John Irwin Clark
University Of Washington
Project start date: 1982-06-01
Project end date: 2014-03-31
Sponsored Links Excellgen http://Excellgen.com
DEVELOPMENT AND MAINTENANCE OF LENS TRANSPARENCY
John Irwin Clark, Professor And Chairman
University Of Washington, Office Of Sponsored Programs, Seattle, Wa 98195-9472
Grant 3R01EY004542-27S1 from National Eye Institute
Abstract: The primary objective of the proposed research is to define the functional mechanism for the protective actions of the stress protein, human ?B crystallin, a small heat shock protein (sHSP) and molecular chaperone. HYPOTHESIS 1 The activity of multiple interactive sequences in human??B crystallin provides protection against protein unfolding, aggregation, and toxicity in aging disorders that include cataract, neurodegeneration, AMD, and possibly, neuromuscular disorders. Synthetic peptides based on the individual interactive domains in ?B crystallin mediate the formation of amyloid fibrils and will be used to characterize the collective mechanism(s) of the interactive domains in the action of ?B crystallin. AIM 1 will optimize the function of human ?B crystallin, the archetype of all small heat shock proteins (sHSP). Functional assays will be used to characterize the effects of new mutants of ?B crystallin on the stress response and protection against protein unfolding and aggregation in vitro. AIM 2 will use slit lamp imaging to conduct phenotype analyses of lenses in transgenic mouse models for neurodegenerative and neuromuscular disorders. This aim addresses HYPOTHESIS 2 The loss of lens transparency is a sensitive indicator of altered protein interactions in neurodegenerative and neuromuscular disorders. The unique accessibility of the lens for non-invasive optical examinations makes lens cells excellent for the study of basic mechanisms of aggregation and amyloid formation in living animals. HYPOTHESIS 3 The proteins responsible for the lens phenotype in neurodegeneration may also be responsible for the lens phenotype in neuromuscular disorders, and AIM 3 will identify critical protein constituents responsible for the structural phenotype in transgenic mouse models for neurodegenerative and neuromuscular disorders. Mass spectrometry of samples obtained using laser capture microdissection (LCM) will be combined with immunocytochemistry, and light and electron microscopy to correlate protein constituents with structural modifications accounting for the phenotype observed in lenses of mouse models for neurodegenerative and neuromuscular disorders. SIGNIFICANCE FOR PUBLIC HEALTH Protein unfolding and amyloid/aggregate formation characterize disorders of aging that include cataract, age related macular degeneration (AMD), neurodegeneration, cardiomyopathy and muscular dystrophy. With the increase in human longevity, the impact of aging diseases is increasing dramatically. Defining the basic mechanism for the protective activity of the stress protein ?B crystallin will be a major advance in biomedical research and has the potential to provide novel therapeutic targets for aging disorders. The lens is ideal for innovative functional studies of ?B crystallin function in vivo and may have significance as a biomarker for the initiation and progression of neurodegenerative and neuromuscular disease. This application is a study of the interactions of the stress protein human ?B crystallin responsible for the fundamental protective mechanism(s) against protein unfolding and aggregation. The approach is to characterize the activity of the interactive domains and quantify their affinities in vitro while studying their influence on the transparent structure of the lens in vivo using the slit lamp. The results will advance biomedical research in understanding the endogenous function of sHSP in diseases of aging that include neurodegeneration, cardiovascular diseases, muscular dystrophy, AMD (age related macular dystrophy) and cataract. This research has the potential for translational applications to the development of novel therapeutics against major diseases of aging. The lens is used for these studies because of its unique accessibility for the in vivo investigation of protein unfolding and aggregation diseases in aging and the well known protective effects of ?B crystallin
Keywords: Accounting; Address; Affinity; Age; Age related macular degeneration; Aging; Amyloid; Amyloid Proteins; Amyloid Substance; Amyloidosis; Animals; Assay; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Biomedical Research; Body Tissues; Brain; Cardiomyopathies; Cardiovascular Diseases; Cataract; Cell Components; Cell Structure; Cells; Cellular Structures; Chaperone; Chimera Protein; Chimeric Proteins; Classification; Crystalline Lens; Crystallins; Cytoskeletal Proteins; Deposit; Deposition; Development; Disease; Disorder; Dissociation; Electron Microscopy; Encephalon; Encephalons; Filament; Fusion Protein; HSP; Heat shock proteins; Human; Human, General; Image; In Vitro; Individual; Investigation; Len, Crystalline; Len, Eye; Length of Life; Lens; Lens Fiber; Lens Proteins; Lens of Eye; Lens, Crystalline; Lens, Eye; Lenses; Life; Light; Link; Literature; Longevity; Maculopathy, Age-Related; Maintenance; Maintenances; Man (Taxonomy); Man, Modern; Mass Spectrum; Mass Spectrum Analysis; Measures; Mediating; Microscopic; Modeling; Modification; Molecular Chaperones; Molecular Interaction; Muscular Dystrophies; Mycocardium Disease; Myocardial Diseases; Myocardial Disorder; Myocardiopathies; Myodystrophica; Myodystrophy; NRVS-SYS; Nerve Cells; Nerve Degeneration; Nerve Unit; Nervous System; Nervous System, Brain; Nervous system structure; Neural Cell; Neurocyte; Neurologic Body System; Neurologic Organ System; Neuromuscular Diseases; Neuron Degeneration; Neurons; Ocular Lens; Optics; Peptides; Phenotype; Photometry/Spectrum Analysis, Mass; Photoradiation; Phylogenetic Analysis; Phylogenetics; Proteins; Public Health; Research; Sampling; Senescence; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Stress Proteins; Structural Protein; Structure; Systematics; Testing; Therapeutic; Tissues; Toxic effect; Toxicities; Transgenic Mice; aging brain; amyloid assembly; amyloid disease; amyloid fibril formation; amyloid formation; base; biological adaptation to stress; biomarker; cardiovascular disorder; cataractogenesis; cataractous lenses; disease/disorder; gene product; imaging; immunocytochemistry; in vitro testing; in vivo; innovate; innovation; innovative; insight; insoluble aggregate; laser capture microdissection; lens; lens transparency; life span; lifespan; light scattering; mouse model; mutant; myocardium disorder; myoneural disorder; neural degeneration; neurodegeneration; neuromuscular disorder; neuronal; neuronal degeneration; new therapeutic target; new therapeutics; next generation therapeutics; novel; novel therapeutics; protective effect; protein B; protein aggregate; protein protein interaction; public health medicine (field); public health relevance; reaction; crisis; senescent; senile macular disease; stress protein; stress response; stress; reaction; synthetic peptide
Relevance: This application is a study of the interactions of the stress protein human ¿B crystallin responsible for the fundamental protective mechanism(s) against protein unfolding and aggregation. The approach is to characterize the activity of the interactive domains and quantify their affinities in vitro while studying their influence on the transparent structure of the lens in vivo using the slit lamp. The results will advance biomedical research in understanding the endogenous function of sHSP in diseases of aging that include neurodegeneration, cardiovascular diseases, muscular dystrophy, AMD (age related macular dystrophy) and cataract. This research has the potential for translational applications to the development of novel therapeutics against major diseases of aging. The lens is used for these studies because of its unique accessibility for the in vivo investigation of protein unfolding and aggregation diseases in aging and the well known protective effects of ¿B crystallin
Project start date: 2009-09-01
Project end date: 2011-08-31
Budget start date: 1-SEP-2009
Budget end date: 31-AUG-2011
PFA/PA: PA-07-070
3R01EY004542-27S1 (2009): $194168
3R01EY004542-26S1 (2008): $69033
Grants awarded to John Irwin Clark
John Irwin Clark, Professor And Chair
University Of Washington Office Of Sponsored Programs Seattle, Wa 98105
Grant 1S10RR022533-01 from National Center For Research Resources IRG: ZRG1
Abstract: We are requesting funds to purchase a Zeiss LSM 510 META NLO on an upright epifluorescence Axioplan 2 microscope. Over the last several years, the invention of multiphoton (two-photon laser scanning) microscopy has spearheaded cellular research into new realms, providing real-time views of cell morphology, function and development within the living animal Multiphoton microscopy uses high-energy infrared pulses to excite fluorophores, therefore enabling imaging of structures deep (hundreds of micrometers) within the tissue. Another advantage of multiphoton imaging is that living specimens can be imaged repeatedly over long periods of time (hours to weeks). This is because excitation is effectively confined to the focal point of the beam, such that fluorophores above and below are not excited. Photobleaching and phototoxicity, common limitations of confocal microscopy, are thus much reduced when using multiphoton microscopy. This microscope will be used to support the ongoing, funded research programs of three primary investigators in two different departments at the University of Washington. Although these studies will focus on basic mechanisms of neural development and plasticity, the findings will have implications for research in neuroscience and neurological diseases, ophthalmology and vision research and deafness and auditory dysfunction. The requested instrument would be housed in the Biological Structure Imaging facility, and would be administered and maintained by funds from the department. The proposed administrative structure and management of the instrument are optimized for the best utilization of the microscope time, thus assuring the maximum benefit to the primary investigators, as well as a broad range of secondary users at the University.
Keywords: microscopy, fluorescent dye /probe, ionophore, university
Project start date: 2006-06-15
Project end date: 2008-06-14
1S10RR022533-01 (2006): $500000
DEVELOPMENT AND MAINTENANCE OF LENS TRANSPARENCY
John Irwin Clark, Professor And Chair
University Of Washington Office Of Sponsored Programs Seattle, Wa 98105
Grant 5R01EY004542-21 from National Eye Institute IRG: VISA
Abstract: Transparent cellular structure is the fundamental property of the biological lens. The next five year application will investigate the molecular basis for the transparent structure of lens cells. The PI s past studies of the collective interactions measured by Tc led to a clinical trial of pantethine as an anticataract agent in humans. Pantethine was used because it was effective against protein aggregation and lens opacification in animal models. During the next five years, the functional motifs of selected human lens proteins that appear to be involved with the interactions important for development and maintenance of lens cell transparency will be investigated. Recombinant human lens crystallins (wild-type and mutant) will be expressed, purified and characterized using biochemical assays for interactions involving human crystallins (AIM 1). Novel quantitative methods for Fourier, power law and fractal analysis will be used to characterize the microscopic structure of lens cells and of model solutions of human lens cytoplasmic proteins (AIM 2). The methods will be used to investigate and quantify the size and order/disorder of major structural components found in lens cytoplasm and will evaluate the fractal dimension of lens cell structure. Preliminary results suggest that alpha B crystallin and cytoskeletal proteins have important functions in the development and maintenance of lens cell transparency. Transgenic and knockout models for cataract will be used for in vivo studies of the relationships between lens crystallins, cytoskeletal proteins and lens opacification (AIM 3). The results of the proposed studies will have direct application in the design and testing of new therapeutic approaches to inhibit protein aggregation and lens opacification in humans.
Keywords: cataract, intermolecular interaction, lens, lens protein, protein structure function, aging, cytoskeletal protein, mathematical model, pathologic process, affinity chromatography, electron microscopy, human tissue, laboratory rat, tissue /cell culture
Project start date: 1982-06-01
Project end date: 2003-05-31
5R01EY004542-21 (2002): $383443
5R01EY004542-20 (2001): $345174
5R01EY004542-19 (2000): $340721
5R01EY004542-18 (1999): $325184
5R01EY004542-11 (1992): $226744
DEVELOPMENT AND MAINTENANCE OF TRANSPARENCY IN LENSES
John Irwin Clark, Professor And Chair
University Of Washington Office Of Sponsored Programs Seattle, Wa 98105
Grant 5R01EY004542-06 from National Eye Institute IRG: VISA
Abstract: The proposed research program will establish the sequential steps in the development of lens transparency. The research will be quantitative study of lens cell growth and development using computer aided 3-dimensional reconstructions to define relationships between structural and chemical parameters and to analyze dimensions of transparent and opaque domains at different stages of development; concentrations and spatial distributions of Ca, Mg, P, S, H, K, Na, Cl; and areas of surfaces labeled with cholesterol and phospholipid probes. Previous research has led to the identification of reversible pathways for cellular restructuring during the early stages of cataract development. I suggest that these mechanisms of restructuring do not appear spontaneously when lens cells are exposed to cataractous conditions, but exist in normal cells before the onset of cataracts and function in the normal development and maintenance of lens transparency. Thus, early stages of cataract restructuring may be understood by studying the mechanisms controlling normal cell restructuring, quite apart from pathological conditions. The study of these mechanisms will use 3-dimensional computer aided imaging to analyze relationships between structure and composition of developing lens cells. New methods of light microspectrometry and LASER/NMR instrumentation will be developed for data collection. I propose to use the information gained from this study as the basis for new methods for prevention of opacification and maintenance of transparency in animal and human eyes.
Keywords: EYE DISORDERS, LENS DISORDERS, CATARACT, EYE, LENS, GROWTH AND DEVELOPMENT, HISTOGENESIS, PHYSICAL PROPERTIES (STATES)(PROCESSES), VISUAL SCIENCES A STUDY SECTION, cell age, ACIDS-BASES, HYDROGEN-ION CONCENTRATION, CHEMICAL STRUCTURE--BIOLOGICAL ACTIVITY, EYE, LENS PROTEINS, MEMBRANE SYNTHESIS AND RECONSTITUTION, MEMBRANE, MEMBRANE (BIOLOGICAL) STRUCTURE, METALS, PHOSPHOLIPIDS, aging, calcium, BIOMEDICAL ENGINEERING, INSTRUMENTATION NOT CLINICALLY ORIENTED, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, LASER, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, MICROSPECTROPHOTOMETRY, CHEMISTRY, ANALYTICAL METHODS, X-RAY STRUCTURE ANALYSIS, COMPUTER PRINTING-GRAPHICS (GENERAL), DYES, EMBRYOLOGY, MAMMALIAN EMBRYOLOGY, HUMAN EMBRYO-FETUS, HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, MAMMALS, RODENTS, MYOMORPHA, RATS (LABORATORY), OPTICS, MICROSCOPY, ELECTRON, OPTICS, MICROSCOPY, ELECTRON SCANNING, OPTICS, NMR IMAGING, PHYSICAL SEPARATION, CHROMATOGRAPHY, HIGH PRESSURE LIQUID, RADIOTRACERS, TEMPERATURE, FREEZE-ETCHING, TISSUE (CELL) CULTURE
Project start date: 1982-06-01
Project end date: 1988-05-31
Sponsored Links Excellgen http://Excellgen.com
DEVELOPMENT AND MAINTENANCE OF LENS TRANSPARENCY
John Irwin Clark, Professor And Chairman
University Of Washington, Office Of Sponsored Programs, Seattle, Wa 98195-9472
Grant 5R01EY004542-28 from National Eye Institute
Abstract: The primary objective of the proposed research is to define the functional mechanism for the protective actions of the stress protein, human ?B crystallin, a small heat shock protein (sHSP) and molecular chaperone. HYPOTHESIS 1 The activity of multiple interactive sequences in human??B crystallin provides protection against protein unfolding, aggregation, and toxicity in aging disorders that include cataract, neurodegeneration, AMD, and possibly, neuromuscular disorders. Synthetic peptides based on the individual interactive domains in ?B crystallin mediate the formation of amyloid fibrils and will be used to characterize the collective mechanism(s) of the interactive domains in the action of ?B crystallin. AIM 1 will optimize the function of human ?B crystallin, the archetype of all small heat shock proteins (sHSP). Functional assays will be used to characterize the effects of new mutants of ?B crystallin on the stress response and protection against protein unfolding and aggregation in vitro. AIM 2 will use slit lamp imaging to conduct phenotype analyses of lenses in transgenic mouse models for neurodegenerative and neuromuscular disorders. This aim addresses HYPOTHESIS 2 The loss of lens transparency is a sensitive indicator of altered protein interactions in neurodegenerative and neuromuscular disorders. The unique accessibility of the lens for non-invasive optical examinations makes lens cells excellent for the study of basic mechanisms of aggregation and amyloid formation in living animals. HYPOTHESIS 3 The proteins responsible for the lens phenotype in neurodegeneration may also be responsible for the lens phenotype in neuromuscular disorders, and AIM 3 will identify critical protein constituents responsible for the structural phenotype in transgenic mouse models for neurodegenerative and neuromuscular disorders. Mass spectrometry of samples obtained using laser capture microdissection (LCM) will be combined with immunocytochemistry, and light and electron microscopy to correlate protein constituents with structural modifications accounting for the phenotype observed in lenses of mouse models for neurodegenerative and neuromuscular disorders. SIGNIFICANCE FOR PUBLIC HEALTH Protein unfolding and amyloid/aggregate formation characterize disorders of aging that include cataract, age related macular degeneration (AMD), neurodegeneration, cardiomyopathy and muscular dystrophy. With the increase in human longevity, the impact of aging diseases is increasing dramatically. Defining the basic mechanism for the protective activity of the stress protein ?B crystallin will be a major advance in biomedical research and has the potential to provide novel therapeutic targets for aging disorders. The lens is ideal for innovative functional studies of ?B crystallin function in vivo and may have significance as a biomarker for the initiation and progression of neurodegenerative and neuromuscular disease. This application is a study of the interactions of the stress protein human ?B crystallin responsible for the fundamental protective mechanism(s) against protein unfolding and aggregation. The approach is to characterize the activity of the interactive domains and quantify their affinities in vitro while studying their influence on the transparent structure of the lens in vivo using the slit lamp. The results will advance biomedical research in understanding the endogenous function of sHSP in diseases of aging that include neurodegeneration, cardiovascular diseases, muscular dystrophy, AMD (age related macular dystrophy) and cataract. This research has the potential for translational applications to the development of novel therapeutics against major diseases of aging. The lens is used for these studies because of its unique accessibility for the in vivo investigation of protein unfolding and aggregation diseases in aging and the well known protective effects of ?B crystallin
Keywords: Accounting; Address; Affinity; Age; Age related macular degeneration; Aging; Amyloid; Amyloid Proteins; Amyloid Substance; Amyloidosis; Animals; Assay; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Biomedical Research; Body Tissues; Brain; Cardiomyopathies; Cardiovascular Diseases; Cataract; Cell Components; Cell Structure; Cells; Cellular Structures; Chaperone; Chimera Protein; Chimeric Proteins; Crystalline Lens; Crystallins; Cytoskeletal Proteins; Deposit; Deposition; Development; Disease; Disorder; Dissociation; Electron Microscopy; Encephalon; Encephalons; Filament; Fusion Protein; HSP; Heat shock proteins; Human; Human, General; Image; In Vitro; Individual; Investigation; Len, Crystalline; Len, Eye; Length of Life; Lens; Lens Fiber; Lens Proteins; Lens of Eye; Lens, Crystalline; Lens, Eye; Lenses; Life; Light; Link; Literature; Longevity; Maculopathy, Age-Related; Maintenance; Maintenances; Man (Taxonomy); Man, Modern; Mass Spectrum; Mass Spectrum Analysis; Measures; Mediating; Mice, Transgenic; Microscopic; Modeling; Modification; Molecular Chaperones; Molecular Interaction; Muscular Dystrophies; Mycocardium Disease; Myocardial Diseases; Myocardial Disorder; Myocardiopathies; Myodystrophica; Myodystrophy; NRVS-SYS; Nerve Cells; Nerve Degeneration; Nerve Unit; Nervous System; Nervous System, Brain; Nervous system structure; Neural Cell; Neurocyte; Neurologic Body System; Neurologic Organ System; Neuromuscular Diseases; Neuron Degeneration; Neurons; Ocular Lens; Optics; Peptides; Phenotype; Photometry/Spectrum Analysis, Mass; Photoradiation; Phylogenetic Analysis; Phylogenetics; Proteins; Public Health; Research; Sampling; Senescence; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Stress Proteins; Structural Protein; Structure; Testing; Therapeutic; Tissues; Toxic effect; Toxicities; Transgenic Mice; aging brain; amyloid assembly; amyloid disease; amyloid fibril formation; amyloid formation; base; biological adaptation to stress; biomarker; cardiovascular disorder; cataractogenesis; cataractous lenses; disease/disorder; gene product; imaging; immunocytochemistry; in vitro testing; in vivo; innovate; innovation; innovative; insight; insoluble aggregate; laser capture microdissection; lens; lens transparency; life span; lifespan; light scattering; mouse model; mutant; myocardium disorder; myoneural disorder; neural degeneration; neurodegeneration; neuromuscular disorder; neuronal; neuronal degeneration; new therapeutic target; new therapeutics; next generation therapeutics; novel; novel therapeutics; protective effect; protein B; protein aggregate; protein protein interaction; public health medicine (field); public health relevance; reaction; crisis; senescent; senile macular disease; stress protein; stress response; stress; reaction; synthetic peptide
Relevance: This application is a study of the interactions of the stress protein human ¿B crystallin responsible for the fundamental protective mechanism(s) against protein unfolding and aggregation. The approach is to characterize the activity of the interactive domains and quantify their affinities in vitro while studying their influence on the transparent structure of the lens in vivo using the slit lamp. The results will advance biomedical research in understanding the endogenous function of sHSP in diseases of aging that include neurodegeneration, cardiovascular diseases, muscular dystrophy, AMD (age related macular dystrophy) and cataract. This research has the potential for translational applications to the development of novel therapeutics against major diseases of aging. The lens is used for these studies because of its unique accessibility for the in vivo investigation of protein unfolding and aggregation diseases in aging and the well known protective effects of ¿B crystallin
Project start date: 1982-06-01
Project end date: 2014-03-31
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
PFA/PA: PA-07-070
5R01EY004542-28 (2010): $592720
2R01EY004542-27 (2009): $598445
SPARC And The Differentiation Of Transparent Lens Fibers
John Irwin Clark, Professor And Chair
University Of Washington Office Of Sponsored Programs Seattle, Wa 98105
Grant 5R01EY013180-02 from National Eye Institute IRG: VISA
Abstract: SPARC (secreted protein acidic and rich in cysteine) is a major component of remodeling tissues and, as such, features prominently in morphogenesis, development, injury, and repair. It belongs to the matricellular class of secreted glycoproteins that, although structurally dissimilar, regulate interactions between cells and extracellular matrix (ECM). SPARC has been shown specifically to a) inhibit the cell cycle, b) prevent or disrupt cell adhesion, c) inactivate cellular responses to certain growth factors including basic fibroblast growth factor (bFGF or FGF2), d) regulate ECM production, e) bind to specific collagens including those of the basement membrane, and f) promote a rounded cell shape and reorganization of the actin cytoskeleton. These properties provide a strong rationale for a targeted disruption of the SPARC gene. Although SPARC -/- mice were viable and fertile, there were phenotypic abnormalities associated with connective tissue. The major, dominant phenotype, however, was the appearance of lenticular opacity at 1-2 mo. after birth and progression to mature cataracts by 8 mo. The explanation for the effect of SPARC on lens transparency in SPARC-null mice is unknown and forms the basis for this proposal. We will test the hypothesis that SPARC regulates lens epithelial cell differentiation via 1) its modulation of signaling pathway(s) activated by bFGF, and 2) its effects on lens cell-cell and cell-ECM (lens capsule) interactions. Our hypothesis is based on the following premises a) throughout lens fiber differentiation, the basal surface of proliferating, migrating, and elongating fiber is directly attached to the ECM of the capsule, which contains SPARC, b) development and fiber differentiation are known to be regulated by FGF2, and c) cell differentiation in the lens is rigorously controlled from early in fetal development throughout the life of the animal (even minor perturban5R01EY013180-05
Keywords: 2005
Project start date: 2001-05-01
Project end date: 2006-04-30
5R01EY013180-02 (2002): $273438
1R01EY013180-01A1 (2001): $286750
5R01EY013180-05 (2005): $273375
5R01EY013180-04 (2004): $273375
CELL AGING AND THE EARLY STAGES OF CATARACT DEVELOPMENT
John Irwin Clark, Professor And Chair
University Of Washington Office Of Sponsored Programs Seattle, Wa 98105
Grant 5R01EY004542-03 from National Eye Institute IRG: VISA
Abstract: Our long term goal is to identify the earliest stages of lens opacification in which the reversal of the cataract may be possible. This could lead to the development of methods to improve transparency in human lenses at the early reversible cataract. A major concern is that morphological and biochemical canges due to the early of opacification are difficult to distinguish from changes due to normal aging. The objective of this proposal is to develop sensitive new quantitative techniques to characterize the parameters associated with cell aging and those associated with early stages of opacification. We will use laser light scattering spectroscopy to differentiate light scattering properties of normal lens aging from light scattering behavior which leads to cataract. In order to study cellular restructuring due to aging and the initial restructuring of opacifying lens cells, we will develop a unique methods of image analysis of electron micrographs. This method will be capable of identifying the subtle morphological changes which lead to the formation of light scattering elements in lens cells. In additon, we will apply high pressure liquid chromatography as a new and highly sensitive method for the analysis of biochemical constituents in aging cells and in developing cataracts. The results of these studies will be used to evaluate the initial morphological and biochemical changes in lens fiber cells that accompany the early stages of cataract development and to differentiate these changes from those which are due to the normal aging process.
Keywords: EYE DISORDERS, LENS DISORDERS, CATARACT, OPTICS, LIGHT SCATTERING, VISUAL SCIENCES A STUDY SECTION, aging, cell age, CHEMICAL STRUCTURE--BIOLOGICAL ACTIVITY, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, LASER, HUMAN, TISSUES, FLUIDS ETC. FROM NON-RELATED SOURCES OUTSIDE IMMEDIATE PROJECT, MAMMALS, LAGOMORPHS, MAMMALS, RODENTS, MYOMORPHA, RATS (LABORATORY), OPTICS, MICROSCOPY, ELECTRON, OPTICS, MICROSCOPY, ELECTRON SCANNING, PHYSICAL SEPARATION, CHROMATOGRAPHY, HIGH PRESSURE LIQUID, TISSUE (CELL) CULTURE
Project start date: 1982-06-01
Project end date: 1985-05-31
John Irwin Clark
University Of Washington, Office Of Sponsored Programs, Seattle, Wa 98195-9472
Grant 5T32EY007031-34 from National Eye Institute
Abstract: Multidisciplinary training in basic science and clinical aspects of vision research will be conducted at both the pre- and post-doctoral levels at the University of Washington. Faculty in 9 departments in the School of Medicine and the School of Arts and Sciences are preceptors for pre- and post-doctoral trainees in structural biology, cell and molecular biology, molecular biophysics, visual function and systems neurobiology using a variety of model systems in vertebrate animals and selected cell and organ culture systems. Vision research at the University of Washington is supported by more than 61 grants and funds from private and public sources. Vision training will emphasize modern technological approaches to clinical problems in the visual system in both normal and abnormal conditions. New faculty are expected to join the Vision Program as preceptors as the vision research program expands. Major additions to the local resources include 2 confocal microscopes and a laser capture microscope. The scientific environment for multidisciplinary science and clinical collaboration is strength of Vision Training at the University of Washington and encourages interdisciplinary interactions that will develop new understanding of the scientific basis for important clinical problems in vision. Education and training in proteomics and informatics will be an emphasis of the next grant period. The Vision Training Program is designed to provide a rigorous education for successful careers in academic and biomedical research in visual sciences
Keywords: Research Training; Vision research
Project start date: 1976-07-01
Project end date: 2012-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
5T32EY007031-34 (2010): $202877
Sponsored Links Excellgen http://Excellgen.com
5T32EY007031-33 (2009): $194930
5T32EY007031-32 (2008): $269099
2T32EY007031-31 (2007): $269099
Development And Maintenance Of Lens Transparency
John Irwin Clark, Professor And Chair
University Of Washington Office Of Sponsored Programs Seattle, Wa 98105
Grant 5R01EY004542-26 from National Eye Institute IRG: ZRG1
Abstract: Human alphaBeta crystallin is the archetype for small heat shock proteins, sHSP, that are involved in protein aggregation and filament assembly diseases including cataracts, neurodegeneration, cardiomyopathy and desmin related myopathy. Interactions between alphaBeta crystallin are necessary for normal filament assembly and organization of crystallins in lens cells. In aim 1, characterization of the interactive sites for subunit assembly, for cytoskeletal proteins and for target peptides on human alphaBeta crystallin, the peptide sequences of the interactive domains on human alphaBeta crystallin will be identified using a protein multipin arrays. The affinities between the interactive domains will be quantified using surface plasmon resonance (SPR) and characterized functionally using in vitro and in vivo assays for chaperone activity. The results are expected to provide new information on the structural basis for the assembly of sHSP subunits to functional complexes and for their interaction with chaperone target proteins and with cellular filaments and cytoskeletal elements. In aim 2, in vivo evaluation of retina - lens relationships that may influence development and maintenance of lens transparency in transgenic mice, the historical hypothesis that a lens - retina relationship is important for normal development of lens cell transparency will be studied. Electroretinograms (ERG) and digital slit lamp recordings of opacity in selected animal models will quantify transparency with retinal function during the development of the lens and during loss of transparency in models for cataract formation. Lastly, the hypothesis that lens cytoskeleton provides a scaffold for development and maintenance of transparent lens fiber structure will be investigated in aim 3, observe the cellular organization of major structural proteins in differentiating lens fibers during development of lens transparency and during loss of transparency in the selenite rat and in selected transgenic mouse models using confocal microscopy and electron microscopy (EM). The patterns and distribution of the cytoskeleton and crystallins during differentiation of transparent lens fibers will be investigated using electron microscopy and confocal immunocytochemistry.
Keywords: cataract, crystallin, histogenesis, lens, lens protein, protein structure function, aging, cytoskeletal protein, cytoskeleton, heat shock protein, mathematical model, molecular assembly /self assembly, pathologic process, protein protein interaction, selenium, clinical research, confocal scanning microscopy, electron microscopy, electroretinography, genetically modified animal, human tissue, laboratory mouse, laboratory rat, surface plasmon resonance, tissue /cell culture
Project start date: 1982-06-01
Project end date: 2008-07-31
5R01EY004542-26 (2007): $569219
5R01EY004542-25 (2006): $555755
5R01EY004542-24 (2005): $555778
5R01EY004542-23 (2004): $542904
2R01EY004542-22 (2003): $623244
5R01EY004542-16 (1997): $304479
5R01EY004542-14 (1995): $276825
Sponsored Links Excellgen http://Excellgen.com
5R01EY004542-13 (1994): $268238
2R01EY004542-12 (1993): $273409
John Irwin Clark, Professor And Chair
University Of Washington Office Of Sponsored Programs Seattle, Wa 98105
Grant 5T32EY007031-30 from National Eye Institute IRG: ZEY1
Abstract: Applicant s ) Multidisciplinary training in basic science and clinical aspects of vision research will be conducted at both the pre- and post-doctoral levels at the University of Washington. Faculty in 11 departments in the School of Medicine and the School of Arts and Sciences will be preceptors for pre- and post-doctoral trainees in structural biology, cell and molecular biology, molecular biophysics, visual function and systems neurobiology using a variety of model systems in vertebrate. animals and selected cell and organ culture systems. Vision research at the University of Washington is supported by more than 88 grants and funds from private and public sources. Vision training will emphasize modem technological approaches to clinical problems in the visual system in both normal and abnormal conditions. Recent additions to the local resources include 2 confocal microscopes and a microarray facility. The unusually strong scientific environment for multidisciplinary and clinical collaboration is a strength of Vision Training at the University of Washington and encourages interdisciplinary interactions that will develop new understanding of the scientific basis of important clinical problems in vision. We propose a program designed to provide a rigorous scientific education for successful careers in academic and biomedical research in visual sciences.
Project start date: 1976-07-01
Project end date: 2007-08-31
5T32EY007031-30 (2006): $99876
5T32EY007031-29 (2005): $296606
5T32EY007031-28 (2004): $296388
5T32EY007031-27 (2003): $296172
2T32EY007031-26 (2002): $296820
John Irwin Clark
University Of Washington
Project start date: 1982-06-01
Project end date: 2014-03-31
John Irwin Clark, Professor And Chair
Biological Structureuniversity Of Washington
office Of Sponsored Programs
seattle, Wa 981959472
Grant 5T32EY007031-25 from National Eye Institute IRG: ZEY1
Project start date: 1976-07-01
Project end date: 2002-08-31
5T32EY007031-25 (2001): $211039
Sponsored Links Excellgen http://Excellgen.com
5T32EY007031-24 (2000): $174488
DEVELOPMENT AND MAINTENANCE OF LENS TRANSPARENCY
John Irwin Clark, Professor And Chair
Biological Structureuniversity Of Washington
office Of Sponsored Programs
seattle, Wa 981959472
Grant 5R01EY004542-15 from National Eye Institute IRG: ZRG1
Project start date: 1982-06-01
Project end date: 1998-05-31
5R01EY004542-15 (1996): $289884