The ubiquinol : cytochrome-c oxidoreductase (cytochrome bcl complex) is a central component of the mitochondrial respiratory chain as well as the respiratory and/or photosynthetic systems of numerous prokaryotic organisms. In Rhodobacter sphaeroides, the bel complex has a dual function. When the cells are grown photosynthetically, the bel complex is present in the intracytoplasmic membrane and is a critical component of the cyclic electron transport system. When the cells are grown in the dark in the presence of oxygen, the same bcl complex is a necessary component of the cytochrome-c2-dependent respiratory chain. The fact that the bel complex from R. sphaeroides has been extensively studied, plus the ability to manipulate this organism genetically, makes this an ideal system for using site-directed mutagenesis to address questions relating to the structure and function of the bel complex.In the current work, the cloning and complete sequence of thejbc operon from R. sphaeroides is reported. As in other bacteria, this operon contains three genes, encoding the Rieske 2Fe-2S subunit, the cytochrome b subunit, and the cytochrome c1 subunit. Recombination techniques were used to delete the entirefie operon from the chromosome. The resulting strain cannot grow photosynthetically, but can grow aerobically utilizing a quinol oxidase. Photosynthetic growth is restored by providingjbc operon on a plasmid, and the reappearance of the protein subunits and the spectroscopic features due to the bel complex are also demonstrated. Finally, a mutation is introduced within the gene encoding the cytochrome b subunit which is predicted to confer resistance to the inhibitor myxothiazol. It is shown that the resulting strain contains a functional bcl complex which, as expected, is resistant to the inhibitor.Hence, this system is suitable for the detailed characterization of the bel complex, combining site-directed mutagenesis with the biochemical and biophysical techniques which have been previously developed for the study of photosynthetic bacteria.The ubiquinol : cytochrome-c oxidoreductase (bel complex) is a redox-linked proton pump which is a central component of the eukaryotic respiratory system as well as the respiratory and/or photosynthetic systems of numerous prokaryotes. In addition, the bcl complex is closely related to the bdcomplex, which is present in chloroplasts and has a similar function as a coupling site in the photosynthetic electron transport chain of green plants (for reviews see [l -31). This family of enzymes couples the oxidation of quinol, within the membrane bilayer, to the electrogenic translocation of protons across the membrane.These enzymes all share a common set of four redox centers. There is a 2Fe-2S cluster (termed the Rieske center), a high-potential b-type cytochrome (bH), a low-potential btype cytochrome (bL) and either cytochrome c1 or cytochrome j : The two b-type cytochromes are contained within a single subunit, which is very highly conserved throughout the family of enzymes [l, 21. The Ries...
3097 Background: BV is a potent inhibitor of vascular endothelial growth factor (VEGF) with broad clinical activity. E is an mTOR (mammalian target of rapamycin) inhibitor in development for cancer and solid organ transplant therapy. VEGF and mTOR inhibitors have anti-tumor and anti-angiogenesis effects alone and in combination in preclinical models. As a combination anti-angiogenesis therapy, we evaluated BV + E in a phase I, pharmacokinetic (PK), biomarker study. Methods: BV was dosed at 10mg/kg IV q14d. E was dosed at 5mg PO QD, escalating to 10mg QD. Cycle length was 28 days. DLT was defined as any grade 4 heme or grade 3/4 non-heme event in Cycle 1 related to treatment. Pts had advanced solid tumors, adequate organ function, and no increased risks for class-related toxicities. Serial blood samples were collected for PK studies of E. Dermal wound angiogenesis assays were performed pre and on treatment for phospho VEGFR2, AKT, mTOR, and S6K. Results: 14 pts have been enrolled (8 F, 6 M), 12 evaluable for toxicity, 14 for efficacy. Median age is 58y (range 29–73). At dose level 1 (BV 10mg/E 5mg) there were no DLT’s in 5 pts. At dose level 2 (BV 10mg/E 10mg), no DLT’s were noted in the initial 3 pts and the cohort was expanded to 9 pts. Side effects were primarily grade 1–2: pain (10/14), mucositis (9/14), anorexia (8/14), rash (7/14), bleeding (7/14), hyperlipidemia (6/14), fatigue (6/14), and HTN (4/14). 1 pt had a myocardial infarction at day 72 and one pt developed nephrotic syndrome at day 70. 7/14 pts had stable disease as best response (70–278d). Conclusions: BV + E is generally well-tolerated. Preliminary clinical activity and class-related side effects were noted. The recommended phase II dose is BV 10mg/kg IV q14d and E 10mg PO QD. [Table: see text]
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