Steven Bassnett
Washington University
Project start date: 1997-04-01
Project end date: 2015-03-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Steven Bassnett
Fiber Cell Formation In Normal And Cataractous Lenses
Steven Bassnett, Associate Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899
Grant 5R01EY009852-14 from National Eye Institute IRG: VISA
Abstract: The programmed elimination of nuclei and other cytoplasmic organelles from cells in the deep cortex of the ocular lens is one of the most striking aspects of terminal differentiation in this tissue. Organelles are light scattering structures and their removal ensures the transparency of the lens substance. The abnormal persistence of organelles is a characteristic feature of cataracts in mice and humans. During the last grant period we completed a morphometric analysis of organelle loss in the developing lens. In the current proposal we will address three related questions that arise directly from these studies What factors trigger organelle loss? What are the biochemical mechanisms underlying this process and, once the organelles have disappeared, what accounts for the extraordinary longevity of certain mRNAs? In considering possible triggering mechanisms, we will evaluate the role of tissue/matrix interactions and lenticular oxygen gradients. There are some striking similarities between organelle loss in the lens and classical apoptosis. We will determine which elements of the apoptotic pathway are utilized during organelle loss. We will also test the putative involvement of 15-lipoxygenase (an enzyme implicated in erythrocyte organelle loss) in fiber cell denucleation. Finally, our preliminary data indicate that following dissolution of the fiber cell nuclei, specific mRNAs are extraordinarily long lived. We will examine the role of cis- and trans-acting factors in stabilizing one such mRNA, delta crystallin. Together these experiments will provide important new information on lens organelle loss, fiber cell differentiation and cataractogenesis.
Keywords: cataract, cell differentiation, fiber cell, lens, cytoskeleton, genetic transcription, intracellular transport, organelle, Primate, chick embryo, chicken, computer program /software, confocal scanning microscopy, dissection, fluorescent dye /probe, laboratory mouse, laboratory rat, microelectrode, polymerase chain reaction, tissue /cell culture
Project start date: 1992-09-30
Project end date: 2006-12-31
CORE GRANT FOR VISION RESEARCH
Steven Bassnett
Washington University, Campus Box 1054, Saint Louis, Mo 63130-4899
Grant 5P30EY002687-31 from National Eye Institute
Abstract: The objective of this application is to provide established NEI-funded vision scientists with additional, shared support to enhance their individual research capabilities. A further goal is to enhance the research capability of Washington University for conducting vision research by encouraging collaborative studies and attracting other scientists to vision research. These objectives will be achieved by operating 5 core modules whose functions will be to provide the following services 1. A morphology core module will provide technical support and expertise in histological procedures such as processing frozen and fixed tissue samples, blocks, and histological and immunological staining. The module will provide expertise in conducting in situ hybridization studies and will coordinate with the imaging module to assist with brightfield and fluorescence microscopy as well as transmission and scanning electron microscopy. 2. An electronics services core module will design and fabricate electronic devices for research investigators, will provide diagnosis, repair and maintenance of research instruments and will assist in modification and/or development of computer software for unique research applications. 3. A molecular biology module will provide assistance in the design and preparation of gene constructs for gene targeting, preparation of DNA clones and probes, design of polymerase chain reaction components, microinjection services for production of transgenic and gene targeted mice, flow cytometry, and training and oversight of shared molecular biology equipment. 4. An imaging core module will provide assistance and expertise in all aspects of conventional light microscopy and confocal microscopy. Additional capabilities to be supported include support for imaging applications involving fluorescence resonance energy transfer and fluorescence correlation spectroscopy. Technical support for image analysis using deconvolution and volume rendering software tools will also be provided. 5. A biostatistics core module will assist in the design and implementation of pilot studies, will provide statistical and methodological expertise in study design, will assist to assure validity of statistical analyses and reported results, and will assist in the training of residents and clinicians in areas of research methodology. Provision of these support services and resources will greatly enhance the research capabilities of investigators at Washington University and will facilitate collaboration among new and established vision scientists
Keywords: No Project Terms available
Project start date: 1997-04-01
Project end date: 2010-03-31
Budget start date: 1-APR-2009
Budget end date: 31-MAR-2010
5P30EY002687-31 (2009): $803856
Steven Bassnett
Washington University, Campus Box 1054, Saint Louis, Mo 63130-4899
Grant 5P30EY002687-31_9005 from National Eye Institute
Abstract: The Imaging Module provides core investigators with advice, training and technical support on the acquisition, analysis and presentation of visual data. The module provides a central resource for investigators interested in fully utilizing the capabilities of imaging technology. The module assists in training individuals in the use of digital imaging devices, analysis and display of digital images, and provides advice on the selection of appropriate image analysis software and training in its use. For the most part, it is impractical for each laboratory to fully develop the expertise necessary for imaging devices because they are used infrequently and the learning curve is steep. Furthermore, working knowledge of an imaging device is not easily communicated from one relatively inexperienced user to another. This unfortunately results in inefficiencies and potentially unintended but inappropriate manipulation of the digital data supporting the image. The environment for vision research is enhanced by access to an experienced graphics specialist who can help investigators make the best and most appropriate use of the sophisticated imaging devices available to them and the software tools with which to display and analyze their results
Keywords: Arts; Biology; CCSG; Cancer Center Support Grant; Collection; Communities; Computer Programs; Computer Software Tools; Computer software; Core Grant; Data; Data Set; Dataset; Environment; Equipment; Image; Image Analyses; Image Analysis; Imaging Device; Imaging Tool; Imaging technology; Individual; Institution; Instrumentation, Other; Investigators; Jobs; Knowledge; Laboratories; Learning; Occupations; P30 Grant; Professional Postions; ROC Analysis; Research Personnel; Research Resources; Researchers; Resources; Scientist; Sight; Software; Software Tools; Specialist; Technology; Tools, Software; Training; Vision; Vision research; Visual; Visual System; Visual system structure; Work; computer imaging; computer program/software; digital; digital imaging; experience; image evaluation; imaging; instrumentation; interest; meetings; visual information
Budget start date: 1-APR-2009
Budget end date: 31-MAR-2010
5P30EY002687-31_9005 (2009): $145912
Sponsored Links Excellgen http://Excellgen.com
LIM2 AND THE LENS CORE SYNCYTIUM
Steven Bassnett
Washington University, Campus Box 1054, Saint Louis, Mo 63130-4899
Grant 5R01EY018185-02 from National Eye Institute
Abstract: Intercellular communication between fiber cells is critical for lens homeostasis and it is well established that gap junctions facilitate the diffusion of small molecules between adjacent fiber cells. Recently, however, we have obtained evidence that large molecules such as proteins can also diffuse between fiber cells. The conduits for intercellular protein diffusion may be regions of limited cell-cell fusion. The presence of fusions between cells in the lens core ensures that the central region of the lens functions as a syncytium. The physiological significance of the core syncytium is not known. Lim2 is the second most abundant integral membrane protein in the lens. It is a member of the claudin super- family and has putative adhesive functions in the lens. In preliminary experiments, the Lim2 locus was disrupted in mice by insertional mutagenesis, resulting in a functional null for Lim2. Significantly, in the absence of Lim2, the lens syncytium did not form. Furthermore, laser analysis of Lim2-null mouse lenses revealed that the internal refractive properties of the lens were profoundly disturbed. These observations suggest a link between the expression of a lens membrane protein (Lim2), syncytial organization, and refractive function in the lens. The current application will examine the molecular mechanism by which macromolecules diffuse between lens cells, the role of Lim2 in the formation of the lens syncytium, and the relationship between syncytial organization and the lens gradient refractive index (GRIN). These studies are expected to provide novel insights into the previously unsuspected link between intercellular diffusion of macromolecules and lens optical quality. This information will, in turn, inform our view of image formation in the eye and the role of the lens in the optical train. The role of the lens is to focus light sharply on the retina. This application will examine how the intercellular movement of protein within the lens contributes to its optical quality
Keywords: Adhesives; Autoregulation; Biological Function; Biological Process; Body Tissues; Cell Membrane Proteins; Cell fusion; Cell membrane; Cell to Cell Communication and Signaling; Cell-Cell Signaling; Cells; Communicating Junction; Communication; Confocal Microscopy; Crystallins; Cytoplasmic Membrane; Data; Diffuse; Diffusion; Electrical Impedance; Electromagnetic, Laser; Ensure; Eye; Eyeball; Family; Gap Junctions; Giant Cells; Homeostasis; Image; Impedance; Insertional Mutagenesis; Integral Membrane Protein; Intrinsic Membrane Protein; Knock-out; Knockout; Knockout Mice; Lasers; Lens Fiber; Lens Proteins; Life; Light; Link; Location; Low-resistance Junction; Mammals, Mice; Maps; Mediating; Membrane Proteins; Membrane-Associated Proteins; Mice; Mice, Knock-out; Mice, Knockout; Microscopy, Confocal; Molecular; Movement; Multinucleated Giant Cells; Murine; Mus; Mutagenesis, Insertional; Nature; Nexus; Nexus Junction; Null Mouse; Optics; Pathway interactions; Performance; Phenotype; Photoradiation; Physiologic; Physiological; Physiological Homeostasis; Physiology; Plasma Membrane; Play; Polykaryocytes; Population; Property; Property, LOINC Axis 2; Proteins; Proteins, Cell Membrane; Publishing; Radiation, Laser; Refractive Indices; Relative; Relative (related person); Retina; Role; Scanning; Shapes; Surface Proteins; Syncytium; System; System, LOINC Axis 4; Testing; Tissues; Training; Transmembrane Protein; Work; base; body movement; cellular pathology; electric impedance; experiment; experimental research; experimental study; fiber cell; gene product; imaging; insight; intercellular communication; lens; macromolecule; member; mutant; novel; pathway; plasmalemma; protein distribution; public health relevance; research study; small molecule; social role
Project start date: 2008-08-01
Project end date: 2013-07-31
Budget start date: 1-AUG-2009
Budget end date: 31-JUL-2010
PFA/PA: PA-07-070
5R01EY018185-02 (2009): $380000
FIBER CELL FORMATION IN NORMAL AND CATARACTOUS LENSES
Steven Bassnett, Associate Professor
Henry M. Jackson Fdn For The Adv Mil/med
advancement Of Military Medicine, Inc.
rockville, Md 20852
Grant 5R01EY009852-03 from National Eye Institute IRG: VISA
Abstract: I will use confocal microscopy and volume rendering to study the cell biology of fiber cell formation in normal and cataractous lenses. I have developed a lens slice preparation, which allows cells lying in the midsagittal plane of the lens to be imaged at high resolution. I will employ the lens slice to study organelle loss during fiber cell maturation, using fluorescent vital-dyes to follow the fate of organelles in living lens tissue. Preliminary studies on the fate of mitochondria and nuclei indicate that these organelles are lost from embryonic fiber cells in an abrupt, coordinated fashion in a restricted domain of the deep cortex. I will investigate the mechanism of mitochondrial degradation and the role of reduced 02 tension in this process. Isolated mitochondria will be used to study the degradative process in vitro. I will extend these studies to other organelles, including the Golgi apparatus, endoplasmic reticulum (ER) and the cytoskeleton. I will compare organelle distribution in adult lenses from several species and two cataract models. Lens fiber cells are among the longest in the body. I have preliminary data that demonstrate that the ER is specifically located near the posterior tips of the fibers. This localization may reflect a requirement for the ER to be located close to the site of protein secretion. I will determine whether collagen mRNAs are also localized at the posterior tips of the fibers, where collagen is secreted during capsule formation. Another expected consequence of the elongated fiber cell morphology is the presence of intracellular transport system. Using time-lapse confocal microscopy, I obtained preliminary data indicating the presence of such mechanisms in lens fiber cells. The kinetics of transport will be characterized in lens slices. Studies will focus on the role of the cytoskeleton in this process. Throughout this project, computerized volume reconstruction (volume rendering) will be used to visualize the inter-relationship of cellular elements in 3-D. This will be particularly important in the final phase of the proposal, where dynamic 3-D growth processes will be imaged and quantitated. The formation of the lens suture is crucial to lens function and is often disturbed in cataracts. I will use confocal microscopy to observe the formation of the suture in real time in living, organ cultured embryonic lenses. This should reveal critical morphological determinants of suture formation. Cell shape and volume are inextricably linked during lens growth. I will test the hypothesis that the cross-sectional shapes of lens fiber cells are determined by volume increases in the epithelial cells from which they are derived. These studies will require careful 3-D reconstruction of embryonic lenses and dynamic imaging of cells that have been stimulated to elongate
Keywords: cataract, cellular, fiber cell, lens Golgi apparatus, biotransformation, cell volume, collagen, cytoskeleton, endoplasmic reticulum, intracellular transport, messenger RNA, mitochondria, organelle, oxygen tension animal tissue, chick embryo, computer simulation, confocal scanning microscopy, embryo /fetus culture, fluorescent dye /probe, immunofluorescence technique, laboratory mouse, tissue /cell preparation
Project start date: 1992-09-30
Project end date: 1995-06-30
5R01EY009852-03 (1994): $110857
5R01EY009852-02 (1993): $106671
1R01EY009852-01 (1992): $125193
Regulation Of Tissue Oxygenation In The Ocular Lens
Steven Bassnett, Associate Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899
Grant 5R01EY015507-03 from National Eye Institute IRG: ZRG1
Abstract: Age-related nuclear cataract is believed to result, at least in part, from accumulated oxidative damage. This proposal explores the role of molecular oxygen in this process. Using a novel optode system, the distribution of oxygen within human and bovine lenses will be measured as a function of temperature and external pO2. The resulting oxygen maps will be related quantitatively to the consumption of oxygen within the tissue and used to calculate the effective diffusion coefficient of oxygen within the lens. The relative contributions of mitochondrial and non-mitochondrial oxygen consumption to the maintenance of hypoxia in the lens core will be evaluated and the role of ascorbate oxidation as an oxygen-consuming process will be tested directly in a scorbutic animal model. It will be determined whether aged human and animal lenses consume less oxygen and, as a result, whether oxygen accumulates in the core of such lenses. The majority of patients who undergo vitrectomy surgery develop nuclear cataracts within 12 months following the surgery. We hypothesize that this is due to oxygen flooding the normally hypoxic core of the lens during the surgical procedure. To test whether this is the case, lens pO2 will be monitored during vitrectomy surgery in a rabbit model. Finally, the preliminary data suggest that fiber cell differentiation occurs on a steep oxygen gradient. As differentiation proceeds, the cells undergo a profound metabolic shift from an aerobic to an anaerobic state. We have developed a mouse lens organ culture system that will allow us to investigate the effects of hypoxia on the pattern of gene expression in differentiating fiber cells. Collectively, these experiments will provide important new information on the role of molecular oxygen in the etiology of cataract and the physiology of the normal lens.
Keywords: aerobiosis, cataract, disease /disorder etiology, disease /disorder proneness /risk, intraocular pressure, lens, oxygen, oxygen transport, aging, cell differentiation, eye surgery, fiber cell, gene expression, hyperoxia, hypoxia, mathematical model, mitochondria, model design /development, temperature, guinea pig, human tissue, laboratory mouse, laboratory rabbit, polymerase chain reaction, tissue /cell culture
Project start date: 2004-05-01
Project end date: 2008-04-30
Steven Bassnett
Washington University
Project start date: 1992-09-30
Project end date: 2017-03-31
Sponsored Links Excellgen http://Excellgen.com
Fiber Cell Formation In Normal And Cataractous Lenses
Steven Bassnett, Associate Professor
Ophthalmology And Visual Scienceswashington University
Grant 5R01EY009852-17 from National Eye Institute IRG: ZRG1
Project start date: 1992-09-30
Project end date: 2011-12-31
Steven Bassnett, Associate Professor
Washington University 1 Brookings Dr, Campus Box 1054 Saint Louis, Mo 631304899
Grant 5P30EY002687-269005 from National Eye Institute IRG: ZEY1
Keywords: biomedical facility, data collection methodology /evaluation, digital imaging, vision, Internet, computer data analysis, computer program /software, confocal scanning microscopy, fluorescence microscopy