Medium-chain fatty acids (FAs), found in storage lipids of certain plants, are an important renewable resource. Seeds of undomesticated California bay accumulate laurate (12:0), and a 12:0-acyl-carrier protein thioesterase (BTE) has been purified from this tissue. Sequencing of BTE enabled the cloning of a complementary DNA coding for a plastid-targeted preprotein. Expression of the complementary DNA in the seeds of Arabidopsis thaliana resulted in BTE activity, and medium chains accumulated at the expense of long-chain (greater than or equal to 16) FAs. Laurate became the most abundant FA species and was deposited in the storage triacylglycerols. These results demonstrate a mechanism for medium-chain FA synthesis in plants.
Molecular gene transfer techniques have been used to engineer the fatty acid composition of Brassica rpa and Brassica napus (canola) oil. Stearoyl-acyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the first desaturation step in seed oil biosynthesis, converting stearoyl-ACP to oleoyl-ACP. Seed-specific antisense gene constructs ofB. rapa stearoyl-ACP desaturase were used to reduce the protein concentration and enzyme activity of stearoyl-ACP desaturase in developing rapeseed embryos during storage lipid biosynthesis. The resulting transgenic plants showed dramatically increased stearate levels in the seeds. A continuous distribution of stearate levels from 2% to 40% was observed in seeds of a transgenic B. napus plant, illustrating the potential to engineer specialized seed oil compositions.Canola and other temperate vegetable oils are composed predominantly of unsaturated 18-carbon fatty acids: the monounsaturated oleic (18:1) and polyunsaturated linoleic (18:2) and linolenic (18:3) acids. In addition to these fatty acids, most oils also contain small but significant amounts of the saturated palmitic (16:0) and stearic (18:0) acids (1). The plastid-localized enzyme stearoyl-acyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the initial desaturation reaction in fatty acid biosynthesis (Fig. 1A) and thus plays a key role in determining the ratio oftotal saturated to unsaturated fatty acids in plants (2,4,5).Specialized fatty acid compositions desired for edible and industrial purposes have been produced in oilseed crops through traditional breeding and selection alone or in combination with mutagenesis programs (6-9). Although the molecular basis for the changes is largely unknown, examples such as the removal of erucic acid from rapeseed oil to create canola (10), reduction of linolenic acid content in flax seed (11), and increases in stearate content of up to six times the wild-type level in safflower (up to 12% stearate) (12) and soybean (up to 30%o stearate) oil (13,14) demonstrate the plasticity of fatty acid composition in seed oil. It should also be possible to modify seed oil composition by the use of genetic engineering techniques (15-17). Antisense RNA technology has proven to be an effective means of reducing the level of specific enzymes in plants (18-21). Because fatty acid biosynthesis is an essential metabolic pathway in all tissues ofthe plant, modification of seed oil biosynthesis may require tissue-specific control of antisense RNA expression. Reduction of stearoyl-ACP desaturase in seeds should alter the ratio of saturated to unsaturated fatty acids and lead to the production of a novel storage oil without compromising the integrity of membrane lipids in leaf and other plant tissues.We report the isolation of a Brassica rapa (syn. Brassica campestris, turnip rape) stearoyl-ACP desaturase cDNAt and expression of antisense stearoyl-ACP desaturase constructs in seeds of B. rapa and Brassica napus. The activity and amount of stearoyl-ACP desaturase...
Key words; Particle disease -septic and aseptic loosening -total joint replacement -array filter analysisThe precise cellular mechariism qf psteolysis in particle disease is still unknown. The aim of the study was to screen for new gene products in macrophages during particle contact. Method: In an established macrophage model THPl-cells (human monocytic cells) were different iated under the influence of vitamin D3 and GM-CSF into macrophage-like cells (MLC). MLCs were incubated each with different concentrations of polyethylen particles, Lipopolysaccharids (LPS) and controls. isolatecl RNA was transcribed into complemeritary radioactive 32 P labeled cDNA. This probe was hybridised on an human cDNA expression array and analysed by autoradiography. To obtain a more reliable method quantifying mRNA, the reverse transcriptase polymerase chain reactioh (KT-PCR) was used. Results: The arrays showed an upregulation of the following genes by particles: TNF-Rezeptor 2, IL-1 Receptor Antagonist, Bone Morphogenic Protein 4 and HM 145. This was proven three times using RT-PCR and statistically significant in comparison to the controls. LPS induced the same upregulation except for HM145 whereas particles caused downregulation of this mRNA expression. Conclusion: Qur results prpve that the model of differentiated THP-1 cells treated with PE particles is a suitable System to analyse differential gene expression patterns, since the induction of the major positive control genes TNF a and iLl« were detected by this approach. BMP 4 is known äs Signal protein which mediates ectopic bone lormation and can also be interpreted äs a contra regulatory gene. HM 145 belongs to the leukocyte chemotactic peptide receptor family. HM 145 seems to be one of the first genes that is enhanced along the septical pathway but less expressed by contact with particles. Analysis of HM 145 expression might help to diagnose septic versus aseptic loosening of prosthesis.Schlüsselwörter. Partikelkrankheit, septische und aseptische Prothesesenlockerung, Totalendoprothese, Analyse von Genprodukten Der zelluläre Mechanismus der Knochenresorption bei der Partikelkrankheit ist unbekannt. Das Ziel der Studie war es, nach neuen Genprodukten zu suchen, die nach Kontakt mit Abriebpartikeln von Mafcrophagen gebildet werden. Methode: In einem etablierten Makrophagenmodell konnten THP-1-Zellen unter dem Einfluß von Vitamin D3 und GM-CSF in Makrophagen-ähnliche Zellen (MäZ) differenziert werden. Die MäZ wurden mit PE-Partikeln und" Lipopolysacchariden (LPS) inkubiert. Die nach Partikelkontakt gebildete mRNA wurde isoliert und in radioaktive 32 P markierte cDNA transkribiert. Danach wurde ein cDNA-Expressions-Array durchgeführt und mit Autoradiographie analysiert. Als zusätzliche Methode wurde eine RT-PCR (reverse transcriptase polymerase chain reaction) angewandt, um die Ergebnisse des Arrays zu überprüfen. Ergebnisse: Der Array zeigte folgende Gene nach Partikelkontakt hochreguliert: TNFRezeptor 2, IL-1-Rezeptor Antagonist, Bone-Morphogenic Protein (BMP) 4 sowie HM 14...
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