Jose A. Rafols - US grants

Cytochrome c oxidase (COX) comprises 13 subunits in mammals; three encoded in mitocondrial (mt) DNA and ten in nuclear DNA. Analysis of the evolution pattern of individual subunits can suggest tests of function and can serve as a model for the evolution of a multisubunit complex. In humans, the COX complex is unique among mammals in lacking the heart isoform of subunit VIII, which was apparently silenced sometime in primate evolution. The lineage in which it was lost will be identified, the promoter regions of COX8 heart and liver isoforms from closely related lineages will be isolated and characterized, and reporter constructs will be used to evaluate their tissue-specificity and activity in differentiated and undifferentiated myoblasts. COX7A isoform genes have mitochondrial targeting presequences which previous results suggest are involved in tissue-specific isoform function. This was unexpected because the presequences are removed during mitochondrial import and are thought not to participate in COX function. To investigate the potential for differential presequence function, the intracellular location of each of the two isoform proteins will be examined in cells that express both. Finally, the nucleotide substitution rates of three selected nuclear subunits will be determined.

COX provides a model system to test whether evolution of nuclear genes is parallel to that of mitochondrial genes with which they come in structural contact or functionally interact.

DESCRIPTION: (Verbatim from the Applicant's Abstract) The endothelial cells which form the vascular wall play a pivotal role in maintaining cerebral microvascular tone through the released of nitric oxide (NO) and endothelin (ET), which relax and constrict, respectively, the vascular smooth muscle. Although factors other than NO and ET participate in maintaining microvascular tone, reciprocal feedback mechanisms between NO and ET are important in the microvascular autoregulation in extracerebral arteries. However such a mechanism in brain microvessels in normal or abnormal states has never been investigated. From the three isoforms that participate in the synthesis of NO, the inducible nitric oxide synthase (iNOS) is expressed only after challenges to the CNS. NO derived from iNOS contributes to excessive amounts of NO released after trauma. Within the endothelin family, endothelin-1 (ET-1) is the most powerful vasoconstrictor produced in a variety of cellular types. Our long term objectives is to understand the interaction between iNOS and ET-1 in the control of microvascular tone during the acute phase post trauma that will ultimately lead to clinically effective therapy in the development of secondary injury. We propose that altered regulation of the genes that encode for iNOS and ET-1 in endothelial cells, at different time points, participate in the abnormal contractility of brain microvessels following TBI. Our specific aims are to: 1) identify vessels with abnormal diameter and the extent to which the endothelial cells that form these vessels synthesize iNOS and ET-1 proteins, 2) characterize quantitatively the expression of iNOS and ET-1 genes (mRNAs), 3) translationally inhibit the expression of iNOS and ET-1 genes in an attempt to restore impaired microcirculation. We will use morphometric analysis in combination with double immunocytochemistry at the ultrastructural level to detect temporal relationships between vascular diameter and protein synthesis of iNOS and ET-1, in situ hybridization for measurement of mRNA synthesis and in vivo intracerebroventricular application of antisense oligonucleotides to attenuate iNOS an ET-1 gene expression. On line laser Doppler flowmetry will be used to assess changes in cortical blood flow and determine how these changes are dependent on structural and molecular alterations as well as to infusions of antisense oligonucleotides. The results will provide valuable information on the expression of iNOS and ET-1 (proteins and mRNAs) and the causal relationship of this expression to alterations of brain perfusion following TBI. Precise time points of the dissociated expression of iNOS and ET-1 will be established which will serve as a baseline for future therapeutic interventions. This study will also provide direct evidence on the therapeutic value of suppressing (or attenuating) activity of genes in cerebral perfusion after TBI.