21 Cross talk between reactive oxygen species producing mitochondria and NADPH oxidase Mariola Kulawiec * , Mohamed M. Desouki, Keshav K. Singh Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, USA Recent studies have suggested that NADPH oxidase (Nox) enzymes are not only present in phagocytic cells but also in various non-phagocytic cells. For example, Nox1 (Mox1, Noh-1) is primarily present in normal colonic epithelium, vascular smooth muscles, uterus and prostate. The NOX family of proteins contains 7 other members: NOX1-5, Doux1 and Doux2. These flavoproteins catalyze the NADPH-dependent reduc- tion of oxygen to superoxide and other related reactive oxygen species (ROS), which are involved in intracellular signaling. Nox enzymes are the second major source of ROS in cells besides oxidative phosphorylation in the mitochondria. Based on these observations, we tested the hypothesis that mitochondria control Nox1 redox signaling, and that the loss of this control contributes to tumorigenesis. Our study revealed that mitochon- dria do indeed control Nox1 expression. Firstly confocal microscopy stud- ies revealed that Nox1 localizes in the perinuclear mitochondria and to a lesser extent in cell membranes. Secondly, inactivation of mitochondrial genes led to a down-regulation of Nox1, and the transfer of wild type mitochondrial genes restored Nox1 expression to a level comparable to that in the parental cell line. Thirdly, exposure of cells to the mitochon- drial inhibitors antimycin and rotenone as well as and uncoupler FCCP caused up-regulation of Nox1. Additionally, our histochemistry studies revealed that Nox1 was highly expressed in breast (86%) and ovarian (71%) tumors. Altogether our studies suggest that mitochondrial regula- tion of Nox1 redox signaling contributes to breast and ovarian tumorigen- esis. Further investigation is underway to identify the genetic mechanisms of mitochondrial regulation of other members of the NOX family and their role in tumorigenesis. doi:10.1016/j.mito.2007.08.025 22 Mitochondrial ND5: A candidate gene for neuromuscular diseases in India A. Vanniarajan a,* , B.H. Jagadish a , Uppin Megha b , P. Govindaraj a , Ajay Kumar a , A.G. Reddy a , C. Sundaram b , A.K. Meena b , N. Gayatri c , S. Dinesh Nayak d a Centre for Cellular and Molecular Biology, Hyderabad, India; b Nizam’s Institute of Medical Sciences, Hyderabad, India; c National Institute of Mental Health and Neurosciences, Bangalore, India; d Sree Chitra Tirunal Institute of Medical Sciences, Trivandru, India Mitochondrial diseases are severely debilitating, often fatal and char- acteristically complex in nature. Impairment of oxidative phosphorylation and normal physiology of mitochondria leads to a myriad number of mitochondrial disorders. The phenotypic consequences caused by the mitochondrial DNA variations are manifested primarily in the major organs that require high-energy, important one being the neuromuscular system. Indian population has a very complex genetic architecture, which makes it suitable to study the genetic diseases, mitochondrial diseases in particular. We have analysed 123 clinically diagnosed individuals having definite neuromuscular symptoms. Tissue samples of 42 patients were processed for histopathology and electron microscopy. Modified Gomori Trichrome staining showed red ragged fibers (RRF), a typical feature of mitochon- drial diseases, in 12 individuals and 7 individuals showed mixed features of the denervation and RRF. Abnormalities in number as well as structure of the mitochondria were observed under the electron microscope. Complete mitochondrial DNA sequencing of 123 patients revealed 870 variations/mutations spread across the mitochondrial genome. A total of 151 of these variants were novel, which were not found in 300 control samples and not reported in the literature. ND5 gene was found to harbor the highest number of variants in these patients. Thirty nine missense mutations were observed in ND5 gene and 21 of them found to alter the secondary structure predicted. Mutations in ND5 region had also been found to affect the enzymatic activity of Complex I. Thus, ND5 has been confirmed to be the candidate gene for the mitochondrial diseases in India and can be used as a primary diagnostic tool for mitochondrial diseases. doi:10.1016/j.mito.2007.08.026 23 Lambda profiling and biocavity laser spectroscopy – Testing a new technology for mitochondrial disease diagnosis Robert K. Naviaux a,* , Paul L. Gourley b a Departments of Medicine and Pediatrics, University of California, San Diego, CA 92103-2071, USA; b Biomolecular Materials and Interface Department, Sandia National Laboratories, Albuquerque, NM 87185, USA The biocavity laser is a gallium arsenide semiconductor nanolaser that was invented by one of us (PLG) as an extension of the vertical cavity sur- face emitting laser (VCSEL). The VCSEL is now used extensively for the high bandwidth transfer of information by fiber optics in the telecommuni- cations industry. Likewise, the biocavity laser provides a high bandwidth of information related to the biophysical composition of cells and mitochon- dria. In a unique collaboration between a physicist (PLG) and a life scientist (RKN), we report the first results of a study designed to test the ability of the biocavity laser to diagnose mitochondrial dysfunction and to measure the cellular responses to this dysfunction. The biocavity laser contains an inte- grated microfluidics chip the size of a dime. When cells or mitochondria flow through the biocavity laser they become part of the resonance cavity and trigger the emission of laser light. The photons of laser light produced have precisely measureable spectral characteristics, such as wavelength (lambda), which can be measured to a precision of greater than 0.02 nm. The numer- ical quantity lambda is a function of the size, shape, organization, and bio- physical composition of mitochondria. When the laser spectra from about 2000 mitochondria are collected and the population analyzed and modeled mathematically, a unique spectral signature results that we call the ‘‘lambda profile’’. The lambda profile correctly identified 6 of 6 normal subjects, and 6 of 8 abnormal subjects with inherited respiratory chain defects in the very first test, without optimization. Complex I and complex IV deficiencies were clearly identified as abnormal by their distinctive lambda profiles. Unlike any other single chemical, enzymologic, or biophysical measurement, lambda profiling is a measure of the overall biophysical state of the cell and mitochondria. This biophysical state of a cell is a reflection of its ‘‘omic’’ profile – its genomics, proteomics, lipidomics, glycomics, and metabolomics integrated as a single numerical parameter – which in turn results from the sum total of changes in biomolecular composition and subcellular organiza- tion of structures within the cell. The biocavity laser is unique in its ability to probe this interface between statistical thermodynamics and metabolism to allow quantitative testing of new biophysical models of the optics of cells and isolated mitochondria. doi:10.1016/j.mito.2007.08.027 24 Reconstructing the tempestuous evolution of the mitochondrial ribosomal proteome Paulien Smits * , Jan A.M. Smeitink, Lambert P. van den Heuvel, Martijn A. Huynen, Thijs J.G. Ettema Nijmegen Center for Mitochondrial Disorders (NCMD) and Center for Molecular and Biomolecular Informatics (CMBI) from the Radboud University Medical Center Nijmegen, The Netherlands 410 Abstracts / Mitochondrion 7 (2007) 404–433