No description available.

No description available.

No description available.

Co-immunoprecipitation (co-IP) followed by proteomic analysis was employed to examine the phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS) endosperm. Earlier studies suggested that immunologically unrelated 107-kDa plant-type and 118-kDa bacterial-type PEPCs (p107/PTPC and p118/BTPC, respectively) are subunits of an unusual ~910-kDa hetero-octameric Class-2 PEPC complex of developing COS. The current results confirm that a tight physical interaction occurs between p118 and p107 since p118 quantitatively co-IP’d with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. No PEPC activity or immunoreactive PTPC or BTPC polypeptides were detected in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation site characteristic of PTPCs, Pro-Q Diamond Phosphoprotein staining, immunoblotting with phospho-(Ser/Thr) Akt substrate IgG, and phosphate-affinity PAGE demonstrated that the co-IP’d p118 was significantly phosphorylated at unique Ser and/or Thr residue(s). The co-IP of p118 and p107 was not influenced by their phosphorylation status. As p118 phosphorylation appeared unchanged 48 h following elimination of photosynthate supply due to COS depodding, the signaling mechanisms responsible for photosynthate-dependent p107 phosphorylation differ from those controlling p118’s in vivo phosphorylation. A third PEPC polypeptide of ~110-kDa (p110; RcPPC1) co-IP’d with p118 and p107 when depodded COS was used. Analysis of RcPpc1’s full-length cDNA sequence revealed p110’s identity with PTPCs, but that a pair of unique amino-acid substitutions occurs in its N-terminal sequence that may render p110 non-phosphorylatable in vivo. The plastidial pyruvate dehydrogenase complex (PDCpl) was identified as a novel PEPC interactor. Subcellular fractionation indicated that p118 and p107 are strictly cytosolic, but that PDCpl is targeted to both the cytosol and leucoplast of developing COS. Thus, a putative cytosolic metabolon involving PEPC and PDCpl could function to channel carbon from phosphoenolpyruvate to acetyl-CoA and/or to recycle CO2 from PDCpl to PEPC. / Thesis (Master, Biology) -- Queen's University, 2007-09-26 15:57:52.216

Abstract Maintenance of the mitochondrial respiratory chain complexes plays crucial role for the aerobic metabolism of the eukaryotes such as unicellular yeasts, for example, Saccharomyces cerevisiae as well as of human being. Mitochondrial respiratory function has been studied using the yeast S. cerevisiae as a model organism. Since yeast cells are also able to grow without respiration by fermentation, identification of the nuclear genes linked to respiratory function is possible by generation of nuclear gene deletions and testing for respiration-deficient phenotype of the yeast deletion strains id est for yeast cells only poorly or not at all growing on the media containing non-fermentable carbon sources. This study reports identification of a novel mitochondrial 2-enoyl thioester reductase from the yeasts Candida tropicalis and S. cerevisiae, Etr1p and Mrf1p, respectively. Examination of the function of these proteins in the respiration-deficient mrf1Δ strain from S. cerevisiae suggests that the reductase is involved in mitochondrial fatty acid synthesis (FAS type II) in the yeast. Site-directed mutagenesis of a conserved tyrosine in the catalytic site of the enzyme indicated that the 2-enoyl thioester reductase activity is critical for mitochondrial respiratory competence. In addition, subcellular localization to mitochondria was required for the complementation of the respiration-deficient phenotype of the yeast reductase deletion strain. The crystal structure for the Etr catalytic site mutant indicated the structural integrity of the mutant supporting the requirement of the tyrosine for the catalysis. Characterization of Etr crystal structures both in apo- and holo-forms containing NADPH established Etr as a member of novel subfamily of enoyl thioester reductases in the superfamily of medium-chain dehydrogenases/reductases (MDR). Two isoforms of Etr with the difference in three amino acids only are encoded by two distinct genes in C. tropicalis, whereas only single gene encodes the reductase functioning in the mitochondria in S. cerevisiae. The presence of two genes in C. tropicalis was taken as an example of genetic redundancy in this yeast, the two genes also shown to be expressed in slightly different ways under various carbon sources available for growth.

Abstract In eukaryotic cells, fatty acids are mainly synthesized in the cytoplasm, but recently, in yeast and in humans, the ability to synthesize fatty acids has been characterized in mitochondria. This mitochondrial pathway is similar to bacterial type II fatty acid synthesis (FAS). The main feature of mitochondrial FAS in yeast is the respiratory deficient phenotype and loss of cytochromes when any of genes encoding enzymes for mitochondrial FAS is deleted. Mitochondrial FAS has been demonstrated to have an important role in lipoic acid production, namely it synthesizes octanoyl-ACP, which is used as a precursor for lipoic acid. However, the role and function of mitochondrial FAS is not yet fully understood. Many components of the mitochondrial FAS pathway in yeast have been identified according to their similarity to bacterial counterparts, but 3-hydroxyacyl-ACP dehydratase does not show any easily recognizable similarity to bacterial dehydratases and thus remained unidentified. In this study 3-hydroxyacyl-ACP dehydratases of mitochondrial FAS were characterized from the yeast Saccharomyces cerevisiae, humans, and the human pathogen Trypanosoma brucei. The yeast 3-hydroxyacyl-ACP dehydratase (Htd2p) was identified by using a genetic screen, and this protein was shown to be encoded by open reading frame (ORF) YHR067w. The product of this gene shows mitochondrial localization and exhibits hydratase 2 activity. The deletion of HTD2 leads to a respiratory deficient phenotype, loss of cytochromes, reduced lipoic acids levels and changes in mitochondrial morphology. The ORF encoding human 3-hydroxyacyl-ACP hydratase (HsHTD2) was identified by functional complementation of the respiratory deficient phenotype of the yeast htd2 mutant with a human cDNA library. The complementing cDNA was previously identified as the RPP14 transcript encoding the 14 kDa subunit of the human RNase P complex. It was found that this transcript contains another 3' ORF, which encodes a protein that displays hydratase 2 activity and has mitochondrial localization. The bicistronic nature of the transcript is conserved in vertebrates and indicates a genetic link between mitochondrial FAS and RNA processing. The mitochondrial 3-hydroxyacyl-ACP hydratase in T. brucei is homologous to human HTD2, can complement the yeast respiratory deficient phenotype, exhibits hydratase 2 activity and localizes to the T. brucei mitochondrion.

Thirty-five volunteers were randomized and twenty-nine completed the study. Mean plasma and red blood cell (RBC) total DHA concentrations, which were analyzed among all participants as a measure of adherence, increased significantly in the DHA-enriched treatment compared to control oil-control flour. The plasma and RBC n-6: n-3 ratio was reduced after consumption of HOCODHA-control flour compared to control oil- control flour. The present study failed to see differences in body composition with the HOCODHA-barley flour treatment versus control oil-control flour treatment. In conclusion, significant increases in plasma EPA and DHA levels, as well as the omega-3 index, provide evidence supporting the cardioprotective effects of HOCODHA. The present study demonstrated that in the context of current Western macronutrient intakes, altering the dietary fatty acid composition and adding β-glucan had no major effect on body composition during the 28 days controlled dietary intervention. / February 2017

No description available.

Considering the major role of insulin signaling on fatty acid synthesis via stimulation of lipogenic enzymes, differential effects of insulin signaling on individual carbon fluxes for fatty acid synthesis have been investigated by comparing the individual lipogenic fluxes in WT and IRS-1 knockout (IRS-1 KO) brown adipocytes. Results from experiments on WT and IRS-1 KO cells incubated with [5-¹³C] glutamine were consistent with the existence of reductive carboxylation pathway. Analysis of isotopomer distribution of nine metabolites related to the lipogenic routes from glucose and glutamine in IRS-1 KO cells using [U-¹³C] glutamine as compared to that in WT cells indicated that flux through reductive carboxylation pathway was diminished while flux through conventional TCA cycle was stimulated due to absence of insulin signaling in IRS-1 KO cells. This observation was confirmed by quantitative estimation of individual lipogenic fluxes in IRS-1 KO cells and their comparison with fluxes in WT cells. Thus, these results suggest that glutamine’s substantial contribution to fatty acid synthesis can be directly manipulated by controlling the flux through reductive carboxylation of alpha-ketoglutarate to citrate using hormone (insulin). / Singapore-MIT Alliance (SMA)

The term "lipid" covers an extremely diverse range of chemical or molecular species. Lipids, defined as molecules that are sparingly soluble in water but readily soluble in organic solvents, are broadly categorized into "neutral " or "apolar" lipids, and "amphiphilic” or "polar" lipids. Neutral lipids will include simple hydrocarbons, carotenes, triacylglycerols, wax esters, sterol eaters, as wel1 as other lipids such as fatty acids, polyprenols, and sterols In which the hydrophilic function has little Impact on the overall molecular characteristics. Polar lipids include phospholipids, glycolipids, sulfolipids, some sphingolipids, oxygenated carotenoids and chlorophylls.