8-hydroxy-2'-deoxyguanosine DNA glycosylase is the first step and rate-limiting enzyme involved in the removal of 8-hydroxy-2'-deoxyguanosine via the base excision repair pathway. Transcriptional regulation of human Ogg1 is sensitive to redox changes via modulation of intracellular glutathione. In response to changes in glutathione, changes in hOgg1 transcription occur similar to genes regulated by the transcription factor Nrf2. It was determined that positions - 47 to - 44 in the hOgg1 promoter are necessary for basal transcription of Ogg1 determined by site-directed deletion. This region is capable of interacting with nuclear protein determined by binding assays. Furthermore, transcription factor Nrf2 is identified as binding to this region determined by parallel, and competition EMSA binding assays. Exposure to arsenic has also been associated with oxidative stress and damage to DNA, specifically oxo8dG. This study identified significant increases in the cellular antioxidant glutathione, and alterations in superoxide dismutase activities subsequent to arsenite exposure in actively dividing and NGF treated PC12 cells. Assessment of Ogg1 activity and mRNA levels demonstrated a significant decrease for both measures subsequent to arsenite exposure. The effect seen was due in large part to alterations in gene transcription since direct testing revealed no effect by arsenite on Ogg1 activity. Levels of oxo8dG did not significantly change subsequent to arsenite exposure, however increased trends were evident. Characterization of Sp1 binding revealed that treatment with sodium arsenite could decrease Sp1 binding at two unique Sp1 sites in the human Ogg1 promoter. In summary, transcription factor Nrf2 is an important factor in the inducible regulation of Ogg1. Transcriptional changes in Ogg1 are further dependent on the redox status of the cell. Despite the role of Nrf2 in response to oxidative stress, sodium arsenite disrupted both the transcription and activity of Ogg1 in PC12 cells. This disruption occurred despite the induction of cellular stress response via increases in GSH and Mn SOD activity. This suggests that arsenite is acting through other mechanisms potentially through disruption of the Sp1 transcription factor.

Persistent pulmonary hypertension of the newborn (PPHN) is a very serious disease affecting nearly 5 in 1000 newborns each year. The development of PPHN has been linked to a decrease in the activity and expression of endothelial nitric oxide synthase (eNOS). Thus, it is critical to understand the mechanisms by which eNOS is regulated to identify new pathways and novel therapies for PPHN. eNOS is dynamically regulated at the transcriptional, post-transcriptional, post-translational, and developmental levels, however mechanisms of this regulation are unresolved. Our data indicates that increases in eNOS expression and activity correlate with increased cellular levels of labile zinc (Zn). In addition, our analysis of the eNOS promoter indicated the presence of a putative heavy metal response element (HRE) in the 5'-flanking sequence. We hypothesized that nitric oxide (NO) may be regulating eNOS expression and activity indirectly through its ability to regulate cellular levels of free Zn. Further, we hypothesized that Zn exerts an effect on eNOS at the transcriptional and post-translational levels. Our results suggested that both NO and hydrogen peroxide (H2O2) activate eNOS in a Zn-dependent manner by liberating cellular stores of Zn and activating the heavy metal-responsive transcription factor 1, MTF-1, which then would bind to the eNOS 5'-regulatory MRE. We found that basal zinc levels in endothelial cells (ECs) helped maintain eNOS promoter activity and total protein levels, but did not affect eNOS dimer levels. However, supplementing ECs with exogenous Zn increased eNOS total and dimer protein levels and activity, mostly through MTF-1. At higher, non-toxic doses of Zn, reactive oxygen species (ROS) production was much higher. eNOS was inhibited by high ROS levels through enzyme fragmentation. Lastly, an eNOS enzyme cofactor, tetrahydrobiopterin (BH4), was found to help maintain eNOS protein due to its high antioxidant potential. In conclusion, eNOS appears to be regulated by NO and ROS through an increase in intracellular Zn, thus activating MTF-1, which binds to the 5' flanking region of the eNOS promoter.

<p>Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that include Creutzfeldt-Jakob disease, bovine spongiform encephalopathy and sheep scrapie. TSE disease pathology and mechanisms within the central nervous system (CNS) of an infected host largely remains unclear. At the cellular level, the uptake of protease resistant prion protein (PrP-res), which strongly correlates with infectivity and is a valid marker for TSE infection, is one of the earliest events that must occur during TSE infection. Given the difficulty of clearly distinguishing input PrP-res from either PrP-res or protease-sensitive PrP (PrP-sen) made by the cell, the uptake of PrP-res from an infectious inoculum into the host cell remains a poorly understood process. Through the development of a novel assay to exclusively detect input PrP-res we hypothesized that the acute infection of cells by PrP-res is mediated through general processes such as endocytosis, whereas internalization, retention, and propagation of PrP-res are dictated by specific characteristics of both the host cell and PrP-res. Using PrP-res tagged with a unique epitope to the mouse monoclonal antibody 3F4, we developed a detection system to specifically follow the acute cellular uptake of PrP-res. Mouse neural and fibroblast cells were exposed to three different mouse scrapie strains and PrP-res from the inoculum monitored. For all strains, PrP-res uptake was rapid and independent of both cellular prion protein expression and cell type. However, only 30%-40% of the cells were able to internalize PrP-res and PrP-res aggregate size influenced PrP-res uptake. Furthermore, infectious brain homogenate PrP-res was taken up more efficiently then PrP-res in either microsome or partially purified preparations. Our results suggest that PrP-res aggregate size, the PrP-res microenvironment, and/or host cell-specific factors can all influence whether or not a cell takes up PrP-res following exposure to TSE infectivity.</p>

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron reductase that catalyzes an NAD(P)H-dependent activation of many quinone-based antitumor agents. NQO1, expressed at high levels in many human solid tumors, can be used as a target for enzyme-directed bioreductive antitumor drug development. We hypothesized that lavendamycins, quinolinedione antitumor antibiotics, can be activated by NQO1 in cancer cells that overexpress NQO1 to exhibit selective toxicity toward those cells. The effects of functional group changes on the metabolism of lavendamycins by recombinant human NQO1 were studied using a spectrophotometric assay. These structure-activity relationship (SAR) studies determined key structural features that were required for lavendamycin substrate specificity. Cytotoxicity toward human colon adenocarcinoma NQO1-deficient (BE) and NQO1-rich (BE-NQ) cells was also determined using colorimetric and clonogenic assays. The best lavendamycin substrates for NQO1 were also the most selectively toxic to the BE-NQ cells compared to BE cells. To facilitate structure-based design of more optimal lavendamycin substrates and NQO1-directed lavendamycin antitumor agent development, we developed a 1H69 crystal structure-based in silico model of the NQO1 active site and performed lavendamycin-docking studies. The docking was performed using the FlexX module of SYBYL software. Lavendamycin analogues were designed as NQO1 substrates utilizing our SAR and docking data as structure-based design criteria. Docking and biological studies on the analogues were performed and were consistent suggesting the in silico model of the enzyme possessed practical predictive power. Our results also suggested practicality of the design criteria resulting in the discovery of good NQO1 substrates with selective toxicity toward BE-NQ cells. The mechanisms of NQO1-mediated selective cytotoxicity of good lavendamycin substrates in BE and BE-NQ cells were also investigated including induction of oxidative stress and apoptosis. Biomarkers of oxidative stress including formation of 8-hydroxy-2'-deoxyguanosine (8-oxo-2dG), an indicator of oxidative DNA damage, and depletion of reduced glutathione (GSH) were examined using an HPLC-based method and a colorimetric assay, respectively. Induction of apoptosis was examined using a colorimetric assay. Our results revealed that oxidative stress and subsequent apoptosis induction by a good lavendamycin substrate was NQO1 dependent and that the poor substrate for NQO1 caused neither oxidative stress nor apoptosis.

As the primary excitatory amino acid, glutamate is essential to proper functioning of the mammalian CNS. Proper regulation of the synaptic release of glutamate, potentially regulated by synaptic vesicle content, is one of many critical aspects to normal excitatory functioning. In particular, the vesicular glutamate transporters (VGLUTs), which load synaptic vesicles with glutamate prior to presynaptic release of neurotransmitter, are distinct from that of the plasma membrane excitatory amino acid transporters (EAAT). The development of a library of compounds which selectively inhibit the uptake of [3H]-L-glutamate into the VGLUTs, has revealed importance of particular structural motifs. Among these structural motifs, one of the most important is that of the "embedded glutamate" which mimics the endogenous substrate of the transporter. With respect to potency, the substitution of a lipophilic moiety at the C6 position seems to be the most important to date, as illustrated by 5,6-napthyl quinoline dicarboxylic acid (5,6-QDC). The structure of this compound strongly resembles that of a steroid molecule. In light of recent research suggesting steroids act within the CNS in a non-genomic manner, this observation prompted the testing of a panel of steroid molecules at VGLUT. These compounds, known as "neuroactive steroids" have been shown to be synthesized, modified, and/or active within the brain. Research from our lab, as well as from the Thompson lab, shows that certain sulfated neuroactive steroids are potent inhibitors of [3H]-L-glutamate uptake into synaptic vesicles. This work identifies pregnenolone sulfate, along with 5,6-QDC, as competitive inhibitors of VGLUT (K i values of 107 and 228 &#956;M, respectively). These two molecules display specificity for VGLUT, with respect to other sites on the synaptic vesicle (i.e., electrochemical gradient), and among other vesicular neurotransmitter transporters (i.e., VGAT, VMAT). Two molecules, 5,6-QDC and Congo Red Fragment (CRF) were aligned to the VGLUT Pharmacophore to illustrate the SAR of these compounds. Biochemical studies have also been conducted to delineate substrate activity of neuroactive steroids and related compounds at VGLUT. The specificity of certain sulfated neuroactive steroids suggest that they could be endogenous regulators of vesicular glutamate uptake.

Low level lead (Pb) exposure is a risk factor for neurological dysfunction including ADHD. How Pb produces these behavioral deficits is unknown, but low-level exposure during development is associated with auditory temporal processing deficits, even though hearing remains normal. Pb disrupts cellular energy metabolism and efficient energy production is crucial for auditory neurons to maintain their high rates of synaptic activity. The voltage dependent ion channel (VDAC) is an ion channel involved in the regulation of mitochondrial physiology and is a critical component in controlling mitochondrial energy production. No studies to date have investigated the effect of Pb on VDAC, therefore the current series of studies examines the interactions between Pb and VDAC. In-vitro studies were used to delineate the effects of Pb on VDAC expression. Both differentiated SH-SY5Y cells and PC-12 cells exposed to 10 μM Pb for 48 h result in a significant decrease in VDAC expression. Exposure to 24 h of hypoxia fails to decrease VDAC expression, suggesting this is a specific effect of Pb. In addition, a corresponding decrease in cellular ATP that is correlated with decreased VDAC expression occurs with Pb. Real-time RT-PCR demonstrated a significant decrease in mRNA levels for VDAC1 isoform, suggesting that Pb decreases VDAC protein expression through decreased transcription. A proteomics approach was then used to confirm that Pb exposure during development results in changes in proteins involved in energy metabolism in auditory regions of the brainstem. CBA mice were exposed to 0 mM (control), 0.01 mM (low), or 2 mM (high) Pb acetate during development. At P21, the ventral brainstem region containing several auditory nuclei, including the Medial Nucleus of the Trapezoid Body, and the medial and lateral superior olivary nuclei, was separated from the total brainstem. Proteomic analysis (isolation and separation of proteins by 2D-PAGE; analysis by MALDI-MS) revealed that chronic Pb exposure alters the expression of proteins involved in the regulation of cellular energy metabolism including VDAC and creatine kinase B. Immunohistochemistry confirms that Pb exposure results in decreased expression of VDAC in auditory nuclei, supporting the hypothesis that Pb disrupts energy metabolism in auditory neurons.

Alveolar macrophages (AM) form the first line of defense against chronic inflammation caused by occupational exposure to environmental particulates such as crystalline silica (CSiO2). The chronic inflammatory process triggered by CSiO2 is known to culminate into a fibrotic response called silicosis in the human lungs. Previous studies have indicated the role of membrane glycoproteins called scavenger receptors in binding of environmental particles. The scavenger receptors are classified into different classes (A-H) based on their structure and function. Class A scavenger receptors are critical in uptake of variety of ligands such as bacteria, acetylated lipoproteins and are typically found on macrophages, dendritic and epithelial cells. One of the members of this family is Macrophage receptor with collagenous structure (MARCO). Recent studies have focused on analyzing the interaction between MARCO and inorganic particles such as CSiO2 and titanium dioxide (TiO2). Both in vivo and in vitro binding studies have identified MARCO as a key receptor in CSiO2 uptake and subsequent cytotoxicity in AM from C57Bl/6 mice. Further in vitro studies using a transfected cell line revealed that the 100 amino acid residues long cysteine-rich (SRCR) domain at the C-terminal end of MARCO is required for binding of inorganic particles such as CSiO2, TiO2 and amorphous silica (ASiO2). Moreover, individual particles bind to SRCR domain of MARCO with unique differences and have varying requirements with respect to need for divalent cations. Our studies demonstrate that physiological absence of MARCO in C57Bl/6 mice leads to a more robust inflammatory response following CSiO2 exposure as compared to wild-type mice. The results suggest that diminished clearance of CSiO2 particles from the MARCO-/- lungs exacerbates the lung inflammation. These findings demonstrate that the involvement of different regions of SRCR domain may distinguish downstream events following particle binding. Taken together, these data establish the role of MARCO in uptake of various inorganic particles and elucidate the protective role of MARCO in CSiO2-induced lung inflammation.

<P>Vesicular glutamate transporters (VGLUTs) are integral membrane proteins that uptake glutamate into synaptic vesicles and are involved in glutamatergic neurotransmission. Since VGLUTs were identified and cloned, efforts have been made to characterize their functional roles. However, due to experimental limitations, the structural features of VGLUT protein remain unclear. In an attempt to better understand VGLUTs, computational and biochemical approaches were employed to characterize them. Plasmid DNA encoding rat VGLUT1 was constructed, amplified and expressed in Pichia pastoris to produce VGLUT1 protein. Immobilized metal affinity chromatography (IMAC) was employed to purify the protein for structural analysis by mass spectrometry and to develop a functional transporting system, VGLUT1 proteoliposomes. Transmembrane topology and homology models of VGLUT1 were generated by web-based and in-house programs. The computational analysis implies that VGLUT1 protein appears to have 12-transmembrane domains. Chemical and enzymatic cleavages and mass spectral analysis of denatured and proteoliposome-reconstituted VGLUT1 protein show that the experimental results are consistent with the computational models. These results provide basic insight into VGLUT protein structure for neuropharmacology studies related to glutamatergic neurotransmission.</P>

Epidemiological studies as well as controlled animal studies have associated exposure to arsenic through drinking water with the development of atherosclerosis. In this study, we have shown for the first time that low and environmentally relevant concentrations of arsenic accelerate atherogenesis. The objective of this study was to elucidate the mechanisms of arsenic-induced atherosclerosis by (1) characterizing the time- and concentration-dependent effects of sodium arsenite [As(III)] on the development of atherosclerosis in ApoE-/- /LDLr-/- mice, (2) determining whether As(III)-induced peroxynitrite activates protein kinase C (PKC) isotypes, α and β, in human aortic endothelial cells (HAECs) and (3) determining the effects of activation of PKC isotypes, α and β, on the endothelial barrier. Accordingly, exposure of ApoE-/- /LDLr-/- mice to As(III) in drinking water showed an increasing trend in atherosclerotic plaque formation in as early as 5 weeks within the innominate arteries. Most remarkable was the evidence that environmentally relevant concentrations of As(III) resulted in significant increase in plaque formation. Initiation of atherosclerosis results from activation/dysfunction of the vascular endothelium that maintains a semipermeable barrier between the blood and vessel wall. To elucidate the mechanism of arsenic-induced atherosclerosis, we analyzed the effect of As(III) on the endothelial monolayer integrity. Endothelial barrier is maintained by proteins of the adherens junction (AJ) such as vascular endothelial cadherin (VE-cadherin) and β-catenin, and their association with the actin cytoskeleton. Treatment of HAECs with As(III) resulted in reorganization of actin filaments into stress fibers and non-uniform VE-cadherin and β-catenin staining at cell-cell junctions. Intercellular gaps were observed with a measured increase in endothelial permeability. In addition, an increase in tyrosine phosphorylation (PY) of β-catenin was observed. These effects were mediated through As(III)-induced activation of PKCα without peroxynitrite formation. No change in PKCβ levels was detected with As(III) treatment. Inhibition of PKCα restored VE-cadherin and β-catenin staining at cell-cell junctions and abolished the formation of intercellular gaps and stress fibers. Endothelial permeability and PY of β-catenin were also reduced to basal levels. These results demonstrate that As(III) induced activation of PKCα causes PY of β-catenin and formation of stress fibers. PY of β-catenin causes weakening of the AJ and this in association with the contractile force generated by stress fibers results in gap formation and increased endothelial permeability. This could potentially accelerate the development of atherosclerosis by increasing the accumulation of oxidized low density lipoproteins and monocytes into the neo-intima of the blood vessel. The findings in this study demonstrate that arsenic disrupts the endothelial monolayer by activation of PKC signaling. Damage to the endothelium plausibly accelerates the process of atherosclerosis at an early stage as evidenced by the increase in atherosclerotic plaques in the ApoE-/- /LDLr-/- mouse model.

<p>APOBEC proteins have evolved in mice and humans as potent innate defences against retroviral infections. APOBEC3G (hA3G) in humans and mouse APOBEC3 (mA3) deaminate cytidine in single-stranded DNA which ultimately results in hypermutation of newly synthesized proviral DNA. Other deaminase-independent mechanisms of inhibition have been identified, such as directly inhibiting reverse transcription. Both HIV and murine leukemia viruses (MuLVs) have evolved mechanisms to evade the action of the APOBEC proteins. HIV encodes the Vif protein which binds to hA3G and facilitates its rapid degradation through the proteasome. The mechanism(s) by which exogenous MuLVs evade mA3 inhibitory activity is unknown.</p> <p>Exogenous MuLVs encode a glycosylated gag protein (gGag) originating from an alternate CUG start site upstream of the AUG start site of the Gag structural polyproteins. gGag is synthesized to similar amounts as the structural Gag polyprotein in MuLV infected cells but is glycosylated in the endoplasmic reticulum and undergoes distinct proteolytic processing. The function(s) of gGag remain unclear, but eliminating its synthesis through mutation markedly impedes in vivo replication of the virus with very little affect on in vitro replication. Endogenous retroviruses have not been found to express gGag and are tightly controlled by mA3. APOBEC3 proteins are expressed in many tissues in the mouse but are not expressed in most in vitro cell lines. These observations are consistent with a link between gGag expression and the evasion of mA3 by MuLVs.</p> <p>Studies described herein demonstrate that gGag is protective against both cellular and virion-associated mA3 in vitro and is protective against mA3 in vivo. While there was no direct interaction between mA3 and gGag in an infected cell, gGag and mA3 are localized in the same compartment in the virion and are able to be coprecipitated together from lysed virions. G-to-A hypermutation is not a mechanism used by mA3 to inhibit gGag-negative MuLV replication. Through an affect on reverse transcription, cellular and virion-associated mA3 reduce viral transcripts in MuLV infected cells in a gGag-dependent manner.</p>