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Peroxisome proliferator-activated receptor alpha (PPAR) plays a crucial role in the transcriptional regulation of myocardial lipid metabolism. In vitro studies on isolated cardiomyocytes showed that PPAR activation induces expression of numerous genes involved in virtually all steps of fatty acid catabolism. However, there is very few data on the effect of PPAR activation on the content and composition of myocardial lipids in vivo. Therefore, our main aim was to examine effects of selective PPAR agonist WY-14643 on the content and fatty acid composition of major lipid classes in the heart of rats fed a standard chow (STD) or a high-fat diet (HFD). In STD rats WY-14643 paradoxically decreased palmitate oxidation rate in the heart, however, in HFD animals such effect was not observed. WY-14643 markedly reduced myocardial free fatty acid and diacylglycerol content in STD rats, whereas in HFD group the opposite effect was observed. These changes reflected alterations in plasma lipid concentration which suggests that effects of WY-14643 on the heart were indirect and secondary to changes in plasma lipid availability induced by the drug. Basal myocardial glucose uptake was not affected by PPAR agonist in either group, however, glycogen content in the heart was markedly increased. WY-14643 exerted profound influence on the fatty acid composition of myocardial phospholipids in both diet groups. These changes included increased percentage of monounsaturated fatty acids and replacement of n-3 polyunsaturated fatty acids (PUFA) by those from the n-6 family. This action of WY-14643 might be detrimental to the heart since n-3 PUFA possess cardioprotective and antiarrhythmic properties.
Mieszaninę oleju rzepakowego i stearyny palmowej (4:6) przeestryfikowywano w obecności biokatalizatora - Lipozyme IM oraz katalizatora chemicznego - metanolami sodu i porównywano ich stabilność oksydatywną. Czyste triacyloglicerole (TAG) wyizolowane z produktu enzymatycznego przeestryfikowania charakteryzowały się stabilnością oksydatywną podobną do TAG z mieszaniny wyjściowej, natomiast stabilność TAG z produktu chemicznego przeestryfikowania była dwukrotnie gorsza. Dodatek frakcji triacyloglicerolowej bogatej w niepełne acyloglicerole do czystych TAG powodował pogorszenie stabilności oksydatywnej produktu, tym większe, im wyższe było stężenie dodanej frakcji, natomiast dodatek handlowego emulgatora 3328 powodował znaczną poprawę tej stabilności.
To obtain sufficient quantities of pure phospholipase C delta 3 (PLC delta 3) necessary for structural and kinetic studies, cDNA of human fibroblast PLC delta 3 was cloned in the pPROEX-1 vector, expressed in E. coli cells as a (6 x His) fusion protein and purified to homogeneity. From 1 L of E. coli culture 8 mg of pure PLC delta 3 was obtained by a two step purification procedure, which includes phosphocellulose and Mono S cation exchange chromatography. The presence of His tag did not affect the catalytic and regulatory properties of PLC delta 3. The K(app) for PIP2 was 142 +/- 11 and 156 +/- 12 microM for His.PLC delta 3 and PLC delta 3, respectively. Recombinant PLC delta 3 showed an absolute requirement for Ca2+. Increasing the free Ca2+ concentration from 0.2 to 0.5 microM resulted in a sharp increase in enzyme activity. In comparison with human recombinant PLC delta 1 the delta 3 isoenzyme was more sensitive to low Ca2+ concentration. The Ca2+ concentration yielding maximal activation of PLC delta 1 and PLC delta 3 was 10 and 1 microM, respectively. The activity of PLC delta 3 was stimulated by polyamines and by basic proteins such as protamine, histone and mellitin. PLC delta 3 was activated most effectively by spermine and histone but the extent of this activation was lower than for PLC delta 1. The data presented indicate that the expression of PLC delta 3 in E. coli cells permits to obtain active enzyme. The catalytic and regulatory properties of PLC delta 3 are similar to those of PLC delta 1.
Nematodes were found to synthesize phosphorylcholine-containing molecules not present in higher organisms, i.e. phosphorylcholine-substituted glycosphingolipids and (glyco)proteins. Investigations on the biosynthesis of these structures provided first biochemical evidence for the presence of the Kennedy and Bremer-Greenberg pathways in the model organism Caenorhabditis elegans.
In plant cells, phospholipids are not only membrane components but also act as second messengers interacting with various proteins and regulating diverse cellular processes, including stress signal transduction. Here, we report studies on the effects of various phospholipids on the activity and expression of maize wound-responsive calcium-dependent protein kinase (ZmCPK11). Our results revealed that in leaves treated with n-butanol, a potent inhibitor of phosphatidic acid (PA) synthesis catalyzed by phospholipase D, a significant decrease of ZmCPK11 activity was observed, indicating contribution of PA in the kinase activation. Using lipid binding assays, we demonstrate that among various phospholipids only saturated acyl species (16 : 0 and 18 : 0) of phosphatidic acid are able to bind to ZmCPK11. Saturated acyl species of PA are also able to stimulate phosphorylation of exogenous substrates by ZmCPK11 and autophosphorylation of the kinase. The level of ZmCPK11 autophosphorylation is correlated with its enzymatic activity. RT-PCR analysis showed that transcript level of ZmCPK11 in maize leaves increased in response to PA treatment. The influence of PA on the activity and transcript level of ZmCPK11 suggests an involvement of this kinase in a PA-mediated wound signal transduction pathway.
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