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University of Maryland Baltimore County

Department of Chemistry and Biochemistry

From left to right: Dr Larry Keefer (NCI Frederick), Dr Nazareno Paolocci (Johns Hopskins Medicine), Dr David Wink (NCI Bethesda), Dr Gerald Rameau (Morgan State Univ), Sir Professor Moncada (Wolfson Institute London), Dr David Roberts (NCI Bethesda), Dr Jeffrey Isenberg (Univ. Pittsburgh), Dr Elsa Garcin (UMBC)

DATE:             November 17, 2010

TIME:              8:30am-3:45pm

LOCATION:   UMBC, Chemistry Building

                       Conference Room 120

                       1000 Hilltop Circle

                        Baltimore, MD 21250


Maps and Directions

The symposium is free and open to the public. Refreshments will be provided. Lunch is not.

For lunch possibilities on campus (cash or credit card), please visit Where to eat 

RSVP is required before October 30th for parking on campus

Please email Elsa Garcin or Jim Fishbein at to RSVP




8:30 AM                      Opening remarks by Geoffrey P. Summers

8:40 AM                      Keynote lecture by Prof. Salvador Moncada
                                   "Nitric oxide and mitochondrial interactions: physiology and pathophysiology"

9:40 AM                      David Wink
                                    "Inducible Nitric Oxide Synthase and Cycloxygenase-2 in Cancer. Identification of Potential Molecular Pathways and Targets that Lead to Poor Prognosis."

10:20 AM                   Coffee Break

10:40 AM                    Larry Keefer
                                    "Nitric oxide-based drug development"

11:20 AM                   Nazareno Paolocci
                                   "Nitroxyl (HNO) ... From Heaven to Heart: Stardust or a New Tool against Heart Failure?"


12:00pm                    Lunch Break


1:30 PM                     David Roberts
                                   "Thrombospondin-1 signaling via CD47 in injury and stress responses"

2:10PM                      Jeffrey Isenberg
                                   "Thrombospondin-1 supports blood pressure by limiting eNOS activation and endothelial-dependent vasorelaxation"

2:50pm                      Gerald Rameau
                                  "Nitric Oxide Regulation of Glucose Transporter 3 (GLUT3)"

3:10pm                      Elsa Garcin
                                  "Structural studies of a mammalian soluble guanylate cyclase. Lessons learnt from nitric oxide synthase"

3:30pm                      Travel Awards

3:40pm                      Closing remarks

3:45pm                      Refreshments and closing of the symposium



[Click on the speaker name to view biography]



Nitric oxide and mitochondrial interactions: physiology and pathophysiology

Prof. Salvador Moncada

Director of the Wolfson Institute for Biomedical Research, University College London, London UK

At physiological concentrations nitric oxide (NO) inhibits mitochondrial cytochrome c oxidase in competition with oxygen. We have developed a technique based on visible light spectroscopy and used it to demonstrate that endogenous NO enhances reduction of the electron transport chain, thus enabling cells to maintain their VO2 at low oxygen concentrations. This favors the release of superoxide anion, which initiates the transcriptional activation of NF-kB as an early stress signaling response. We have also used this technique to demonstrate that NO is inactivated by cytochrome c oxidase in its oxidised state and have proposed that cessation of such inactivation at low oxygen concentrations may account for hypoxic vasodilatation.

Many cells respond to a decrease in oxygen availability via stabilisation of hypoxia-inducible factor-1a (HIF-1a), whose accumulation is normally prevented by the action of prolyl hydroxylases. We have found that inhibition of mitochondrial respiration by low concentrations of NO leads to inhibition of HIF-1a stabilisation. This prevents the cell from registering hypoxia at low oxygen concentrations, which would otherwise result in upregulation of defensive genes, including those for glycolysis and angiogenesis. Furthermore, inhibition of mitochondrial respiration in hypoxia leads to redistribution of available oxygen toward non-respiratory oxygen-dependent targets.

In addition to its interaction with cytochrome c oxidase, NO can signal for mitochondrial biogenesis via a cyclic GMP-dependent mechanism. Furthermore, Increases in NO beyond physiological levels lead to persistent inhibition of other key enzymes in the mitochondria and this may account for NO-dependent initiation of cell pathology.


Inducible Nitric Oxide Synthase and Cycloxygenase-2 in Cancer. Identification of Potential Molecular Pathways and Targets that Lead to Poor Prognosis

Dr. David Wink

National Institutes of Health, National Cancer Institute, Radiation Biology Branch, Bethesda USA

Epidemiological studies have found that inflammatory proteins iNOS and COX-2 are poor prognostic indicators for many cancers.  We have been investigating the chemical mechanism of chemistry of NO and other reactive species associated with biological mechanisms in cancer.  These studies have shown that specific concentrations of NO determine the pro or anti-tumorigenic behavior. When prolonged exposure to ┬ÁM amounts of NO, there is an increase in the phosphorylation of p53 and cystostasis.  However, when cells are exposed to 100 nM of NO, there is increase in protumorigenic molecular pathways such as MAPK, pAkt and HIF1a. Several of these pathways are also activated by PGE2. In ER negative breast cancer patients we found that if either iNOS and COX-2 was highly expressed there was a decrease in survival.  When both were present there was a dramatic decreased survival.  From this epidemiological data we have been able to develop cellular models to determine if we can find compounds that will reverse these mechanisms that result in poor phenotypes.  We have found a class of thiol and HNO-based compounds that activate a tumor suppressor protein reversing the molecular pathways associated with iNOS and COX-2 in ER negative breast cancer.  This talk will focus on the understanding the chemical biology of nitric oxide and how it increases cancer risk and describe some potential new molecular targets for the treatment of cancer. 


Nitric oxide-based drug development

Dr. Larry Keefer

National Cancer Institute Frederick, Laboratory of Comparative Carcinogenesis, Frederick USA

Nitric oxide (NO) is a central player in the inflammatory process. To investigate its many roles in normal as well as pathophysiology, caged NO donors of the diazeniumdiolate class (also known as NONOates) have come into wide use as research tools, offering a range of reliable half-lives from 2 seconds to 20 hours for spontaneously generating authentic NO into aqueous media at physiological temperature and pH. Current work is aimed at exploring the utility of this caged NO chemistry in designing improved drugs and biomedical devices. My presentation will highlight a selection of recent advances in this area.


Nitroxyl (HNO) ... From Heaven to Heart: Stardust or a New Tool against Heart Failure?

Dr. Nazareno Paolocci

Johns Hopkins Medicine, Johns Hopkins Heart and Vascular Institute, Baltimore USA

Nitroxyl (HNO) is the one electron-reduction product of NO.. HNO was first discovered in the clouds of interstellar space. Yet its endogenous formation in mammals still awaits definitive proof. However, the pharmacological properties of HNO donors are emerging as neatly distinct from those exhibited by NO., nitrogen-related or oxygen-derived reactive species, particularly in the cardiovascular system. In vivo, HNO elicits both vasodilation and cardiac function enhancement, namely positive inotropy/lusitropy. In vitro, HNO cardiac properties are fully independent from cGMP/PKG and cAMP/PKA signaling, but relying on the availability of critical thiols, namely cysteines, situated in key components of the electro-contraction coupling machinery of the heart. HNO enhances Ca2+ cycling at the sarcoplasmic reticulum (SR) level without affecting Ca2+ level at rest. Moreover, it sensitizes myofilaments to Ca2+. The involvement of critical "redox-switches" in HNO cardiac effects is supported by the fact that HNO action is prevented when the intracellular amount of free-floating thiols rises (or reversed when more reducing equivalents are provided) and by cysteine-to-alanine mutagenesis experiments. The fact that HNO action is fully retained in vivo hearts and in vitro myocytes suffering from heart failure (HF), both typically displaying increased oxidative stress, suggests that HNO may target a selected population of highly reactive cysteines, not easily accessible for other ROS/RNS. In conclusion, HNO donors may be advantageous for treating HF patients that have impaired cardiac contraction, volume overload and high peripheral vascular resistance, and in whose hearts oxidative stress and altered Ca2+ handling coexist.


Thrombospondin-1 signaling via CD47 in injury and stress responses

Dr. David Roberts

National Institutes of Health, National Cancer Institute, Laboratory of Pathology, Bethesda USA

The thrombospondin-1 receptor CD47 controls angiogenesis, vascular tone, and survival of ischemic stress by regulating nitric oxide (NO) signaling.  CD47 ligation redundantly inhibits signaling upstream and downstream of NO. Soft tissues in thrombospondin-1- and CD47-null mice are highly resistant to ischemia and ischemia/reperfusion injuries.  Remarkably, this protection extends to high-dose radiation injury.  Similar protection can be achieved in wild type animals by treatment with antagonists of thrombospondin-1-CD47 interaction.  Tumors in mice treated with CD47-blocking agents, however, become more sensitive to radiation.  Some of these therapeutic activities can be reproduced by NO donors or agents that elevate tissue cGMP levels, but this is not the case for ischemia/reperfusion and radiation injury responses.  This implies that targets of CD47 other than the NO/cGMP pathway are involved.  We are examining the role of additional signaling pathways in vascular and immune cells that are regulated by CD47 in order to optimize these therapeutic activities.


Thrombospondin-1 supports blood pressure by limiting eNOS activation and endothelial-dependent vasorelaxation

Dr. Jeffrey Isenberg

University of Pittsburgh, Vascular Medicine Institute, Pittsburgh USA

Thrombospondin-1 (TSP1), via its necessary receptor CD47, inhibits nitric oxide (NO)-stimulated soluble guanylate cyclase activation in vascular smooth muscle cells, and TSP1-null mice have increased shear-dependent blood flow compared with wild-type mice. Yet, the endothelial basement membrane should in theory function as a barrier to diffusion of soluble TSP1 into the arterial smooth muscle cell layer. These findings suggested that endothelial-dependent differences in blood flow in TSP1-null mice may be the result of direct modulation of endothelial NO synthase (eNOS) activation by circulating TSP1. Here we tested the hypothesis that TSP1 inhibits eNOS activation and endothelial-dependent arterial relaxation. Acetylcholine (ACh)-stimulated activation of eNOS and agonist-driven calcium transients in endothelial cells were inhibited by TSP1. TSP1 also inhibited eNOS phosphorylation at serine1177. TSP1 treatment of the endothelium of wild-type and TSP1-null but not CD47-null arteries inhibited ACh-stimulated relaxation. TSP1-null vessels demonstrated greater endothelial-dependent vasorelaxation compared with the wild type. Conversely, TSP1-null arteries demonstrated less vasoconstriction to phenylephrine compared with the wild type, which was corrected upon inhibition of eNOS. In TSP1-null mice, intravenous TSP1 blocked ACh-stimulated decreases in blood pressure, and both intravenous TSP1 and a CD47 agonist antibody acutely elevated blood pressure in mice.TSP1, via CD47, inhibits eNOS activation and endothelial-dependent arterial relaxation and limits ACh-driven decreases in blood pressure. Conversely, intravenous TSP1 and a CD47 antibody increase blood pressure. These findings suggest that circulating TSP1, by limiting endogenous NO production, functions as a pressor agent supporting blood pressure.



Nitric Oxide Regulation of Glucose Transporter 3 (GLUT3)

Dr. Gerald Rameau

Morgan State University, Baltimore USA

The glucose transporter type 3 (GLUT3) regulates glucose uptake, which provides neurons with an energy substrate. However, the mechanism by which synaptic activity regulates GLUT3 trafficking has not been elucidated. To examine activity dependent regulation of GLUT3, primary cultured neurons were stimulated with bicuculline, which induced synaptic activation of N-Methyl-D-Aspartate receptor (NMDAR). Stimulation of NMDAR increases the production of NO by neuronal nitric oxide synthase (nNOS). This regulation is achieved by Ca2+-Calmodulin binding and phosphorylation. nNOS is phosphorylated by Akt at S1412 (positive regulation) and by CaMKII at S847 (negative regulation). We found that activation of NMDAR induces a program of positive and negative phosphorylation of nNOS, which suggests physiological function. This activation increased surface GLUT3 via a NO/cGK pathway that correlated with increased glucose uptake. In total, our results suggest a mechanism that regulates surface GLUT3 that may serve homeostatic strategies allowing neuronal cells to meet the changing energy demands brought about by increased synaptic activity.



Structural studies of a mammalian soluble guanylate cyclase. Lessons learnt from nitric oxide synthase

Dr. Elsa Garcin

University of Maryland Baltimore County, Department of Chemistry and Biochemistry, Baltimore USA

Soluble guanylate cyclase (sGC) is the direct sensor and mediator of nitric oxide (NO) signal transduction via cyclic GMP (cGMP).  NO-induced vasodilation depends primarily on the activation of sGC, which produces cGMP whose effects are mediated by cGMP-dependent kinases, ion channels, and phosphodiesterases. Compounds that activate cGMP production by sGC have outstanding clinical potential for treating cardiovascular and other diseases due to impaired blood flow. To elucidate the structural details of sGC assembly and determine the dynamic events associated with NO-induced sGC activation, we have initiated a multidisciplinary approach using biochemical, mutagenesis studies, combined with structural methods.

Working with mammalian sGC is not trivial. First, obtaining large quantities of pure protein has been a major bottleneck in the sGC field. Second, purification is complicated by (i) the fact that the sGC heme is extremely labile, (ii) the presence of 34 cysteine residues, and (iii) sensitivity to proteolysis. Nitric oxide synthase posed similar challenges to studying its assembly, catalysis and regulation, therefore our laboratory will apply lessons learned from the biophysical characterization of NOS to shed light on sGC structure and function.

Our first step was to design a bacterial overexpression system for bovine sGC. To our knowledge, we have developed the first bacterial overexpression system yielding soluble and active full-length mammalian sGC, as well as truncated constructs. Here, I will present our recent advances towards structural studies of bovine sGC.



Sponsor: DOW Chemical Company

Organizers: Elsa Garcin & Jim Fishbein