Background: Chloromethyl-X-rosamine (CMXRos) and MitoTracker Green (MTG) have proved to be useful dyes with which to measure mitochondrial function. CMXRos is a lipophilic cationic fluorescent dye that is concentrated inside mitochondria by their negative mitochondrial membrane potential (MMP). MTG fluorescence has been used as a measure of mitochondrial mass independent of MMP. The fluorescence ratio of the two dyes is a relative measure of the MMP independent of mitochondrial mass. Because MTG was recently reported to be sensitive to MMP, we have reevaluated the effects of loss of MMP on MTG and CMXRos fluorescence, using both flow cytometry and laser scanning confocal microscopy (LSCM). Methods: Using flow cytometry, the relative fluorescence of CMXRos, R123, and MTG was determined in human lymphoblastoid cell lines (LCLs) with or without carbonyl cyanide p-trifluoromethoxylphenyl-hydrazone (FCCP), used to collapse the MMP. LSCM analysis was also used to evaluate the effect of FCCP on MTG and CMXRos fluorescence of mouse cells and viable lenses in culture. The cytotoxicity of the dyes was determined using flow anal-

SummaryDietary restriction increases lifespan and slows the onset of age-associated disease in organisms from yeast to mammals. In humans, several age-related diseases are associated with aberrant protein folding or aggregation, including neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases. We report here that dietary restriction dramatically suppresses ageassociated paralysis in three nematode models of proteotoxicity. Similar to its longevity-enhancing properties, dietary restriction protects against proteotoxicity by a mechanism distinct from reduced insulin/IGF-1-like signaling. Instead, the heat shock transcription factor, hsf-1 , is required for enhanced thermotolerance, suppression of proteotoxicity, and lifespan extension by dietary restriction. These findings demonstrate that dietary restriction confers a general protective effect against proteotoxicity and promotes longevity by a mechanism involving hsf-1 .

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In a study comparing animal life spans and in vitro clonal proliferative capacity of skin fibroblasts in groupings of small, middle, large, and very large breeds of dogs of specific ages, the following results were obtained: (1) their life spans were inversely correlated to the frame sizes of the breeds; (2) the percent of large clones present in clone size distributions from the small dogs was inversely proportional to the age of the subjects (this was not true for the large breeds; however, animals older than 8 years were not available in those breeds); and (3) the group composed of the two largest breeds (Great Dane and Irish Wolfhound) had the shortest life spans and also had significantly smaller percentages of large skin fibroblast clones formed in vitro than either of the two groupings of smaller dogs at any age studied. It appears that within the domestic dogs the large body size is accompanied by shorter life span and, in the two largest breeds, decreased cellular growth potential.

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