Insight into the Carboxyl Transferase Domain Mechanism of Pyruvate Carboxylase from \u3cem\u3eRhizobium etli\u3c/em\u3e

The effects of mutations in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase have been determined for the forward reaction to form oxaloacetate, the reverse reaction to form MgATP, the oxamate-induced decarboxylation of oxaloacetate, the phosphorylation of MgADP by carbamoyl phosphate, and the bicarbonate-dependent ATPase reaction. Additional studies with these mutants examined the effect of pyruvate and oxamate on the reactions of the biotin carboxylase domain. From these mutagenic studies, putative roles for catalytically relevant active site residues were assigned and a more accurate description of the mechanism of the carboxyl transferase domain is presented. The T882A mutant showed no catalytic activity for reactions involving the carboxyl transferase domain but surprisingly showed 7- and 3.5-fold increases in activity, as compared to that of the wild-type enzyme, for the ADP phosphorylation and bicarbonate-dependent ATPase reactions, respectively. Furthermore, the partial inhibition of the T882A-catalyzed BC domain reactions by oxamate and pyruvate further supports the critical role of Thr882 in the proton transfer between biotin and pyruvate in the carboxyl transferase domain. The catalytic mechanism appears to involve the decarboxylation of carboxybiotin and removal of a proton from Thr882 by the resulting biotin enolate with either a concerted or subsequent transfer of a proton from pyruvate to Thr882. The resulting enolpyruvate then reacts with CO2 to form oxaloacetate and complete the reaction

Probing the Catalytic Roles of Arg548 and Gln552 in the Carboxyl Transferase Domain of the \u3cem\u3eRhizobium etli\u3c/em\u3e Pyruvate Carboxylase by Site-directed Mutagenesis

The roles of Arg548 and Gln552 residues in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase were investigated using site-directed mutagenesis. Mutation of Arg548 to alanine or glutamine resulted in the destabilization of the quaternary structure of the enzyme, suggesting that this residue has a structural role. Mutations R548K, Q552N, and Q552A resulted in a loss of the ability to catalyze pyruvate carboxylation, biotin-dependent decarboxylation of oxaloacetate, and the exchange of protons between pyruvate and water. These mutants retained the ability to catalyze reactions that occur at the active site of the biotin carboxylase domain, i.e., bicarbonate-dependent ATP cleavage and ADP phosphorylation by carbamoyl phosphate. The effects of oxamate on the catalysis in the biotin carboxylase domain by the R548K and Q552N mutants were similar to those on the catalysis of reactions by the wild-type enzyme. However, the presence of oxamate had no effect on the reactions catalyzed by the Q552A mutant. We propose that Arg548 and Gln552 facilitate the binding of pyruvate and the subsequent transfer of protons between pyruvate and biotin in the partial reaction catalyzed in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase

Effect of venting range hood flow rate on size-resolved ultrafine particle concentrations from gas stove cooking

Cooking is the main source of ultrafine particles (UFP) in homes. This study investigated the effect of venting range hood flow rate on size-resolved UFP concentrations from gas stove cooking. The same cooking protocol was conducted 60 times using three venting range hoods operated at six flow rates in twin research houses. Size-resolved particle (10–420 nm) concentrations were monitored using a NanoScan scanning mobility particle sizer (SMPS) from 15 min before cooking to 3 h after the cooking had stopped. Cooking increased the background total UFP number concentrations to 1.3 × 103 particles/cm3 on average, with a mean exposure-relevant source strength of 1.8 × 1012 particles/min. Total particle peak reductions ranged from 25% at the lowest fan flow rate of 36 L/s to 98% at the highest rate of 146 L/s. During the operation of a venting range hood, particle removal by deposition was less significant compared to the increasing air exchange rate driven by exhaust ventilation. Exposure to total particles due to cooking varied from 0.9 to 5.8 × 104 particles/cm3·h, 3 h after cooking ended. Compared to the 36 L/s range hood, higher flow rates of 120 and 146 L/s reduced the first-hour post-cooking exposure by 76% and 85%, respectively. © 2018 Crown Copyright. Published with license by Taylor & Francis Group, LLC

Mitochondria directly influence fertilisation outcome in the pig

The mitochondrion is explicitly involved in cytoplasmic regulation and is the cell's major generator of ATP. Our aim was to determine whether mitochondria alone could influence fertilisation outcome. In vitro, oocyte competence can be assessed through the presence of glucose-6-phosphate dehydrogenase (G6PD) as indicated by the dye, brilliant cresyl blue (BCB). Using porcine in vitro fertilisation (IVF), we have assessed oocyte maturation, cytoplasmic volume, fertilisation outcome, mitochondrial number as determined by mtDNA copy number, and whether mitochondria are uniformly distributed between blastomeres of each embryo. After staining with BCB, we observed a significant difference in cytoplasmic volume between BCB positive (BCB+) and BCB negative (BCB-) oocytes. There was also a significant difference in mtDNA copy number between fertilised and unfertilised oocytes and unequal mitochondrial segregation between blastomeres during early cleavage stages. Furthermore, we have supplemented BCB- oocytes with mitochondria from maternal relatives and observed a significant difference in fertilisation outcomes following both IVF and intracytoplasmic sperm injection (ICSI) between supplemented, sham-injected and non-treated BCB- oocytes. We have therefore demonstrated a relationship between oocyte maturity, cytoplasmic volume, and fertilisation outcome and mitochondrial content. These data suggest that mitochondrial number is important for fertilisation outcome and embryonic development. Furthermore, a mitochondrial pre-fertilisation threshold may ensure that, as mitochondria are diluted out during post-fertilisation cleavage, there are sufficient copies of mtDNA per blastomere to allow transmission of mtDNA to each cell of the post-implantation embryo after the initiation of mtDNA replication during the early postimplantation stages

Transmission of mitochondrial DNA following assisted reproduction and nuclear transfer

Review of the articleMitochondria are the organelles responsible for producing the majority of a cell's ATP and also play an essential role in gamete maturation and embryo development. ATP production within the mitochondria is dependent on proteins encoded by both the nuclear and the mitochondrial genomes, therefore co-ordination between the two genomes is vital for cell survival. To assist with this co-ordination, cells normally contain only one type of mitochondrial DNA (mtDNA) termed homoplasmy. Occasionally, however, two or more types of mtDNA are present termed heteroplasmy. This can result from a combination of mutant and wild-type mtDNA molecules or from a combination of wild-type mtDNA variants. As heteroplasmy can result in mitochondrial disease, various mechanisms exist in the natural fertilization process to ensure the maternal-only transmission of mtDNA and the maintenance of homoplasmy in future generations. However, there is now an increasing use of invasive oocyte reconstruction protocols, which tend to bypass mechanisms for the maintenance of homoplasmy, potentially resulting in the transmission of either form of mtDNA heteroplasmy. Indeed, heteroplasmy caused by combinations of wild-type variants has been reported following cytoplasmic transfer (CT) in the human and following nuclear transfer (NT) in various animal species. Other techniques, such as germinal vesicle transfer and pronuclei transfer, have been proposed as methods of preventing transmission of mitochondrial diseases to future generations. However, resulting embryos and offspring may contain mtDNA heteroplasmy, which itself could result in mitochondrial disease. It is therefore essential that uniparental transmission of mtDNA is ensured before these techniques are used therapeutically

G-Quadruplex Dynamics Contribute To Regulation Of Mitochondrial Gene Expression

Single-stranded DNA or RNA sequences rich in guanine (G) can adopt non-canonical structures known as G-quadruplexes (G4). Mitochondrial DNA (mtDNA) sequences that are predicted to form G4 are enriched on the heavy-strand and have been associated with formation of deletion breakpoints. Increasing evidence supports the ability of mtDNA to form G4 in cancer cells; however, the functional roles of G4 structures in regulating mitochondrial nucleic acid homeostasis in non-cancerous cells remain unclear. Here, we demonstrate by live cell imaging that the G4-ligand RHPS4 localizes primarily to mitochondria at low doses. We find that low doses of RHPS4 do not induce a nuclear DNA damage response but do cause an acute inhibition of mitochondrial transcript elongation, leading to respiratory complex depletion. We also observe that RHPS4 interferes with mtDNA levels or synthesis both in cells and isolated mitochondria. Importantly, a mtDNA variant that increases G4 stability and anti-parallel G4-forming character shows a stronger respiratory defect in response to RHPS4, supporting the conclusion that mitochondrial sensitivity to RHPS4 is G4-mediated. Taken together, our results indicate a direct role for G4 perturbation in mitochondrial genome replication, transcription processivity, and respiratory function in normal cells

Can changing the timing of outdoor air intake reduce indoor concentrations of traffic-related pollutants in schools?

Traffic emissions have been associated with a wide range of adverse health effects. Many schools are situated close to major roads, and as children spend much of their day in school, methods to reduce traffic‐related air pollutant concentrations in the school environment are warranted. One promising method to reduce pollutant concentrations in schools is to alter the timing of the ventilation so that high ventilation time periods do not correspond to rush hour traffic. Health Canada, in collaboration with the Ottawa‐Carleton District School Board, tested the effect of this action by collecting traffic‐related air pollution data from four schools in Ottawa, Canada, during October and November 2013. A baseline and intervention period was assessed in each school. There were statistically significant (P < 0.05) reductions in concentrations of most of the pollutants measured at the two late‐start (9 AM start) schools, after adjusting for outdoor concentrations and the absolute indoor–outdoor temperature difference. The intervention at the early‐start (8 AM start) schools did not have significant reductions in pollutant concentrations. Based on these findings, changing the timing of the ventilation may be a cost‐effective mechanism of reducing traffic‐related pollutants in late‐start schools located near major roads

Can changing the timing of outdoor air intake reduce indoor concentrations of traffic-related pollutants in schools?

Traffic emissions have been associated with a wide range of adverse health effects. Many schools are situated close to major roads, and as children spend much of their day in school, methods to reduce traffic-related air pollutant concentrations in the school environment are warranted. One promising method to reduce pollutant concentrations in schools is to alter the timing of the ventilation so that high ventilation time periods do not correspond to rush hour traffic. Health Canada, in collaboration with the Ottawa-Carleton District School Board, tested the effect of this action by collecting traffic-related air pollution data from four schools in Ottawa, Canada, during October and November 2013. A baseline and intervention period was assessed in each school. There were statistically significant (P \u3c 0.05) reductions in concentrations of most of the pollutants measured at the two late-start (9 AM start) schools, after adjusting for outdoor concentrations and the absolute indoor–outdoor temperature difference. The intervention at the early-start (8 AM start) schools did not have significant reductions in pollutant concentrations. Based on these findings, changing the timing of the ventilation may be a cost-effective mechanism of reducing traffic-related pollutants in late-start schools located near major roads. © 2015 Her Majesty the Queen in Right of Canada. Indoor Air published by John Wiley & Sons Ltd. Reproduced with the permission of the Minister of Health Canada