National Biomedical EPR Center (Hyde, PI, P41 EB001980)
The broad aim of the National Biomedical EPR Center is to create and maintain a comprehensive center with balance in all five categories of a Research Resource:
(1) Resonator development
(2) W-band enhancement
(3) Broadband digital detection, signal analysis, and archiving in EPR spectroscopy
(4) Microwave oscillator development
(5) H1||H0 (parallel mode) EPR at Q- and W-band
(6) X-band/Q-band spin trapping methodology development: Structure/function aspects of NOS-generated radicals
Dissemination, including one workshop during the funded period
(1) Graduate and post-graduate training
(2) Training established investigators
(3) Training of young investigators
The functions of a Research Resource will be accomplished with expertise in the three main application areas of EPR spectroscopy: free radicals, transition metals, and spin labels and with competence in EPR instrumental development.
Development of Biomedical EPR Instrumentation (Hyde, PI, R01 EB002052)
The proposal contains five projects that are concerned with pulse (or time-domain) saturation-recovery electron-spin resonance instrumentation and methodology. 1) Digital signal processors and improved time response. 2) 2D saturation recovery. 3) Multifrequency saturation recovery and FIDs. 4) Loop-gap resonators for saturation recovery. 5) Multiquantum readout of saturation recover.
Advanced Instrumental Development Core: Medical College of Wisconsin
DARTMOUTH COLLEGE Project Number: 5U19AI091173-03 Sub-Project ID: 6786
Project Leader: HYDE, JAMES S
This core provides input from what is generally agreed to be the leading EPR instrumental development site in the world, the National Biomedical EPR Center at the Medical College of Wisconsin (MCW). The role of this core will be to provide leadership in the development of specific aspects of the technology needed to accomplish the goals of the CMCR. These developments will be carried out in collaboration with the projects and with the instrumental Core at Dartmouth, to facilitate the development of the best possible o prototype instruments for EPR dosimetry. The contributions of the core at MCW are described in four specific aims. Specific Aim 1 (50% of effort) is the Development of resonators for measurements at X-Band in vivo for nails and teeth; this will be the most extensive project and will focus on exploiting the breakthrough achieved in initial collaborative studies where for the first time, resonators that operate at the very sensitive X-Band frequency were successfully used to make measurements in vivo. This development is at the heart of project 3. During the course of the grant this core will attempt to make analogous resonators that can be used for in vivo tooth dosimetry, which could dramatically improve the already impressive sensitivity of this approach (Project 1). Specific Aim 2 (25% of effort) - Development of instrumentation to support measurements at X-Band, will especially focus on development of specifically designed microwave bridges that will facilitate both Projects 2 &3. These developments will be carried out in collaboration with a longstanding collaborative partner of both Dr. Hyde and Dr. Swartz, Dr. Froncisz of the Jagiellonian University in Krakow (Dr. Froncisz's support will come via a purchase agreement. Specific Aim 3 (20% of effort) - Improvements in resonators and bridges for measurements at L-Band. This core will similarly work with Dartmouth and Krakow in the development of improved L-Band bridges, in support of Project 1. It also will facilitate resonator development in project 1 by use of the very sophisticated modeling techniques that are highly developed at MCW, which enable different configurations of resonators to e=be rigorously evaluated so that construction can be focused on designs that are likely to be successful Specific Aim 4 -(5 % of effort) support of multifrequency studies will available an exceptional set of EPR spectrometers that can be of value to all three projects in helping to elucidate the characteristics of potentially overlapping components of EPR spectra.
Advanced Research and Development of Chemical, Biological, Radiological, and Nuclear Medical Countermeasures (Hyde HHSO100201100024C)
Mitochondria-Targeted Agents in Breast Cancer (Kalyanaraman, PI, R01 CA152810)
ABSTRACT We will develop mitochondria-targeted antioxidants (MTAs) and imaging probes that will mitigate cardiotoxicity and enhance antitumor efficacies of chemotherapeutic drugs. We will use doxorubicin (DOX), a front-line antitumor agent in breast cancer treatment. DOX causes delayed dose-dependent cardiotoxicity. Clinically, this side effect is managed with conventional antioxidants and iron chelators. This proposal provides a new adjuvant approach in breast cancer chemotherapy. Its genesis is based upon the following discoveries: 1) MTAs (e.g., Mito-Q, a synthetic drug analog of an endogenous antioxidant, Co-enzyme-Q, present in the mitochondrial respiratory chain) inhibit DOX-mediated cardiotoxicity in a preclinical animal model and in cardiomyocytes, and 2) MTAs (Mito-Q and Mito-CP, a nitroxide targeted to mitochondria) cause antiproliferative and cytotoxic effects in breast cancer cells (MCF-7 and MDA-MB-231) but not in non- transformed breast epithelial cells (MCF-10A) and significantly enhance DOX-induced breast cancer cell toxicity. We hypothesize that mitochondria-targeted antioxidants enhance DOX-mediated antitumor effects but attenuate DOX cardiotoxicity. Response to chemotherapy will be monitored by using the mitochondria-targeted technetium-labeled imaging agents (99mTc-Mito10-MAG3) in a chemically-induced breast carcinoma animal model. Specifically, we will: (i) Investigate the cytotoxic effects of MTAs alone and with DOX in breast cancer cells, (ii) Assess the cytotoxic effects of MTAs and DOX in breast cancer cells overexpressing multi-drug resistant protein, (iii) Evaluate the adjuvant chemotherapeutic effects of MTAs and DOX in an in vivo breast cancer model, and (iv) Assess the cardioprotective and oxy-radical scavenging effects of MTAs in DOX- treated cardiomyocytes and in DOX-treated rat cardiomyopathy model. These aims will be accomplished using HPLC-fluorescence and HPLC-electrochemical detection techniques, scintimammography and echocardiography. Abnormal generation of reactive oxygen species will be detected using novel species- and target-specific probes. We will develop innovative MTA-based adjuvant therapy that can be used to inhibit DOX-induced cardiotoxicity. This research may potentially lead to novel ways for improving the therapeutic efficacy of DOX and other antitumor agents used in breast cancer treatment.
The long-term goal of this multiple PI proposal is to develop neuroprotective strategies that involve synthesis and testing of mitochondria-targeted antioxidants in a preclinical mouse model of Parkinson's Disease (PD) that can be subsequently translated to patients with PD. In this proposal, we have combined the expertise and experience of individuals in synthetic organic chemistry, free radical biology, neuropharmacology, and neurotoxicology from two institutions, the Medical College of Wisconsin and Iowa State University. This proposal is based on the discovery that mitochondria- targeted antioxidants (MTAs) inhibit oxidative stress and neuronal damage in 1-methyl-4- phenylpyridinium (MPP+) treated neuronal cell culture models of PD as well as in 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) animal models of PD. We hypothesize that MTAs provide an effective neuroprotective strategy for treatment of PD. As a corollary, we propose that MTAs attenuate mitochondria-derived reactive oxygen and nitrogen species (ROS/RNS), thereby protecting against inactivation of key redox sensors in response to mitochondrial neurotoxin exposure. Specifically, we will: (i) Design and synthesize MTAs and assess their cytoprotection in neuronal cell culture models of PD, (ii) Determine the molecular mechanisms of cytoprotection of MTAs in neuronal cell culture models of PD, (iii) Assess the neuroprotective effects of MTAs in a well-established preclinical MPTP mouse model of PD, (iv) Determine the activation/inactivation of key redox targets in mitochondria in response to MTA treatment in the preclinical MPTP mouse model, and (iv) Investigate the long-term tolerability of MTAs during chronic treatment in the mouse model. We will use several analytical techniques (low-temperature EPR, HPLC-fluorescence and electrochemical detection, HPLC/MS, proteomics) to detect and quantitate ROS/RNS, molecular biological approaches (apoptosis measurements, transcription factor translocation) to define the molecular mechanisms, and neurobehavioral and histopathological analyses to evaluate the neuroprotective effects. Abnormal generation of mitochondrial ROS/RNS in response to environmental toxins has been implicated in the pathogenesis of PD. Numerous antioxidants and iron chelators have been used with partial success in experimental animal models of PD. Emerging literature suggests that antioxidants specifically targeted to mitochondria might serve as promising neuroprotectants for treatment of PD. In this proposal, we will assess the neuroprotective efficacy of several novel MTAs in a cell culture and preclinical mouse model of PD. Systematic characterization of neuroprotective properties and long-term tolerability of novel MTAs in both cell culture and animal models will yield comprehensive preclinical data for the clinical development of efficacious mitochondria-targeted antioxidant therapies for PD. PUBLIC HEALTH RELEVANCE: Parkinson's Disease (PD) is a debilitating neurodegenerative disease. Effective treatment to intervene the progression of neurodegenerative processes in PD remains unavailable. Using a cell culture and a mouse model of PD, we propose to develop a "mitochondria-targeted" antioxidant - based neuroprotective strategy for treating PD. Proposed studies which bring together chemical and neuropharmacological expertise from two institutions (Medical College of Wisconsin and Iowa State University), will help us develop efficacious mitochondria-targeted antioxidants for treatment of PD as well as understand the possible neuroprotective mechanisms of these novel class of agents.
Parkinson's Disease (PD) is a debilitating neurodegenerative disease. Effective treatment to intervene the progression of neurodegenerative processes in PD remains unavailable. Using a cell culture and a mouse model of PD, we propose to develop a "mitochondria-targeted" antioxidant - based neuroprotective strategy for treating PD. Proposed studies which bring together chemical and neuropharmacological expertise from two institutions (Medical College of Wisconsin and Iowa State University), will help us develop efficacious mitochondria-targeted antioxidants for treatment of PD as well as understand the possible neuroprotective mechanisms of these novel class of agents.
LPTA-MEDIATED TRANSPORT OF LPS (KLUG, PI, R01 GM108817)
DESCRIPTION (provided by applicant): Lipopolysaccharide (LPS) is the major component of the outer leaflet of the outer membrane (OM) of Gram-negative bacteria such as Escherichia coli, Salmonella typhimurium and many other important pathogens. LPS, also referred to as endotoxin, is essential for survival in this large class of bacteria and serves as a first line of defense against hostile environments encountered during host infection. Given the essential role of LPS in the survival of Gram-negative bacteria - i.e., the bacterial cells die if any step o LPS transport does not occur - and the unique cell surface it creates, a detailed understanding of the proteins and mechanisms involved in LPS synthesis and transport will be the foundation on which to develop novel antibiotics against these promising new drug targets. Many of the proteins involved in LPS transport have been identified through recent genetics studies, suggesting that a set of seven inner membrane (IM), periplasmic, and OM proteins (named LptA, LptB, LptC, LptD, LptE, LptF, and LptG) are directly involved in moving LPS from the IM to the OM. However, the mechanism of how this group of proteins transports LPS to the OM is yet unknown. One of the most striking questions about this process is how the hydrophobic domain of LPS crosses the periplasm. Therefore, the proposed studies will focus on how the periplasmic protein LptA receives LPS from the IM-associated protein LptC, how LptA protects the hydrophobic acyl chains of LPS as it crosses the periplasm, and how LptA delivers LPS to LptDE at the OM. The successful completion of the proposed studies will include the development of a novel functional assessment tool for LptA, the creation of a comprehensive library of in vivo growth assay results to identify LptA amino acids critical for its structure or function, the identification of the specific LptA sites and conformational changes involved in LPS binding, and the characterization of the interactions between LptA and its binding partners LptC, LptDE, and LPS. The results of the novel genetic screenings, the laser light scattering analyses, the innovative electron paramagnetic resonance (EPR) spectroscopy studies, and the isothermal titration calorimetry measurements will provide detailed insights into the mechanism of LPS transport across the periplasm of Gram-negative bacteria. This unique knowledge will greatly enhance our growing understanding of LPS transport in bacteria and set the stage for future studies on the other Lpt proteins of unknown structure and function.
The cell membrane has a 2-dimensional liquid-like structure containing domains that form and disperse continuously on various time and space scales. Rafts are membrane domains that require lipid interactions for their formation. The long-term objective of this proposal is to better understand the molecular mechanisms by which rafts form, are maintained and disintegrate in biological membranes, in particular in the plasma membrane of fiber cells of the eye lens. Detergent insolubility, which has been used to define rafts biochemically, does not reflect pre-existing structures and organization of the membrane. Furthermore, such an approach is not useful for understanding the size, lifetime and dynamics of the raft-constituent molecules and the raft itself. To address these issues, it is proposed to apply the pulse EPR spin labeling technique "discrimination by oxygen transport (DOT)" for in situ studies of rafts in both model and cell membranes. Since the spin-lattice relaxation time of spin labels is sufficiently long, membrane dynamics can be observed on the time scale 0.1 - 100 mu s. The DOT method permits discrimination of different membrane domains because the collision rate between O2 and the nitroxide moiety of spin labels (oxygen diffusion-concentration product) can be quite different in these domains. Additionally, membrane domains can be characterized by profiles of the oxygen diffusion concentration product in situ without the need for separation. This method is especially suitable for obtaining time-space characteristics of small/transient domains. It is hypothesized that rafts form liquid-ordered domains in the plasma membrane liquid-disordered environment. Membrane lipid composition as well as protein content is expected to modulate raft size and dynamics. The DOT method will be used to test the hypothesis on well-defined model systems in which domain size and the lipid exchange rate will be controlled by membrane lipid composition, selected protein and peptide content, and temperature. Furthermore, it will be used to study domain structure in cell membranes. These studies will include mature and aged fiber cell membranes in which the increased cholesterol/lipid ratio and elevated level of sphingomyelin create conditions favoring the formation of rafts. It is proposed: 1) to detect coexisting liquid-ordered and liquid-disordered domains in membranes containing cholesterol; 2) to evaluate the size and stability of the raft domains in model membranes made from raft-forming mixtures; 3) to examine how membrane anchored proteins and transmembrane alpha-helical peptides affect the organization and dynamics of these lipid raft domains; and 4) to apply the DOT method to look for raft domains in fiber cell plasma membranes of the eye lens during maturation and aging, as well as membrane models of mature, aged and cataractous lenses. Age-related nuclear cataract is a primary cause of blindness in the elderly in third world countries.