Identification and functional characterization of the mitochondrial adenine nucleotide carriers of Trypanosoma brucei

The Mitochondrial Carrier Family encloses a group of transmembrane proteins that transport metabolites across the mitochondrial inner membrane. The ADP/ATP carrier is the most widely studied MCF protein. It catalyzes the counter exchange of ADP for ATP in the mitochondrion of all eukaryotes. In the genome of the kinetoplastid parasite Trypanosoma brucei, three putative ADP/ATP carrier sequences (MCP5, MCP15 and MCP16) and one GDP/GTP (MCP13) entries were analyzed by sequence analyses and phylogenetic reconstruction. AACs phylogenetic reconstruction proved a strong association with yeast, funghi and plant clades, whilst separates from those AACs and from metazoans. MCP13 groups with GGCs, seems to be present only on lower eukaryotes and do not seem to present any homologues in metazoans. Gene deletion studies were performed to assess the roles of MCP5, MCP15, MCP16 and 13. A conditional double knockout cell line, with an inducible myc-tagged rescue copy was constructed for MCP5, which proves the essentiality of the protein for the parasite. Growth curves of the mutant cell line proved a growth defect phenotype in various carbon sources conditions. Mitochondrial ATP production assays were performed in the mutant cell line, in presence and absence of the inducible protein, using permeabilized cells with digitonin that confirmed the ADP/ATP transport activity of the carrier. For invitro activity assays, the carriers were cloned and expressed in Escherichia coli and Spodoptera frugiperda, solubilised and reconstituted into liposomes. Unfortunately, the reconstitution was unsuccessful and the conditions and methodologies are discussed

Protective Effect of Modified Human Fibroblast Growth Factor on Diabetic Nephropathy

Oxidative stress is a key mechanism causing Diabetic Nephropathy (DN). Acidic fibroblast growth factor (aFGF) is known to confer protection from oxidative stress. However, it also has significant angiogenic activity. Hence, we have generated a mutated human acidic FGF (maFGF), with intact antioxidant properties but devoid of angiogenic activities. Recent evidence shows that maFGF treatment prevented diabetic cardiomyopathy and further in vitro studies suggest that this prevention is mediated by suppression of cardiac oxidative stress, hypertrophy and fibrosis. We hypothesized that maFGF treatment has a protective effect in DN. We show that maFGF treatment did not affect body weight and blood sugar levels in a type 1 diabetic mouse model. However maFGF prevented renal functional alterations in diabetes and decreased renal hypertrophy following long-term diabetes. maFGF also prevented diabetes–induced DNA damage, upregulation of angiotensinogen, oxidative stress marker heme oxygenase 1, and alteration of endothelial nitric oxide synthase (eNOS). Surprisingly, it failed to prevent upregulation of the fibrogenic cytokine transforming growth factor β1 mRNA expression. However, long term administration of maFGF partially prevented diabetes-induced extracellular matrix proteins accumulation. Further analyses showed similar results in high glucose-induced alterations in podocytes and microvascular endothelial cells. Likewise, maFGF showed prevention of diabetes- induced decreased nitric oxide (NO) production and apoptosis in vivo and in vitro. These results were consistent with the prevention of long term diabetes- induced down regulation of eNOS enzyme. Data from these experiments suggest that the preventative effects of maFGF treatment in DN are probably via alteration of NO production, and indicate a potential therapeutic role of maFGF in DN

The dual nature of mismatch repair as antimutator and mutator:for better or for worse

DNA is constantly under attack by a number of both exogenous and endogenous agents that challenge its integrity. Among the mechanisms that have evolved to counteract this deleterious action, mismatch repair (MMR) has specialized in removing DNA biosynthetic errors that occur when replicating the genome. Malfunction or inactivation of this system results in an increase in spontaneous mutability and a strong predisposition to tumor development. Besides this key corrective role, MMR proteins are involved in other pathways of DNA metabolism such as mitotic and meiotic recombination and processing of oxidative damage. Surprisingly, MMR is also required for certain mutagenic processes. The mutagenic MMR has beneficial consequences contributing to the generation of a vast repertoire of antibodies through class switch recombination and somatic hypermutation processes. However, this non-canonical mutagenic MMR also has detrimental effects; it promotes repeat expansions associated with neuromuscular and neurodegenerative diseases and may contribute to cancer/disease-related aberrant mutations and translocations. The reaction responsible for replication error correction has been the most thoroughly studied and it is the subject to numerous reviews. This review describes briefly the biochemistry of MMR and focuses primarily on the non-canonical MMR activities described in mammals as well as emerging research implicating interplay of MMR and chromatin