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 μM, 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>

Glutamate is the major excitatory neurotransmitter in the brain and is crucial for processes such as learning and memory. Due its importance as a signaling molecule, the extracellular glutamate concentration is tightly regulated, largely by the excitatory amino-acid transporters (EAATs). In these studies, we investigated the role of EAAT1-3 in synaptic transmission at the Schaffer-CA1 synapse in acute hippocampal brain slices. My results demonstrated that transport block by L-TBA resulted in glutamate spillover and activation of NMDARs. Further investigation showed that L-TBA-mediated activation of NMDARs was facilitated by the Mg2+ unblock of the receptor. Furthermore our data indicate that NMDAR signaling was controlled by the interplay between several factors, including synaptic frequency, glutamate transport, Mg2+ block, and NMDAR channel kinetics. We propose that the observed theta frequency threshold for enhanced NMDAR signaling observed in physiological conditions is a consequence of a phase shifted signal at rhythms limited by NMDAR channel kinetics. We also found that dense fiber recruitment created conditions of spillover and glutamate pooling and therefore resulted in an increase in AMPAR desensitization at the hippocampal Schaffer-CA1 synapse. Overall my studies focused on the effects of glutamate spillover onto both NMDA and AMPA receptors at the Schaffer-CA1 synapse in hippocampal slice.

The System xc- antiporter is plasma membrane transporter that mediates the exchange of extracellular L-cystine with intracellular L-glutamate. This exchange is significant within the context of the CNS because the import of L-cystine is required for the synthesis of the antioxidant glutathione, while the efflux of L-glutamate has the potential to contribute to either excitatory signaling or excitotoxic pathology. Changes in the activity of the transport system have been suggested to contribute to the underlying pathological mechanisms of a variety of CNS disorders, one of the most prominent of which is it highly enriched expression glial brain tumors. In an effort to produce more potent System xc- blocker, we have been using amino-3-carboxy-5-methylisoxazole propionic acid (ACPA) as a scaffold for inhibitor development. We previously demonstrated that the addition of lipophilic aryl groups to either the #4 or #5 position on the isoxazole ring markedly increased the inhibitory activity at System xc-. In the present work a novel series of analogues has been prepared in which aryl groups have been introduced at both the #4 and #5 positions. In contrast to the competitive action of the mono-substituted analogues, kinetic analyses indicate that the di-substituted isoxazoles block System XC--mediated uptake of 3H-L-glutamate into SNB-19 activity by a noncompetitive mechanism. These new analogues appear to be the first noncompetitive inhibitors identified for this transport system, as well as being among the most potent blockers identified to date. These diaryl-isoxazoles should be of value in assessing the physiological roles and molecular structure of System xc-.

The development of multidrug resistance in tumor cells has been recognized as a major obstacle to successful cancer treatment. Tumor cells in vitro and in vivo can develop multidrug resistance (MDR) to the lethal effects of a variety of cytotoxic drugs used to treat cancerous tumor cells. The over expression of multiple drug resistance gene 1 has been correlated with the expression of multi drug resistance protein 1(MDR1, also known as P-glycoprotein or P-gp). MDR1's role in altering uptake, distribution and bioavailability is considered a significant factor when examining drugs for clinical administration, and represents a viable drug target for the reversal of MDR. MDR1 is driven by ATP hydrolysis and as such it shares both sequence and structural homology with proteins that are energy-dependent efflux transporters driven by ATP hydrolysis, making MDR1 a member of the ATP binding cassette (ABC) super family. Because, MDR1 transports substrates that are often toxic xenobiotics, MDR1 is thought to fulfill a cellular detoxification function. As such it is expressed in several tissues in the body such as the liver, pancreas, kidney, colon, intestinal mucosa, and in the blood brain barrier. Due to its presences in a wide variety of cells it has been suggested that MDR1 is involved in protection of the organism as a whole. Consequently, the overproduction of MDR1 is seen in cancer cells. MDR1 has been shown to transport a wide variety of lipophilic agents, of importance, MDR1 effluxes chemotherapeutics agents out of the cell resulting in a low and ineffective intracellular drug concentration. Thus, the over production of MDR1 in cancer cells can then be thought of as a protective factor for cancer cells, and as an effect causes MDR cancer cells. Therefore, understanding MDR1âs function is important for controlling the bioavailability of drugs and for improving anticancer chemotherapy. Reversal of multidrug resistance is of interest, and MDR reversing agents have been under intensive investigation. The 4-Isoxazolyl-1, 4-Dihydropyridines (IDHPâs) have been shown to exhibit inhibition of MDR1. A novel series of IDHP compounds have been prepared and found to inhibit MDR1. The synthesis, MDR1 assay results, and relevant controls will be discussed. If successful, halting the function of MDR1 will stop the outward efflux of chemotherapeutic agents. In combination with chemotherapeutic treatments IDHP agents could allow for greater effectiveness of pharmaceutical intervention. This research would give an option in the treatment of cancer that would normally never exist for MDR cancer patients, and would allow for far more effective treatment via pharmaceutical means.

Genomic DNA is organized around the double-stranded of B-form DNA, which is both durable and flexible enough to store and pass on genetic information. Once freed from the associations of an extended complimentary sequence, single stranded DNA and RNA can adopt a vast array of stable secondary structure motifs, such as stem-loop, pseudo-knots, and tetra-loops, ideal for its involvement in biological settings other than as a store of genetic information. Originally, alkylating agents were used as "mustard gas" and related chemical weapons in World War I. Alkylating agents, in general, can react with one or two different 7-N-guanine residues and could potentially result in the cross-linkage of DNA strands, preventing uncoiling of the DNA double helix leading to cell death. More recent evidence show that guanine-rich nucleic acids can fold into distinctive four-stranded conformers found in telomeric DNA repeats (i.e. TTAGGG), also known as G-quadruplexes (G4), as well as in sequences in the promoter and other regulatory regions of genes, especially those involved in cellular proliferation. Small molecules that induce the formation of, and selectively bind to, G4 structures are of interest for development as potential anticancer therapeutics. Novel 10-oxoanthracene and substituted anthracenyl isoxazole esters (AIEs) were synthesized and characterized based on NMR studies. To date, quarfloxin is the only G-quadruplex ligand from the large number developed to progress to clinical evaluation. The synthesis, structural characterization, and biological studies will be presented.