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Proteomics
Deb Chakravarti
Bulbul Chakravarti The proteome is the complete set of proteins present at any given time in a cell or tissue. Knowledge of the proteome provides crucial insights into various physiological as well as pathological cellular processes, since most of these are carried out by proteins. Using current proteomics technology, such as two dimensional gel electrophoresis, difference gel electrophoresis and tandem mass spectrometry, the laboratory of Drs. Bulbul and Deb Chakravarti, which is part of KGI's Proteomics Center, is involved in various collaborative research projects as follows: - Proteome Profiling of Aging in Mouse Models: Aging is a time dependent complex biological phenomenon observed in all living organisms; it is controlled by different biochemical pathways. The goal of the project is to characterize at the level of the proteome the molecular basis of aging in different organs of mice from different age groups. A specific emphasis of the project involves the relationship between aging and the damages to proteins caused by reactive oxygen species derived from atmospheric oxygen which is essential to life.
- Proteomics in Hearing Research: The organ of Corti is the auditory organ in the inner ear of mammals, including humans. This organ contains hair cells, specialized sensory cells that transduce sound into electrical impulses that are then transmitted to the brain, and Hensen cells (HC) whose function is unknown, even though they are one of the most prominent cell types in the organ of Corti. In collaboration with Dr. Federico Kalinec and colleagues at the House Ear Institute in Los Angeles, this project aims to identify the full set of proteins expressed by guinea pig HC. We would also like to understand the structural and functional roles of HC in the auditory process with particular emphasis on their role in the protection of auditory hair cells from acoustic trauma.
- Chromatin Condensation in Tetrahymena: Chromatin, a complex of DNA and proteins, condenses during cell division to form chromosomes. Little is currently known about the factors mediating chromatin condensation. Using Tetrahymena thermophila, proteins that are present in the highly condensed, transcriptionally silent micronucleus, but absent in the decondensed, transcriptionally active macronucleus are being identified. These proteins are considered candidates for chromatin condensation factors that merit further study. This project is carried out jointly with Dr. Emily Wiley from the Joint Science Department of the Claremont University Consortium.
- Early Diagnosis of Pancreatic Cancer: Ductal adenocarcinoma is the major form of pancreatic cancer and is usually diagnosed at an advanced stage of the disease. A reliable diagnostic test which can detect this cancer at its early stage can aid significantly in patient survival. In collaboration with Dr. Joseph Kim of the City of Hope, Duarte, California and Prof. Bijay K. Pal of the California State Polytechnic University, Pomona, researchers at the Proteomics Center are engaged in finding a set of serum biomarkers for the early detection of pancreatic cancer. Human serum contains a wide variety of proteins. Changes in the relative abundance as well as post-translational modifications of these proteins may indicate pathological status of different organs and tissues.
- Development of New Instrumentation for Proteomics Research: Another collaborative project at the Proteomics Center, carried out jointly with Dr. Sean Gallagher of Ultraviolet Products (UVP), LLC, Upland, California, involves the development and validation of new instrumentation for proteomics research. One of the main methods of proteomics employs electrophoresis in gel matrices to separate protein molecules by size and charge. The goal of this project is to develop a new imaging system for difference gel electrophoresis analysis (DIGE) using fiber optic arrays. Such systems will make the DIGE system affordable for academic laboratories. Earlier, a highly uniform UV illumination system developed by UVP was validated at KGI's Proteomics Center for quantitative DNA and protein analysis. The value of this collaboration was recognized by the Photonics Circle of Excellence Award in 2004 that acknowledged the 25 most technically innovative new products of the year.
- Educational Outreach Activity at the Proteomics Center: Funded by the National Science Foundation, the Proteomics Center is involved in outreach activity to train high school students, high school teachers and undergraduate students in current proteomics technology. Recently, two high school students from Upland High School were awarded gold medals in the regional Inland Area Science and Engineering Fair, 2007.
Stem Cells
Ian Phillips
Yao-Liang Tang Cardiac ischemia frequently results in irreversible damage to cardiomyocytes and cardiac function which can lead to heart failure and death. A potential method to remedy myocardial damage is replacement therapy with stem cells. Drs. Phillips and Tang and their co-workers are engaged in several projects related to gene modification, stem cells and cardiovascular effects. There are three studies currently underway: - The lab is investigating ways to attract stem cells such as those in adult bone marrow to mouse hearts damaged by ischemia by a study on binding of homing factors (SDF-1a) to chemokine receptors (CXCR4) on stem cells.
- Drs. Phillips and Tang are pioneering gene modification of stem cells. These studies which involve inserting gene switches and transgenes into stem cells are focused on enhancing the survival of these cells after transplantation.
- They are also examining the possibility that adult stem cells within the heart itself may be a source of new cardiomyocytes to replace damaged tissue. They are studying the molecular events that stimulate the differentiation of cultured adult stem cells from mouse hearts.
In addition the lab is investigating a new method for preventing blood loss in battlefield injuries. The method is based on a "vigilant vector" that the lab has developed to switch on blood clotting when blood vessels are damaged. Gene Expression Systems
James Cregg Dr. Cregg's research is aimed at understanding and improving the P. pastoris expression system. P. pastoris can be grown rapidly on inexpensive media and offers a subcellular environment conducive to the folding of eukaryotic proteins with the ability to perform post-translational modifications such as proteolytic processing, disulfide bridge formation and glycosylation. A major advantage of the P. pastoris system is the promoter used to control the function of foreign genes; the promoter is derived from the methanol-regulated alcohol oxidase I gene (AOX1) and is among the most powerful and tightly regulated promoters known. The basic molecular biology of the promoter is being studied with the goal of developing stronger versions of the AOX1 promoter and P. pastoris strains that are able to hyper-induce expression from the promoter. Dr. Cregg is also examining P. pastoris as a model system to investigate the molecular mechanisms controlling the assembly of the subcellular organelles known as peroxisomes. P. pastoris is an ideal model system for studying peroxisomes because the organelles contain several key enzymes required for methanol metabolism; when Pichia is grown on methanol, the peroxisomes become unusually large and numerous. Mutants of the yeast with defects in the assembly of the organelles are not able to grow on methanol but can grow on other carbon sources. Dr. Cregg and other investigators have utilized these P. pastoris mutants, called pex mutants, to clone approximately 20 PEX genes which with their protein products have provided critical insights into how peroxisomes function. In addition, the amino acid sequences of the yeast PEX proteins have been utilized to search human DNA sequence databases and identify the human PEX gene equivalents, many of which have turned out to be genes affected in patients with Zellweger syndrome, a family of lethal genetic diseases characterized by defects in the assembly of peroxisomes.
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