| Chapter 1. The balance between oxidized and reduced couples, i.e., glutathione (GSH/GSSG), pyridine dinucleotides (NADH/NAD, NADPH/NADP), thioredoxin reduced/thioredoxinoxidized, dihydrolipoic acid/alpha-lipoic acid, and lactate/pyruvate determines the cellular redox status. Oxidative stress and altered redox status are widely considered as major components of aging and age-related diseases. The isolation of mitochondria from organs is a widely used tool to study mitochondrial biology. However, inherent in the long isolation process, are alterations in mitochondrial redox status. Previous work from our laboratory has shown that different isolation methods can alter the redox status with respect to the glutathione pool. Substrate supplementation of isolated mitochondria resulted in higher buffering capacity against H2O2 challenges, in part due to increased GSH levels. The aim of the present study is to extend our previous work and monitor changes in the pyridine dinucleotide pool. Upon substrate supplementation, changes in the pyridine dinucleotide pool were quantified using HPLC and changes in membrane potential have been monitored using fluorescent dye Rhodamine-123. Our data show that substrate supplementation shifts the mitochondrial redox status towards a more reduced state. These data are in agreement with our previous work and show changes in redox status of pyridine dinucleotides when supplemented with mitochondrial energy substrates.;Chapter 2. The present paper reviews the use of protein splicing for the biosynthesis of backbone cyclic polypeptides. This general method allows the in vivo and in vitro biosynthesis of cyclic polypeptides using recombinant DNA expression techniques. Biosynthetic access to backbone cyclic peptides opens the possibility to generate cell-based combinatorial libraries that can be screened inside living cells for their ability to attenuate or inhibit cellular processes thus providing a new way for finding therapeutic agent. |
