymph nodes, suggesting a role for Aire in extra-thymic tolerance induction. Molecular and functional studies conducted in this dissertation revealed that Aire induction in bone marrow derived DC results in expression of immunosuppressive cytokines and decreased expression of co-stimulatory molecules. Similar results were also obtained from lenti-virus-mediated Aire overexpression in the DC2.4 dendritic cell line. It was further shown that DC from Aire'7' mice exhibited greater antigen presenting function both in vitro and in vivo. These DC were more potent stimulators of T cell proliferation leading to increased IL-2 and IFNy production. These studies suggest that Aire7' DC may play a role in exacerbating the autoimmunity seen in Aire7' mice. DC over-expressing Aire were shown to suppress activation and proliferation of naive T cells and promote activation-induced cell death of activated T cells. Furthermore, we demonstrate that Aire also controls transcription of tissue-specific antigens in DC. These results, together, suggest that Aire plays an important role in the tolerogenic function of DC.
    • Autolytic enzymes associated with cell walls of mycobacterium smegmatis

      Kilburn, James Otis; Department of Cell and Molecular Biology (1974-06)