• NAE1-MEDIATED NEDDYLATION IS REQUIRED FOR POSTNATAL LIVER DEVELOPMENT AND FUNCTION

      Sahay, Khushboo; Department of Physiology (7/26/2018)
      Liver disease is an important health concern and a significant source of morbidity and mortality in the United States and worldwide. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is a novel ubiquitin-like protein modifier. The conjugation of NEDD8 to target proteins, termed neddylation, requires NEDD8 specific E1, E2 and E3 ligases. Neddylation participates in various cellular processes. However, whether neddylation regulates liver development and function is completely unknown. We created mice with hepatocyte specific deletion of NAE1, a subunit of the only NEDD8 E1 enzyme, and identified that they display severe hepatomegaly, hypertriglyceridemia, and hypercholesterolemia from 10 days after birth. By postnatal 14 days, their liver cytoarchitecture is completely disrupted, along with formation of numerous biliary cysts, fibrosis and hypoglycemia, which ultimately result in liver failure and premature death by 6 weeks. Mechanistically, NAE1 deficiency in hepatocytes caused reduced hepatocytespecific gene expression but increased biliary/oval cell gene expression in liver. In vitro, NAE1 inhibition by MLN4924 and CRISPR/Cas9-mediated NAE1 deletion in HepG2 cells recapitulated in vivo findings with repressed expression of hepatocyte specific genes but elevated biliary/oval cell gene expression. Together, these data highlight an essential role for neddylation in regulating hepatocyte lineage commitment and function as well as polycyst formation through trans/de-differentiation of hepatocytes.
    • Needle Bore Size and The Degree of Hemolysis During Blood Specimen Acquisition

      Carr, Rebecca Lamb; Department of Physiological and Technological Nursing (1983-05)
      This study examined the degree of detectable hemolysis in- blood specimens _as-pirated with needles'p~ :small and 1.arge bore_ size. A quasiexperimental design was used to test the resea;ch hypothesis that blood . specimens. aspira~ed_~tth a 22~gauge_needle will have a higher degree of -detectable-hemolysis than blood specimens asp:irated .with a 20-gauge needle. Data collectionconsisted of aspirating from each subject - (n=31) one blood-·speci~en--wi-th a 22-gauge-needle and-one-:.blood specimen·: _ _, . ,.,. : . . . . ' . . with a 20~gauge ·needle~- .. Th~- -plasma -hemoglobin df each specimen was . . . . . . '• . mea~ured and data were 'analyzed using the independent t~test~ The - results- indicated- that the mean difference in the plasma hemogl_obin· . ' . . . . . - ·between the two blood specimens w·as not stattstically · si'gnif1cant', -t-hus ·· .. the research hypothesis was not supported .. Thefindi~gs sug\Jested that . . '·· heme lysis was not influenced by the bore si z~ of the needle us.ed during blood specimen acq-uisition.
    • Neonatal Nurses' Knowledge of Their Standards of Practice: A Reflection of Accountability

      Chadwich, Jean; Department of Nursing (1984-02)
      The purpose of this study was to examine the neonatal nurses'- knowledge of standards of nursing practice. It was post~lated the degree to which a nurse understands and accepts her professional . accou~ta.bility depend·s :upon her own understanding of her status~ The level of knowledge was obtained from the investigator's selfdesigned tool. Additionally, the study .examined··whether or not a ' . ' ' . difference existed between the knowledge of nu'rses who care for . . . infants who require varying degree~ of 'nursing care, as evidenced by the t:y-pe of hospital ·they are employed in· und~r :regionalization guide~ lines. It further examined the relationship be~ween ·the personal -· variable of type of basic nursing education with the t·otal and subscale scores on the tool. A descriptive--correlational design was ·used to examine the study data generated from· 61 registered nurses, distri~uted _among 15 hospitals in one Southeastern stat_e. Analysis of the study .data using descriptive stati.stics indicated that the neonatal nurses wereknowledgeable of their standards of practice. Pe~rson correlation coefficients revealed no significant difference exis~ed amongst the nurses in Level I,. II _and III neonatal care facilities, nor was there any correlation between the nurses' educational preparation and their level of knowledge of the standards of practice. Additionally~ Pearson correlation coefficients· and Cronhach' s alpha ·were. used to determine the statistic:al validity and r~liabi.li ty es-timates of the tool •..
    • Neuregulin1 promotes excitatory synapse development specifically in GABAergic interneurons

      Tin, Kin Lai; Department of Neuroscience and Regenerative Medicine (2010-03)
      Neuregulin 1 (NRG1) and its receptor ErbB4 are both susceptibility genes of schizophrenia. However, little is known about the underlying mechanisms of their malfunction. Although ErbB4 is enriched in GABAergic interneurons, the role of NRG1 in excitatory synapse formation in these neurons remains poorly understood. We showed that NRG1 increased both the number and size of PSD- 95 puncta in GABAergic interneurons, indicating that NRG1 stimulates the formation of new synapses and strengthens existing synapses. In contrast, NRG1 treatment had no consistent effect on either the number or size of excitatory synapses in glutamatergic neurons, suggesting its synaptogenic effect is specific to GABAergic interneurons. Ecto-ErbB4 treatment diminished both the number and size of excitatory synapses, suggesting that endogenous NRG1 may be critical for basal synapse formation. NRG1 could stimulate the stability of PSD-95 in the manner that requires tyrosine kinase activity of ErbB4. Finally, deletion of ErbB4 in parvalbumin-positive interneurons led to reduced amplitude of mEPSCs, providing in vivo evidence that ErbB4 is important in postsynaptic differentiation in interneurons. Taken together, our findings suggested a novel synaptogenic role of NRG1 in excitatory synapse development, possibly via stabilizing PSD-95, and this effect is specific to GABAergic interneurons. In light of the association of the genes of both NRG1 and ErbB4 with schizophrenia and dysfunction of GABAergic system in this disorder, these results provide insight into its potential pathological mechanism.
    • Neuregulin3 Regulation of Glutamatergic Transmission

      Figueiredo, Dwight; Department of Neuroscience and Regenerative Medicine (2015-12)
      Synapses are fundamental communication units in the brain, essential for meaningful response to stimuli received from the environment. Abnormal synaptic communication leads to mental disorders. My studies focus on Neuregulin3, a member of the Neuregulin family. Single Nucleotide Polymorphisms (SNPs) within the NRG3 gene are associated with schizophrenia in different populations. Analysis of postmortem human brain samples of schizophrenia patients revealed abnormal levels of NRG3. However, unlike its well-studied family member NRG1, NRG3’s role in synaptic transmission is not understood. I studied how depletion of Nrg3 protein in the brain could affect synaptic transmission. I measured the amplitude and the frequency of spontaneous as well as miniature Excitatory Post Synaptic Currents (sEPSC and mEPSC, respectively) at hippocampal CA1 neurons of GFAP
    • Neuro-vascular Communication in the Hypothalamic Supraoptic Nucleus in Rats. Do nitric oxide and vasopressin play a role?

      Du, Wenting; Department of Physiology (2015-03)
      The classical model of neurovascular coupling (NVC) proposes that activity-dependent synaptically released glutamate dilates arterioles. However, whether this model is also applicable to brain areas that use less conventional neurotransmitters, such as neuropeptides, is currently unknown. To this end, we studied NVC in the hypothalamic magnocellular neurosecretory system (MNS) of the supraoptic nucleus (SON), in which dendritically released vasopressin (VP) can be found. Bath-applied VP significantly constricted SON arterioles via activation of the V ia receptor subtype. Vasoconstriction was also observed in response to single VP neuronal stimulation, an effect prevented by V ia receptor blockade (V2255). Conversely, osmotically-driven magnocellular neurosecretory neuronal population activity leads to a predominant nitric oxide (NO)- mediated vasodilation. Activity-dependent vasodilation was followed by a VP-mediated vasoconstriction, which acted to reset vascular tone. Taken together, our results unveiled a unique and complex form of NVC in the MNS, supporting a competitive balance between activity-dependent dendritic released VP and NO, in the generation of proper NVC responses.
    • Neuron-Glia interaction and role of Nrf2 in hyperhomocysteinemic retina

      Navneet, Soumya; Biomedical Sciences (Augusta University, 2019-05)
      Elevated level of the excitatory amino acid homocysteine (Hcy) or hyperhomocysteinemia (Hhcy) has been reported in patients with glaucoma, a disease characterized by increased oxidative stress and retinal ganglion cell (RGC) degeneration. Whether Hhcy is causative or merely a biomarker for glaucoma is not known. Primary RGCs exhibit acute sensitivity to Hcy exposure, while in vivo murine models of Hhcy demonstrate a more modest RGC loss (∼20%) over a period of several months. This differential response to Hhcy in isolated cells and the intact retina suggests the presence a buffering mechanism invoked by the retinal milieu. Oxidative stress has been implicated as a mechanism of Hcy-induced neuronal loss. Owing to the key role of Müller glial cells (MCs) in retinal antioxidant defense we hypothesized that MCs protect RGCs under conditions of Hhcy via the NRF2 antioxidant pathway. Compared to RGCs, MCs were less sensitive to Hcy. Hcy exposure increased oxidative stress and induced apoptosis in RGCs, whereas in MCs Hcy evoked several cytoprotective responses including reduced oxidative stress, increased antioxidant levels and improved mitochondrial function. Hcy upregulated the expression of Nrf2 and several downstream antioxidant targets including glutathione in MCs. To investigate the role of NRF2 in Hcy-induced RGC degeneration, we crossed Nrf2-/- mice with two mouse models of chronic Hhcy (Cbs+/- and Mthfr+/- mice) and generated Cbs+/-Nrf2-/- and Mthfr+/-Nrf2-/- mouse models and analyzed their retinas. Absence of NRF2 reduced inner retinal thickness and visual acuity, accelerated RGC loss and increased gliosis in Hhcy mice. To understand the role of Müller glial specific NRF2 in RGC survival, we established an ex-vivo indirect co-culture system using primary RGCs and MCs. Apoptosis induced by Hcy exposure in primary RGCs were abrogated when the RGCs were co-cultured with wild type (WT) MCs but not with Nrf2-/- MCs. Hhcy induced robust mitochondrial and glycolytic response in WT MCs, but not in Nrf2-/- MCs. Altogether, the in vivo and in vitro data here suggest that the deleterious effects of Hhcy on RGCs are likely dependent upon the health of retinal glial cells and the availability of retinal antioxidant response mechanism.
    • Neuronal and Astroglial Injury and Recovery from Stroke-Induced Depolarizations

      Risher, William Christopher; Department of Neurosurgery (2010-12)
    • Neurotransmitter Gases as Modulators of GnRH and the Preovulatory LH Surge

      Lamar, Charisee; Institute of Molecular Medicine and Genetics (1998-04)
      Until recently nitric oxide (NO) and carbon monoxide (CO) were viewed only as toxic substances. However, there has been a substantial amount of evidence in the past decade that has redefined these gaseous molecules as physiological messengers. Along these lines, NO and CO are now recognized as modulators of immunological defense, vasodilation, endocrine signaling, and neurotransmission (1-8). As neurotransmitters, NO and CO are unique when compared to classical neurotransmitters. For instance, unlike all other classical neurotransmitters NO and CO are, 1) lipophilic gases with short half lives, 2) not stored in synaptic vesicles, 3) their effects are not mediated through classic receptor proteins - rather their effects result from NO and CO binding to the heme moiety of heme-proteins such as guanylate cyclase and cyclooxygenase, and 4) their effects are terminated by diffusion from target tissues ( 1,2,5,7,9). Production of NO and CO relies on the activity of the enzymes nitric oxide synthase (NOS) and heme oxygenase (HO), respectively. NOS uses the substrate Larginine to generate NO (2, 10), while HO uses the substrate heme to generate CO (9,11-16). NOS exists as three isoforms, macrophage NOS (mNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) (17-21). HO also exists as three isoforms, heme oxygenase-1 (HO-1), heme oxygenase-2 (HO-2), and heme oxygenase-3 (HO-3) HO-1 is inducible, while HO-2 and HO-3 are constitutive enzymes; however, HO-3 is currently viewed as a poor heme catalyst (12,13,22). There is a growing body of evidence that suggests that NO and CO regulate hypothalamic function. For example, recent studies have shown that the hypothalamus produces significant quantities of NO, primarily through the actions of nNOS (23,24). Likewise, the hypothalamus possesses one of the largest production rates of CO in the brain (25,26). That NO and CO can regulate neurohormone release from the hypothalamus is evidenced by findings demonstrating that NO and CO regulate corticotropin-releasing hormone (CRH) (27-33), vasopressin (29-31,34,35), and oxytocin secretion (29,36). With respect to reproductive function, numerous studies (23,37-45) have provided evidence for a significant role of NO in the control of the hypothalamic releasing factor, gonadotropin-releasing hormone (GnRH). For instance, it has been demonstrated (37,46,47) that NO neurons are located in close proximity to GnRH generating cells bodies in the hypothalamus and thus strategically located to exert regulatory effects over GnRH neurons. That NO can exert regulatory effects on GnRH neurons was demonstrated in studies where exogenously applied NO markedly stimulated GnRH release from male hypothalamic fragments (1,48,49) and immortalized GnRH (GT1-7) cells in vitro (1,48). A physiological role for NO in the steroid-induced luteinizing hormone (LH) surge has been suggested based on studies in which the LH surge was attenuated by the administration of NOS inhibitors (43,44) or NOS antisense oligonucleotides (38) No studies, however, have reported nNOS mRNA, protein, or NOS activity levels in the hypothalamus during the LH surge so as to verify that an increased NO tone actually occurs during this critical time. To address this deficit, Aim 1 of this study was designed to examine NO tone on proestrus in the cycling female rat-the day the natural preovulatory LH surge occurs. Since heme oxygenase, the enzyme that produces CO from heme molecule metabolism, is located in the hypothalamus (2,11,12,14,15), and CO production in the hypothalamus is one of the highest in the brain (25,26), it is conceivable that CO could play a role in regulating GnRH secretion. This possibility has not been investigated and thus studies on this issue appear warranted. Therefore, Aims 2-4 of this study were designed to assess the possible role of CO in the control of GnRH and LH secretion.
    • Neurotransmitter receptor binding and protein phosphorylation in the rabbit iris smooth muscle

      Taft, William C; Department of Cell and Molecular Biology (1982-06)
    • Neutrophil function studies with respect to antibiotic tolerant staphylococcus aureus and patients with recurring infection

      Raynor, Robert H; Depatment of Cell and Molecular Biology (1980-08)
      Polymorphonuclear neutrophils (PMNs) from s~ven patients who experien<;: ed recurring staphylococcal infections, were tested in order to establish if measurements of phagocytic and bactericidal capacity would reveal a functional cause for the repeated episodes of disease. Using a microprocedure developed for this 'purpose, the results for six of these patients were found to be comparable to normal values. PMNs from one patient, however, showed a deficiency in their ability to both phagocytize and kill s. aureus. This defect could not be attributed to serum deficiencies or ascribed to any previously defined class of nel.ltrophil function. In other studies, six clinical isolates of Staphylococcus aureus were compared for their relative susceptibilities to the killing effects of oxacillin. Three of the strains had minimum bactericidal concentrations which were >10 times the minimum bacteriostatic concentration for this antibiotic and were designated tolerant (Tol+). The other st;r-ains had minimum bactericidal concentrations which were comparable to the minimum bacteriostatic concentration (Tol-). Lysis curves of these strains revealed that the Tal+ strains exhibited a diminished rate of lysis when inhibited by oxacillin. This reduced rate of lysis was reflected also in a reduced rate of viability loss when the cells were exposed to oxacillin. Cells of each phenotype, previously labeled with [ 14C]Glycerol, secreted radioactivity when inhibited by oxacillin. However,. the Tal+ strains rele.ased over twice as much labe 1 as the Talstrains. The behavior of 60 to 65%_ of the labeled material released by. inhibited cells during both sodium dodecyl sulfate gel electrophoresis and Sepharose 6B chromatography corresponded to that of lipoteichoic acid. When the major component of secreted material was added to oxacillin-inhibited Tol- strains, an inhibition of the lytic response was observed. These results suggest that oxacillin tolerance in S. aureus could be related to the enhanced secretion of an autolysin inhibitor, such as lipoteichoic acid. Several investigators have recently reported an increase in the severity of infections due to Tol+ strains. Since the enhanced'excretion of LTA by Tol+ staphylococci is the only knownphenotypJ.c difference between these two strains, the effect of this molecule on neutrophil function was measured using the microprocedure. The addition of LTA to the incubation mix·ture res1..1lted in a dose dependent inhibition of phagocytosis •. The decrease~ uptake of. S. aureus in the presence of LTA was accompanied by a corresponding increase in the number of organisms surviving in the presence of the phagocytes. Additional studies suggested that LTA interferes with the process' of opsonization, prior to engulfment.
    • A New Method For Analyzing 1:N Matched Case Control Studies With Incomplete Data

      Jin, Chan; Department of Biostatisctics and Epidemiology (5/8/2017)
      1:n matched case-control studies are commonly used to evaluate the association between the exposure to a risk factor and a disease, where one case is matched to up till n controls. The odds ratio is typically used to quantify such association. Difficulties in estimating the true odds ratio arise, when the exposure status is unknown for at least one individual in a group. In the case where the exposure status is known for all individuals in a group, the true odds ratio is estimated as the ratio of the counts in the discordant cells of the observed two-by-two table. In the case where all data are independent, the odds ratio is estimated using the cross-product ratio from the observed table. Conditional logistic regression estimates are used for incomplete matching data. In this dissertation we suggest a simple method for estimating the odds ratio when the sample consists of a combination of paired and unpaired observations, with 1:n matching. This method uses a weighted average of the odds ratio calculations described above. This dissertation compares the new method to existing methods via simulation.
    • Nitric Oxide Synthase Regulation in Inner Medullary Collecting Duct Cells

      Cai, Zheqing; Department of Internal Medicine (2001-06)
      Nitric oxide (NO) is a key regulator of sodium and water excretion in the kidney. It has been shown that renal tubules contain abundant nitric oxide synthase (NOS); however, little is known about the regulation of NOS expression and NOS activity in renal tubular cells. In the renal medulla, collecting duct cells produce a high level of endothelin-1 (ET-1), express caveolin-1 and protein tyrosine kinases (PTKs), and under certain conditions are exposed to high flows, resulting in an increased shear stress. In the current study, we hypothesize that ET-1 regulates expression of NOS isoform(s) and NOS activity is modulated by caveolin-1, tyrosine phosphorylation and shear stress. Western blot analysis and immunofluorescent staining showed that all three NOS isoforms were shown to be present in inner medullary collecting duct (IMCD) cells, a mouse IMCD cell line. After the IMCD cells were treated with 50 nM ET-1, NOS 1 was significantly and specifically increased, but not NOS 2 and NOS 3 expression. ET-1 also increased phosphorylation of p42/p44 MAPK in the IMCD cells. Genistein, a protein tyrosine kinase inhibitor, and PD 98059, a Mekl inhibitor, reduced the effects of ET-1 on phosphorylation of p42/p44 MAPK and up-regulation of NOS 1; furthermore, the ETA receptor antagonist, A 127722, rather than the ETB receptor antagonist, A 192621. inhibited the ET-1 effects in a concentration-dependent manner. The IMCD cells also Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. express caveolin-1, but none of the NOS isoforms appear to be associated with caveolin-1 by co-immunoprecipitation experiments, suggesting that caveolin-1 does not regulate NOS activity in the IMCD cells. NOS I is regulated by tyrosine phosphorylation and is shown to be phosphorylated at basal conditions. The non-specific inhibition of protein tyrosine kinases with 100 pM erbstatin A significantly increased nitrite production in the IMCD cell media. The tyrosine phosphorylation of NOS 1 was reduced by erbstatin A, and enhanced by vanadate, a protein tyrosine phosphatase inhibitor. When the IMCD cells were exposed to three levels of shear stress, 30, 10, 3.3 dyn/cnr for 1 hour, a significant increase in nitrite production was detected. L-NAME, a non-specific NOS inhibitor, completely blocked the effect of shear stress on nitrite production in IMCD cells. Therefore, in IMCD cells, NOS1 expression is up-regulated by ET-1 through activation of the ETA receptor and p42/p44 MAPK pathway; NO production is stimulated by tyrosine dephosphorylation, and activated by shear stress, but does not appear to be regulated by caveolin-1.
    • Nitroxyl anion as a novel relaxant molecule in the rat pudendal artery and metformin as treatment for angiotensin II-induced erectile dysfunction

      Labazi, Hicham; Department of Physiology (2012)
      Cardiovascular diseases, which are the leading cause of illness and death in the United States, frequently share similar risk factors (hypertension, smoking, aging, etc…) as erectile dysfunction (ED). Hypertension is an important risk factor for both heart failure and ED; hypertension and ED are closely intertwined diseases, which have vascular and endothelial dysfunction as a common cause. Recently it has been shown that ED is an independent predictor of cardiovascular diseases (CVD). Thus, studying mechanisms of erectile function and ED will be of great importance for developing treatments for ED, as well as reducing the burden of CVD. In this dissertation, the mechanisms of angiotensin II (AngII)-induced ED was investigated. In addition, we also investigated the effect of metformin on erectile function in an AngII-hypertensive model of ED. We hypothesized that AngII infusion for 4 weeks results in ED and treatment with metformin improves erectile function in the AngII-infused rats. We observed that AngII infusion resulted in ED, which was accompanied by an increased contraction and decreased relaxation response of the corpus cavernosum and pudendal arteries. Furthermore, it was observed that ERK1/2 activation contributes to ED in the AngIIhypertensive model of ED. Treatment with metformin restored erectile function in AngIIhypertensive rats, with a reversal of the increased contractility and decreased relaxation seen in both the corpus cavernosum and pudendal arteries of the hypertensive rats. Our data suggest that metformin may have potential therapeutic effects in ED, independent of its anti-diabetic effects. Additionally we investigated the contribution of nitroxyl anion (HNO), a NO congener, to erectile function. We observed that the endothelium mediated relaxation in pudendal arteries was partially mediated through HNO, and that this relaxation was soluble guanylyl cyclase-dependent (sGC), resulting in activation of voltage-dependent potassium channels (KV +) and large conductance calcium-activated potassium channel (BKCa). The identification of this novel pathway will enhance our understanding of erectile function and possibly allow for development of therapeutic agents for the treatment of ED.
    • NLRP3 Inflammasome-Mediated Uncoupling of Hippocampal Vasoneuronal Communications in Diabetes: Relevance to Cognitive Impairment and Stroke

      Ward, Rebecca; Department of Neuroscience and Regenerative Medicine (5/22/2018)
      Diabetes is a prevalent chronic disease that affects over 29 million individuals in the United States and 422 million people worldwide as of 2017. Given the high mortality and morbidity associated with diabetes due to its complications including retinopathy, chronic kidney disease, peripheral neuropathy, heart disease and stroke, this increase in incidence of diabetes creates many clinical, social and economic problems. A silent, but unrecognized complication of diabetes is cognitive impairment, which ranges from mild cognitive impairment to dementia. The increased risk and incidence of stroke amplifies these cognitive deficits. The objectives of this dissertation were to 1) determine the role and mechanism(s) by which diabetes worsens cognitive decline and 2) determine the extent and mechanism by which NLRP3 activation contributes to poor cognitive function after stroke in diabetes. To investigate these objectives, feeding rats a high fat diet and administering a low dose of streptozotocin was used as a clinically relevant diet-enhanced model of diabetes. Stroke was induced through either transient MCAO (60 or 90 min) or embolic MCAO. Embolic stroke caused more severe hippocampal neurovascular injury, microglial activation and cognitive decline in diabetes as compared to stroke induced by a shorter 60 min suture occlusion of the MCA. Diabetic females were more sensitive to ischemic injury than males. Furthermore, hippocampal vascularization patterns at baseline and after ischemic injury differed in males and females and despite these sex differences in the extent and patterns of hippocampal neurovascular injury, diabetes worsened cognitive outcomes in both sexes. Collectively, these first studies provide a preclinical foundation for future studies addressing cognitive impairment in diabetes in both sexes. NLRP3 inflammasome, which cleaves IL-1β and IL-18 into their active forms, was upregulated in diabetes and amplified following ischemia. Inhibition of the NLRP3 inflammasome with MCC950, a specific small molecule inhibitor of NLRP3 activation, improved post-stroke cognition, reduced hippocampal cell death, was associated with less leaky vasculature, and blunted chronic inflammation in the hippocampus after 90-min MCAO. MCC950 did not seem to provide neuroprotection to the neuron through the mBDNF, but did reduce cell death after hypoxia/reoxygenation in vitro. These results are the first to provide essential data showing that MCC950 has the potential to become a therapeutic agent to prevent neurovascular remodeling and worsened cognitive decline in diabetic patients following stroke. Collectively, this work may provide a piece of the puzzle explaining how diabetes leads to cognitive impairment and worsens outcome following acute ischemic injury, and it provides a potential therapeutic target to treat cognitive impairment after stroke, especially in diabetic patients.
    • Notch3 Signaling Mediates Heterotypic Cell Interactions During Blood Vessel Formation

      Liu, Hua; Vascular Biology Center (2010-11)
      Blood vessel formation is essential for embryogenesis, wound healing, menstruation, and pregnancy [1, 2]. While much emphasis has been placed on understanding the initial event of endothelial-tube formation, relatively little attention has been paid to the interactions of endothelial cells and the surrounding mural cells (pericytes, smooth muscle cells and fibroblasts). Increasing evidence suggests that the communication of endothelial cells and mural cells is crucial for the assembly, subsequent maturation, and stabilization of blood vessels [3-5]. Abnormal interactions between these two cell types have been implicated in many pathological conditions, including tumor angiogenesis, diabetic microangiopathy, tissue calcification and stroke. However, the molecules mediating the heterotypic interaction are still largely unknown. Our previous studies have shown that in a three-dimensional (3-D) angiogenesis assay, mural cells enhance blood vessel formation and directly interact with endothelial cells [6]. During this process, Notch3 is one gene that is strongly induced in mural cells upon coculture with endothelial cells [6]. Notch3, the causative gene of the neurovascular disorder CADASIL [7], belongs to an evolutionarily conserved family of transmembrane receptors that are known to govern cell fate determination in diverse cell types [8]. Given that Notch receptors and ligands are expressed on both endothelial and mural cells and - 2 - Notch3 is upregulated in mural cells by coculturing with endothelial cells, it is reasonable to assume that the Notch3 receptor might regulate the association of endothelial and mural cells through receptor-ligand interaction during blood vessel formation. The goal of my thesis is to investigate how Notch3 gene expression is regulated in mural cells by endothelial cells and whether the Notch3 receptor is involved in the communication between endothelial and mural cells during blood vessel formation. To achieve these goals, three aims were proposed: Specific Aim 1: To define how Notch3 expression in mural cells is upregulated by endothelial cells. Specific Aim 2: To determine if endothelial cell-induced Notch3 expression is critical for mural cell differentiation. Specific Aim 3: To determine whether Notch3 expression in mural cells modulates blood vessel formation under both physiological and pathological conditions.
    • A Novel Function of ADP-Ribosylation Factor 1 in Prostate Cancer Cell Proliferation through Activating the Mitogen-Activated Protein Kinase Pathway

      Davis, Jason E.; Department of Pharmacology and Toxicology (8/23/2016)
      The enhanced activation of the mitogen-activated protein kinase (MAPK) Raf-MEK-ERK1/2 pathway directly correlates with the growth, androgen-independence, and poor prognosis of prostate cancer. However, the underlying molecular mechanisms remain poorly understood. Here, we have demonstrated that ADP-ribosylation factor 1 (ARF1), a Ras-like small GTPase, was highly expressed in human prostate cancer cells and tissues. In addition, ARF1 was markedly activated in prostate cancer cells. More interestingly, oncogenic G protein-coupled receptors (GPCRs) strongly activated ARF1 and the activation was mediated through Gβγ subunits. These data indicate that GPCRs and heterotrimeric G proteins are the upstream activators of ARF1 in prostate cancer cells. Next, we determined the role of ARF1 in the MAPK activation and proliferation in prostate cancer cells. Lentiviral-mediated overexpression of ARF1 remarkably enhanced, whereas shRNA-mediated depletion of ARF1 dramatically reduced ERK1/2 activation in prostate cancer cells. In addition, disruption of both the Golgi localization of ARF1 and the Golgi structure substantially attenuated ERK1/2 activation in prostate cancer cells. In parallel with their effects on the MAPK activation, ARF1 overexpression greatly enhanced and ARF1 knockdown inhibited the proliferation of prostate cancer cells. These data suggest that ARF1, by its ability to activate the MAPK pathway likely at the Golgi, controls prostate cancer cell proliferation. We then investigated the consequence of pharmacologically inhibiting ARF1 activation. Small molecule inhibitors including brefeldin A, golgicide A, and Exo2 that specifically target Golgi-localized ARF1 markedly reduced both ERK1/2 activation and proliferation in prostate cancer cells. These results further indicate an important role of ARF1 activation in regulating the MAPK pathway and prostate cancer cell proliferation. Altogether, our data suggest a possible GPCR-G-ARF1-MAPK signaling pathway, which may be responsible for the hyperactivation of the MAPK ERK1/2 in prostate cancer and contributes to prostate cancer progression. Our results also imply a novel approach for prostate cancer therapy by targeting ARF1 activation
    • Novel Nitric Oxide Synthase-Dependent Mechanism of Vasorelaxation in Small Arteries from Hypertensive Rats

      Kang, Kyu-Tae; Department of Medicine (2007-10)
      Endothelial dysfunction in hypertension is associated with impaired endothelium-dependent vasorelaxation, which is consistently observed in conduit vessels. However, the controversial observation of either impaired or intact vasorelaxation of small resistance arteries from hypertensive animals suggests that the mechanism(s) of endothelium-dependent vasorelaxation in small resistance arteries may be different from that observed in conduit vessels under hypertensive condition. Vasorelaxation in small resistance arteries is mediated via multiple pathways including nitric oxide synthase (NOS)-, cyclooxygenase (COX)-, and endothelium-derived hyperpolarizing factor (EDHF)-mediated pathway. Therefore, the overall goal of these studies was to determine the mechanism(s) involving vasorelaxation of small arteries from hypertensive rats. For these studies, normotensive (NORM), angiotensin II-infused (ANG), high salt (HS), ANG high salt (ANG/HS), placebo, and deoxycorticosterone acetate-salt (DOCA) rats were studied. The studies with pharmacological blockade of each pathway demonstrated that the NOS-dependent component was increased to maintain acetylcholine (ACh)-induced vasorelaxation in small mesenteric arteries from hypertensive rats. Furthermore, increased NOS-dependent pathway appears to compensate for the dysfunctional Ca2+-activated K+ channel-sensitive EDHF pathway in small mesenteric arteries from ANG compared to NORM. These results led us to design further experiments to test the hypothesis that both NO and H2O2 serve as NOS-dependent mediators to maintain vasorelaxation in small mesenteric arteries from hypertensive rats. In small arteries from ANG, ACh increased NOS-dependent cGMP production. ACh also increased NOS3 phosphorylation at Ser 633 and decreased phosphorylation at Thr 495. While, NOS3 phosphorylation at Ser 1177 was impaired in response to ACh in ANG, which was accompanied by reduced basal and a less extended ACh-stimulated cGMP production in ANG compared to NORM. To investigate the alteration of signal transduction pathways related to impaired NOS3 phosphorylation at Ser 1177 in response to ACh, Akt phosphorylation at Ser 473 and VASP phosphorylation at Ser 239 were tested. These pathways were not changed by ACh in the small mesenteric arteries from ANG. Our results indicate that the NO/cGMP signaling is present in response to ACh in small mesenteric arteries from ANG, however this signaling pathway-mediating vasorelaxation may be facilitated via neither Akt nor PKG. On the other hand, ACh stimulated L-NAME-sensitive H2O2 production in small mesenteric arteries from ANG, but not NORM. H2O2 induced vasorelaxation and catalase blunted ACh-mediated vasorelaxation in small mesenteric arteries from ANG. Reduced BH4/BH2 ratio was observed in small mesenteric arteries from ANG compared to NORM, which might be one of the mechanisms of NOS-mediated H2O2 production. Antioxidant enzyme capacity was also determined in small mesenteric arteries from ANG and NORM. Total superoxide dismutase (SOD) activity and protein expression of CuZn SOD and ecSOD were reduced in ANG compared to NORM, while Mn SOD expression was comparable between groups. Interestingly, both activity and expression of catalase were reduced in ANG compared to NORM, whereas GPx activity and expression were not changed. These results indicate that reduced catalase activity and expression may contribute to the augmentation of H2O2 in small mesenteric arteries from ANG, whereas reduced SOD does not greatly influence the H2O2 production in both basal and ACh-stimulated condition. In conclusion, the NOS pathway appears to be the primary endothelium-derived relaxing factor (EDRF) pathway in small mesenteric arteries from experimental animal models of hypertension. The increased dependence on the NOS pathway in ACh-induced vasorelaxation is mediated by both NOS-derived NO/cGMP signaling and NOS-mediated H2O2.
    • Novel Peroxisomal Localization of Sirtuin 3 and its Biological Implications

      Padia, Ravi N.; Department of Biochemistry and Molecular Biology (2014-04)
      Post translational modifications (PTMs) such as phosphorylation, acetylation, ubiquitination, methylation, SUMOylation etc. serve to add another level of complexity and functional diversity to the proteome. These PTMs serve to generate a distinct signal which can then regulate the functionality of the protein harboring one or more of these PTMs and hence ultimately regulating the associated signaling/biological pathway. Some of the ways in which PTMs can bring about the change in the functionality of proteome is by influencing the sub-cellular localization of proteins, protein-protein, protein-DNA, protein-RNA interactions, activity of enzymes, protein turnover, oligomerization of proteins etc. Further, a combination of two or more PTMs can bring about another layer of regulation where presence of a particular combination of PTMs is obligatory to elicit the regulatory message (Yang and Seto 2008a).
    • Novel Role of Heat Shock Protein (HSP) 90 in Regulating ATR-CHK1 DNA Damage Response Pathway in Cancer Cells

      Ha, Kyungsoo; Institute of Molecular Medicine and Genetics (2011-10)
      DNA damage caused by environmental mutagens or reactive metabolic byproducts induces DNA damage response (DDR), which regulates cell cycle transit, DNA repair and apoptosis. DDR involves the phosphorylation and activation of Ataxia Telangiectasia Mutated (ATM) and ATM and RAD3-related (ATR) proteins. ATR regulates the firing of the replication forks during S phase, and the repair of damaged replication forks to prevent premature onset of mitosis. ATR phosphorylates and activates CHK1 which phosphorylates and inactivates CDC25, thereby inhibiting CDK1 activation and cell cycle progression. In the present studies, we determined that treatment with an hsp90 inhibitor AUY922, without affecting the mRNA levels, dose-dependently depletes the protein levels of p-ATR (Ser 428), ATR and CHK1 in human breast and cervical cancer cells. Additionally, treatment with the pan-histone deacetylase inhibitor panobinostat (PS), which is known to induce hyperacetylation and inhibition of hsp90 function, also depleted ATR and CHK1 levels in cancer cells. Co-treatment with the proteasome inhibitor bortezomib (BZ) partially reversed AUY922- or PS-mediated depletion of ATR and CHK1 expression, indicating proteasome-mediated degradation of ATR and CHK1. Treatment with either AUY922 or PS markedly inhibited the binding of ATR with hsp90, induced polyubiquitylation of ATR, and decreased the half-life of both ATR and CHK1 proteins. Treatment with AUY922 also abrogated ionizing radiation (IR)-induced cell cycle arrest and increased the amount of DNA damage in the cancer cells following IR. Treatment with AUY922 also inhibited the recruitment of p-ATR, ATR and 53BP1 to the site of DNA damage. In addition, HDAC3 binds to and deacetylates hsp90 in the nucleus. Depletion of HDAC3 by either short hairpin RNA or genetic knockout induced hyperacetylation of nuclear hsp90, resulting in the inhibition of chaperone association of ATR with hsp90 and depletion of ATR. These findings demonstrate that 1) ATR is chaperoned by hsp90, 2) Inhibition of chaperone function of hsp90 results in proteasomal degradation of ATR and inhibition of DDR, 3) pan-HDAC inhibitors abrogate ATRCHK1 cell cycle checkpoint pathway by modulating chaperone activity of hsp90 and 4) HD AC3 plays a critical role in the regulation of DNA damage response by stabilizing the chaperone activity of nuclear hsp90.