Gaining incite into normal and pathophysiological processes at the molecular level in the central nervous system is one of the laboratory?s primary goals. To further our studies it is essential to develop methods for studying proteins expressed in individual cells. The cellular heterogeneity of the brain, in which glial cells generally surround and outnumber neurons at a ratio of 10 to 1 and the existence of different types of neurons in close proximity to one another requires a technology that permits the study of proteins from single neurons and single glial cells. Current 2-D protein electrophoresis methodology has a major inadequacy caused by the limits of sensitivity for protein detection. While silver staining can detect as little as 0.01 nanogram of protein, for an average protein of molecular weight 30kD this represents approximately 200 million molecules. At this level of detection it is not possible to detect many proteins from a single cell. One of the techniques that we are investigating uses confocal laser technology. To facilitate this effort we continue our collaborative efforts with the researchers in the NIST Optical and Analytical Chemistry Division. With help from these collaborators we have developed a micro gel chamber 10 microns thick. This is approximately 10x thinner than existing micro gels. The first prototypes are in the testing phase. The separation chamber is thin to prevent quenching of the laser dyes. Unlike CE (capillary electrophoresis) techniques our detection dye is added after separation, avoiding mobility changes by bound fluorescent dye molecules. The trailing edge of the extended stack zone compresses all the protein in a microscopically thin and highly ordered (anisotropic) zone. Our immediate goal with the micro gel chamber is to resolve numerous proteins with unique dye characteristics with a detection sensitivity approaching single molecules. In addition we are also engaged in fundamental studies to facilitate protein separations. SDS-PAGE gels are run everyday but the underlying mechanisms of SDS-PAGE are not clearly understood. Up to 40% of proteins in the first dimension of a 2-D gel do not enter the second dimension. Proteins >200 kD and <10 kD are not well resolved. We hope to dramatically improve the SDS protein gel dimension separation and detection. These studies are ongoing with the collaboration of Lori Goldner's group at NIST. Experiments are being conducted in two key areas of interest: (1) The stacking mechanism is being studied using mass spectroscopy to measure the sodium ion and sulfur ion content in critical zones on the gel. (2) The structure of the """"""""extended stack"""""""" ES is being studied in collaboration with the Optical Division at NIST. We are examining the structure of the detergent complex in the stack. A manuscript titled ?Trailing buffer ion conditions effect dodecyl sulfate detergent properties and protein sieving properties in SDS-PAGE.?(Giulian, Merril, Hwang, Goldner)is in preparation. In addition we are exploring methods to extend the number of proteins that can be characterized by 2-Dimensional electrophoresis. One method of exploration is prefractionation of samples by micro solution isoelectrofocusing. The use of SYPRO Ruby Red as a sensitive protein stain in 2-D electrophoresis is also being explored. This fluorescent dye claimes to have the same sensitivity as silver; and it is reported to be linear over a 1000-fold range. Its employment as a detection method replaces a lengthy, expensive technique which produces a heavy metal waste product. The SYPRO stain should prove to be quicker, less expensive, and more environmentally friendly. A Human Proteome Organization has been established to identify the proteins expressed in various human tissues and fluids. This laboratory is participating in the Plasma Pilot Study of this organization. Data from 2-DE gels on 4 standard plasma preparations were analyzed and forwarded to this organization. We continue to examine the long-term effects of lithium on the proteome of a defined CNS neuronal cell line. We have collected new data from HCN-1a cell lines, human cultured trabecular meshwork (Alverado, USSF) and human glial cells using the mRNA gene chip with or without Lithium treatment at therapeutic levels. The early results are very encouraging and suggest new potential targets for Li therapy including eye & cortical Aquaporins. A manuscript ?Does the Lithium-induced down regulation of the human aquaporin mRNAs account for many of the known Lithium human side-effects?? (Giulian and Merril)is in preparation regarding this work. Another pattern is emerging from gene chip data and the miRNA target list that another potential target for Li at therapeutic levels is at the level of short, regulatory RNAs. We are begining experiments at NIST and NIH to explore the new model. A manuscript ?mRNA array analysis of human neuronal and glial cell culture after therapeutic treatment levels of Li salts for 1 and 17 days.? (Giulian, Merril, Brownstein, von Kollmar, Xiang) is in preparation from preliminary experimental results. Protein patterns from primary neuronal cultures, derived from cortical, striatal and hippocampal brain regions dissected from 19 day Sprague-Dawley rat embryos, are being compared with each other and with tissue from similar regions from an adult brain. Currently a manuscript is in preparation for submission to Electrophoresis. The goal of this effort is to develop a rat neuronal protein database on the WWW and to use this type of neuronal culture system as a model for studying the effects of stress such as hypoxia, growth factors, toxins, and as a guide to interpret protein patterns with the protein detection system being developed which may permit detection of proteins from single neurons. This database will be linked with the SWISSPROT database. Our high-resolution two-dimensional protein electrophoresis methodologies, will help us to pursue clinical and basic studies of the proteome more effectively than we were able to in the past. A collaborative study, using 2D electrophoretic and mass spectroscopy techniques, has also been initiated with Howard Nash to study protein variation in fruit flies with resistance to anesthesia.
| Zullo, S J; Parks, W T; Chloupkova, M et al. (2005) Stable transformation of CHO Cells and human NARP cybrids confers oligomycin resistance (oli(r)) following transfer of a mitochondrial DNA-encoded oli(r) ATPase6 gene to the nuclear genome: a model system for mtDNA gene therapy. Rejuvenation Res 8:18-28 |
