Modified acyl-ACP desaturase

DOEpatents

Cahoon, E.B.; Shanklin, J.; Lindgvist, Y.; Schneider, G.

1998-01-06

Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 1 fig.

Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.).

PubMed

Thambugala, Dinushika; Cloutier, Sylvie

2014-11-01

Little is known about the relationship between expression levels of fatty acid desaturase genes during seed development and fatty acid (FA) composition in flax. In the present study, we looked at promoter structural variations of six FA desaturase genes and their relative expression throughout seed development. Computational analysis of the nucleotide sequences of the sad1, sad2, fad2a, fad2b, fad3a and fad3b promoters showed several basic transcriptional elements including CAAT and TATA boxes, and several putative target-binding sites for transcription factors, which have been reported to be involved in the regulation of lipid metabolism. Using semi-quantitative reverse transcriptase PCR, the expression patterns throughout seed development of the six FA desaturase genes were measured in six flax genotypes that differed for FA composition but that carried the same desaturase isoforms. FA composition data were determined by phenotyping the field grown genotypes over four years in two environments. All six genes displayed a bell-shaped pattern of expression peaking at 20 or 24 days after anthesis. Sad2 was the most highly expressed. The expression of all six desaturase genes did not differ significantly between genotypes (P = 0.1400), hence there were no correlations between FA desaturase gene expression and variations in FA composition in relatively low, intermediate and high linolenic acid genotypes expressing identical isoforms for all six desaturases. These results provide further clues towards understanding the genetic factors responsible for FA composition in flax.

Effects of saturated and unsaturated fatty acids on estimated desaturase activities during a controlled dietary intervention.

PubMed

Warensjö, Eva; Risérus, Ulf; Gustafsson, Inga-Britt; Mohsen, Rawya; Cederholm, Tommy; Vessby, Bengt

2008-12-01

Direct measurement of desaturase activities are difficult to obtain in humans. Consequently, surrogate measures of desaturase activity (estimated desaturase activities) have been frequently used in observational studies, and estimated Delta(9)- (or stearoyl-CoA-desaturase (SCD)), Delta(6)- and Delta(5)-desaturase activities have been associated with cardiometabolic disease. Data on how the markers of desaturase activities are modified by changes in dietary fat quality are lacking and therefore warrant examination. In a two-period (three weeks) strictly controlled cross-over study, 20 subjects (six women and 14 men) consumed a diet high in saturated fat (SAT-diet) and a rapeseed oil diet (RO-diet), rich in oleic acid (OA), linoleic acid (LA) and alpha-linolenic acid (ALA). Estimated desaturase activities were calculated as precursor to product FA ratios in serum cholesteryl esters and phospholipids. The estimated SCD [16:1 n-7/16:0] and Delta(6)-desaturase [20:3 n-6/18:2 n-6] was significantly higher while Delta(5)-desaturase [20:4 n-6/20:3 n-6] was significantly lower in the SAT-diet (P<0.001 for all), compared to the RO-diet. The serum proportions of palmitic, stearic, palmitoleic and dihomo-gamma-linolenic acids were significantly higher in the SAT-diet while the proportions of LA and ALA were significantly higher in the RO-diet. This is the first study to demonstrate that surrogate measures of desaturase activities change as a consequence of an alteration in dietary fat quality. Both the [16:1/16:0]-ratio and 16:1 seem to reflect changes in saturated fat intake and may be useful markers of saturated fat intake in Western countries.

An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van De Loo, F.J.; Broun, P.; Turner, S.

1995-07-18

Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 andmore » with {approx}67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution. 26 refs., 6 figs., 1 tab.« less

Handmade Cloned Transgenic Sheep Rich in Omega-3 Fatty Acids

PubMed Central

Dou, Hongwei; Chen, Lei; Chen, Longxin; Lin, Lin; Tan, Pingping; Vajta, Gabor; Gao, Jianfeng; Du, Yutao; Ma, Runlin Z.

2013-01-01

Technology of somatic cell nuclear transfer (SCNT) has been adapted worldwide to generate transgenic animals, although the traditional procedure relies largely on instrumental micromanipulation. In this study, we used the modified handmade cloning (HMC) established in cattle and pig to produce transgenic sheep with elevated levels of omega-3 (n−3) fatty acids. Codon-optimized nematode mfat-1 was inserted into a eukaryotic expression vector and was transferred into the genome of primary ovine fibroblast cells from a male Chinese merino sheep. Reverse transcriptase PCR, gas chromatography, and chromosome analyses were performed to select nuclear donor cells capable of converting omega-6 (n−6) into n−3 fatty acids. Blastocysts developed after 7 days of in vitro culture were surgically transplanted into the uterus of female ovine recipients of a local sheep breed in Xinjiang. For the HMC, approximately 8.9% (n  = 925) of reconstructed embryos developed to the blastocyst stage. Four recipients became pregnant after 53 blastocysts were transplanted into 29 naturally cycling females, and a total of 3 live transgenic lambs were produced. Detailed analyses on one of the transgenic lambs revealed a single integration of the modified nematode mfat-1 gene at sheep chromosome 5. The transgenic sheep expressed functional n−3 fatty acid desaturase, accompanied by more than 2-folds reduction of n−6/n−3 ratio in the muscle (p<0.01) and other major organs/tissues (p<0.05). To our knowledge, this is the first report of transgenic sheep produced by the HMC. Compared to the traditional SCNT method, HMC showed an equivalent efficiency but proved cheaper and easier in operation. PMID:23437077

Production of γ-linolenic acid and stearidonic acid by Synechococcus sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

NASA Astrophysics Data System (ADS)

Dong, Xuewei; He, Qingfang; Peng, Zhenying; Yu, Jinhui; Bian, Fei; Li, Youzhi; Bi, Yuping

2016-07-01

Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

Development of marker-free transgenic Jatropha plants with increased levels of seed oleic acid

PubMed Central

2012-01-01

Background Jatropha curcas is recognized as a new energy crop due to the presence of the high amount of oil in its seeds that can be converted into biodiesel. The quality and performance of the biodiesel depends on the chemical composition of the fatty acids present in the oil. The fatty acids profile of the oil has a direct impact on ignition quality, heat of combustion and oxidative stability. An ideal biodiesel composition should have more monounsaturated fatty acids and less polyunsaturated acids. Jatropha seed oil contains 30% to 50% polyunsaturated fatty acids (mainly linoleic acid) which negatively impacts the oxidative stability and causes high rate of nitrogen oxides emission. Results The enzyme 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in plants. We identified three putative delta 12 fatty acid desaturase genes in Jatropha (JcFAD2s) through genome-wide analysis and downregulated the expression of one of these genes, JcFAD2-1, in a seed-specific manner by RNA interference technology. The resulting JcFAD2-1 RNA interference transgenic plants showed a dramatic increase of oleic acid (> 78%) and a corresponding reduction in polyunsaturated fatty acids (< 3%) in its seed oil. The control Jatropha had around 37% oleic acid and 41% polyunsaturated fatty acids. This indicates that FAD2-1 is the major enzyme responsible for converting oleic acid to linoleic acid in Jatropha. Due to the changes in the fatty acids profile, the oil of the JcFAD2-1 RNA interference seed was estimated to yield a cetane number as high as 60.2, which is similar to the required cetane number for conventional premium diesel fuels (60) in Europe. The presence of high seed oleic acid did not have a negative impact on other Jatropha agronomic traits based on our preliminary data of the original plants under greenhouse conditions. Further, we developed a marker-free system to generate the transgenic

TALEN-mediated targeted mutagenesis of fatty acid desaturase 2 (FAD2) in peanut (Arachis hypogaea L.) promotes the accumulation of oleic acid.

PubMed

Wen, Shijie; Liu, Hao; Li, Xingyu; Chen, Xiaoping; Hong, Yanbin; Li, Haifen; Lu, Qing; Liang, Xuanqiang

2018-05-01

A first creation of high oleic acid peanut varieties by using transcription activator-like effecter nucleases (TALENs) mediated targeted mutagenesis of Fatty Acid Desaturase 2 (FAD2). Transcription activator like effector nucleases (TALENs), which allow the precise editing of DNA, have already been developed and applied for genome engineering in diverse organisms. However, they are scarcely used in higher plant study and crop improvement, especially in allopolyploid plants. In the present study, we aimed to create targeted mutagenesis by TALENs in peanut. Targeted mutations in the conserved coding sequence of Arachis hypogaea fatty acid desaturase 2 (AhFAD2) were created by TALENs. Genetic stability of AhFAD2 mutations was identified by DNA sequencing in up to 9.52 and 4.11% of the regeneration plants at two different targeted sites, respectively. Mutation frequencies among AhFAD2 mutant lines were significantly correlated to oleic acid accumulation. Genetically, stable individuals of positive mutant lines displayed a 0.5-2 fold increase in the oleic acid content compared with non-transgenic controls. This finding suggested that TALEN-mediated targeted mutagenesis could increase the oleic acid content in edible peanut oil. Furthermore, this was the first report on peanut genome editing event, and the obtained high oleic mutants could serve for peanut breeding project.

Alternation of plasma fatty acids composition and desaturase activities in children with liver steatosis

PubMed Central

Su, Hui-Min; Yao, Tsung-Chieh; Kuo, Ming-Ling; Lai, Ming-Wei; Tsai, Ming-Han; Huang, Jing-Long

2017-01-01

Objective The aim of this study was to investigate changes in plasma fatty acids proportions and estimated desaturase activities for variable grading of liver steatosis in children. Methods In total, 111 schoolchildren (aged 8–18 years) were included in the analysis from March 2015 to August 2016. Anthropometric evaluation, liver ultrasound examination and scoring for nonalcoholic fatty liver disease (NAFLD score = 0–6), and biochemical and plasma fatty acids analysis were performed. We compared the composition ratio of fatty acids between children with high-grade liver steatosis (NAFLD score = 4–6), low-grade liver steatosis (NAFLD score = 1–3), and healthy controls (NAFLD score = 0). In addition, correlation coefficients (r) between NAFLD score, metabolic variables, and estimated activity of desaturase indices (stearoyl-coenzyme A desaturase-1 (SCD1), delta-5 and delta-6 desaturase) were calculated. Results Compared with healthy controls, children with liver steatosis showed a higher proportion of monounsaturated fatty acids (21.16 ± 2.81% vs. 19.68 ± 2.71%, p = 0.024). In addition, children with high- grade liver steatosis exhibited higher proportions of palmitic acid (C16:0), palmitoleic acid (C16:1n-7), dihomo-γ-linolenic acid (C20:3n-6), adrenic acid (C22:4n-6), and docosapentaenoic acid (C22:5n-6); and lower proportions of eicosapentaenoic acid (C20:5n-3) (P< 0.05). In all subjects, the NAFLD score was positively correlated with body mass index (BMI) (kg/m2) (r = 0.696), homeostasis model of assessment ratio–index (HOMA-IR) (r = 0.510), SCD1(16) (r = 0.273), and the delta-6 index (r = 0.494); and inversely associated with the delta-5 index (r = -0.443). Conclusion Our current data suggested that children with liver steatosis was highly associated with obesity, and insulin resistance. In addition, increased endogenous lipogenesis through altered desaturase activity may contribute to the progression of liver steatosis in children. PMID:28759573

Conferring high-temperature tolerance to nontransgenic tomato scions using graft transmission of RNA silencing of the fatty acid desaturase gene.

PubMed

Nakamura, Shinya; Hondo, Kana; Kawara, Tomoko; Okazaki, Yozo; Saito, Kazuki; Kobayashi, Kappei; Yaeno, Takashi; Yamaoka, Naoto; Nishiguchi, Masamichi

2016-02-01

We investigated graft transmission of high-temperature tolerance in tomato scions to nontransgenic scions from transgenic rootstocks, where the fatty acid desaturase gene (LeFAD7) was RNA-silenced. Tomato was transformed with a plasmid carrying an inverted repeat of LeFAD7 by Agrobacterium. Several transgenic lines showed the lower amounts of LeFAD7 RNA and unsaturated fatty acids, while nontransgenic control did not, and siRNA was detected in the transgenic lines, but not in control. These lines grew under conditions of high temperature, while nontransgenic control did not. Further, the nontransgenic plants were grafted onto the silenced transgenic plants. The scions showed less of the target gene RNA, and siRNA was detected. Under high-temperature conditions, these grafted plants grew, while control grafted plants did not. Thus, it was shown that high-temperature tolerance was conferred in the nontransgenic scions after grafting onto the silenced rootstocks. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Identification of Delta5-fatty acid desaturase from the cellular slime mold dictyostelium discoideum.

PubMed

Saito, T; Ochiai, H

1999-10-01

cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project. Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase. The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N-terminal cytochrome b5 domain. The whole sequence was 38.6% identical to the initially identified Delta5-desaturase of Mortierella alpina. We have confirmed its function as Delta5-desaturase by over expression mutation in D. discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae. Analysis of the lipids from transformed D. discoideum and yeast demonstrated the accumulation of Delta5-desaturated products. This is the first report concering fatty acid desaturase in cellular slime molds.

Improved γ-linolenic acid production in Mucor circinelloides by homologous overexpressing of delta-12 and delta-6 desaturases.

PubMed

Zhang, Yao; Luan, Xiao; Zhang, Huaiyuan; Garre, Victoriano; Song, Yuanda; Ratledge, Colin

2017-06-21

γ-Linolenic acid (GLA) is important because of its nutritional value and medicinal applications. Although the biosynthetic pathways of some plant and microbial GLA have been deciphered, current understanding of the correlation between desaturases and GLA synthesis in oleaginous fungi is incomplete. In previous work, we found that a large amount of oleic acid (OA) had not been converted to linoleic acid (LA) or GLA in Mucor circinelloides CBS 277.49, which may be due to inadequate activities of the delta-12 or delta-6 desaturases, and thus leading to the accumulation of OA and LA. Thus, it is necessary to explore the main contributing factor during the process of GLA biosynthesis in M. circinelloides. To enhance GLA production in M. circinelloides, homologous overexpression of delta-12 and two delta-6 desaturases (named delta-6-1 and delta-6-2, respectively) were analyzed. When delta-6 desaturase were overexpressed in M. circinelloides, up to 43% GLA was produced in the total fatty acids, and the yield of GLA reached 180 mg/l, which were, respectively, 38 and 33% higher than the control strain. These findings revealed that delta-6 desaturase (especially for delta-6-1 desaturase) plays an important role in GLA synthesis by M. circinelloides. The strain overexpressing delta-6-1 desaturase may have potential application in microbial GLA production.

Biosynthesis of 10,12-dienoic fatty acids by a bifunctional Delta11 desaturase in Spodoptera littoralis.

PubMed

Serra, Montserrat; Piña, Benjami; Bujons, Jordi; Camps, Francisco; Fabriàs, Gemma

2006-08-01

In the biosynthetic pathway of Spodoptera littoralis sex pheromone, (E,E)-10,12-tetradecadienoic acid is produced from (Z)-11-tetradecenoic acid by desaturation and concomitant migration of the precursor double bond. With the aim of identifying the enzyme involved in this biotransformation, yeast Deltaelo1/Deltaole mutants, which are both elongase 1 and Delta9 desaturase-deficient, were transformed with the S. littoralis Delta11 desaturase gene using a Cu+2 inducible expression vector. The transformants produced a recombinant polyhistidine-tagged Delta11 desaturase that could be detected by immunoblotting from cell lysates. Lipid analysis revealed that besides producing large quantities of C11-monounsaturated fatty acids, mainly (Z)-11-hexadecenoic acid, (E,E)-10,12-tetradecadienoic acid and minor amounts of (E,Z)-10,12-hexadecadienoic acid were also produced, as well as very low quantities of another tetradecadienoate, which was tentatively identified as the (E,Z)-10,12-tetradecadienoic isomer. None of these dienes was detected with the Delta11 desaturase gene of Trichoplusia ni, which does not produce conjugated dienes as pheromone components. We conclude that the Delta11 desaturase of S. littoralis is a bifunctional enzyme with both Delta11 and Delta10,12 desaturation activities. The relationship between the substrate structure and the stereochemical outcome of the reaction is discussed.

Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants.

PubMed

Berdichevets, Iryna N; Shimshilashvili, Hristina R; Gerasymenko, Iryna M; Sindarovska, Yana R; Sheludko, Yuriy V; Goldenkova-Pavlova, Irina V

2010-07-01

Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.

Functional screening of a novel Δ15 fatty acid desaturase from the coccolithophorid Emiliania huxleyi.

PubMed

Kotajima, Tomonori; Shiraiwa, Yoshihiro; Suzuki, Iwane

2014-10-01

The coccolithophorid Emiliania huxleyi is a bloom-forming marine phytoplankton thought to play a key role as a biological pump that transfers carbon from the surface to the bottom of the ocean, thus contributing to the global carbon cycle. This alga is also known to accumulate a variety of polyunsaturated fatty acids. At 25°C, E. huxleyi produces mainly 14:0, 18:4n-3, 18:5n-3 and 22:6n-3. When the cells were transferred from 25°C to 15°C, the amount of unsaturated fatty acids, i.e. 18:1n-9, 18:3n-3 and 18:5n-3, gradually increased. Among the predicted desaturase genes whose expression levels were up-regulated at low temperature, we identified a gene encoding novel ∆15 fatty acid desaturase, EhDES15, involved in the production of n-3 polyunsaturated fatty acids in E. huxleyi. This desaturase contains a putative transit sequence for localization in chloroplasts and a ∆6 desaturase-like domain, but it does not contain a cytochrome b5 domain nor typical His-boxes found in ∆15 desaturases. Heterologous expression of EhDES15 cDNA in cyanobacterium Synechocystis sp. PCC 6803 cells increased the level of n-3 fatty acid species, which are produced at low levels in wild-type cells grown at 30°C. The orthologous genes are only conserved in the genomes of prasinophytes and cryptophytes. The His-boxes conserved in orthologues varied from that of the canonical ∆15 desaturases. These results suggested the gene encodes a novel ∆15 desaturase responsible for the synthesis of 18:3n-3 from 18:2n-6 in E. huxleyi. Copyright © 2014 Elsevier B.V. All rights reserved.

INHIBITION OF FATTY ACID DESATURASES IN Drosophila melanogaster LARVAE BLOCKS FEEDING AND DEVELOPMENTAL PROGRESSION.

PubMed

Wang, Yiwen; da Cruz, Tina Correia; Pulfemuller, Alicia; Grégoire, Stéphane; Ferveur, Jean-François; Moussian, Bernard

2016-05-01

Fatty acid desaturases are metabolic setscrews. To study their systemic impact on growth in Drosophila melanogaster, we inhibited fatty acid desaturases using the inhibitor CAY10566. As expected, the amount of desaturated lipids is reduced in larvae fed with CAY10566. These animals cease feeding soon after hatching, and their growth is strongly attenuated. A starvation program is not launched, but the expression of distinct metabolic genes is activated, possibly to mobilize storage material. Without attaining the normal size, inhibitor-fed larvae molt to the next stage indicating that the steroid hormone ecdysone triggers molting correctly. Nevertheless, after molting, expression of ecdysone-dependent regulators is not induced. While control larvae molt a second time, these larvae fail to do so and die after few days of straying. These effects are similar to those observed in experiments using larvae deficient for the fatty acid desaturase1 gene. Based on these data, we propose that the ratio of saturated to unsaturated fatty acids adjusts a sensor system that directs feeding behavior. We also hypothesize that loss of fatty acid desaturase activity leads to a block of the genetic program of development progression indirectly by switching on a metabolic compensation program. © 2016 Wiley Periodicals, Inc.

Novel Genes Encoding Hexadecanoic Acid Δ6-Desaturase Activity in a Rhodococcus sp.

PubMed

Araki, Hiroyuki; Hagihara, Hiroshi; Takigawa, Hirofumi; Tsujino, Yukiharu; Ozaki, Katsuya

2016-11-01

cis-6-Hexadecenoic acid, a major component of human sebaceous lipids, is involved in the defense mechanism against Staphylococcus aureus infection in healthy skin and closely related to atopic dermatitis. Previously, Koike et al. (Biosci Biotechnol Biochem 64:1064-1066, 2000) reported that a mutant strain of Rhodococcus sp. produced cis-6-hexadecenoate derivatives from palmitate alkyl esters. From the mutant Rhodococcus strain, we identified and sequenced two open reading frames present in an amplified 5.7-kb region; these open reading frames encoded tandemly repeated Δ6-desaturase-like genes, Rdes1 and Rdes2. A phylogenetic tree indicated that Rdes1 and Rdes2 were different from previously known Δ6-desaturase genes, and that they formed a new cluster. Rdes1 and Rdes2 were each introduced into vectors and then expressed separately in Escherichia coli, and the fatty acid composition of the transformed cells was analyzed by gas chromatography and mass spectrometry. The amount of cis-6-hexadecenoic acid was significantly higher in Rdes1- or Rdes2-transformed E. coli cells (twofold and threefold, respectively) than in vector-only control cells. These results showed that cis-6-hexadecenoic acid was produced in E. coli cells by the rhodococcal Δ6-desaturase-like proteins.

Loss of Function of FATTY ACID DESATURASE7 in Tomato Enhances Basal Aphid Resistance in a Salicylate-Dependent Manner1[W][OA

PubMed Central

Avila, Carlos A.; Arévalo-Soliz, Lirio M.; Jia, Lingling; Navarre, Duroy A.; Chen, Zhaorigetu; Howe, Gregg A.; Meng, Qing-Wei; Smith, Jonathon E.; Goggin, Fiona L.

2012-01-01

We report here that disruption of function of the ω-3 FATTY ACID DESATURASE7 (FAD7) enhances plant defenses against aphids. The suppressor of prosystemin-mediated responses2 (spr2) mutation in tomato (Solanum lycopersicum), which eliminates the function of FAD7, reduces the settling behavior, survival, and fecundity of the potato aphid (Macrosiphum euphorbiae). Likewise, the antisense suppression of LeFAD7 expression in wild-type tomato plants reduces aphid infestations. Aphid resistance in the spr2 mutant is associated with enhanced levels of salicylic acid (SA) and mRNA encoding the pathogenesis-related protein P4. Introduction of the Naphthalene/salicylate hydroxylase transgene, which suppresses SA accumulation, restores wild-type levels of aphid susceptibility to spr2. Resistance in spr2 is also lost when we utilize virus-induced gene silencing to suppress the expression of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1), a positive regulator of many SA-dependent defenses. These results indicate that FAD7 suppresses defenses against aphids that are mediated through SA and NPR1. Although loss of function of FAD7 also inhibits the synthesis of jasmonate (JA), the effects of this desaturase on aphid resistance are not dependent on JA; other mutants impaired in JA synthesis (acx1) or perception (jai1-1) show wild-type levels of aphid susceptibility, and spr2 retains aphid resistance when treated with methyl jasmonate. Thus, FAD7 may influence JA-dependent defenses against chewing insects and SA-dependent defenses against aphids through independent effects on JA synthesis and SA signaling. The Arabidopsis (Arabidopsis thaliana) mutants Atfad7-2 and Atfad7-1fad8 also show enhanced resistance to the green peach aphid (Myzus persicae) compared with wild-type controls, indicating that FAD7 influences plant-aphid interactions in at least two plant families. PMID:22291202

Cloned transgenic heart-healthy pork?

PubMed

Prather, Randall S

2006-08-01

Here I comment on the production and uses of swine that express a humanized fat-1 gene. The gene product is a fatty acid desaturase that converts omega-6 fatty acids to omega-3 fatty acids. Omega-3 fatty acids have been implicated as being important for reproductive success, maintaining a healthy cardiovascular system, sustaining a functional immune system, and even preventing depression and cancer. The descendants of these hfat-1 transgenic swine will be very useful as models of the human condition, and if they are permitted to enter the food chain, they may improve human health.

Modified Acyl-ACP desaturase

DOEpatents

Cahoon, Edgar B.; Shanklin, John; Lindqvist, Ylva; Schneider, Gunter

1999-03-30

Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

Modified acyl-ACP desaturase

DOEpatents

Cahoon, Edgar B.; Shanklin, John; Lindgvist, Ylva; Schneider, Gunter

1998-01-06

Disclosed is a methods for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

USDA-ARS?s Scientific Manuscript database

In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

Modified Acyl-ACP desaturase

DOEpatents

Cahoon, E.B.; Shanklin, J.; Lindqvist, Y.; Schneider, G.

1999-03-30

Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 2 figs.

Identification and characterization of a plastidial ω-3 fatty acid desaturase EgFAD8 from oil palm (Elaeis guineensis Jacq.) and its promoter response to light and low temperature

PubMed Central

Chen, Lizhi; Wang, Lei; Wang, Herong; Sun, Ruhao; You, Lili; Zheng, Yusheng; Yuan, Yijun

2018-01-01

In higher plants, ω-3 fatty acid desaturases are the key enzymes in the biosynthesis of alpha-linolenic acid (18:3), which plays key roles in plant metabolism as a structural component of both storage and membrane lipids. Here, the first ω-3 fatty acid desaturase gene was identified and characterized from oil palm. The bioinformatic analysis indicated it encodes a temperature-sensitive chloroplast ω-3 fatty acid desaturase, designated as EgFAD8. The expression analysis revealed that EgFAD8 is highly expressed in the oil palm leaves, when compared with the expression in the mesocarp. The heterologous expression of EgFAD8 in yeast resulted in the production of a novel fatty acid 18:3 (about 0.27%), when fed with 18:2 in the induction culture. Furthermore, to detect whether EgFAD8 could be induced by the environment stress, we detected the expression efficiency of the EgFAD8 promoter in transgenic Arabidopsis treated with low temperature and darkness, respectively. The results indicated that the promoter of EgFAD8 gene could be significantly induced by low temperature and slightly induced by darkness. These results reveal the function of EgFAD8 and the feature of its promoter from oil palm fruits, which will be useful for understanding the fuction and regulation of plastidial ω-3 fatty acid desaturases in higher plants. PMID:29698515

Reducing Isozyme Competition Increases Target Fatty Acid Accumulation in Seed Triacylglycerols of Transgenic Arabidopsis1[OPEN

PubMed Central

van Erp, Harrie; Shockey, Jay; Zhang, Meng; Adhikari, Neil D.; Browse, John

2015-01-01

One goal of green chemistry is the production of industrially useful fatty acids (FAs) in crop plants. We focus on hydroxy fatty acids (HFAs) and conjugated polyenoic FAs (α-eleostearic acids [ESAs]) using Arabidopsis (Arabidopsis thaliana) as a model. These FAs are found naturally in seed oils of castor (Ricinus communis) and tung tree (Vernicia fordii), respectively, and used for the production of lubricants, nylon, and paints. Transgenic oils typically contain less target FA than that produced in the source species. We hypothesized that competition between endogenous and transgenic isozymes for substrates limits accumulation of unique FAs in Arabidopsis seeds. This hypothesis was tested by introducing a mutation in Arabidopsis diacylglycerol acyltransferase1 (AtDGAT1) in a line expressing castor FA hydroxylase and acyl-Coenzyme A:RcDGAT2 in its seeds. This led to a 17% increase in the proportion of HFA in seed oil. Expression of castor phospholipid:diacylglycerol acyltransferase 1A in this line increased the proportion of HFA by an additional 12%. To determine if our observations are more widely applicable, we investigated if isozyme competition influenced production of ESA. Expression of tung tree FA conjugase/desaturase in Arabidopsis produced approximately 7.5% ESA in seed lipids. Coexpression of VfDGAT2 increased ESA levels to approximately 11%. Overexpression of VfDGAT2 combined with suppression of AtDGAT1 increased ESA accumulation to 14% to 15%. Our results indicate that isozyme competition is a limiting factor in the engineering of unusual FAs in heterologous plant systems and that reduction of competition through mutation and RNA suppression may be a useful component of seed metabolic engineering strategies. PMID:25739701

Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

PubMed

Wu, Pingzhi; Zhang, Sheng; Zhang, Lin; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

2013-10-15

Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants. Copyright © 2013 Elsevier GmbH. All rights reserved.

Identification and evaluation of ω-3 fatty acid desaturase genes for hyperfortifying α-linolenic acid in transgenic rice seed.

PubMed

Liu, Hua Liang; Yin, Zhi Jie; Xiao, Li; Xu, Yi Nong; Qu, Le Qing

2012-05-01

α-Linolenic acid (ALA) deficiency and a skewed of ω6:ω3 fatty acid ratio in the diet are a major explanation for the prevalence of cardiovascular diseases and inflammatory/autoimmune diseases. There is a need to enhance the ALA content and to reduce the ratio of linoleic acid (LA) to ALA. Six ω-3 (Δ-15) fatty acid desaturase (FAD) genes were cloned from rice and soybean. The subcellular localizations of the proteins were identified. The FAD genes were introduced into rice under the control of an endosperm-specific promoter, GluC, or a Ubi-1 promoter to evaluate their potential in increasing the ALA content in seeds. The ALA contents in the seeds of endoplasmic reticulum (ER)-localized GmFAD3-1 and OsFAD3 overexpression lines increased from 0.36 mg g⁻¹ to 8.57 mg g⁻¹ and 10.06 mg g⁻¹, respectively, which was 23.8- and 27.9-fold higher than that of non-transformants. The trait of high ALA content was stably inheritable over three generations. Homologous OsFAD3 is more active than GmFAD3-1 in catalysing LA conversion to ALA in rice seeds. Overexpression of ER-localized GmFAD3-2/3 and chloroplast-localized OsFAD7/8 had less effect on increasing the ALA content in rice seeds. The GluC promoter is advantageous compared with Ubi-1 in this experimental system. The enhanced ALA was preferentially located at the sn-2 position in triacylglycerols. A meal-size portion of high ALA rice would meet >80% of the daily adult ALA requirement. The ALA-rich rice could be expected to ameliorate much of the global dietary ALA deficiency.

Altered fatty acid metabolism and reduced stearoyl-coenzyme a desaturase activity in asthma.

PubMed

Rodriguez-Perez, N; Schiavi, E; Frei, R; Ferstl, R; Wawrzyniak, P; Smolinska, S; Sokolowska, M; Sievi, N A; Kohler, M; Schmid-Grendelmeier, P; Michalovich, D; Simpson, K D; Hessel, E M; Jutel, M; Martin-Fontecha, M; Palomares, O; Akdis, C A; O'Mahony, L

2017-11-01

Fatty acids and lipid mediator signaling play an important role in the pathogenesis of asthma, yet this area remains largely underexplored. The aims of this study were (i) to examine fatty acid levels and their metabolism in obese and nonobese asthma patients and (ii) to determine the functional effects of altered fatty acid metabolism in experimental models. Medium- and long-chain fatty acid levels were quantified in serum from 161 human volunteers by LC/MS. Changes in stearoyl-coenzyme A desaturase (SCD) expression and activity were evaluated in the ovalbumin (OVA) and house dust mite (HDM) murine models. Primary human bronchial epithelial cells from asthma patients and controls were evaluated for SCD expression and activity. The serum desaturation index (an indirect measure of SCD) was significantly reduced in nonobese asthma patients and in the OVA murine model. SCD1 gene expression was significantly reduced within the lungs following OVA or HDM challenge. Inhibition of SCD in mice promoted airway hyper-responsiveness. SCD1 expression was suppressed in bronchial epithelial cells from asthma patients. IL-4 and IL-13 reduced epithelial cell SCD1 expression. Inhibition of SCD reduced surfactant protein C expression and suppressed rhinovirus-induced IP-10 secretion, which was associated with increased viral titers. This is the first study to demonstrate decreased fatty acid desaturase activity in humans with asthma. Experimental models in mice and human epithelial cells suggest that inhibition of desaturase activity leads to airway hyper-responsiveness and reduced antiviral defense. SCD may represent a new target for therapeutic intervention in asthma patients. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

delta 6 Hexadecenoic acid is synthesized by the activity of a soluble delta 6 palmitoyl-acyl carrier protein desaturase in Thunbergia alata endosperm.

PubMed

Cahoon, E B; Cranmer, A M; Shanklin, J; Ohlrogge, J B

1994-11-04

delta 6 Hexadecenoic acid (16:1 delta 6) composes more than 80% of the seed oil of Thunbergia alata. Studies were conducted to determine the biosynthetic origin of the double bond of this unusual fatty acid. Assays of fractions of developing T. alata seed endosperm with [1-14C]palmitoyl (16:0)-acyl carrier protein (ACP) revealed the presence of a soluble delta 6 desaturase activity. This activity was greatest when 16:0-ACP was provided as a substrate, whereas no desaturation of the coenzyme A ester of this fatty acid was detected. In addition, delta 6 16:0-ACP desaturase activity in T. alata endosperm extracts was dependent on the presence of ferredoxin and molecular oxygen and was stimulated by catalase. To further characterize this enzyme, a cDNA encoding a diverged acyl-ACP desaturase was isolated from a T. alata endosperm cDNA library using polymerase chain reaction with degenerate oligonucleotides corresponding to conserved amino acid sequences in delta 9 stearoyl (18:0)- and delta 4 16:0-ACP desaturases. The primary structure of the mature peptide encoded by this cDNA shares 66% identity with the mature castor delta 9 18:0-ACP desaturase and 57% identity with the mature coriander delta 4 16:0-ACP desaturase. Extracts of Escherichia coli that express the T. alata cDNA catalyzed the delta 6 desaturation of 16:0-ACP. These results demonstrate that 16:1 delta 6 in T. alata endosperm is formed by the activity of a soluble delta 6 16:0-ACP desaturase that is structurally related to the delta 9 18:0- and delta 4 16:0-ACP desaturases. Implications of this work to an understanding of active site structures of acyl-ACP desaturases are discussed.

A multifunctional desaturase involved in the biosynthesis of the processionary moth sex pheromone

PubMed Central

Serra, Montserrat; Piña, Benjamin; Abad, José Luis; Camps, Francisco; Fabriàs, Gemma

2007-01-01

The sex pheromone of the female processionary moth, Thaumetopoea pityocampa, is a unique C16 enyne acetate that is biosynthesized from palmitic acid. Three consecutive desaturation reactions transform this saturated precursor into the triunsaturated fatty acyl intermediate: formation of (Z)-11-hexadecenoic acid, acetylenation to 11-hexadecynoic acid, and final Δ13 desaturation to (Z)-13-hexadecen-11-ynoic acid. By using degenerate primers common to all reported insect desaturases, a single cDNA sequence was isolated from total RNA of T. pityocampa female pheromone glands. The full-length transcript of this putative desaturase was expressed in elo1Δ/ole1Δ yeast mutants (both elongase 1 and Δ9 desaturase-deficient) for functional assays. The construct fully rescued the Δole1 yeast phenotype, confirming its desaturase activity. Analysis of the unsaturated products from transformed yeast extracts demonstrated that the cloned enzyme showed Δ11 desaturase, Δ11 acetylenase, and Δ13 desaturase activities. Therefore, this single desaturase may account for the three desaturation steps involved in the sex pheromone biosynthetic pathway of the processionary moth. PMID:17921252

[Desaturases of fatty acids (FADS) and their physiological and clinical implication].

PubMed

Žák, Aleš; Slabý, Adolf; Tvrzická, Eva; Jáchymová, Marie; Macášek, Jaroslav; Vecka, Marek; Zeman, Miroslav; Staňková, Barbora

States associated with insulin resistance, as overweight/obesity, type 2 diabetes mellitus (DM2), cardiovascular diseases (CVD), some cancers and neuropsychiatric diseases are characterized with a decrease of long-chain polyunsaturated fatty acids (LC-PUFA) levels. Amounts of LC-PUFA depend on the exogenous intake of their precursors [linoleic (LA) and α-linolenic acid (ALA)] and by rate of their metabolism, which is influenced by activities of enzymes, such as Δ6-desaturase (D6D, FADS2), D5D, FADS1, elongases (Elovl2, -5, 6).Altered activities of D5D/D6D were described in plenty of diseases, e.g. neuropsychiatric (depressive disorders, bipolar disorder, dementia), metabolic (obesity, metabolic syndrome, DM2) and cardiovascular diseases (arterial hypertension, coronary heart disease), inflammatory states and allergy (Crohns disease, atopic eczema) or some malignancies. Similar results were obtained in studies dealing with the associations between genotypes/haplotypes of FADS1/FADS2 and above mentioned diseases, or interactions of dietary intake of LA and ALA on one hand and of the polymorphisms of minor allels of FADS1/FADS2, usually characterized by lower activities, on the other hand.The decrease of the desaturases activities leads to decreased concentrations of products with concomitant increased concentrations of substrates. Associations of some SNP FADS with coronary heart disease, concentrations of plasma lipids, oxidative stress, glucose homeostasis, and inflammatory reaction, were described. Experimental studies on animal models and occurrence of rare diseases, associated with missing or with marked fall activities of D5D/D6D emphasized the significance of desaturases for healthy development of organism as well as for pathogenesis of some disease.

Serum Fatty Acids, Desaturase Activities and Abdominal Obesity – A Population-Based Study of 60-Year Old Men and Women

PubMed Central

Alsharari, Zayed D.; Risérus, Ulf; Leander, Karin; Sjögren, Per; Carlsson, Axel C.; Vikström, Max; Laguzzi, Federica; Gigante, Bruna; Cederholm, Tommy; De Faire, Ulf; Hellénius, Mai-Lis

2017-01-01

Abdominal obesity is a key contributor of metabolic disease. Recent trials suggest that dietary fat quality affects abdominal fat content, where palmitic acid and linoleic acid influence abdominal obesity differently, while effects of n-3 polyunsaturated fatty acids are less studied. Also, fatty acid desaturation may be altered in abdominal obesity. We aimed to investigate cross-sectional associations of serum fatty acids and desaturases with abdominal obesity prevalence in a population-based cohort study. Serum cholesteryl ester fatty acids composition was measured by gas chromatography in 60-year old men (n = 1883) and women (n = 2015). Cross-sectional associations of fatty acids with abdominal obesity prevalence and anthropometric measures (e.g., sagittal abdominal diameter) were evaluated in multivariable-adjusted logistic and linear regression models, respectively. Similar models were employed to investigate relations between desaturase activities (estimated by fatty acid ratios) and abdominal obesity. In logistic regression analyses, palmitic acid, stearoyl-CoA-desaturase and Δ6-desaturase indices were associated with abdominal obesity; multivariable-adjusted odds ratios (95% confidence intervals) for highest versus lowest quartiles were 1.45 (1.19–1.76), 4.06 (3.27–5.05), and 3.07 (2.51–3.75), respectively. Linoleic acid, α-linolenic acid, docohexaenoic acid, and Δ5-desaturase were inversely associated with abdominal obesity; multivariable-adjusted odds ratios (95% confidence intervals): 0.39 (0.32–0.48), 0.74 (0.61–0.89), 0.76 (0.62–0.93), and 0.40 (0.33–0.49), respectively. Eicosapentaenoic acid was not associated with abdominal obesity. Similar results were obtained from linear regression models evaluating associations with different anthropometric measures. Sex-specific and linear associations were mainly observed for n3-polyunsaturated fatty acids, while associations of the other exposures were generally non-linear and similar across

Fatty acid desaturase 1 knockout mice are lean with improved glycemic control and decreased development of atheromatous plaque

PubMed Central

Powell, David R; Gay, Jason P; Smith, Melinda; Wilganowski, Nathaniel; Harris, Angela; Holland, Autumn; Reyes, Maricela; Kirkham, Laura; Kirkpatrick, Laura L; Zambrowicz, Brian; Hansen, Gwenn; Platt, Kenneth A; van Sligtenhorst, Isaac; Ding, Zhi-Ming; Desai, Urvi

2016-01-01

Delta-5 desaturase (D5D) and delta-6 desaturase (D6D), encoded by fatty acid desaturase 1 (FADS1) and FADS2 genes, respectively, are enzymes in the synthetic pathways for ω3, ω6, and ω9 polyunsaturated fatty acids (PUFAs). Although PUFAs appear to be involved in mammalian metabolic pathways, the physiologic effect of isolated D5D deficiency on these pathways is unclear. After generating >4,650 knockouts (KOs) of independent mouse genes and analyzing them in our high-throughput phenotypic screen, we found that Fads1 KO mice were among the leanest of 3,651 chow-fed KO lines analyzed for body composition and were among the most glucose tolerant of 2,489 high-fat-diet-fed KO lines analyzed by oral glucose tolerance test. In confirmatory studies, chow- or high-fat-diet-fed Fads1 KO mice were leaner than wild-type (WT) littermates; when data from multiple cohorts of adult mice were combined, body fat was 38% and 31% lower in Fads1 male and female KO mice, respectively. Fads1 KO mice also had lower glucose and insulin excursions during oral glucose tolerance tests along with lower fasting glucose, insulin, triglyceride, and total cholesterol levels. In additional studies using a vascular injury model, Fads1 KO mice had significantly decreased femoral artery intima/media ratios consistent with a decreased inflammatory response in their arterial wall. Based on this result, we bred Fads1 KO and WT mice onto an ApoE KO background and fed them a Western diet for 14 weeks; in this atherogenic environment, aortic trees of Fads1 KO mice had 40% less atheromatous plaque compared to WT littermates. Importantly, PUFA levels measured in brain and liver phospholipid fractions of Fads1 KO mice were consistent with decreased D5D activity and normal D6D activity. The beneficial metabolic phenotype demonstrated in Fads1 KO mice suggests that selective D5D inhibitors may be useful in the treatment of human obesity, diabetes, and atherosclerotic cardiovascular disease. PMID:27382320

Characterization of Stearoyl-CoA Desaturases from a Psychrophilic Antarctic Copepod, Tigriopus kingsejongensis.

PubMed

Jung, Woongsic; Kim, Eun Jae; Han, Se Jong; Choi, Han-Gu; Kim, Sanghee

2016-10-01

Stearoyl-CoA desaturase is a key regulator in fatty acid metabolism that catalyzes the desaturation of stearic acid to oleic acid and controls the intracellular levels of monounsaturated fatty acids (MUFAs). Two stearoyl-CoA desaturases (SCD, Δ9 desaturases) genes were identified in an Antarctic copepod, Tigriopus kingsejongensis, that was collected in a tidal pool near the King Sejong Station, King George Island, Antarctica. Full-length complementary DNA (cDNA) sequences of two T. kingsejongensis SCDs (TkSCDs) were obtained from next-generation sequencing and isolated by reverse transcription PCR. DNA sequence lengths of the open reading frames of TkSCD-1 and TkSCD-2 were determined to be 1110 and 681 bp, respectively. The molecular weights deduced from the corresponding genes were estimated to be 43.1 kDa (TkSCD-1) and 26.1 kDa (TkSCD-2). The amino acid sequences were compared with those of fatty acid desaturases and sterol desaturases from various organisms and used to analyze the relationships among TkSCDs. As assessed by heterologous expression of recombinant proteins in Escherichia coli, the enzymatic functions of both stearoyl-CoA desaturases revealed that the amount of C16:1 and C18:1 fatty acids increased by greater than 3-fold after induction with isopropyl β-D-thiogalactopyranoside. In particular, C18:1 fatty acid production increased greater than 10-fold in E. coli expressing TkSCD-1 and TkSCD-2. The results of this study suggest that both SCD genes from an Antarctic marine copepod encode a functional desaturase that is capable of increasing the amounts of palmitoleic acid and oleic acid in a prokaryotic expression system.

Differential Contribution of Endoplasmic Reticulum and Chloroplast ω-3 Fatty Acid Desaturase Genes to the Linolenic Acid Content of Olive (Olea europaea) Fruit.

PubMed

Hernández, M Luisa; Sicardo, M Dolores; Martínez-Rivas, José M

2016-01-01

Linolenic acid is a polyunsaturated fatty acid present in plant lipids, which plays key roles in plant metabolism as a structural component of storage and membrane lipids, and as a precursor of signaling molecules. The synthesis of linolenic acid is catalyzed by two different ω-3 fatty acid desaturases, which correspond to microsomal- (FAD3) and chloroplast- (FAD7 and FAD8) localized enzymes. We have investigated the specific contribution of each enzyme to the linolenic acid content in olive fruit. With that aim, we isolated two different cDNA clones encoding two ω-3 fatty acid desaturases from olive (Olea europaea cv. Picual). Sequence analysis indicates that they code for microsomal (OepFAD3B) and chloroplast (OepFAD7-2) ω-3 fatty acid desaturase enzymes, different from the previously characterized OekFAD3A and OekFAD7-1 genes. Functional expression in yeast of the corresponding OepFAD3A and OepFAD3B cDNAs confirmed that they encode microsomal ω-3 fatty acid desaturases. The linolenic acid content and transcript levels of olive FAD3 and FAD7 genes were measured in different tissues of Picual and Arbequina cultivars, including mesocarp and seed during development and ripening of olive fruit. Gene expression and lipid analysis indicate that FAD3A is the gene mainly responsible for the linolenic acid present in the seed, while FAD7-1 and FAD7-2 contribute mostly to the linolenic acid present in the mesocarp and, therefore, in the olive oil. These results also indicate the relevance of lipid trafficking between the endoplasmic reticulum and chloroplast in determining the linolenic acid content of membrane and storage lipids in oil-accumulating photosynthetic tissues. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Functional identification and regulatory analysis of Δ6-fatty acid desaturase from the oleaginous fungus Mucor sp. EIM-10.

PubMed

Jiang, Xianzhang; Liu, Hongjiao; Niu, Yongchao; Qi, Feng; Zhang, Mingliang; Huang, Jianzhong

2017-03-01

To enlarge the diversity of the desaturases associated with PUFA biosynthesis and to better understand the transcriptional regulation of desaturases, a Δ 6 -desaturase gene (Md6) from Mucor sp. and its 5'-upstream sequence was functionally identified in Saccharomyces cerevisiae. Expression of the Δ 6 -fatty acid desaturase (Md6) in S. cerevisiae showed that Md6 could convert linolenic acid to γ-linolenic acid. Computational analysis of the promoter of Md6 suggested it contains several eukaryotic fundamental transcription regulatory elements. In vivo functional analysis of the promoter showed the 5'-upstream sequence of Md6 could initiate expression of GFP and Md6 itself in S. cerevisiae. A series deletion analysis of the promoter suggested that sequence between -919 to -784 bp (relative to start site) named as eMd6 is the key factor for high activity of Δ 6 -desaturase. The activity of Δ 6 -desaturase was increased by 2.8-fold and 2.5-fold when the eMd6 sequence was placed upstream of -434 with forward or reverse orientations respectively. To our best knowledge, the native promoter of Md6 from Mucor is the strongest promoter for Δ 6 -desaturase reported so far and the sequence between -919 to -784 bp is an enhancer for Δ 6 -desaturase activity.

Plant fatty acid hydroxylases

DOEpatents

Somerville, Chris; Broun, Pierre; van de Loo, Frank

2001-01-01

This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

Regulation of yeast fatty acid desaturase in response to iron deficiency.

PubMed

Romero, Antonia María; Jordá, Tania; Rozès, Nicolas; Martínez-Pastor, María Teresa; Puig, Sergi

2018-06-01

Unsaturated fatty acids (UFA) are essential components of phospholipids that greatly contribute to the biophysical properties of cellular membranes. Biosynthesis of UFAs relies on a conserved family of iron-dependent fatty acid desaturases, whose representative in the model yeast Saccharomyces cerevisiae is Ole1. OLE1 expression is tightly regulated to adapt UFA biosynthesis and lipid bilayer properties to changes in temperature, and in UFA or oxygen availability. Despite iron deficiency being the most extended nutritional disorder worldwide, very little is known about the mechanisms and the biological relevance of fatty acid desaturases regulation in response to iron starvation. In this report, we show that endoplasmic reticulum-anchored transcription factor Mga2 activates OLE1 transcription in response to nutritional and genetic iron deficiencies. Cells lacking MGA2 display low UFA levels and do not grow under iron-limited conditions, unless UFAs are supplemented or OLE1 is overexpressed. The proteasome, E3 ubiquitin ligase Rsp5 and the Cdc48 Npl4/Ufd1 complex are required for OLE1 activation during iron depletion. Interestingly, Mga2 also activates the transcription of its own mRNA in response to iron deficiency, hypoxia, low temperature and low UFAs. MGA2 up-regulation contributes to increase OLE1 expression in these situations. These results reveal the mechanism of OLE1 regulation when iron is scarce and identify the MGA2 auto-regulation as a potential activation strategy in multiple stresses. Copyright © 2018 Elsevier B.V. All rights reserved.

[The isozymes of stearil-coenzymeA-desaturase and insulin activity in the light of phylogenetic theory of pathology. Oleic fatty acid and realization of biologic functions of trophology and locomotion].

PubMed

2013-11-01

The formation of function of isozymes of stearil-coenzymeA-desaturases occured at the different stages of phylogeny under realization of biologic function of trophology (stearil-coenzymeA-desaturase 1) and biologic function of locomotion, insulin system (stearil-coenzymeA-desaturase 2) billions years later. The stearil-coenzymeA-desaturase 1 transforms in C 18:1 oleic fatty acid only exogenous C 16:0 palmitinic saturated fatty acid. The stearil-coenzymeA-desaturase 2 transforms only endogenic palmitinic saturated fatty acid, synthesized form glucose. The biologic role of insulin is in energy support of biologic function of locomotion. Insulin through expressing stearil-coenzymeA-desaturase 2 transforms energetically non-optimal palmitinic variation of metabolism of substrates into highly effective oleic variation for cells' groundwork of energy (saturated fatty acid and mono fatty acid). The surplus of palmitinic saturated fatty acid in food is enabled in pathogenesis of resistance to insulin and derangement of synthesis of hormone by beta-cells of islets. The resistance to insulin and diabetes mellitus are primarily the derangement of metabolism of saturated fatty acids with mono fatty acids, energy problems of organism and only afterwards the derangement of metabolism of carbohydrates. It is desirable to restrict food intake of exogenous palmitinic saturated fatty acid. The reasons are low expression of independent of insulin stearil-coenzymeA-desaturase 2, marked lipotoxicity of polar form of palmitinic saturated fatty acid and synthesis of non-optimal palmitinic triglycerides instead of physiologic and more energetically more effective oleic triglycerides.

Homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerant Pseudomonas sp. AMS8

PubMed Central

Garba, Lawal; Mohamad Yussoff, Mohamad Ariff; Abd Halim, Khairul Bariyyah; Ishak, Siti Nor Hasmah; Mohamad Ali, Mohd Shukuri; Oslan, Siti Nurbaya

2018-01-01

Membrane-bound fatty acid desaturases perform oxygenated desaturation reactions to insert double bonds within fatty acyl chains in regioselective and stereoselective manners. The Δ9-fatty acid desaturase strictly creates the first double bond between C9 and 10 positions of most saturated substrates. As the three-dimensional structures of the bacterial membrane fatty acid desaturases are not available, relevant information about the enzymes are derived from their amino acid sequences, site-directed mutagenesis and domain swapping in similar membrane-bound desaturases. The cold-tolerant Pseudomonas sp. AMS8 was found to produce high amount of monounsaturated fatty acids at low temperature. Subsequently, an active Δ9-fatty acid desaturase was isolated and functionally expressed in Escherichia coli. In this paper we report homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerant Pseudomonas sp. AMS8 for the first time to the best of our knowledge. Three dimensional structure of the enzyme was built using MODELLER version 9.18 using a suitable template. The protein model contained the three conserved-histidine residues typical for all membrane-bound desaturase catalytic activity. The structure was subjected to energy minimization and checked for correctness using Ramachandran plots and ERRAT, which showed a good quality model of 91.6 and 65.0%, respectively. The protein model was used to preform MD simulation and docking of palmitic acid using CHARMM36 force field in GROMACS Version 5 and Autodock tool Version 4.2, respectively. The docking simulation with the lowest binding energy, −6.8 kcal/mol had a number of residues in close contact with the docked palmitic acid namely, Ile26, Tyr95, Val179, Gly180, Pro64, Glu203, His34, His206, His71, Arg182, Thr85, Lys98 and His177. Interestingly, among the binding residues are His34, His71 and His206 from the first, second, and third conserved histidine motif, respectively, which constitute

Expression and evolution of delta9 and delta11 desaturase genes in the moth Spodoptera littoralis.

PubMed

Rodríguez, Sergio; Hao, Guixia; Liu, Weitian; Piña, Benjamín; Rooney, Alejandro P; Camps, Francisco; Roelofs, Wendell L; Fabriàs, Gemma

2004-12-01

Desaturation of fatty acids is a key reaction in the biosynthesis of moth sex pheromones. The main component of Spodoptera littoralis sex pheromone blend is produced by the action of Delta11 and Delta9 desaturases. In this article, we report on the cloning of four desaturase-like genes in this species: one from the fat body (Sls-FL1) and three (Sls-FL2, Sls-FL3 and Sls-FL4) from the pheromone gland. By means of a computational/phylogenetic method, as well as functional assays, the desaturase gene products have been characterized. The fat body gene expressed a Delta9 desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1:4.5) ratio, whereas the pheromone gland Sls-FL2 expressed a Delta9 desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1.5:1) ratio. Although both Delta9 desaturases produced (Z)-9-tetradecenoic acid from myristic acid, transformed yeast grown in the presence of a mixture of myristic and (E)-11-tetradecenoic acids produced (Z,E)-9,11-tetradecadienoic acid, but not (Z)-9-tetradecenoic acid. The Sls-FL3 gene expressed a protein that produced a mixture of (E)-11-tetradecenoic, (Z)-11-tetradecenoic, (Z)-11-hexadecenoic and (Z)-11-octadecenoic acids in a 5:4:60:31 ratio. Despite having all the characteristics of a desaturase gene, no function could be found for Sls-FL4.

Plasma lipid fatty acid composition, desaturase activities and insulin sensitivity in Amerindian women.

PubMed

Vessby, B; Ahrén, B; Warensjö, E; Lindgärde, F

2012-03-01

Two Amerindian populations--Shuar women living in the Amazonian rain forest under traditional conditions and urbanized women in a suburb of Lima were studied. The fatty acid composition in plasma lipids and the relationships between fatty acid composition and metabolic variables were studied, as well as in a reference group of Swedish women. Fasting plasma was used for analyses of glucose, insulin, leptin and fatty acid composition. Women in Lima had more body fat, higher fasting insulin and leptin and lower insulin sensitivity than the Shuar women, who had insulin sensitivity similar to Swedish women. Shuar women had very high proportions (mean; SD) of palmitoleic (13.2; 3.9%) and oleic (33.9; 3.7%) acids in the plasma cholesteryl esters with very low levels of linoleic acid (29.1; 6.1 3%), as expected on a low fat, high carbohydrate diet. The estimated activity of delta 9 (SCD-1) desaturase was about twice as high in the Shuar compared with Lima women, suggesting neo lipogenesis, while the delta 5 desaturase activity did not differ. The Lima women, as well as the Swedish, showed strong positive correlations between SCD-1 activity on the one hand and fasting insulin and HOMA index on the other. These associations were absent in the Shuar women. The high SCD-1 activity in the Shuar women may reflect increased lipogenesis in adipose tissue. It also illustrates how a low fat diet rich in non-refined carbohydrates can be linked to a good metabolic situation. Copyright © 2010. Published by Elsevier B.V.

Cardiometabolic risk factors are influenced by Stearoyl-CoA Desaturase (SCD) -1 gene polymorphisms and n-3 polyunsaturated fatty acid supplementation.

PubMed

Rudkowska, Iwona; Julien, Pierre; Couture, Patrick; Lemieux, Simone; Tchernof, André; Barbier, Olivier; Vohl, Marie-Claude

2014-05-01

To determine if single nucleotide polymorphisms (SNPs) in stearoyl-CoA desaturase (SCD)-1 gene that encodes a key enzyme for fatty acid metabolism are associated with the response of cardiometabolic risk factors to n-3 PUFA supplementation. Two hundred and ten subjects completed a 2-week run-in period followed by 6-week supplementation with 5 g of fish oil (1.9-2.2 g eicosapentaenoic acid and 1.1 g docosahexaenoic acid). Risk factors were measured pre and post n-3 supplementation. Fatty acid composition of plasma phospholipids was analyzed by GC and the desaturase indices SCD16 (16:1n-7/16:0) and SCD18 (18:1n-9/18:0) were calculated. Genotyping of eight SNPs of the SCD1 gene was performed. N-3 PUFA supplementation decreased plasma triglycerides, as well as SCD16 and SCD18 indices, but increased fasting plasma glucose concentrations. SNPs in SCD1-modified cardiometabolic risk factors pre and post n-3 PUFA supplementation: triglyceride (rs508384, p = 0.0086), IL6 (rs3071, p = 0.0485), C-reactive protein (rs3829160, p = 0.0489), and SCD18 indices (rs2234970, p = 0.0337). A significant interaction effect between the SNP and n-3 PUFA supplementation was also observed for fasting plasma glucose levels (rs508384, p = 0.0262). These results suggest that cardiometabolic risk factors are modulated by genetic variations in the SCD1 gene alone or in combination with n-3 PUFA supplementation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Map-based cloning of a gene controlling Omega-3 fatty acid desaturation in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arondel, V.; Lemieux, B.; Hwang, I.

1992-11-20

A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase. 24 refs., 2 figs., 1more » tabs.« less

Genetic variation of six desaturase genes in flax and their impact on fatty acid composition.

PubMed

Thambugala, Dinushika; Duguid, Scott; Loewen, Evelyn; Rowland, Gordon; Booker, Helen; You, Frank M; Cloutier, Sylvie

2013-10-01

Flax (Linum usitatissimum L.) is one of the richest plant sources of omega-3 fatty acids praised for their health benefits. In this study, the extent of the genetic variability of genes encoding stearoyl-ACP desaturase (SAD), and fatty acid desaturase 2 (FAD2) and 3 (FAD3) was determined by sequencing the six paralogous genes from 120 flax accessions representing a broad range of germplasm including some EMS mutant lines. A total of 6 alleles for sad1 and sad2, 21 for fad2a, 5 for fad2b, 15 for fad3a and 18 for fad3b were identified. Deduced amino acid sequences of the alleles predicted 4, 2, 3, 4, 6 and 7 isoforms, respectively. Allele frequencies varied greatly across genes. Fad3a, with 110 SNPs and 19 indels, and fad3b, with 50 SNPs and 5 indels, showed the highest levels of genetic variations. While most of the SNPs and all the indels were silent mutations, both genes carried nonsense SNP mutations resulting in premature stop codons, a feature not observed in sad and fad2 genes. Some alleles and isoforms discovered in induced mutant lines were absent in the natural germplasm. Correlation of these genotypic data with fatty acid composition data of 120 flax accessions phenotyped in six field experiments revealed statistically significant effects of some of the SAD and FAD isoforms on fatty acid composition, oil content and iodine value. The novel allelic variants and isoforms identified for the six desaturases will be a resource for the development of oilseed flax with unique and useful fatty acid profiles.

Sex-related differences in the enhancing effects of perfluoro-octanoic acid on stearoyl-CoA desaturase and its influence on the acyl composition of phospholipid in rat liver. Comparison with clofibric acid and tiadenol.

PubMed Central

Kawashima, Y; Uy-Yu, N; Kozuka, H

1989-01-01

The effects of the peroxisome proliferators clofibric acid (p-chlorophenoxyisobutyric acid), tiadenol [2,2'-(decamethylenedithio)diethanol] and perfluoro-octanoic acid (PFOA) on hepatic stearoyl-CoA desaturation in male and female rats were compared. Treatment of male rats with the three peroxisome proliferators increased markedly the activity of stearoyl-CoA desaturase. Administration of clofibric acid or tiadenol to female rats increased greatly the hepatic activity of stearoyl-CoA desaturase, the extent of the increases being slightly less pronounced than those of male rats. In contrast with the other two peroxisome proliferators, however, PFOA did not change the activity of stearoyl-CoA desaturase in female rats. Hormonal manipulations revealed that this sex-related difference in the effect of PFOA on stearoyl-CoA desaturase activity is strongly dependent on testosterone. The increase in stearoyl-CoA desaturase activity by peroxisome proliferators was not accompanied by any notable increases in the microsomal content of cytochrome b5 or the activity of NADH: cytochrome b5 reductase. The administration of the peroxisome proliferators greatly altered the acyl composition of hepatic phosphatidylcholine and phosphatidylethanolamine (namely the proportions of C18:1 and C20:3,n-9 fatty acids increased in both phospholipids), and the alterations were partially associated with the increase in stearoyl-CoA desaturase activity. PMID:2574572

Biosynthesis of Polyunsaturated Fatty Acids in Octopus vulgaris: Molecular Cloning and Functional Characterisation of a Stearoyl-CoA Desaturase and an Elongation of Very Long-Chain Fatty Acid 4 Protein

PubMed Central

Monroig, Óscar; de Llanos, Rosa; Varó, Inmaculada; Hontoria, Francisco; Tocher, Douglas R.; Puig, Sergi; Navarro, Juan C.

2017-01-01

Polyunsaturated fatty acids (PUFAs) have been acknowledged as essential nutrients for cephalopods but the specific PUFAs that satisfy the physiological requirements are unknown. To expand our previous investigations on characterisation of desaturases and elongases involved in the biosynthesis of PUFAs and hence determine the dietary PUFA requirements in cephalopods, this study aimed to investigate the roles that a stearoyl-CoA desaturase (Scd) and an elongation of very long-chain fatty acid 4 (Elovl4) protein play in the biosynthesis of essential fatty acids (FAs). Our results confirmed the Octopus vulgaris Scd is a ∆9 desaturase with relatively high affinity towards saturated FAs with ≥ C18 chain lengths. Scd was unable to desaturate 20:1n-15 (∆520:1) suggesting that its role in the biosynthesis of non-methylene interrupted FAs (NMI FAs) is limited to the introduction of the first unsaturation at ∆9 position. Interestingly, the previously characterised ∆5 fatty acyl desaturase was indeed able to convert 20:1n-9 (∆1120:1) to ∆5,1120:2, an NMI FA previously detected in octopus nephridium. Additionally, Elovl4 was able to mediate the production of 24:5n-3 and thus can contribute to docosahexaenoic acid (DHA) biosynthesis through the Sprecher pathway. Moreover, the octopus Elovl4 was confirmed to play a key role in the biosynthesis of very long-chain (>C24) PUFAs. PMID:28335553

Biosynthesis of Polyunsaturated Fatty Acids in Octopus vulgaris: Molecular Cloning and Functional Characterisation of a Stearoyl-CoA Desaturase and an Elongation of Very Long-Chain Fatty Acid 4 Protein.

PubMed

Monroig, Óscar; de Llanos, Rosa; Varó, Inmaculada; Hontoria, Francisco; Tocher, Douglas R; Puig, Sergi; Navarro, Juan C

2017-03-21

Polyunsaturated fatty acids (PUFAs) have been acknowledged as essential nutrients for cephalopods but the specific PUFAs that satisfy the physiological requirements are unknown. To expand our previous investigations on characterisation of desaturases and elongases involved in the biosynthesis of PUFAs and hence determine the dietary PUFA requirements in cephalopods, this study aimed to investigate the roles that a stearoyl-CoA desaturase (Scd) and an elongation of very long-chain fatty acid 4 (Elovl4) protein play in the biosynthesis of essential fatty acids (FAs). Our results confirmed the Octopus vulgaris Scd is a ∆9 desaturase with relatively high affinity towards saturated FAs with ≥ C 18 chain lengths. Scd was unable to desaturate 20:1 n- 15 ( ∆5 20:1) suggesting that its role in the biosynthesis of non-methylene interrupted FAs (NMI FAs) is limited to the introduction of the first unsaturation at ∆9 position. Interestingly, the previously characterised ∆5 fatty acyl desaturase was indeed able to convert 20:1 n- 9 ( ∆11 20:1) to ∆5,11 20:2, an NMI FA previously detected in octopus nephridium. Additionally, Elovl4 was able to mediate the production of 24:5 n- 3 and thus can contribute to docosahexaenoic acid (DHA) biosynthesis through the Sprecher pathway. Moreover, the octopus Elovl4 was confirmed to play a key role in the biosynthesis of very long-chain (>C 24 ) PUFAs.

Molecular cloning and sequence analysis of stearoyl-CoA desaturase in milkfish, Chanos chanos.

PubMed

Hsieh, S L; Liao, W L; Kuo, C M

2001-12-01

Stearoyl-CoA desaturase (EC 1.14.99.5) is a key enzyme in the biosynthesis of polyunsaturated fatty acids and the maintenance of the homeoviscous fluidity of biological membranes. The stearoyl-CoA desaturase cDNA in milkfish (Chanos chanos) was cloned by RT-PCR and RACE, and it was compared with the stearoyl-CoA desaturase in cold-tolerant teleosts, common carp and grass carp. Nucleotide sequence analysis revealed that the cDNA clone has a 972-bp open reading frame encoding 323 amino acid residues. Alignments of the deduced amino acid sequence showed that the milkfish stearoyl-CoA desaturase shares 79% and 75% identity with common carp and grass carp, and 63%-64% with other vertebrates such as sheep, hamsters, rats, mice, and humans. Like common carp and grass carp, the deduced amino acid sequence in milkfish well conserves three histidine cluster motifs (one HXXXXH and two HXXHH) that are essential for catalysis of stearoyl-CoA desaturase activity. However, RT-PCR analysis showed that stearoyl-CoA desaturase expression in milkfish is detected in the tissues of liver, muscle, kidney, brain, and gill, and more expression sites were found in milkfish than in common carp and grass carp. Phylogenic relationships among the deduced stearoyl-CoA desaturase amino acid sequence in milkfish and those in other vertebrates showed that the milkfish stearoyl-CoA desaturase amino acid sequence is phylogenetically closer to those of common carp and grass carp than to other higher vertebrates.

Production of hydroxylated fatty acids in genetically modified plants

DOEpatents

Somerville, Chris [Portola Valley, CA; Broun, Pierre [Burlingame, CA; van de Loo, Frank [Weston, AU; Boddupalli, Sekhar S [Manchester, MI

2011-08-23

This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

Production of hydroxylated fatty acids in genetically modified plants

DOEpatents

Somerville, Chris; Broun, Pierre; van de Loo, Frank; Boddupalli, Sekhar S.

2005-08-30

This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

Functional analysis of the omega-6 fatty acid desaturase (CaFAD2) gene family of the oil seed crop Crambe abyssinica

PubMed Central

2013-01-01

Background Crambe abyssinica produces high erucic acid (C22:1, 55-60%) in the seed oil, which can be further increased by reduction of polyunsaturated fatty acid (PUFA) levels. The omega-6 fatty acid desaturase enzyme (FAD2) is known to be involved in PUFA biosynthesis. In crambe, three CaFAD2 genes, CaFAD2-C1, CaFAD2-C2 and CaFAD2-C3 are expressed. Results The individual effect of each CaFAD2 gene on oil composition was investigated through studying transgenic lines (CaFAD2-RNAi) for differential expression levels in relation to the composition of seed-oil. Six first generation transgenic plants (T1) showed C18:1 increase (by 6% to 10.5%) and PUFA reduction (by 8.6% to 10.2%). The silencing effect in these T1-plants ranged from the moderate silencing (40% to 50% reduction) of all three CaFAD2 genes to strong silencing (95% reduction) of CaFAD2-C3 alone. The progeny of two T1-plants (WG4-4 and WG19-6) was further analysed. Four or five transgene insertions are characterized in the progeny (T2) of WG19-6 in contrast to a single insertion in the T2 progeny of WG4-4. For the individual T2-plants of both families (WG19-6 and WG4-4), seed-specific silencing of CaFAD2-C1 and CaFAD2-C2 was observed in several individual T2-plants but, on average in both families, the level of silencing of these genes was not significant. A significant reduction in expression level (P < 0.01) in both families was only observed for CaFAD2-C3 together with significantly different C18:1 and PUFA levels in oil. Conclusions CaFAD2-C3 expression is highly correlated to levels of C18:1 (r = -0.78) and PUFA (r = 0.75), which suggests that CaFAD2-C3 is the most important one for changing the oil composition of crambe. PMID:24083776

Pomegranate seed oil influences the fatty acids profile and reduces the activity of desaturases in livers of Sprague-Dawley rats.

PubMed

Białek, Agnieszka; Stawarska, Agnieszka; Bodecka, Joanna; Białek, Małgorzata; Tokarz, Andrzej

2017-07-01

The aim of our study was to compare the influence of diet supplementation with pomegranate seed oil - as conjugated linolenic acids (CLnA) source, or conjugated linoleic acids (CLA) and to examine the mechanism of their activity. The content of fatty acids, levels of biomarkers of lipids' oxidation and the activity of key enzymes catalyzing lipids metabolism were measured. Obtained results revealed that conjugated fatty acids significantly decrease the activity of Δ5-desaturase (p=0.0001) and Δ6-desaturase (p=0.0008) and pomegranate seed oil reduces their activity in the most potent way. We confirmed that diet supplementation with pomegranate seed oil - a rich source of punicic acid leads to the increase of cis-9, trans-11 CLA content in livers (p=0.0003). Lack of side effects and beneficial influence on desaturases activity and fatty acids profile claim pomegranate seed oil to become interesting alternative for CLA as functional food. Copyright © 2017 Elsevier Inc. All rights reserved.

Stearoyl-CoA desaturase expression and fatty acid composition in milkfish (Chanos chanos) and grass carp (Ctenopharyngodon idella) during cold acclimation.

PubMed

Hsieh, S L; Kuo, C-M

2005-05-01

Desaturation of fatty acids is an important adaptation mechanism for fish to maintain membrane fluidity under thermal stress. To comprehend the temperature adaptation mechanism in fish, we investigated the difference in the changes of stearoyl-CoA desaturase expression and fatty acid composition between milkfish and grass carp under cold acclimation. We find that in both fish the proportions of unsaturated fatty acids at 15 degrees C are all higher than those at 25 degrees C. In milkfish Delta(9)-desaturation index (ratios of 16:1/16:0 and 18:1/18:0) increases significantly in the beginning of cold acclimation at 15 degrees C and decreases afterward, but in grass carp it increases slightly in the beginning of cold acclimation followed by a sustained dramatic increase. Similarly, activity of stearoyl-CoA desaturase in milkfish increases significantly in the beginning, peaks at day 4, and then decreases constantly, but in grass carp it increases gradually in the first week, rises dramatically afterward, and then maintains a very high level. The change of stearoyl-CoA desaturase activity is parallel to the change of Delta(9)-desaturation index in both milkfish and grass carp, but it is one day earlier than Delta(9)-desaturation index in milkfish. The difference of adaptation capability between milkfish and grass carp under cold stress is further evidenced by RT-PCR and Northern blot analysis of stearoyl-CoA desaturase gene expression.

Associations between a fatty acid desaturase gene polymorphism and blood arachidonic acid compositions in Japanese elderly.

PubMed

Horiguchi, Sayaka; Nakayama, Kazuhiro; Iwamoto, Sadahiko; Ishijima, Akiko; Minezaki, Takayuki; Baba, Mamiko; Kontai, Yoshiko; Horikawa, Chika; Kawashima, Hiroshi; Shibata, Hiroshi; Kagawa, Yasuo; Kawabata, Terue

2016-02-01

We investigated whether the single nucleotide polymorphism rs174547 (T/C) of the fatty acid desaturase-1 gene, FADS1, is associated with changes in erythrocyte membrane and plasma phospholipid (PL) long-chain polyunsaturated fatty acid (LCPUFA) composition in elderly Japanese participants (n=124; 65 years or older; self-feeding and oral intake). The rs174547 C-allele carriers had significantly lower arachidonic acid (ARA; n-6 PUFA) and higher linoleic acid (LA, n-6 PUFA precursor) levels in erythrocyte membrane and plasma PL (15% and 6% ARA reduction, respectively, per C-allele), suggesting a low LA to ARA conversion rate in erythrocyte membrane and plasma PL of C-allele carriers. α-linolenic acid (n-3 PUFA precursor) levels were higher in the plasma PL of C-allele carriers, whereas levels of the n-3 LCPUFAs eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) were unchanged in erythrocyte membrane and plasma PL. Thus, rs174547 genotypes were significantly associated with different ARA compositions of the blood of elderly Japanese. Copyright © 2015 Elsevier Ltd. All rights reserved.

Method for production of petroselinic acid and OMEGA12 hexadecanoic acid in transgenic plants

DOEpatents

Ohlrogge, John B.; Cahoon, Edgar B.; Shanklin, John; Somerville, Christopher R.

1995-01-01

The present invention relates to a process for producing lipids containing the fatty acid petroselinic acid in plants. The production of petroselinic acid is accomplished by genetically transforming plants which do not normally accumulate petroselinic acid with a gene for a .omega.12 desaturase from another species which does normally accumulate petroselinic acid.

Temporal and tissue-specific regulation of a Brassica napus stearoyl-acyl carrier protein desaturase gene.

PubMed Central

Slocombe, S P; Piffanelli, P; Fairbairn, D; Bowra, S; Hatzopoulos, P; Tsiantis, M; Murphy, D J

1994-01-01

The nucleotide sequence of a Brassica napus stearoyl-acyl carrier protein desaturase gene (Bn10) is presented. This gene is one member of a family of four closely related genes expressed in oilseed rape. The expression of the promoter of this gene in transgenic tobacco was found to be temporally regulated in the developing seed tissues. However, the promoter was also particularly active in other oleogenic tissues such as the tapetum and pollen grains. This raises the interesting question of whether seed-expressed lipid synthesis genes are regulated by separate tissue-specific determinants or by a single factor common to all oleogenic tissues. Parts of the plants undergoing rapid development such as the components of immature flowers and seedlings also exhibited high levels of promoter activity. These tissues are likely to have an elevated requirement for membrane lipid synthesis. Stearoyl-acyl carrier protein desaturase transcript levels have previously been shown to be temporally regulated in the B. napus embryo (S.P. Slocombe, I. Cummins, R.P. Jarvis, D.J. Murphy [1992] Plant Mol Biol 20: 151-155). Evidence is presented demonstrating the induction of desaturase mRNA by abscisic acid in the embryo. PMID:8016261

Method for production of petroselinic acid and OMEGA12 hexadecanoic acid in transgenic plants

DOEpatents

Ohlrogge, J.B.; Cahoon, E.B.; Shanklin, J.; Somerville, C.R.

1995-07-04

The present invention relates to a process for producing lipids containing the fatty acid, petroselinic acid, in plants. The production of petroselinic acid is accomplished by genetically transforming plants which do not normally accumulate petroselinic acid with a gene for a {omega}12 desaturase from another species which does normally accumulate petroselinic acid. 19 figs.

Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans

PubMed Central

Hulver, Matthew W.; Berggren, Jason R.; Carper, Michael J.; Miyazaki, Makoto; Ntambi, James M.; Hoffman, Eric P.; Thyfault, John P.; Stevens, Robert; Dohm, G. Lynis; Houmard, Joseph A.; Muoio, Deborah M.

2014-01-01

Summary Obesity and type 2 diabetes are strongly associated with abnormal lipid metabolism and accumulation of intramyocellular triacylglycerol, but the underlying cause of these perturbations are yet unknown. Herein, we show that the lipogenic gene, stearoyl-CoA desaturase 1 (SCD1), is robustly up-regulated in skeletal muscle from extremely obese humans. High expression and activity of SCD1, an enzyme that catalyzes the synthesis of monounsaturated fatty acids, corresponded with low rates of fatty acid oxidation, increased triacylglycerol synthesis and increased monounsaturation of muscle lipids. Elevated SCD1 expression and abnormal lipid partitioning were retained in primary skeletal myocytes derived from obese compared to lean donors, implying that these traits might be driven by epigenetic and/or heritable mechanisms. Overexpression of human SCD1 in myotubes from lean subjects was sufficient to mimic the obese phenotype. These results suggest that elevated expression of SCD1 in skeletal muscle contributes to abnormal lipid metabolism and progression of obesity. PMID:16213227

Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans.

PubMed

Hulver, Matthew W; Berggren, Jason R; Carper, Michael J; Miyazaki, Makoto; Ntambi, James M; Hoffman, Eric P; Thyfault, John P; Stevens, Robert; Dohm, G Lynis; Houmard, Joseph A; Muoio, Deborah M

2005-10-01

Obesity and type 2 diabetes are strongly associated with abnormal lipid metabolism and accumulation of intramyocellular triacylglycerol, but the underlying cause of these perturbations are yet unknown. Herein, we show that the lipogenic gene, stearoyl-CoA desaturase 1 (SCD1), is robustly up-regulated in skeletal muscle from extremely obese humans. High expression and activity of SCD1, an enzyme that catalyzes the synthesis of monounsaturated fatty acids, corresponded with low rates of fatty acid oxidation, increased triacylglycerol synthesis and increased monounsaturation of muscle lipids. Elevated SCD1 expression and abnormal lipid partitioning were retained in primary skeletal myocytes derived from obese compared to lean donors, implying that these traits might be driven by epigenetic and/or heritable mechanisms. Overexpression of human SCD1 in myotubes from lean subjects was sufficient to mimic the obese phenotype. These results suggest that elevated expression of SCD1 in skeletal muscle contributes to abnormal lipid metabolism and progression of obesity.

Amino Acids Regulate Transgene Expression in MDCK Cells

PubMed Central

Torrente, Marta; Guetg, Adriano; Sass, Jörn Oliver; Arps, Lisa; Ruckstuhl, Lisa; Camargo, Simone M. R.; Verrey, François

2014-01-01

Gene expression and cell growth rely on the intracellular concentration of amino acids, which in metazoans depends on extracellular amino acid availability and transmembrane transport. To investigate the impact of extracellular amino acid concentrations on the expression of a concentrative amino acid transporter, we overexpressed the main kidney proximal tubule luminal neutral amino acid transporter B0AT1-collectrin (SLC6A19-TMEM27) in MDCK cell epithelia. Exogenously expressed proteins co-localized at the luminal membrane and mediated neutral amino acid uptake. However, the transgenes were lost over few cell culture passages. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2α phosphorylation may trigger transgene derepression. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes possibly via stimulation of the GCN2 pathway. PMID:24797296

Production of Fatty Acid Components of Meadowfoam Oil in Somatic Soybean Embryos

PubMed Central

Cahoon, Edgar B.; Marillia, Elizabeth-France; Stecca, Kevin L.; Hall, Sarah E.; Taylor, David C.; Kinney, Anthony J.

2000-01-01

The seed oil of meadowfoam (Limnanthes alba) and other Limnanthes spp. is enriched in the unusual fatty acid Δ5-eicosenoic acid (20:1Δ5). This fatty acid has physical and chemical properties that make the seed oil of these plants useful for a number of industrial applications. An expressed sequence tag approach was used to identify cDNAs for enzymes involved in the biosynthesis of 20:1Δ5). By random sequencing of a library prepared from developing Limnanthes douglasii seeds, a class of cDNAs was identified that encode a homolog of acyl-coenzyme A (CoA) desaturases found in animals, fungi, and cyanobacteria. Expression of a cDNA for the L. douglasii acyl-CoA desaturase homolog in somatic soybean (Glycine max) embryos behind a strong seed-specific promoter resulted in the accumulation of Δ5-hexadecenoic acid to amounts of 2% to 3% (w/w) of the total fatty acids of single embryos. Δ5-Octadecenoic acid and 20:1Δ5 also composed <1% (w/w) each of the total fatty acids of these embryos. In addition, cDNAs were identified from the L. douglasii expressed sequence tags that encode a homolog of fatty acid elongase 1 (FAE1), a β-ketoacyl-CoA synthase that catalyzes the initial step of very long-chain fatty acid synthesis. Expression of the L. douglassi FAE1 homolog in somatic soybean embryos was accompanied by the accumulation of C20 and C22 fatty acids, principally as eicosanoic acid, to amounts of 18% (w/w) of the total fatty acids of single embryos. To partially reconstruct the biosynthetic pathway of 20:1Δ5 in transgenic plant tissues, cDNAs for the L. douglasii acyl-CoA desaturase and FAE1 were co-expressed in somatic soybean embryos. In the resulting transgenic embryos, 20:1Δ5 and Δ5-docosenoic acid composed up to 12% of the total fatty acids. PMID:10982439

Production of fatty acid components of meadowfoam oil in somatic soybean embryos.

PubMed

Cahoon, E B; Marillia, E F; Stecca, K L; Hall, S E; Taylor, D C; Kinney, A J

2000-09-01

The seed oil of meadowfoam (Limnanthes alba) and other Limnanthes spp. is enriched in the unusual fatty acid Delta(5)-eicosenoic acid (20:1Delta(5)). This fatty acid has physical and chemical properties that make the seed oil of these plants useful for a number of industrial applications. An expressed sequence tag approach was used to identify cDNAs for enzymes involved in the biosynthesis of 20:1Delta(5)). By random sequencing of a library prepared from developing Limnanthes douglasii seeds, a class of cDNAs was identified that encode a homolog of acyl-coenzyme A (CoA) desaturases found in animals, fungi, and cyanobacteria. Expression of a cDNA for the L. douglasii acyl-CoA desaturase homolog in somatic soybean (Glycine max) embryos behind a strong seed-specific promoter resulted in the accumulation of Delta(5)-hexadecenoic acid to amounts of 2% to 3% (w/w) of the total fatty acids of single embryos. Delta(5)-Octadecenoic acid and 20:1Delta(5) also composed <1% (w/w) each of the total fatty acids of these embryos. In addition, cDNAs were identified from the L. douglasii expressed sequence tags that encode a homolog of fatty acid elongase 1 (FAE1), a beta-ketoacyl-CoA synthase that catalyzes the initial step of very long-chain fatty acid synthesis. Expression of the L. douglassi FAE1 homolog in somatic soybean embryos was accompanied by the accumulation of C(20) and C(22) fatty acids, principally as eicosanoic acid, to amounts of 18% (w/w) of the total fatty acids of single embryos. To partially reconstruct the biosynthetic pathway of 20:1Delta(5) in transgenic plant tissues, cDNAs for the L. douglasii acyl-CoA desaturase and FAE1 were co-expressed in somatic soybean embryos. In the resulting transgenic embryos, 20:1Delta(5) and Delta(5)-docosenoic acid composed up to 12% of the total fatty acids.

Associations between plasma fatty acids, desaturase and elongase, and insulin resistance in children

USDA-ARS?s Scientific Manuscript database

Background and Objectives - Fatty acid profiles, desaturase (SCD-16, SCD018, D5D, D6D) and elongase (ELOVL6) enzyme activity have been associated with adiposity and metabolic disease. While this has been studied in adults, few studies have evaluated children. The objective of this study was to evalu...

Fatty acid desaturase gene variants, cardiovascular risk factors, and myocardial infarction in the costa rica study

USDA-ARS?s Scientific Manuscript database

Genetic variation in fatty acid desaturases (FADS) has previously been linked to long-chain polyunsaturated fatty acids (PUFAs) in adipose tissue and cardiovascular risk. The goal of our study was to test associations between six common FADS polymorphisms (rs174556, rs3834458, rs174570, rs2524299, r...

Amino Acid Change in an Orchid Desaturase Enables Mimicry of the Pollinator's Sex Pheromone.

PubMed

Sedeek, Khalid E M; Whittle, Edward; Guthörl, Daniela; Grossniklaus, Ueli; Shanklin, John; Schlüter, Philipp M

2016-06-06

Mimicry illustrates the power of selection to produce phenotypic convergence in biology [1]. A striking example is the imitation of female insects by plants that are pollinated by sexual deception of males of the same insect species [2-4]. This involves mimicry of visual, tactile, and chemical signals of females [2-7], especially their sex pheromones [8-11]. The Mediterranean orchid Ophrys exaltata employs chemical mimicry of cuticular hydrocarbons, particularly the 7-alkenes, in an insect sex pheromone to attract and elicit mating behavior in its pollinators, males of the cellophane bee Colletes cunicularius [11-13]. A difference in alkene double-bond positions is responsible for reproductive isolation between O. exaltata and closely related species, such as O. sphegodes [13-16]. We show that these 7-alkenes are likely determined by the action of the stearoyl-acyl-carrier-protein desaturase (SAD) homolog SAD5. After gene duplication, changes in subcellular localization relative to the ancestral housekeeping desaturase may have allowed proto-SAD5's reaction products to undergo further biosynthesis to both 7- and 9-alkenes. Such ancestral coproduction of two alkene classes may have led to pollinator-mediated deleterious pleiotropy. Despite possible evolutionary intermediates with reduced activity, amino acid changes at the bottom of the substrate-binding cavity have conferred enzyme specificity for 7-alkene biosynthesis by preventing the binding of longer-chained fatty acid (FA) precursors by the enzyme. This change in desaturase function enabled the orchid to perfect its chemical mimicry of pollinator sex pheromones by escape from deleterious pleiotropy, supporting a role of pleiotropy in determining the possible trajectories of adaptive evolution. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species.

PubMed

Zheng, X; Seiliez, I; Hastings, N; Tocher, D R; Panserat, S; Dickson, C A; Bergot, P; Teale, A J

2004-10-01

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which

Rapid, transient, and highly localized induction of plastidial ω-3 fatty acid desaturase mRNA at fungal infection sites in Petroselinum crispum

PubMed Central

Kirsch, Christoph; Takamiya-Wik, Monica; Reinold, Susanne; Hahlbrock, Klaus; Somssich, Imre E.

1997-01-01

Parsley (Petroselinum crispum) plants and suspension-cultured cells have been used extensively for studies of non-host-resistance mechanisms in plant/pathogen interactions. We now show that treatment of cultured parsley cells with a defined peptide elicitor of fungal origin causes rapid and large changes in the levels of various unsaturated fatty acids. While linoleic acid decreased and linolenic acid increased steadily for several hours, comparatively sharp increases in oleic acid followed a biphasic time course. In contrast, the overall level of stearic acid remained unaffected. Using a PCR-based approach, a parsley cDNA was isolated sharing high sequence similarity with ω-3 fatty acid desaturases. Subsequent isolation and characterization of a full-length cDNA enabled its functional identification as a plastid-localized ω-3 fatty acid desaturase by complementation of the Arabidopsis thaliana fad7/8 double mutant which is low in trienoic fatty acids. ω-3 Fatty acid desaturase mRNA accumulated rapidly and transiently in elicitor-treated cultured parsley cells, protoplasts, and leaves, as well as highly localized around fungal infection sites in parsley leaf buds. These results indicate that unsaturated fatty acid metabolism is yet another component of the highly complex, transcriptionally regulated pathogen defense response in plants. PMID:9050908

Effect of seafood mediated PCB exposure on desaturase activity and PUFA profile in Faroese septuagenarians.

PubMed

Tøttenborg, Sandra Søgaard; Choi, Anna L; Bjerve, Kristian S; Weihe, Pal; Grandjean, Philippe

2015-07-01

Polychlorinated biphenyl (PCB) exposure may affect serum concentrations of polyunsaturated fatty acids (PUFAs) by inhibiting desaturases ∆5 and ∆6 that drive their synthesis from precursor fatty acids. Such changes in the composition of fatty acids may affect cardiovascular disease risk, which is thought to increase at elevated PCB exposures. This population-based cross-sectional study examined 712 Faroese men and women aged 70-74 years. The serum phospholipid fraction of fasting blood samples was used to determine the PUFA profile, including linoleic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosatrienoic acid, and other relevant fatty acids. Ratios between precursor and metabolite fatty acids were used as proxies for ∆5 and ∆6 desaturase activity. Tertiles of serum-PCB concentrations were used in multiple regression analyses to determine the association between the exposure and desaturase activity. In multiple regression models, PCB exposure was inversely related to the estimated Δ6 desaturase activity resulting in accumulation of precursor fatty acids and decrease in the corresponding product PUFAs. A positive association between PCB and Δ5 desaturation was also found. A relative increase in EA was also observed, though only in the third tertile of PCB exposure. Non-linear relationships between the exposure and the desaturase activity were not found. Consuming fish and seafood may not be translated into beneficial fatty acid profiles if the diet simultaneously causes exposure to PCBs. Although the desaturase estimates were likely influenced by dietary intakes of product PUFAs, the association between PCB exposure and ∆6 desaturase activity is plausible and may affect cardiovascular disease risk. Copyright © 2015 Elsevier Inc. All rights reserved.

Half-of-the-Sites Reactivity of the Castor Δ9-18:0-Acyl Carrier Protein Desaturase1[OPEN

PubMed Central

Liu, Qin; Chai, Jin; Moche, Martin; Guy, Jodie; Lindqvist, Ylva; Shanklin, John

2015-01-01

Fatty acid desaturases regulate the unsaturation status of cellular lipids. They comprise two distinct evolutionary lineages, a soluble class found in the plastids of higher plants and an integral membrane class found in plants, yeast (Saccharomyces cerevisiae), animals, and bacteria. Both classes exhibit a dimeric quaternary structure. Here, we test the functional significance of dimeric organization of the soluble castor Δ9-18:0-acyl carrier protein desaturase, specifically, the hypothesis that the enzyme uses an alternating subunit half-of-the-sites reactivity mechanism whereby substrate binding to one subunit is coordinated with product release from the other subunit. Using a fluorescence resonance energy transfer assay, we demonstrated that dimers stably associate at concentrations typical of desaturase assays. An active site mutant T104K/S202E, designed to occlude the substrate binding cavity, was expressed, purified, and its properties validated by x-ray crystallography, size exclusion chromatography, and activity assay. Heterodimers comprising distinctly tagged wild-type and inactive mutant subunits were purified at 1:1 stoichiometry. Despite having only one-half the number of active sites, purified heterodimers exhibit equivalent activity to wild-type homodimers, consistent with half-of-the-sites reactivity. However, because multiple rounds of turnover were observed, we conclude that substrate binding to one subunit is not required to facilitate product release from the second subunit. The observed half-of-the-sites reactivity could potentially buffer desaturase activity from oxidative inactivation. That soluble desaturases require only one active subunit per dimer for full activity represents a mechanistic difference from the membrane class of desaturases such as the Δ9-acyl-CoA, Ole1p, from yeast, which requires two catalytically competent subunits for activity. PMID:26224800

An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

PubMed Central

Murakami, Akira; Nagao, Kohjiro; Juni, Naoto; Hara, Yuji; Umeda, Masato

2017-01-01

The Δ9-fatty acid desaturase introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA and regulates the cellular levels of unsaturated fatty acids. However, it is unclear how Δ9-desaturase expression is regulated in response to changes in the levels of fatty acid desaturation. In this study, we found that the degradation of DESAT1, the sole Δ9-desaturase in the Drosophila cell line S2, was significantly enhanced when the amounts of unsaturated acyl chains of membrane phospholipids were increased by supplementation with unsaturated fatty acids, such as oleic and linoleic acids. In contrast, inhibition of DESAT1 activity remarkably suppressed its degradation. Of note, removal of the DESAT1 N-terminal domain abolished the responsiveness of DESAT1 degradation to the level of fatty acid unsaturation. Further truncation and amino acid replacement analyses revealed that two sequential prolines, the second and third residues of DESAT1, were responsible for the unsaturated fatty acid–dependent degradation. Although degradation of mouse stearoyl-CoA desaturase 1 (SCD1) was unaffected by changes in fatty acid unsaturation, introduction of the N-terminal sequential proline residues into SCD1 conferred responsiveness to unsaturated fatty acid–dependent degradation. Furthermore, we also found that the Ca2+-dependent cysteine protease calpain is involved in the sequential proline–dependent degradation of DESAT1. In light of these findings, we designated the sequential prolines at the second and third positions of DESAT1 as a “di-proline motif,” which plays a crucial role in the regulation of Δ9-desaturase expression in response to changes in the level of cellular unsaturated fatty acids. PMID:28972163

Functional desaturase Fads1 (Δ5) and Fads2 (Δ6) orthologues evolved before the origin of jawed vertebrates.

PubMed

Castro, Luís Filipe Costa; Monroig, Óscar; Leaver, Michael J; Wilson, Jonathan; Cunha, Isabel; Tocher, Douglas R

2012-01-01

Long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are essential components of biomembranes, particularly in neural tissues. Endogenous synthesis of ARA, EPA and DHA occurs from precursor dietary essential fatty acids such as linoleic and α-linolenic acid through elongation and Δ5 and Δ6 desaturations. With respect to desaturation activities some noteworthy differences have been noted in vertebrate classes. In mammals, the Δ5 activity is allocated to the Fads1 gene, while Fads2 is a Δ6 desaturase. In contrast, teleosts show distinct combinations of desaturase activities (e.g. bifunctional or separate Δ5 and Δ6 desaturases) apparently allocated to Fads2-type genes. To determine the timing of Fads1-Δ5 and Fads2-Δ6 evolution in vertebrates we used a combination of comparative and functional genomics with the analysis of key phylogenetic species. Our data show that Fads1 and Fads2 genes with Δ5 and Δ6 activities respectively, evolved before gnathostome radiation, since the catshark Scyliorhinus canicula has functional orthologues of both gene families. Consequently, the loss of Fads1 in teleosts is a secondary episode, while the existence of Δ5 activities in the same group most likely occurred through independent mutations into Fads2 type genes. Unexpectedly, we also establish that events of Fads1 gene expansion have taken place in birds and reptiles. Finally, a fourth Fads gene (Fads4) was found with an exclusive occurrence in mammalian genomes. Our findings enlighten the history of a crucially important gene family in vertebrate fatty acid metabolism and physiology and provide an explanation of how observed lineage-specific gene duplications, losses and diversifications might be linked to habitat-specific food web structures in different environments and over geological timescales.

Isolation and Characterization of the Diatom Phaeodactylum Δ5-Elongase Gene for Transgenic LC-PUFA Production in Pichia pastoris

PubMed Central

Jiang, Mulan; Guo, Bing; Wan, Xia; Gong, Yangmin; Zhang, Yinbo; Hu, Chuanjiong

2014-01-01

The diatom Phaeodactylum tricornutum can accumulate eicosapentaenoic acid (EPA) up to 30% of the total fatty acids. This species has been targeted for isolating gene encoding desaturases and elongases for long-chain polyunsaturated fatty acid (LC-PUFA) metabolic engineering. Here we first report the cloning and characterization of Δ5-elongase gene in P. tricornutum. A full-length cDNA sequence, designated PhtELO5, was shown to contain a 1110 bp open reading frame encoding a 369 amino acid polypeptide. The putative protein contains seven transmembrane regions and two elongase characteristic motifs of FLHXYHH and MYSYY, the latter being typical for microalgal Δ5-elongases. Phylogenetic analysis indicated that PhtELO5 belongs to the ELO5 group, tightly clustered with the counterpart of Thalassiosira pseudonana. Heterologous expression of PhtELO5 in Pichia pastoris confirmed that it encodes a specific Δ5-elongase capable of elongating arachidonic acid and eicosapentaenoic acid. Co-expression of PhtELO5 and IsFAD4 (a ∆4-desaturase from Isochrysis sphaerica) demonstrated that the high-efficiency biosynthetic pathway of docosahexaenoic acid was assembled in the transgenic yeast. Substrate competition revealed that PhtELO5 exhibited higher activity towards n-3 PUFA than n-6 PUFA. It is hypothesized that Phaeodactylum ELO5 may preferentially participate in biosynthesis of transgenic LC-PUFA via a n-3 pathway in the yeast host. PMID:24608969

Increasing dietary EPA and DHA influence estimated fatty acid desaturase activity in systemic organs which is reflected in the red blood cell in mice.

PubMed

Davidson, Emily A; Pickens, C Austin; Fenton, Jenifer I

2018-03-01

Delta-5 (D5D) and delta-6 (D6D) desaturase are key enzymes in fatty acid (FA) metabolism. Dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may alter tissue FA composition via D5D and D6D. The purpose was to determine the relationship between dietary EPA + DHA, estimated desaturase activities of various tissues and the reflection of desaturase activity in the red blood cell (RBC). Mice were fed diets with increasing percent of energy from EPA + DHA. Phospholipid FA composition of heart, muscle, spleen, lung, adipose tissues and RBC were analysed. D5D and D6D enzyme activity estimates (EAE) were calculated as the ratio of 20:4/20:3 and 20:3/18:2, respectively. D5D EAE decreased in all tissues, except muscle, with increasing dietary EPA + DHA. RBC D5D EAE positively correlated with D5D EAE in all tissues. RBC D6D EAE positively correlated with muscle and inversely correlated with adipose D6D EAE. Our findings suggest differential influence of dietary EPA + DHA upon tissue desaturase activities.

Desaturase and elongase-limiting endogenous long-chain polyunsaturated fatty acid biosynthesis.

PubMed

Zhang, Ji Yao; Kothapalli, Kumar S D; Brenna, J Thomas

2016-03-01

Endogenous synthesis of the long-chain polyunsaturated fatty acids (LCPUFAs) is mediated by the fatty acid desaturase (FADS) gene cluster (11q12-13.1) and elongation of very long-chain fatty acids 2 (ELOVL2) (6p24.2) and ELOVL5 (6p12.1). Although older biochemical work identified the product of one gene, FADS2, rate limiting for LCPUFA synthesis, recent studies suggest that polymorphisms in any of these genes can limit accumulation of product LCPUFA. Genome-wide association study (GWAS) of Greenland Inuit shows strong adaptation signals within FADS gene cluster, attributed to high omega-3 fatty acid intake, while GWAS found ELOVL2 associated with sleep duration, age and DNA methylation. ELOVL5 coding mutations cause spinocerebellar ataxia 38, and epigenetic marks were associated with depression and suicide risk. Two sterol response element binding sites were found on ELOVL5, a SREBP-1c target gene. Minor allele carriers of a 3 single nucleotide polymorphism (SNP) haplotype in ELOVL2 have decreased 22 : 6n-3 levels. Unequivocal molecular evidence shows mammalian FADS2 catalyzes direct Δ4-desaturation to yield 22 : 6n-3 and 22 : 5n-6. An SNP near FADS1 influences the levels of 5-lipoxygenase products and epigenetic alteration. Genetic polymorphisms within FADS and ELOVL can limit LCPUFA product accumulation at any step of the biosynthetic pathway.

Transgenic Production of Epoxy Fatty Acids by Expression of a Cytochrome P450 Enzyme from Euphorbia lagascae Seed

PubMed Central

Cahoon, Edgar B.; Ripp, Kevin G.; Hall, Sarah E.; McGonigle, Brian

2002-01-01

Seed oils of a number of Asteraceae and Euphorbiaceae species are enriched in 12-epoxyoctadeca-cis-9-enoic acid (vernolic acid), an unusual 18-carbon Δ12-epoxy fatty acid with potential industrial value. It has been previously demonstrated that the epoxy group of vernolic acid is synthesized by the activity of a Δ12-oleic acid desaturase-like enzyme in seeds of the Asteraceae Crepis palaestina and Vernonia galamensis. In contrast, results from metabolic studies have suggested the involvement of a cytochrome P450 enzyme in vernolic acid synthesis in seeds of the Euphorbiaceae species Euphorbia lagascae. To clarify the biosynthetic origin of vernolic acid in E. lagascae seed, an expressed sequence tag analysis was conducted. Among 1,006 randomly sequenced cDNAs from developing E. lagascae seeds, two identical expressed sequence tags were identified that encode a cytochrome P450 enzyme classified as CYP726A1. Consistent with the seed-specific occurrence of vernolic acid in E. lagascae, mRNA corresponding to the CYP726A1 gene was abundant in developing seeds, but was not detected in leaves. In addition, expression of the E. lagascae CYP726A1 cDNA in Saccharomyces cerevisiae was accompanied by production of vernolic acid in cultures supplied with linoleic acid and an epoxy fatty acid tentatively identified as 12-epoxyoctadeca-9,15-dienoic acid (12-epoxy-18:2Δ9,15) in cultures supplied with α-linolenic acid. Consistent with this, expression of CYP726A1 in transgenic tobacco (Nicotiana tabacum) callus or somatic soybean (Glycine max) embryos resulted in the accumulation of vernolic acid and 12-epoxy-18:2Δ9,15. Overall, these results conclusively demonstrate that Asteraceae species and the Euphorbiaceae E. lagascae have evolved structurally unrelated enzymes to generate the Δ12-epoxy group of vernolic acid. PMID:11842164

Identification and functional analysis of delta-9 desaturase, a key enzyme in PUFA Synthesis, isolated from the oleaginous diatom Fistulifera.

PubMed

Muto, Masaki; Kubota, Chihiro; Tanaka, Masayoshi; Satoh, Akira; Matsumoto, Mitsufumi; Yoshino, Tomoko; Tanaka, Tsuyoshi

2013-01-01

Oleaginous microalgae are one of the promising resource of nonedible biodiesel fuel (BDF) feed stock alternatives. Now a challenge task is the decrease of the long-chain polyunsaturated fatty acids (PUFAs) content affecting on the BDF oxidative stability by using gene manipulation techniques. However, only the limited knowledge has been available concerning the fatty acid and PUFA synthesis pathways in microalgae. Especially, the function of Δ9 desaturase, which is a key enzyme in PUFA synthesis pathway, has not been determined in diatom. In this study, 4 Δ(9) desaturase genes (fD9desA, fD9desB, fD9desC and fD9desD) from the oleaginous diatom Fistulifera were newly isolated and functionally characterized. The putative Δ(9) acyl-CoA desaturases in the endoplasmic reticulum (ER) showed 3 histidine clusters that are well-conserved motifs in the typical Δ(9) desaturase. Furthermore, the function of these Δ(9) desaturases was confirmed in the Saccharomyces cerevisiae ole1 gene deletion mutant (Δole1). All the putative Δ(9) acyl-CoA desaturases showed Δ(9) desaturation activity for C16∶0 fatty acids; fD9desA and fD9desB also showed desaturation activity for C18∶0 fatty acids. This study represents the first functional analysis of Δ(9) desaturases from oleaginous microalgae and from diatoms as the first enzyme to introduce a double bond in saturated fatty acids during PUFA synthesis. The findings will provide beneficial insights into applying metabolic engineering processes to suppressing PUFA synthesis in this oleaginous microalgal strain.

Polymorphisms in Fatty Acid Desaturase (FADS) Gene Cluster: Effects on Glycemic Controls Following an Omega-3 Polyunsaturated Fatty Acids (PUFA) Supplementation.

PubMed

Cormier, Hubert; Rudkowska, Iwona; Thifault, Elisabeth; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

2013-09-10

Changes in desaturase activity are associated with insulin sensitivity and may be associated with type 2 diabetes mellitus (T2DM). Polymorphisms (SNPs) in the fatty acid desaturase (FADS) gene cluster have been associated with the homeostasis model assessment of insulin sensitivity (HOMA-IS) and serum fatty acid composition. To investigate whether common genetic variations in the FADS gene cluster influence fasting glucose (FG) and fasting insulin (FI) responses following a 6-week n-3 polyunsaturated fatty acids (PUFA) supplementation. 210 subjects completed a 2-week run-in period followed by a 6-week supplementation with 5 g/d of fish oil (providing 1.9 g-2.2 g of EPA + 1.1 g of DHA). Genotyping of 18 SNPs of the FADS gene cluster covering 90% of all common genetic variations (minor allele frequency ≥ 0.03) was performed. Carriers of the minor allele for rs482548 (FADS2) had increased plasma FG levels after the n-3 PUFA supplementation in a model adjusted for FG levels at baseline, age, sex, and BMI. A significant genotype*supplementation interaction effect on FG levels was observed for rs482548 (p = 0.008). For FI levels, a genotype effect was observed with one SNP (rs174456). For HOMA-IS, several genotype*supplementation interaction effects were observed for rs7394871, rs174602, rs174570, rs7482316 and rs482548 (p = 0.03, p = 0.01, p = 0.03, p = 0.05 and p = 0.07; respectively). RESULTS suggest that SNPs in the FADS gene cluster may modulate plasma FG, FI and HOMA-IS levels in response to n-3 PUFA supplementation.

Polymorphisms in Fatty Acid Desaturase (FADS) Gene Cluster: Effects on Glycemic Controls Following an Omega-3 Polyunsaturated Fatty Acids (PUFA) Supplementation

PubMed Central

Cormier, Hubert; Rudkowska, Iwona; Thifault, Elisabeth; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

2013-01-01

Changes in desaturase activity are associated with insulin sensitivity and may be associated with type 2 diabetes mellitus (T2DM). Polymorphisms (SNPs) in the fatty acid desaturase (FADS) gene cluster have been associated with the homeostasis model assessment of insulin sensitivity (HOMA-IS) and serum fatty acid composition. Objective: To investigate whether common genetic variations in the FADS gene cluster influence fasting glucose (FG) and fasting insulin (FI) responses following a 6-week n-3 polyunsaturated fatty acids (PUFA) supplementation. Methods: 210 subjects completed a 2-week run-in period followed by a 6-week supplementation with 5 g/d of fish oil (providing 1.9 g–2.2 g of EPA + 1.1 g of DHA). Genotyping of 18 SNPs of the FADS gene cluster covering 90% of all common genetic variations (minor allele frequency ≥ 0.03) was performed. Results: Carriers of the minor allele for rs482548 (FADS2) had increased plasma FG levels after the n-3 PUFA supplementation in a model adjusted for FG levels at baseline, age, sex, and BMI. A significant genotype*supplementation interaction effect on FG levels was observed for rs482548 (p = 0.008). For FI levels, a genotype effect was observed with one SNP (rs174456). For HOMA-IS, several genotype*supplementation interaction effects were observed for rs7394871, rs174602, rs174570, rs7482316 and rs482548 (p = 0.03, p = 0.01, p = 0.03, p = 0.05 and p = 0.07; respectively). Conclusion: Results suggest that SNPs in the FADS gene cluster may modulate plasma FG, FI and HOMA-IS levels in response to n-3 PUFA supplementation. PMID:24705214

Lipopolysaccharide derived from the rumen down-regulates stearoyl-CoA desaturase 1 expression and alters fatty acid composition in the liver of dairy cows fed a high-concentrate diet.

PubMed

Xu, Tianle; Tao, Hui; Chang, Guangjun; Zhang, Kai; Xu, Lei; Shen, Xiangzhen

2015-03-07

Dairy cows are often fed a high-concentrate diet to meet lactating demands, yet long-term concentrate feeding induces subacute ruminal acidosis (SARA) and leads to a decrease in milk fat. Stearoyl-CoA desaturase1 (SCD1) participates in fatty acid biosynthesis in the liver of lactating ruminants. Here, we conducted this study to investigate the impact of lipopolysaccharide derived from the rumen on SCD1 expression and on fatty acid composition in the liver of dairy cows fed a high-concentrate diet. Eight multiparous mid-lactating Holstein cows (455 ± 28 kg) were randomly assigned into two groups in the experiment and were fed a low-concentrate diet (LC) or high-concentrate diet (HC) for 18 weeks. The results showed that the total volatile fatty acids and lactic acid accumulated in the rumen, leading to a decreased rumen pH and elevated lipopolysaccharides (LPSs) in the HC group. The long chain fatty acid profile in the rumen and hepatic vein was remarkably altered in the animals fed the HC diet. The triglyceride (TG), non-esterified fatty acid (NEFA) and total cholesterol (TCH) content in the plasma was significantly decreased, whereas plasma glucose and insulin levels were increased. The expression of SCD1 in the liver was significantly down-regulated in the HC group. In regards to transcriptional regulators, the expression of sterol regulatory element binding transcription factors (SREBF1c, SREBF2) and SREBP cleavage activating protein (SCAP) was down-regulated, while peroxisome proliferator-activated receptor α (PPARα) was up-regulated. These data indicate that lipopolysaccharide derived from the rumen down-regulates stearoyl-CoA desaturase 1 expression and alters fatty acid composition in the liver of dairy cows fed a high-concentrate diet.

TEMPERATURE-SENSITIVE, POST-TRANSLATIONAL REGULATION OF PLANT OMEGA-3 FATTY ACID DESATURASES IS MEDIATED BY THE ER-ASSOCIATED DEGRADATION PATHWAY

USDA-ARS?s Scientific Manuscript database

In plants, the endoplasmic reticulum (ER)-localized omega-3 fatty acid desaturases (Fad3s) increase the production of polyunsaturated fatty acids at cooler temperatures, but the FAD3 genes themselves are typically not upregulated during this adaptive response. Here, we expressed two closely related ...

Accumulation of eicosapolyenoic acids enhances sensitivity to abscisic acid and mitigates the effects of drought in transgenic Arabidopsis thaliana

PubMed Central

Qi, Baoxiu

2014-01-01

IgASE1, a C18 Δ9-specific polyunsaturated fatty acid elongase from the marine microalga Isochrysis galbana, is able to convert linoleic acid and α-linolenic acid to eicosadienoic acid and eicosatrienoic acid in Arabidopsis. Eicosadienoic acid and eicosatrienoic acid are precursors of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, which are synthesized via the Δ8 desaturation biosynthetic pathways. This study shows that the IgASE1-expressing transgenic Arabidopsis exhibited altered morphology (decreased leaf area and biomass) and enhanced drought resistance compared to wild-type plants. The transgenic Arabidopsis were hypersensitive to abscisic acid (ABA) during seed germination, post-germination growth, and seedling development. They had elevated leaf ABA levels under well-watered and dehydrated conditions and their stomata were more sensitive to ABA. Exogenous application of eicosadienoic acid and eicosatrienoic acid can mimic ABA and drought responses in the wild type plants, similar to that found in the transgenic ones. The transcript levels of genes involved in the biosynthesis of ABA (NCED3, ABA1, AAO3) as well as other stress-related genes were upregulated in this transgenic line upon osmotic stress (300mM mannitol). Taken together, these results indicate that these two eicosapolyenoic acids or their derived metabolites can mitigate the effects of drought in transgenic Arabidopsis, at least in part, through the action of ABA. PMID:24609499

Accumulation of eicosapolyenoic acids enhances sensitivity to abscisic acid and mitigates the effects of drought in transgenic Arabidopsis thaliana.

PubMed

Yuan, Xiaowei; Li, Yaxiao; Liu, Shiyang; Xia, Fei; Li, Xinzheng; Qi, Baoxiu

2014-04-01

IgASE1, a C₁₈ Δ(9)-specific polyunsaturated fatty acid elongase from the marine microalga Isochrysis galbana, is able to convert linoleic acid and α-linolenic acid to eicosadienoic acid and eicosatrienoic acid in Arabidopsis. Eicosadienoic acid and eicosatrienoic acid are precursors of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, which are synthesized via the Δ(8) desaturation biosynthetic pathways. This study shows that the IgASE1-expressing transgenic Arabidopsis exhibited altered morphology (decreased leaf area and biomass) and enhanced drought resistance compared to wild-type plants. The transgenic Arabidopsis were hypersensitive to abscisic acid (ABA) during seed germination, post-germination growth, and seedling development. They had elevated leaf ABA levels under well-watered and dehydrated conditions and their stomata were more sensitive to ABA. Exogenous application of eicosadienoic acid and eicosatrienoic acid can mimic ABA and drought responses in the wild type plants, similar to that found in the transgenic ones. The transcript levels of genes involved in the biosynthesis of ABA (NCED3, ABA1, AAO3) as well as other stress-related genes were upregulated in this transgenic line upon osmotic stress (300 mM mannitol). Taken together, these results indicate that these two eicosapolyenoic acids or their derived metabolites can mitigate the effects of drought in transgenic Arabidopsis, at least in part, through the action of ABA.

Effect of tanniniferous Terminalia chebula extract on rumen biohydrogenation, ∆(9)-desaturase activity, CLA content and fatty acid composition in longissimus dorsi muscle of kids.

PubMed

Rana, Madhu Suman; Tyagi, A; Hossain, Sk Asraf; Tyagi, A K

2012-03-01

Conjugated linoleic acid, a fatty acid found in milk fat and ruminant meat is one of the functional food components. Modifying fatty acid composition so as to increase CLA and other beneficial PUFA/MUFA level and reducing SFA levels might be a key to enhance the neutraceutical and therapeutic value of ruminant-derived food products. In the present experiment, the effect of supplementation of polyphenol rich Terminalia chebula plant extract at different concentrations (1.06g/kg and 3.18g/kg of body weight in T1 and T2 groups, respectively) was investigated on fatty acid composition of rumen fluid, plasma, intramuscular fat and Δ9-desaturase activity in longissimus dorsi muscle of crossbred kids. Total MUFA and PUFA content in muscle were enhanced by 25 and 35%, respectively, whereas SFA was reduced by 20% thereby improving the desaturation index. Δ9-desaturase activity also increased by 47% resulting in an enhancement of total CLA content (58.73%) in muscle. Copyright © 2011 Elsevier Ltd. All rights reserved.

Amino Acid Change in an Orchid Desaturase Enables Mimicry of the Pollinator’s Sex Pheromone

DOE PAGES

Sedeek, Khalid E. M.; Whittle, Edward; Guthörl, Daniela; ...

2016-05-19

Here, we show that mimicry illustrates the power of selection to produce phenotypic convergence in biology. A striking example is the imitation of female insects by plants that are pollinated by sexual deception of males of the same insect species. This involves mimicry of visual, tactile, and chemical signals of females, especially their sex pheromones. The Mediterranean orchid Ophrys exaltata employs chemical mimicry of cuticular hydrocarbons, particularly the 7-alkenes, in an insect sex pheromone to attract and elicit mating behavior in its pollinators, males of the cellophane bee Colletes cunicularius. A difference in alkene double-bond positions is responsible for reproductivemore » isolation between O. exaltata and closely related species, such as O. sphegodes. We show that these 7-alkenes are likely determined by the action of the stearoyl-acyl-carrier-protein desaturase (SAD) homolog SAD5. After gene duplication, changes in subcellular localization relative to the ancestral housekeeping desaturase may have allowed proto-SAD5’s reaction products to undergo further biosynthesis to both 7- and 9-alkenes. Such ancestral coproduction of two alkene classes may have led to pollinator-mediated deleterious pleiotropy. Despite possible evolutionary intermediates with reduced activity, amino acid changes at the bottom of the substrate-binding cavity have conferred enzyme specificity for 7-alkene biosynthesis by preventing the binding of longer-chained fatty acid (FA) precursors by the enzyme. In conclusion, this change in desaturase function enabled the orchid to perfect its chemical mimicry of pollinator sex pheromones by escape from deleterious pleiotropy, supporting a role of pleiotropy in determining the possible trajectories of adaptive evolution.« less

Amino Acid Change in an Orchid Desaturase Enables Mimicry of the Pollinator’s Sex Pheromone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sedeek, Khalid E. M.; Whittle, Edward; Guthörl, Daniela

Here, we show that mimicry illustrates the power of selection to produce phenotypic convergence in biology. A striking example is the imitation of female insects by plants that are pollinated by sexual deception of males of the same insect species. This involves mimicry of visual, tactile, and chemical signals of females, especially their sex pheromones. The Mediterranean orchid Ophrys exaltata employs chemical mimicry of cuticular hydrocarbons, particularly the 7-alkenes, in an insect sex pheromone to attract and elicit mating behavior in its pollinators, males of the cellophane bee Colletes cunicularius. A difference in alkene double-bond positions is responsible for reproductivemore » isolation between O. exaltata and closely related species, such as O. sphegodes. We show that these 7-alkenes are likely determined by the action of the stearoyl-acyl-carrier-protein desaturase (SAD) homolog SAD5. After gene duplication, changes in subcellular localization relative to the ancestral housekeeping desaturase may have allowed proto-SAD5’s reaction products to undergo further biosynthesis to both 7- and 9-alkenes. Such ancestral coproduction of two alkene classes may have led to pollinator-mediated deleterious pleiotropy. Despite possible evolutionary intermediates with reduced activity, amino acid changes at the bottom of the substrate-binding cavity have conferred enzyme specificity for 7-alkene biosynthesis by preventing the binding of longer-chained fatty acid (FA) precursors by the enzyme. In conclusion, this change in desaturase function enabled the orchid to perfect its chemical mimicry of pollinator sex pheromones by escape from deleterious pleiotropy, supporting a role of pleiotropy in determining the possible trajectories of adaptive evolution.« less

Overexpression of alpha(1)-acid glycoprotein in transgenic mice leads to sensitisation to acute colitis.

PubMed

Hochepied, T; Wullaert, A; Berger, F G; Baumann, H; Brouckaert, P; Steidler, L; Libert, C

2002-09-01

alpha(1)-Acid glycoprotein (alpha(1)-AGP) is an acute phase protein in most mammalian species whose concentration rises 2-5-fold during an acute phase reaction. Its serum concentration has often been used as a marker of disease, including inflammatory bowel disease (IBD). High alpha(1)-AGP levels were found to have a prognostic value for an increased risk of relapse in IBD. To investigate a possible role for increased serum levels of alpha(1)-AGP in the development of IBD. Dextran sodium sulphate (DSS) 2% was added to the drinking water of transgenic mice, overexpressing the rat alpha(1)-AGP gene, to induce acute colitis, thus mimicking the conditions of relapse. Clinical parameters, inflammatory parameters, and histological analyses on colon sections were performed. Homozygous alpha(1)-AGP-transgenic mice started losing weight and showed rectal bleeding significantly earlier than heterozygous transgenic or wild-type mice. Survival time of homozygous transgenic mice was significantly shorter compared with heterozygous and wild-type mice. The higher susceptibility of homozygous alpha(1)-AGP-transgenic mice to DSS induced acute colitis was also reflected in higher local myeloperoxidase levels, higher inflammation scores of the colon, and higher systemic levels of interleukin 6 and serum amyloid P component. Local inflammatory parameters were also significantly different in heterozygous transgenic mice compared with wild-type mice, indicating a local dosage effect. In homozygous transgenic mice, significantly higher amounts of bacteria were found in organs but IgA levels were only slightly lower than those of control mice. Sufficiently high serum levels of alpha(1)-AGP result in a more aggressive development of acute colitis.

Plasma fatty acid composition, estimated desaturase activities, and their relation with the metabolic syndrome in a population at high risk of cardiovascular disease.

PubMed

Mayneris-Perxachs, Jordi; Guerendiain, Marcela; Castellote, Ana I; Estruch, Ramón; Covas, María Isabel; Fitó, Montserrat; Salas-Salvadó, Jordi; Martínez-González, Miguel A; Aros, Fernando; Lamuela-Raventós, Rosa M; López-Sabater, M Carmen

2014-02-01

The metabolic syndrome (MetS) is a clustering of various metabolic abnormalities which is associated with increased risk of cardiovascular disease (CVD) and type 2 diabetes mellitus. Due to its increasing prevalence, it has become an important public health concern. Altered fatty acid (FA) composition and desaturase activities have been associated with several metabolic diseases, including MetS. The aim of the present study was to evaluate the relationship of the plasma FA profile and desaturase activities with the MetS in a Mediterranean population at high risk of CVD. Baseline data from 427 participants aged 55-80 years who took part in the interventional PREDIMED study were obtained. Individual FA was determined in plasma and desaturase activities were estimated from product/precursor ratios. Odds ratios (OR) and partial correlation coefficients were used to examine these relations with MetS and its components, respectively. We found higher levels of C14:0, C16:0, C16:1n-7, estimated Δ(9)- or stearoyl-CoA desaturase (SCD), and estimated Δ(6) desaturase (D6D), and lower levels of C18:2n-6 in people with MetS compared to those without it. After adjustment for several confounders, only higher quartiles of C14:0, C16:0, C16:1n-7, and D6D were found to be associated with an increasing prevalence of MetS, while higher quartiles of C18:2n-6 were inversely associated with MetS. High proportions of C14:0, C16:0, C16:1n-7, C20:3n-6, SCD, and D6D, and decreased proportions of C18:2n-6 and estimated Δ(5)-desaturase (D5D) were associated with adverse profiles of several metabolic risk factors. Women showed more unhealthy FA pattern and lipid profiles than men, but only among those with MetS. A FA composition and estimated desaturase activities consisting in high levels of SFA, SCD and D6D, and low levels of PUFA and D5D are associated with increased MetS probability and are characteristic of people presenting MetS, especially women. These findings support those observed

Mutations in a delta9-Stearoyl-ACP-Desaturase Gene Are Associated with Enhanced Stearic Acid Levels in Soybean Seeds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, P.; Shanklin, J.; Burton, J. W.

2008-11-01

Stearic acid (18:0) is typically a minor component of soybean [Glycine max (L.) Merr.] oil, accounting for only 2 to 4% of the total fatty acid content. Increasing stearic acid levels of soybean oil would lead to enhanced oxidative stability, potentially reducing the need for hydrogenation, a process leading to the formation of undesirable trans fatty acids. Although mutagenesis strategies have been successful in developing soybean germplasm with elevated 18:0 levels in the seed oil, the specific gene mutations responsible for this phenotype were not known. We report a newly identified soybean gene, designated SACPD-C, that encodes a unique isoformmore » of {Delta}{sup 9}-stearoyl-ACP-desaturase, the enzyme responsible for converting stearic acid to oleic acid (18:1). High levels of SACPD-C transcript were only detected in developing seed tissue, suggesting that the encoded desaturase functions to enhance oleic acid biosynthetic capacity as the immature seed is actively engaged in triacylglycerol production and storage. The participation of SACPD-C in storage triacylglycerol synthesis is further supported by the observation of mutations in this gene in two independent sources of elevated 18:0 soybean germplasm, A6 (30% 18:0) and FAM94-41 (9% 18:0). A molecular marker diagnostic for the FAM94-41 SACPD-C gene mutation strictly associates with the elevated 18:0 phenotype in a segregating population, and could thus serve as a useful tool in the development of cultivars with oils possessing enhanced oxidative stability.« less

[Functional expression of an omega-3 fatty acid desaturase gene from Glycine max in Saccharomyces cerevisiae].

PubMed

Zhang, Hong-Tao; Yang, Jia-Sen; Shan, Lei; Bi, Yu-Ping

2006-01-01

Alpha-linolenic acid(ALA, C18:3delta9,12,15 ) is an essential fatty acid which has many sanitary functions to human. However, its contents in diets are often not enough. In plants, omega-3 fatty acid desaturases(FAD) catalyze linoleic acid(LA, C18:2delta9,12) into ALA. The seed oil of Glycine max contains high level of ALA. To investigate the functions of Glycine max omega-3FAD, the cDNA of GmFAD3 C was amplified by RT-PCR from immature seeds, then cloned into the shuttle expression vector p416 to generate the recombinant vector p4GFAD3C. The resulting vector was transformed into Saccharomyces cerevisiae K601 throuth LiAc method. The positive clones were screened on the CM(Ura-) medium and identified by PCR, and then cultured in CM (Ura-) liquid medium with exogenous LA in 20 degrees C for three days. The intracellular fatty acid composition of the engineering strain Kp416 and Kp4GFAD3C was analyzed by gas chromatography (GC). A novel peak in strain Kp4GFAD3C was detected,which was not detectable in control, Comparison of the retention times of the newly yielded peak with that of authentic standard indicated that the fatty acid is ALA. The content of ALA reached to 3.1% of the total fatty acid in recombinant strain, the content of LA correspondingly decreased from 22% to 16.2% by contrast. It was suggested that the protein encoded by GmFAD3 C can specifically catalyze 18 carbon PUFA substrate of LA into ALA by taking off hydrogen atoms at delta15 location. In this study, we expressed a Glycine max omega-3 fatty acid desaturase gene in S. cerevisiae; An efficient and economical yeast expressing system(K601-p416 system) which is suitable for the expression of FAD was built.

Variants in CPT1A, FADS1, and FADS2 are Associated with Higher Levels of Estimated Plasma and Erythrocyte Delta-5 Desaturases in Alaskan Eskimos.

PubMed

Voruganti, V Saroja; Higgins, Paul B; Ebbesson, Sven O E; Kennish, John; Göring, Harald H H; Haack, Karin; Laston, Sandra; Drigalenko, Eugene; Wenger, Charlotte R; Harris, William S; Fabsitz, Richard R; Devereux, Richard B; Maccluer, Jean W; Curran, Joanne E; Carless, Melanie A; Johnson, Matthew P; Moses, Eric K; Blangero, John; Umans, Jason G; Howard, Barbara V; Cole, Shelley A; Comuzzie, Anthony Gean

2012-01-01

The delta-5 and delta-6 desaturases (D5D and D6D), encoded by fatty acid desaturase 1 (FADS1) and 2 (FADS2) genes, respectively, are rate-limiting enzymes in the metabolism of ω-3 and ω-6 fatty acids. The objective of this study was to identify genes influencing variation in estimated D5D and D6D activities in plasma and erythrocytes in Alaskan Eskimos (n = 761) participating in the genetics of coronary artery disease in Alaska Natives (GOCADAN) study. Desaturase activity was estimated by product: precursor ratio of polyunsaturated fatty acids. We found evidence of linkage for estimated erythrocyte D5D (eD5D) on chromosome 11q12-q13 (logarithm of odds score = 3.5). The confidence interval contains candidate genes FADS1, FADS2, 7-dehydrocholesterol reductase (DHCR7), and carnitine palmitoyl transferase 1A, liver (CPT1A). Measured genotype analysis found association between CPT1A, FADS1, and FADS2 single-nucleotide polymorphisms (SNPs) and estimated eD5D activity (p-values between 10(-28) and 10(-5)). A Bayesian quantitative trait nucleotide analysis showed that rs3019594 in CPT1A, rs174541 in FADS1, and rs174568 in FADS2 had posterior probabilities > 0.8, thereby demonstrating significant statistical support for a functional effect on eD5D activity. Highly significant associations of FADS1, FADS2, and CPT1A transcripts with their respective SNPs (p-values between 10(-75) and 10(-7)) in Mexican Americans of the San Antonio Family Heart Study corroborated our results. These findings strongly suggest a functional role for FADS1, FADS2, and CPT1A SNPs in the variation in eD5D activity.

The Mycobacterium tuberculosis desaturase DesA1 (Rv0824c) is a Ca{sup 2+} binding protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeruva, Veena C., E-mail: veenachaitanya@ccmb.res.in; Savanagouder, Mamata; Khandelwal, Radhika

The hallmark feature of Mycobacterium tuberculosis (M.tb) the causative agent of human tuberculosis, is its complex lipid rich cell wall comprised primarily of mycolic acids, long chain fatty acids that play a key role in structural stability and permeability of the cell wall. In addition, they are involved in inhibiting phagosome-lysosome fusion and aid in granuloma formation during the pathogenic process. M.tb DesA1 is an essential acyl-acyl carrier protein desaturase predicted to catalyze the introduction of position specific double bonds during the biosynthesis of mycolic acids. This protein is one among three annotated desaturases (DesA1-3) in the M.tb genome butmore » is unique in containing a βγ-crystallin Greek key signature motif, a well-characterized fold known to mediate Ca{sup 2+} binding in both prokaryotic and eukaryotic organisms. Using Isothermal Titration Calorimetry and {sup 45}CaCl{sub 2} overlay, we demonstrate that Ca{sup 2+} binds to DesA1. Spectroscopic measurements suggested that this binding induces changes in protein conformation but does not lead to significant alterations in the secondary structure of the protein, a feature common to several βγ-crystallins. An M. smegmatis strain over-expressing M.tb desA1 showed a Ca{sup 2+} dependent variation in surface phenotype, revealing a functional role for Ca{sup 2+}in DesA1 activity. This study represents the first identification of a Ca{sup 2+} binding βγ-crystallin in M.tb, emphasizing the implicit role of Ca{sup 2+} in the pathogenesis of M.tb. - Highlights: • Mycobacterium tuberculosis DesA1 is an essential acyl-ACP desaturase. • DesA1 was identified to contain a βγ-crystallin Greek key signature motif. • Ca{sup 2+} binds to DesA1 with an affinity of 53 μM and induces changes in its conformation. • M. smegmatis overexpressing M.tb DesA1 shows a Ca{sup 2+} dependent phenotype. • Targetting the Ca{sup 2+} dependent function of DesA1 could be of therapeutic value.« less

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases.

PubMed

Guy, Jodie E; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

2011-10-04

Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals.

Omega-6 polyunsaturated fatty acids, serum zinc, delta-5- and delta-6-desaturase activities and incident metabolic syndrome.

PubMed

Yary, T; Voutilainen, S; Tuomainen, T-P; Ruusunen, A; Nurmi, T; Virtanen, J K

2017-08-01

The associations of n-6 polyunsaturated fatty acids (PUFA) with metabolic syndrome have been poorly explored. We investigated the associations of the serum n-6 PUFA and the activities of enzymes involved in the PUFA metabolism, delta-5-desaturase (D5D) and delta-6-desaturase (D6D) with risk of incident metabolic syndrome. We also investigated whether zinc, a cofactor for these enzymes, modifies these associations. A prospective follow-up study was conducted on 661 men who were aged 42-60 years old at baseline in 1984-1989 and who were re-examined in 1998-2001. Men in the highest versus the lowest serum total omega-6 PUFA tertile had a 70% lower multivariate-adjusted risk of incident metabolic syndrome [odds ratio (OR) = 0.30; 95% confidence interval (CI) = 0.18-0.51, P trend < 0.001]. Inverse associations were also observed for linoleic acid, arachidonic acid and D5D activity. By contrast, men in the highest tertile of D6D activity had an 84% higher risk (OR = 1.84; 95% CI = 1.15-2.94, P trend = 0.008). Similar associations were observed with many of the metabolic syndrome components at the re-examinations. Most associations were attenuated after adjustment for body mass index. Finally, the associations of D6D and LA were stronger among those with a higher serum zinc concentration. Higher serum total n-6 PUFA, linoleic acid and arachidonic acid concentrations and D5D activity were associated with a lower risk of developing metabolic syndrome and higher D6D activity was associated with a higher risk. The role of zinc also needs to be investigated in other populations. © 2016 The British Dietetic Association Ltd.

Hydroxytyrosol prevents reduction in liver activity of Δ-5 and Δ-6 desaturases, oxidative stress, and depletion in long chain polyunsaturated fatty acid content in different tissues of high-fat diet fed mice.

PubMed

Valenzuela, Rodrigo; Echeverria, Francisca; Ortiz, Macarena; Rincón-Cervera, Miguel Ángel; Espinosa, Alejandra; Hernandez-Rodas, María Catalina; Illesca, Paola; Valenzuela, Alfonso; Videla, Luis A

2017-04-11

Eicosapentaenoic acid (EPA, C20:5n-3), docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (AA, C20:4n-6) are long-chain polyunsaturated fatty acids (LCPUFAs) with relevant roles in the organism. EPA and DHA are synthesized from the precursor alpha-linolenic acid (ALA, C18:3n-3), whereas AA is produced from linoleic acid (LA, C18:2n-6) through the action of Δ5 and Δ6-desaturases. High-fat diet (HFD) decreases the activity of both desaturases and LCPUFA accretion in liver and other tissues. Hydroxytyrosol (HT), a natural antioxidant, has an important cytoprotective effects in different cells and tissues. Male mice C57BL/6 J were fed a control diet (CD) (10% fat, 20% protein, 70% carbohydrates) or a HFD (60% fat, 20% protein, 20% carbohydrates) for 12 weeks. Animals were daily supplemented with saline (CD) or 5 mg HT (HFD), and blood and the studied tissues were analyzed after the HT intervention. Parameters studied included liver histology (optical microscopy), activity of hepatic desaturases 5 and 6 (gas-liquid chromatography of methyl esters derivatives) and antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase by spectrophotometry), oxidative stress indicators (glutathione, thiobarbituric acid reactants, and the antioxidant capacity of plasma), gene expression assays for sterol regulatory element-binding protein 1c (SREBP-1c) (qPCR and ELISA), and LCPUFA profiles in liver, erythrocyte, brain, heart, and testicle (gas-liquid chromatography). HFD led to insulin resistance and liver steatosis associated with SREBP-1c upregulation, with enhancement in plasma and liver oxidative stress status and diminution in the synthesis and storage of n-6 and n-3 LCPUFAs in the studied tissues, compared to animals given control diet. HT supplementation significantly reduced fat accumulation in liver and plasma as well as tissue metabolic alterations induced by HFD. Furthermore, a normalization of desaturase activities

Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alloatti, Andres; Gupta, Shreedhara; Gualdron-Lopez, Melisa

2011-08-26

Highlights: {yields} Inhibiting {Delta}9 desaturase drastically changes T. brucei's fatty-acid composition. {yields} Isoxyl specifically inhibits the {Delta}9 desaturase causing a growth arrest. {yields} RNA interference of desaturase expression causes a similar effect. {yields} Feeding T. brucei-infected mice with Isoxyl decreases the parasitemia. {yields} 70% of Isoxyl-treated mice survived the trypanosome infection. -- Abstract: Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes namedmore » desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei's survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC{sub 50}) of PCF was 1.0 {+-} 0.2 {mu}M for Isoxyl and 5 {+-} 2 {mu}M for 10-TS, whereas BSF appeared more susceptible with EC{sub 50} values 0.10 {+-} 0.03 {mu}M (Isoxyl) and 1.0 {+-} 0.6 {mu}M (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents' survival.« less

Dietary arachidonic acid and docosahexaenoic acid regulate liver fatty acid desaturase (FADS) alternative transcript expression in suckling piglets.

PubMed

Wijendran, Vasuki; Downs, Ian; Srigley, Cynthia Tyburczy; Kothapalli, Kumar S D; Park, Woo Jung; Blank, Bryant S; Zimmer, J Paul; Butt, C M; Salem, Norman; Brenna, J Thomas

2013-10-01

Molecular regulation of fatty acid desaturase (Fads) gene expression by dietary arachidonic acid (ARA) and docosahexaenoic acid (DHA) during early post-natal period, when the demand for long chain polyunsaturated fatty acids (LC-PUFA) is very high, has not been well defined. The objective of the current study was to determine regulation of liver Fads1, Fads2 and Fads3 classical (CS) and alternative transcripts (AT) expression by dietary ARA and DHA, within the physiological range present in human breast milk, in suckling piglets. Piglets were fed one of six milk replacer formula diets (formula-reared groups, FR) with varying ARA and DHA content from days 3-28 of age. The ARA/DHA levels of the six formula diets were as follows (% total fatty acid, FA/FA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3-D3) 0.69/1.0; (A4) 1.1/1.0; (D2) 0.67/0.62; and (D1) 0.66/0.33. The control maternal-reared (MR) group remained with the dam. Fads1 expression was not significantly different between FR and MR groups. Fads2 expression was down-regulated significantly in diets with 1:1 ratio of ARA:DHA, compared to MR. Fads2 AT1 expression was highly correlated to Fads2 expression. Fads3 AT7 was the only Fads3 transcript sensitive to dietary LC-PUFA intake and was up-regulated in the formula diets with lowest ARA and DHA contents compared to MR. Thus, the present study provides evidence that the proportion of dietary ARA:DHA is a significant determinant of Fads2 expression and LC-PUFA metabolism during the early postnatal period. Further, the data suggest that Fads3 AT7 may have functional significance when dietary supply of ARA and DHA are low during early development. © 2013 Elsevier Ltd. All rights reserved.

A Transgenic Camelina sativa Seed Oil Effectively Replaces Fish Oil as a Dietary Source of Eicosapentaenoic Acid in Mice123

PubMed Central

Tejera, Noemi; Vauzour, David; Betancor, Monica B; Sayanova, Olga; Usher, Sarah; Cochard, Marianne; Rigby, Neil; Ruiz-Lopez, Noemi; Menoyo, David; Tocher, Douglas R; Napier, Johnathan A; Minihane, Anne Marie

2016-01-01

Background: Fish currently supplies only 40% of the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) required to allow all individuals globally to meet the minimum intake recommendation of 500 mg/d. Therefore, alternative sustainable sources are needed. Objective: The main objective was to investigate the ability of genetically engineered Camelina sativa (20% EPA) oil (CO) to enrich tissue EPA and DHA relative to an EPA-rich fish oil (FO) in mammals. Methods: Six-week-old male C57BL/6J mice were fed for 10 wk either a palm oil–containing control (C) diet or diets supplemented with EPA-CO or FO, with the C, low-EPA CO (COL), high-EPA CO (COH), low-EPA FO (FOL), and high-EPA FO (FOH) diets providing 0, 0.4, 3.4, 0.3, and 2.9 g EPA/kg diet, respectively. Liver, muscle, and brain were collected for fatty acid analysis, and blood glucose and serum lipids were quantified. The expression of selected hepatic genes involved in EPA and DHA biosynthesis and in modulating their cellular impact was determined. Results: The oils were well tolerated, with significantly greater weight gain in the COH and FOH groups relative to the C group (P < 0.001). Significantly lower (36–38%) blood glucose concentrations were evident in the FOH and COH mice relative to C mice (P < 0.01). Hepatic EPA concentrations were higher in all EPA groups relative to the C group (P < 0.001), with concentrations of 0.0, 0.4, 2.9, 0.2, and 3.6 g/100 g liver total lipids in the C, COL, COH, FOL, and FOH groups, respectively. Comparable dose-independent enrichments of liver DHA were observed in mice fed CO and FO diets (P < 0.001). Relative to the C group, lower fatty acid desaturase 1 (Fads1) expression (P < 0.005) was observed in the COH and FOH groups. Higher fatty acid desaturase 2 (Fads2), peroxisome proliferator–activated receptor α (Ppara), and peroxisome proliferator–activated receptor γ (Pparg) (P < 0.005) expressions were induced by CO. No impact of treatment on liver X receptor

Association of polyunsaturated fatty acids in breast milk with fatty acid desaturase gene polymorphisms among Chinese lactating mothers.

PubMed

Ding, Zhen; Liu, Guo-Liang; Li, Xiang; Chen, Xue-Yan; Wu, Yi-Xia; Cui, Can-Can; Zhang, Xi; Yang, Guang; Xie, Lin

2016-06-01

The fatty acid desaturase (FADS) controls polyunsaturated fatty acid (PUFA) synthesis in human tissues and breast milk. Evaluate the influence of 10 single nucleotide polymorphisms (SNPs) and various haplotypes in the FADS gene cluster (FADS1, FADS2, FADS3) on PUFA concentration in the breast milk of 209 healthy Chinese women. PUFA concentrations were measured in breast milk using gas chromatography and genotyping was performed using the Sequenom Mass Array system. A SNP (rs1535) and 2-locus haplotypes (rs3834458-rs1535, rs1535-rs174575) in the FADS2 gene were associated with concentrations of γ-linoleic acid (GLA) and arachidonic acid (AA) in breast milk. Likewise, in the FADS1 gene, a 2-locus constructed haplotype (rs174547-rs174553) also affected GLA and AA concentration (P<0.05 for all). Minor allele carriers of the SNP and haplotypes described above had lower concentrations of GLA and AA. In the FADS2 gene, the 3-locus haplotype rs3834458-rs1535-rs174575, significantly affected concentrations of GLA but not AA. Pairwise comparison showed that individuals major homozygous for the SNP rs1000778 in the FADS3 gene had lower concentrations of ALA and linoleic acid (LA) in their breast milk. Polymorphisms in the FADS gene cluster influence PUFA concentrations in the breast milk of Chinese Han lactating women. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Bacillus subtilis Acyl Lipid Desaturase Is a Δ5 Desaturase

PubMed Central

Altabe, Silvia G.; Aguilar, Pablo; Caballero, Gerardo M.; de Mendoza, Diego

2003-01-01

Bacillus subtilis was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions Δ5, Δ7, and Δ9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the B. subtilis acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by B. subtilis is located exclusively at the Δ5 position, regardless of the growth temperature and the length chain of the fatty acids. PMID:12730185

Stearoyl-CoA Desaturase and its Relation to High-Carbohydrate Diets and Obesity

PubMed Central

Flowers, Matthew T.; Ntambi, James M.

2009-01-01

Obesity is currently a worldwide epidemic and public health burden that increases the risk for developing insulin resistance and several chronic diseases such as diabetes, cardiovascular diseases and non-alcoholic fatty liver disease. The multifactorial causes of obesity include several genetic, dietary and lifestyle variables that together result in an imbalance between energy intake and energy expenditure. Dietary approaches to limit fat intake are commonly prescribed to achieve the hypocaloric conditions necessary for weight loss. But dietary fat restriction is often accompanied by increased carbohydrate intake, which can dramatically increase endogenous fatty acid synthesis depending upon carbohydrate composition. Since both dietary and endogenously synthesized fatty acids contribute to the whole-body fatty acid pool, obesity can therefore result from excessive fat or carbohydrate consumption. Stearoyl-Coenzyme A desaturase-1 (SCD1) is a delta-9 fatty acid desaturase that converts saturated fatty acids into monounsaturated fatty acids (MUFA) and this activity is elevated by dietary carbohydrate. Mice lacking Scd1 are protected from obesity and insulin resistance and are characterized by decreased fatty acid synthesis and increased fatty acid oxidation. In this review, we address the association of high-carbohydrate diets with increased SCD activity and summarize the current literature on the subject of SCD1 and body weight regulation. PMID:19166967

A Bacillus subtilis Gene Induced by Cold Shock Encodes a Membrane Phospholipid Desaturase

PubMed Central

Aguilar, Pablo S.; Cronan, John E.; de Mendoza, Diego

1998-01-01

Bacillus subtilis grown at 37°C synthesizes saturated fatty acids with only traces of unsaturated fatty acids (UFAs). However, when cultures growing at 37°C are transferred to 20°C, UFA synthesis is induced. We report the identification and characterization of the gene encoding the fatty acid desaturase of B. subtilis. This gene, called des, was isolated by complementation of Escherichia coli strains with mutations in either of two different genes of UFA synthesis. The des gene encodes a polypeptide of 352 amino acid residues containing the three conserved histidine cluster motifs and two putative membrane-spanning domains characteristic of the membrane-bound desaturases of plants and cyanobacteria. Expression of the des gene in E. coli resulted in desaturation of palmitic acid moieties of the membrane phospholipids to give the novel mono-UFA cis-5-hexadecenoic acid, indicating that the B. subtilis des gene product is a Δ5 acyl-lipid desaturase. The des gene was disrupted, and the resulting null mutant strains were unable to synthesize UFAs upon a shift to low growth temperatures. The des null mutant strain grew as well as its congenic parent at 20 or 37°C but showed severely reduced survival during stationary phase. Analysis of operon fusions in which the des promoter directed the synthesis of a lacZ reporter gene showed that des expression is repressed at 37°C, but a shift of cultures from 37 to 20°C resulted in a 10- to 15-fold increase in transcription. This is the first report of a membrane phospholipid desaturase in a nonphotosynthetic organism and the first direct evidence for cold induction of a desaturase. PMID:9555904

Genetic variants in desaturase gene, erythrocyte fatty acids, and risk for type 2 diabetes in Chinese Hans.

PubMed

Huang, Tao; Sun, Jianqin; Chen, Yanqiu; Xie, Hua; Xu, Danfeng; Huang, Jinyan; Li, Duo

2014-01-01

The aim of this study was to examine the association of the genetic variants in the fatty acid desaturase (FADS) gene cluster with erythrocyte phospholipid fatty acids (PLFA), and their relation to risk for type 2 diabetes mellitus (T2DM) in Han Chinese. Seven hundred and fifty-eight patients with T2DM and 400 healthy individuals were recruited. The erythrocyte PLFA and single-nucleotide polymorphism were determined by standard method. Minor allele homozygotes and heterozygotes of rs174575 and rs174537 had lower PL 20:4 ω-6 levels in healthy individuals. Minor allele homozygotes and heterozygotes of rs174455 in FADS3 gene had lower levels of 22:5 ω-3, 20:4 ω-6, and Δ5desaturase activity in patients with T2DM. Erythrocyte membrane PL 18:3 ω-3 (P for trend = 0.002), 22:5 ω-3 (P for trend < 0.001), ω-3 polyunsaturated fatty acid (P for trend < 0.001), and ω-3:ω-6 (P for trend < 0.001) were significantly inversely associated with risk for T2DM. Genetic variants in the FADS gene cluster are associated with altered erythrocyte PLFAs. High levels of PL 18:3 ω-3, 22:5 ω-3, and total ω-3 polyunsaturated fatty acid were associated with low risk for T2DM. Copyright © 2014 Elsevier Inc. All rights reserved.

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

PubMed Central

Guy, Jodie E.; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

2011-01-01

Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals. PMID:21930947

Association of desaturase activity and C-reactive protein in European children.

PubMed

Wolters, Maike; Börnhorst, Claudia; Schwarz, Heike; Risé, Patrizia; Galli, Claudio; Moreno, Luis A; Pala, Valeria; Russo, Paola; Veidebaum, Toomas; Tornaritis, Michael; Fraterman, Arno; De Henauw, Stefaan; Eiben, Gabriele; Lissner, Lauren; Molnár, Dénes; Ahrens, Wolfgang

2017-01-01

Desaturase enzymes influence the fatty acid (FA) composition of body tissues and their activity affects the conversion rate of saturated to monounsaturated FA and of polyunsaturated FA (PUFA) to long-chain PUFA. Desaturase activity has further been shown to be associated with inflammation. We investigate the association between delta-9 (D9D), delta-6 (D6D) and delta-5 desaturase (D5D) activity and high-sensitive C-reactive protein (CRP) in young children. In the IDEFICS (Identification and prevention of dietary- and lifestyle-induced health effects in children and infants) cohort study children were examined at baseline (T0) and after 2 y (T1). D9D, D6D, and D5D activities were estimated from T0 product-precursor FA ratios. CRP was measured at T0 and T1. In a subsample of 1,943 children with available information on FA, CRP, and covariates, the cross-sectional and longitudinal associations of desaturase activity and CRP were analyzed. Cross-sectionally, a D9D increase of 0.01 units was associated with a 11% higher risk of having a serum CRP ≥ Percentile 75 (P75) (OR, 99% CI: 1.11 (1.01; 1.22)) whereas D6D and D5D were not associated with CRP. No significant associations were observed between baseline desaturase activity and CRP 2 y later. Cross-sectionally, our results indicate a positive association of D9D and CRP independent of weight status. High D9D activity may increase the risk of subclinical inflammation which is associated with metabolic disorders. As D9D expression increases with higher intake of saturated FA and carbohydrates, dietary changes may influence D9D activity and thus CRP. However, it remains to be investigated whether there is a causal relationship between D9D activity and CRP.

Inducing effect of clofibric acid on stearoyl-CoA desaturase in intestinal mucosa of rats.

PubMed

Yamazaki, Tohru; Kadokura, Makiko; Mutoh, Yuki; Sakamoto, Takeshi; Okazaki, Mari; Mitsumoto, Atsushi; Kawashima, Yoichi; Kudo, Naomi

2014-12-01

Fibrates have been reported to elevate the hepatic proportion of oleic acid (18:1n-9) through inducing stearoyl-CoA desaturase (SCD). Despite abundant studies on the regulation of SCD in the liver, little is known about this issue in the small intestine. The present study aimed to investigate the effect of clofibric acid on the fatty acid profile, particularly monounsaturated fatty acids (MUFA), and the SCD expression in intestinal mucosa. Treatment of rats with a diet containing 0.5% (w/w) clofibric acid for 7 days changed the MUFA profile of total lipids in intestinal mucosa; the proportion of 18:1n-9 was significantly increased, whereas those of palmitoleic (16:1n-7) and cis-vaccenic (18:1n-7) acids were not changed. Upon the treatment with clofibric acid, SCD was induced and the gene expression of SCD1, SCD2, and fatty acid elongase (Elovl) 6 was up-regulated, but that of Elovl5 was unaffected. Fat-free diet feeding for 28 days increased the proportions of 16:1n-7 and 18:1n-7, but did not effectively change that of 18:1n-9, in intestinal mucosa. Fat-free diet feeding up-regulated the gene expression of SCD1, but not that of SCD2, Elovl6, or Elovl5. These results indicate that intestinal mucosa significantly changes its MUFA profile in response to challenges by clofibric acid and a fat-free diet and suggest that up-regulation of the gene expression of SCD along with Elovl6 is indispensable to elevate the proportion of 18:1n-9 in intestinal mucosa.

Characterization of a stearoyl-acyl carrier protein desaturase gene family from chocolate tree, Theobroma cacao L.

PubMed

Zhang, Yufan; Maximova, Siela N; Guiltinan, Mark J

2015-01-01

In plants, the conversion of stearoyl-ACP to oleoyol-ACP is catalyzed by a plastid-localized soluble stearoyl-acyl carrier protein (ACP) desaturase (SAD). The activity of SAD significantly impacts the ratio of saturated and unsaturated fatty acids, and is thus a major determinant of fatty acid composition. The cacao genome contains eight putative SAD isoforms with high amino acid sequence similarities and functional domain conservation with SAD genes from other species. Sequence variation in known functional domains between different SAD family members suggested that these eight SAD isoforms might have distinct functions in plant development, a hypothesis supported by their diverse expression patterns in various cacao tissues. Notably, TcSAD1 is universally expressed across all the tissues, and its expression pattern in seeds is highly correlated with the dramatic change in fatty acid composition during seed maturation. Interestingly, TcSAD3 and TcSAD4 appear to be exclusively and highly expressed in flowers, functions of which remain unknown. To test the function of TcSAD1 in vivo, transgenic complementation of the Arabidopsis ssi2 mutant was performed, demonstrating that TcSAD1 successfully rescued all AtSSI2 related phenotypes further supporting the functional orthology between these two genes. The identification of the major SAD gene responsible for cocoa butter biosynthesis provides new strategies for screening for novel genotypes with desirable fatty acid compositions, and for use in breeding programs to help pyramid genes for quality and other traits such as disease resistance.

Transgenic expression of omega-3 PUFA synthesis genes improves zebrafish survival during Vibrio vulnificus infection.

PubMed

Cheng, Chih-Lun; Huang, Shin-Jie; Wu, Chih-Lu; Gong, Hong-Yi; Ken, Chuian-Fu; Hu, Shao-Yang; Wu, Jen-Leih

2015-11-17

Highly desaturated n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are synthesized by desaturases and elongase. They exert hepatoprotective effects to prevent alcoholic fatty liver syndrome or cholestatic liver injury. However, it is unclear how n-3 PUFAs improve immune function in liver. Vibrio vulnificus, a gram-negative bacterial pathogen, causes high mortality of aquaculture fishes upon infection. Humans can become infected with V. vulnificus through open wounds or by eating raw seafood, and such infections may result in systemic septicemia. Moreover, patients with liver diseases are vulnerable to infection, and are more likely than healthy persons to present with liver inflammation following infection. This study quantified n-3 PUFAs and their anti-bacterial effects in Fadsd6 and Elvol5a transgenic zebrafish. Two transgenic zebrafish strains with strong liver specific expression of Fadsd6 and Elvol5a (driven by the zebrafish Fabp10 promoter) were established using the Tol2 system. Synthesis of n-3 PUFAs in these strains were increased by 2.5-fold as compared to wild type (Wt) fish. The survival rate in 24 h following challenge with V. vulnificus was 20 % in Wt, but 70 % in the transgenic strains. In addition, the bacteria counts in transgenic fish strains were significantly decreased. The expression levels of pro-inflammatory genes, such as TNF-α, IL-1β, and NF-κB, were suppressed between 9 and 12 h after challenge. This study confirms the anti-bacterial function of n-3 PUFAs in a transgenic zebrafish model. Fadsd6 and Elvol5a transgenic zebrafish are more resistant to V. vulnificus infection, and enhance survival by diminishing the attendant inflammatory response.

Improved eicosapentaenoic acid production in Pythium splendens RBB-5 based on metabolic regulation analysis.

PubMed

Ren, Liang; Zhou, Pengpeng; Zhu, Yuanmin; Zhang, Ruijiao; Yu, Longjiang

2017-05-01

Eicosapentaenoic acid (EPA) is an essential polyunsaturated fatty acid for human beings. At present, the production of commercially available long-chain polyunsaturated fatty acids, mainly from wild-caught ocean fish, is struggling to meet the increasing demand for EPA. Production of EPA by microorganisms may be an alternative, effective and economical method. The oleaginous fungus Pythium splendens RBB-5 is a potential source of EPA, and thanks to the simple culture conditions required, high yields can be achieved in a facile manner. In the study, lipid metabolomics was performed in an attempt to enhance EPA biosynthesis in Pythium splendens. Synthetic, metabolic regulation and gene expression analyses were conducted to clarify the mechanism of EPA biosynthesis, and guide optimization of EPA production. The results showed that the Δ 6 desaturase pathway is the main EPA biosynthetic route in this organism, and ∆ 6 , ∆ 12 and Δ 17 desaturases are the rate-limiting enzymes. All the three desaturase genes were separately introduced into the parent strain to increase the flow of fatty acids into the Δ 6 desaturase pathway. Enhanced expression of these key enzymes, in combination with improved regulation of metabolism, resulted in a maximum yield of 1.43 g/L in the D12 transgenic strain, which represents a tenfold increase over the parent strain before optimization. This is the higher EPA production yield yet reported for a microbial system. Our findings may allow the production of EPA at an industrial scale, and the strategy employed could be used to increase the production of EPA or other lipids in oleaginous microorganisms.

Suppression of the rice fatty-acid desaturase gene OsSSI2 enhances resistance to blast and leaf blight diseases in rice.

PubMed

Jiang, Chang-Jie; Shimono, Masaki; Maeda, Satoru; Inoue, Haruhiko; Mori, Masaki; Hasegawa, Morifumi; Sugano, Shoji; Takatsuji, Hiroshi

2009-07-01

Fatty acids and their derivatives play important signaling roles in plant defense responses. It has been shown that suppressing a gene for stearoyl acyl carrier protein fatty-acid desaturase (SACPD) enhances the resistance of Arabidopsis (SSI2) and soybean to multiple pathogens. In this study, we present functional analyses of a rice homolog of SSI2 (OsSSI2) in disease resistance of rice plants. A transposon insertion mutation (Osssi2-Tos17) and RNAi-mediated knockdown of OsSSI2 (OsSSI2-kd) reduced the oleic acid (18:1) level and increased that of stearic acid (18:0), indicating that OsSSI2 is responsible for fatty-acid desaturase activity. These plants displayed spontaneous lesion formation in leaf blades, retarded growth, slight increase in endogenous free salicylic acid (SA) levels, and SA/benzothiadiazole (BTH)-specific inducible genes, including WRKY45, a key regulator of SA/BTH-induced resistance, in rice. Moreover, the OsSSI2-kd plants showed markedly enhanced resistance to the blast fungus Magnaporthe grisea and leaf-blight bacteria Xanthomonas oryzae pv. oryzae. These results suggest that OsSSI2 is involved in the negative regulation of defense responses in rice, as are its Arabidopsis and soybean counterparts. Microarray analyses identified 406 genes that were differentially expressed (>or=2-fold) in OsSSI2-kd rice plants compared with wild-type rice and, of these, approximately 39% were BTH responsive. Taken together, our results suggest that induction of SA-responsive genes, including WRKY45, is likely responsible for enhanced disease resistance in OsSSI2-kd rice plants.

Dietary fish oil supplements increase tissue n-3 fatty acid composition and expression of delta-6 desaturase and elongase-2 in Jade Tiger hybrid abalone.

PubMed

Mateos, Hintsa T; Lewandowski, Paul A; Su, Xiao Q

2011-08-01

This study was conducted to investigate the effects of fish oil (FO) supplements on fatty acid composition and the expression of ∆6 desaturase and elongase 2 genes in Jade Tiger abalone. Five test diets were formulated to contain 0.5, 1.0, 1.5, 2.0 and 2.5% of FO respectively, and the control diet was the normal commercial abalone diet with no additional FO supplement. The muscle, gonad and digestive glands (DG) of abalone fed with all of the five test diets showed significantly high levels of total n-3 polyunsaturated fatty acid (PUFA), eicosapentaenoic acid (EPA), docosapentaenoic acid n-3 (DPAn-3), and docosahexaenoic acid (DHA) than the control group. In all three types of tissue, abalone fed diet supplemented with 1.5% FO showed the highest level of these fatty acids (P < 0.05). For DPAn-3 the higher level was also found in muscle and gonad of abalone fed diet supplemented with 2% FO (P < 0.05). Elongase 2 expression was markedly higher in the muscle of abalone fed diet supplemented with 1.5% FO (P < 0.05), followed by the diet containing 2% FO supplement. For ∆6 desaturase, significantly higher expression was observed in muscle of abalone fed with diet containing 0.5% FO supplement (P < 0.05). Supplementation with FO in the normal commercial diet can significantly improve long chain n-3 PUFA level in cultured abalone, with 1.5% being the most effective supplementation level.

Selection in Europeans on Fatty Acid Desaturases Associated with Dietary Changes

PubMed Central

Buckley, Matthew T.; Racimo, Fernando; Allentoft, Morten E.; Jensen, Majken K.; Jonsson, Anna; Huang, Hongyan; Hormozdiari, Farhad; Sikora, Martin; Marnetto, Davide; Eskin, Eleazar; Jørgensen, Marit E.; Grarup, Niels; Pedersen, Oluf; Hansen, Torben; Kraft, Peter; Willerslev, Eske

2017-01-01

Abstract FADS genes encode fatty acid desaturases that are important for the conversion of short chain polyunsaturated fatty acids (PUFAs) to long chain fatty acids. Prior studies indicate that the FADS genes have been subjected to strong positive selection in Africa, South Asia, Greenland, and Europe. By comparing FADS sequencing data from present-day and Bronze Age (5–3k years ago) Europeans, we identify possible targets of selection in the European population, which suggest that selection has targeted different alleles in the FADS genes in Europe than it has in South Asia or Greenland. The alleles showing the strongest changes in allele frequency since the Bronze Age show associations with expression changes and multiple lipid-related phenotypes. Furthermore, the selected alleles are associated with a decrease in linoleic acid and an increase in arachidonic and eicosapentaenoic acids among Europeans; this is an opposite effect of that observed for selected alleles in Inuit from Greenland. We show that multiple SNPs in the region affect expression levels and PUFA synthesis. Additionally, we find evidence for a gene–environment interaction influencing low-density lipoprotein (LDL) levels between alleles affecting PUFA synthesis and PUFA dietary intake: carriers of the derived allele display lower LDL cholesterol levels with a higher intake of PUFAs. We hypothesize that the selective patterns observed in Europeans were driven by a change in dietary composition of fatty acids following the transition to agriculture, resulting in a lower intake of arachidonic acid and eicosapentaenoic acid, but a higher intake of linoleic acid and α-linolenic acid. PMID:28333262

Cloning and functional expression of a cDNA encoding stearoyl-ACP Δ9-desaturase from the endosperm of coconut (Cocos nucifera L.).

PubMed

Gao, Lingchao; Sun, Ruhao; Liang, Yuanxue; Zhang, Mengdan; Zheng, Yusheng; Li, Dongdong

2014-10-01

Coconut (Cocos nucifera L.) is an economically tropical fruit tree with special fatty acid compositions. The stearoyl-acyl carrier protein (ACP) desaturase (SAD) plays a key role in the properties of the majority of cellular glycerolipids. In this paper, a full-length cDNA of a stearoyl-acyl carrier protein desaturase, designated CocoFAD, was isolated from cDNA library prepared from the endosperm of coconut (C. nucifera L.). An 1176 bp cDNA from overlapped PCR products containing ORF encoding a 391-amino acid (aa) protein was obtained. The coded protein was virtually identical and shared the homology to other Δ9-desaturase plant sequences (greater than 80% as similarity to that of Elaeis guineensis Jacq). The real-time fluorescent quantitative PCR result indicated that the yield of CocoFAD was the highest in the endosperm of 8-month-old coconut and leaf, and the yield was reduced to 50% of the highest level in the endosperm of 15-month-old coconut. The coding region showed heterologous expression in strain INVSc1 of yeast (Saccharomyces cerevisiae). GC-MS analysis showed that the levels of palmitoleic acid (16:1) and oleic acid (18:1) were improved significantly; meanwhile stearic acid (18:0) was reduced. These results indicated that the plastidial Δ9 desaturase from the endosperm of coconut was involved in the biosynthesis of hexadecenoic acid and octadecenoic acid, which was similar with other plants. These results may be valuable for understanding the mechanism of fatty acid metabolism and the genetic improvement of CocoFAD gene in palm plants in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

Cloning and characterization of a delta-6 desaturase encoding gene from Nannochloropsis oculata

NASA Astrophysics Data System (ADS)

Ma, Xiaolei; Yu, Jianzhong; Zhu, Baohua; Pan, Kehou; Pan, Jin; Yang, Guanpin

2011-03-01

A gene ( NANOC-D6D) encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata (Droop) D. J. Hibberd (Eustigmatophyceae). The unicellular marine microalga N. oculata synthesizes rich long chain polyunsaturated fatty acids (LCPUFAs), including eicosapentaenoic acid (20:5n-3, EPA). The deduced protein contains 474 amino acids that fold into 4 trans-membrane domains. The neighbor-joining phylogenetic tree indicates that NANOC-D6D is phylogenetically close to the delta-6 fatty acid desaturase of marine microalgae such as Glossomastix chrysoplasta, Thalassiosira pseudonana, and Phaeodactylum tricornutum. The gene was expressed in Saccharomyces cerevisiae INVScl to verify the substrate specificity of NANOC-D6D. Our results suggest that the recombinant NANOC-D6D simultaneously desaturates linoleic acid (LA) and α-linolenic acid (ALA).

[Overexpression of four fatty acid synthase genes elevated the efficiency of long-chain polyunsaturated fatty acids biosynthesis in mammalian cells].

PubMed

Zhu, Guiming; Saleh, Abdulmomen Ali Mohammed; Bahwal, Said Ahmed; Wang, Kunfu; Wang, Mingfu; Wang, Didi; Ge, Tangdong; Sun, Jie

2014-09-01

Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian A6/A5 fatty acid desaturases and multiple-genes expression vector carrying mammalian Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.

Characterization of a stearoyl-acyl carrier protein desaturase gene family from chocolate tree, Theobroma cacao L

PubMed Central

Zhang, Yufan; Maximova, Siela N.; Guiltinan, Mark J.

2015-01-01

In plants, the conversion of stearoyl-ACP to oleoyol-ACP is catalyzed by a plastid-localized soluble stearoyl-acyl carrier protein (ACP) desaturase (SAD). The activity of SAD significantly impacts the ratio of saturated and unsaturated fatty acids, and is thus a major determinant of fatty acid composition. The cacao genome contains eight putative SAD isoforms with high amino acid sequence similarities and functional domain conservation with SAD genes from other species. Sequence variation in known functional domains between different SAD family members suggested that these eight SAD isoforms might have distinct functions in plant development, a hypothesis supported by their diverse expression patterns in various cacao tissues. Notably, TcSAD1 is universally expressed across all the tissues, and its expression pattern in seeds is highly correlated with the dramatic change in fatty acid composition during seed maturation. Interestingly, TcSAD3 and TcSAD4 appear to be exclusively and highly expressed in flowers, functions of which remain unknown. To test the function of TcSAD1 in vivo, transgenic complementation of the Arabidopsis ssi2 mutant was performed, demonstrating that TcSAD1 successfully rescued all AtSSI2 related phenotypes further supporting the functional orthology between these two genes. The identification of the major SAD gene responsible for cocoa butter biosynthesis provides new strategies for screening for novel genotypes with desirable fatty acid compositions, and for use in breeding programs to help pyramid genes for quality and other traits such as disease resistance. PMID:25926841

Production of Delta(1)-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris.

PubMed

Taura, Futoshi; Dono, Emi; Sirikantaramas, Supaart; Yoshimura, Kohji; Shoyama, Yukihiro; Morimoto, Satoshi

2007-09-28

Delta(1)-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of Delta(1)-tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted approximately 1.32nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a approximately 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure-function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.

Menhaden oil, but not safflower or soybean oil, aids in restoring the polyunsaturated fatty acid profile in the novel delta-6-desaturase null mouse

PubMed Central

2012-01-01

Background Polyunsaturated fatty acids (PUFA) have diverse biological effects, from promoting inflammation to preventing cancer and heart disease. Growing evidence suggests that individual PUFA may have independent effects in health and disease. The individual roles of the two essential PUFA, linoleic acid (LA) and α-linolenic acid (ALA), have been difficult to discern from the actions of their highly unsaturated fatty acid (HUFA) downstream metabolites. This issue has recently been addressed through the development of the Δ-6 desaturase knock out (D6KO) mouse, which lacks the rate limiting Δ-6 desaturase enzyme and therefore cannot metabolize LA or ALA. However, a potential confounder in this model is the production of novel Δ-5 desaturase (D5D) derived fatty acids when D6KO mice are fed diets containing LA and ALA, but void of arachidonic acid. Objective The aim of the present study was to characterize how the D6KO model differentially responds to diets containing the essential n-6 and n-3 PUFA, and whether the direct provision of downstream HUFA can rescue the phenotype and prevent the production of D5D fatty acids. Methodology Liver and serum phospholipid (PL) fatty acid composition was examined in D6KO and wild type mice fed i) 10% safflower oil diet (SF, LA rich) ii) 10% soy diet (SO, LA+ALA) or iii) 3% menhaden oil +7% SF diet (MD, HUFA rich) for 28 days (n = 3-7/group). Results Novel D5D fatty acids were found in liver PL of D6KO fed SF or SO-fed mice, but differed in the type of D5D fatty acid depending on diet. Conversely, MD-fed D6KO mice had a liver PL fatty acid profile similar to wild-type mice. Conclusions Through careful consideration of the dietary fatty acid composition, and especially the HUFA content in order to prevent the synthesis of D5D fatty acids, the D6KO model has the potential to elucidate the independent biological and health effects of the parent n-6 and n-3 fatty acids, LA and ALA. PMID:22642787

Expression of a desaturase gene, desat1, in neural and nonneural tissues separately affects perception and emission of sex pheromones in Drosophila

PubMed Central

Bousquet, François; Nojima, Tetsuya; Houot, Benjamin; Chauvel, Isabelle; Chaudy, Sylvie; Dupas, Stéphane; Yamamoto, Daisuke; Ferveur, Jean-François

2012-01-01

Animals often use sex pheromones for mate choice and reproduction. As for other signals, the genetic control of the emission and perception of sex pheromones must be tightly coadapted, and yet we still have no worked-out example of how these two aspects interact. Most models suggest that emission and perception rely on separate genetic control. We have identified a Drosophila melanogaster gene, desat1, that is involved in both the emission and the perception of sex pheromones. To explore the mechanism whereby these two aspects of communication interact, we investigated the relationship between the molecular structure, tissue-specific expression, and pheromonal phenotypes of desat1. We characterized the five desat1 transcripts—all of which yielded the same desaturase protein—and constructed transgenes with the different desat1 putative regulatory regions. Each region was used to target reporter transgenes with either (i) the fluorescent GFP marker to reveal desat1 tissue expression, or (ii) the desat1 RNAi sequence to determine the effects of genetic down-regulation on pheromonal phenotypes. We found that desat1 is expressed in a variety of neural and nonneural tissues, most of which are involved in reproductive functions. Our results suggest that distinct desat1 putative regulatory regions independently drive the expression in nonneural and in neural cells, such that the emission and perception of sex pheromones are precisely coordinated in this species. PMID:22114190

ω-3 Long-Chain Polyunsaturated Fatty Acids and Fatty Acid Desaturase Activity Ratios as Eventual Endophenotypes for ADHD.

PubMed

Henríquez-Henríquez, Marcela; Solari, Sandra; Várgas, Gisela; Vásquez, Luis; Allende, Fidel; Castañón S, Carla; Tenorio, Marcela; Quiroga Gutiérrez, Teresa

2015-11-01

Epidemiological studies suggest that long-chain polyunsaturated fatty acids (LC-PUFAs) may be suitable as endophenotypes for ADHD. To be appropriated vulnerability traits, endophenotypes should be altered in unaffected relatives of index cases. Serum profiles of LC-PUFAs in unaffected relatives of ADHD patients remain understudied. The main objective of this study was to compare serum LC-PUFAs in ADHD patients, unaffected relatives of index cases, and general-population unaffected participants. LC-PUFA profiles of 72 participants (27 ADHD patients, 27 unaffected relatives, and 18 general-population participants) were obtained by gas chromatography-mass spectrometry (GC-MS). Groups were compared by parametrical statistics. Unaffected females from the general population presented lower Docosapentaenoic acid (DPA; p = .0012) and a-linolenic acid (ALA; p = .0091) levels compared with ADHD females and unaffected relatives. In addition, docosahexaenoic acid (DHA)/ALA and DHA/DPA ratios, addressing desaturase activity, were significantly lower in ADHD patients and unaffected relatives of ADHD patients in the female-subgroup (p = .022 and .04, respectively). DHA/ALA, DHA/DPA, serum DPA, and serum ALA may be suitable as endophenotypes for ADHD women. © The Author(s) 2012.

Factors affecting the palmitoyl-coenzyme A desaturase of Saccharomyces cerevisiae

NASA Technical Reports Server (NTRS)

Klein, H. P.; Volkmann, C. M.

1975-01-01

The activity and stability of the palmitoyl-coenzyme A (CoA) desaturase complex of Saccharomyces cerevisiae was influenced by several factors. Cells, grown nonaerobically and then incubated with glucose, either in air or under N2, showed a marked increase in desaturase activity. Cycloheximide, added during such incubations, prevented the increase in activity, suggesting de novo synthesis. The stability of the desaturase from cells grown nonaerobically was affected by subsequent treatment of the cells; enzyme from freshly harvested cells, or from cells that were then shaken under nitrogen, readily lost activity upon washing or during density gradient analysis, whereas aerated cells, in the presence or absence of glucose, yielded stable enzyme preparations. The loss of activity in nonaerobic preparations could be reversed by adding soluble supernatant from these homogenates and could be prevented by growing the cells in the presence of palmitoleic acid and ergosterol, but not with several other lipids tested.

StearoylCoA Desaturase-5: A Novel Regulator of Neuronal Cell Proliferation and Differentiation

PubMed Central

Sinner, Debora I.; Kim, Gretchun J.; Henderson, Gregory C.; Igal, R. Ariel

2012-01-01

Recent studies have demonstrated that human stearoylCoA desaturase-1 (SCD1), a Δ9-desaturase that converts saturated fatty acids (SFA) into monounsaturated fatty acids, controls the rate of lipogenesis, cell proliferation and tumorigenic capacity in cancer cells. However, the biological function of stearoylCoA desaturase-5 (SCD5), a second isoform of human SCD that is highly expressed in brain, as well as its potential role in human disease, remains unknown. In this study we report that the constitutive overexpression of human SCD5 in mouse Neuro2a cells, a widely used cell model of neuronal growth and differentiation, displayed a greater n-7 MUFA-to-SFA ratio in cell lipids compared to empty-vector transfected cells (controls). De novo synthesis of phosphatidylcholine and cholesterolesters was increased whereas phosphatidylethanolamine and triacylglycerol formation was reduced in SCD5-expressing cells with respect to their controls, suggesting a differential use of SCD5 products for lipogenic reactions. We also observed that SCD5 expression markedly accelerated the rate of cell proliferation and suppressed the induction of neurite outgrowth, a typical marker of neuronal differentiation, by retinoic acid indicating that the desaturase plays a key role in the mechanisms of cell division and differentiation. Critical signal transduction pathways that are known to modulate these processes, such epidermal growth factor receptor (EGFR)Akt/ERK and Wnt, were affected by SCD5 expression. Epidermal growth factor-induced phosphorylation of EGFR, Akt and ERK was markedly blunted in SCD5-expressing cells. Furthermore, the activity of canonical Wnt was reduced whereas the non-canonical Wnt was increased by the presence of SCD5 activity. Finally, SCD5 expression increased the secretion of recombinant Wnt5a, a non-canonical Wnt, whereas it reduced the cellular and secreted levels of canonical Wnt7b. Our data suggest that, by a coordinated modulation of key lipogenic pathways and

A single mutation in the castor Delta9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry.

PubMed

Guy, Jodie E; Abreu, Isabel A; Moche, Martin; Lindqvist, Ylva; Whittle, Edward; Shanklin, John

2006-11-14

Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by approximately 2 x 10(3)-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-A crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme.

A single mutation in the castor Δ9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry

PubMed Central

Guy, Jodie E.; Abreu, Isabel A.; Moche, Martin; Lindqvist, Ylva; Whittle, Edward; Shanklin, John

2006-01-01

Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by ≈2 × 103-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-Å crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme. PMID:17088542

Half-of-the-Sites Reactivity of the Castor Δ9-18:0-Acyl Carrier Protein Desaturase.

PubMed

Liu, Qin; Chai, Jin; Moche, Martin; Guy, Jodie; Lindqvist, Ylva; Shanklin, John

2015-09-01

Fatty acid desaturases regulate the unsaturation status of cellular lipids. They comprise two distinct evolutionary lineages, a soluble class found in the plastids of higher plants and an integral membrane class found in plants, yeast (Saccharomyces cerevisiae), animals, and bacteria. Both classes exhibit a dimeric quaternary structure. Here, we test the functional significance of dimeric organization of the soluble castor Δ9-18:0-acyl carrier protein desaturase, specifically, the hypothesis that the enzyme uses an alternating subunit half-of-the-sites reactivity mechanism whereby substrate binding to one subunit is coordinated with product release from the other subunit. Using a fluorescence resonance energy transfer assay, we demonstrated that dimers stably associate at concentrations typical of desaturase assays. An active site mutant T104K/S202E, designed to occlude the substrate binding cavity, was expressed, purified, and its properties validated by x-ray crystallography, size exclusion chromatography, and activity assay. Heterodimers comprising distinctly tagged wild-type and inactive mutant subunits were purified at 1:1 stoichiometry. Despite having only one-half the number of active sites, purified heterodimers exhibit equivalent activity to wild-type homodimers, consistent with half-of-the-sites reactivity. However, because multiple rounds of turnover were observed, we conclude that substrate binding to one subunit is not required to facilitate product release from the second subunit. The observed half-of-the-sites reactivity could potentially buffer desaturase activity from oxidative inactivation. That soluble desaturases require only one active subunit per dimer for full activity represents a mechanistic difference from the membrane class of desaturases such as the Δ9-acyl-CoA, Ole1p, from yeast, which requires two catalytically competent subunits for activity. © 2015 American Society of Plant Biologists. All Rights Reserved.

SCD1 activity in muscle increases triglyceride PUFA content, exercise capacity, and PPARδ expression in mice[S

PubMed Central

Rogowski, Michael P.; Flowers, Matthew T.; Stamatikos, Alexis D.; Ntambi, James M.; Paton, Chad M.

2013-01-01

Stearoyl-CoA desaturase (SCD)1 converts saturated fatty acids into monounsaturated fatty acids. Using muscle overexpression, we sought to determine the role of SCD1 expression in glucose and lipid metabolism and its effects on exercise capacity in mice. Wild-type C57Bl/6 (WT) and SCD1 muscle transgenic (SCD1-Tg) mice were generated, and expression of the SCD1 transgene was restricted to skeletal muscle. SCD1 overexpression was associated with increased triglyceride (TG) content. The fatty acid composition of the muscle revealed a significant increase in polyunsaturated fatty acid (PUFA) content of TG, including linoleate (18:2n6). Untrained SCD1-Tg mice also displayed significantly increased treadmill exercise capacity (WT = 6.6 ± 3 min, Tg = 71.9 ± 9.5 min; P = 0.0009). SCD1-Tg mice had decreased fasting plasma glucose, glucose transporter (GLUT)1 mRNA, fatty acid oxidation, mitochondrial content, and increased peroxisome proliferator-activated receptor (PPAR)δ and Pgc-1 protein expression in skeletal muscle. In vitro studies in C2C12 myocytes revealed that linoleate (18:2n6) and not oleate (18:1n9) caused a 3-fold increase in PPARδ and a 9-fold increase in CPT-1b with a subsequent increase in fat oxidation. The present model suggests that increasing delta-9 desaturase activity of muscle increases metabolic function, exercise capacity, and lipid oxidation likely through increased PUFA content, which increases PPARδ expression and activity. However, the mechanism of action that results in increased PUFA content of SCD1-Tg mice remains to be elucidated. PMID:23918045

Obesity resistance and deregulation of lipogenesis in Δ6-fatty acid desaturase (FADS2) deficiency.

PubMed

Stoffel, Wilhelm; Hammels, Ina; Jenke, Britta; Binczek, Erika; Schmidt-Soltau, Inga; Brodesser, Susanne; Odenthal, Margarete; Thevis, Mario

2014-01-01

Δ-6-fatty acid desaturase (FADS2) is the key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs), the essential structural determinants of mammalian membrane lipid-bilayers. We developed the auxotrophic fads2(-/-) mouse mutant to assess the enigmatic role of ω3- and ω6-PUFAs in lipid homeostasis, membrane structure and function. Obesity resistance is another major phenotype of the fads2(-/-) mutant, the molecular basis of which is unknown. Phospholipidomic profiling of membrane systems of fads2(-/-)mice revealed diacylglycerol-structures, deprived of PUFAs but substituted with surrogate eicosa-5,11,14-trienoic acid. ω6-Arachidonic (AA) and ω3-docosahexaenoic acid (DHA) supplemented diets transformed fads2(-/-) into AA-fads2(-/-) and DHA-fads2(-/-) mutants. Severely altered phospholipid-bilayer structures of subcellular membranes of fads2(-/-) liver specifically interfered with maturation of transcription factor sterol-regulatory-element-binding protein, the key regulator of lipogenesis and lipid homeostasis. This study strengthens the concept that specific PUFA-substituted membrane phospholipid species are critical constituents of the structural platform operative in lipid homeostasis in normal and disease conditions.

Formation of conjugated delta8,delta10-double bonds by delta12-oleic-acid desaturase-related enzymes: biosynthetic origin of calendic acid.

PubMed

Cahoon, E B; Ripp, K G; Hall, S E; Kinney, A J

2001-01-26

Divergent forms of the plant Delta(12)-oleic-acid desaturase (FAD2) have previously been shown to catalyze the formation of acetylenic bonds, epoxy groups, and conjugated Delta(11),Delta(13)-double bonds by modification of an existing Delta(12)-double bond in C(18) fatty acids. Here, we report a class of FAD2-related enzymes that modifies a Delta(9)-double bond to produce the conjugated trans-Delta(8),trans-Delta(10)-double bonds found in calendic acid (18:3Delta(8trans,10trans,12cis)), the major component of the seed oil of Calendula officinalis. Using an expressed sequence tag approach, cDNAs for two closely related FAD2-like enzymes, designated CoFADX-1 and CoFADX-2, were identified from a C. officinalis developing seed cDNA library. The deduced amino acid sequences of these polypeptides share 40-50% identity with those of other FAD2 and FAD2-related enzymes. Expression of either CoFADX-1 or CoFADX-2 in somatic soybean embryos resulted in the production of calendic acid. In embryos expressing CoFADX-2, calendic acid accumulated to as high as 22% (w/w) of the total fatty acids. In addition, expression of CoFADX-1 and CoFADX-2 in Saccharomyces cerevisiae was accompanied by calendic acid accumulation when induced cells were supplied exogenous linoleic acid (18:2Delta(9cis,12cis)). These results are thus consistent with a route of calendic acid synthesis involving modification of the Delta(9)-double bond of linoleic acid. Regiospecificity for Delta(9)-double bonds is unprecedented among FAD2-related enzymes and further expands the functional diversity found in this family of enzymes.

Cloning and expression analysis of phytoene desaturase and ζ-carotene desaturase genes in Carica papaya.

PubMed

Yan, P; Gao, X Z; Shen, W T; Zhou, P

2011-02-01

The fruit flesh color of papaya is an important nutritional quality trait and is due to the accumulation of carotenoid. To elucidate the carotenoid biosynthesis pathway in Carica papaya, the phytoene desaturase (PDS) and the ζ-carotene desaturase (ZDS) genes were isolated from papaya (named CpPDS and CpZDS) using the rapid amplification of cDNA ends (RACE) approach, and their expression levels were investigated in red- and yellow-fleshed papaya varieties. CpPDS contains a 1749 bp open reading frame coding for 583 amino acids, while CpZDS contains a 1716 bp open reading frame coding for 572 amino acids. The deduced CpPDS and CpZDS proteins contain a conserved dinucleotide-binding site at the N-terminus and a carotenoid-binding domain at the C-terminus. Papaya genome sequence analysis revealed that CpPDS and CpZDS are single copy; the CpPDS was mapped to papaya chromosome LG6, and the CpZDS was mapped to chromosome LG3. Quantitative PCR showed that both CpPDS and CpZDS were expressed in all tissues examined with the highest expression in maturing fruits, and that the expression of CpPDS and CpZDS were higher in red-fleshed fruits than in yellow-fleshed fruits. These results indicated that the differential accumulation of carotenoids in red- and yellow-fleshed papaya varieties might be partly explained by the transcriptional level of CpPDS and CpZDS.

Determination of fatty acid composition in seed oil of rapeseed (Brassica napus L.) by mutated alleles of the FAD3 desaturase genes.

PubMed

Bocianowski, Jan; Mikołajczyk, Katarzyna; Bartkowiak-Broda, Iwona

2012-02-01

One of the goals in oilseed rape programs is to develop genotypes producing oil with low linolenic acid content (C18:3, ≤3%). Low linolenic mutant lines of canola rapeseed were obtained via chemical mutagenesis at the Plant Breeding and Acclimatization Institute - NRI, in Poznan, Poland, and allele-specific SNP markers were designed for monitoring of two statistically important single nucleotide polymorphisms detected by SNaPshot analysis in two FAD3 desaturase genes, BnaA.FAD3 and BnaC.FAD3, respectively. Strong negative correlation between the presence of mutant alleles of the genes and linolenic acid content was revealed by analysis of variance. In this paper we present detailed characteristics of the markers by estimation of the additive and dominance effects of the FAD3 genes with respect to particular fatty acid content in seed oil, as well as by calculation of the phenotypic variation of seed oil fatty acid composition accounted by particular allele-specific marker. The obtained percentage of variation in fatty acid composition was considerable only for linolenic acid content and equaled 35.6% for BnaA.FAD3 and 39.3% for BnaC.FAD3, whereas the total percentage of variation in linolenic acid content was 53.2% when accounted for mutations in both genes simultaneously. Our results revealed high specificity of the markers for effective monitoring of the wild-type and mutated alleles of the Brassica napus FAD3 desaturase genes in the low linolenic mutant recombinants in breeding programs.

Perfluorodecanoic acid enhances the formation of oleic acid in rat liver.

PubMed Central

Yamamoto, A; Kawashima, Y

1997-01-01

The feeding of perfluorodecanoic acid (PFDA) to male rats at a dietary concentration of 0.005% (w/w) for 7 days resulted in a marked increase in the activity of microsomal stearoyl-CoA desaturation in the liver. This increase in the overall desaturation activity was due to the induction of terminal desaturase among the components comprising the desaturation system. In contrast, PFDA inhibited desaturation in vitro, seemingly due to interference with electron transport through the desaturation system. Accordingly, PFDA can be an inducer and also an inhibitor of delta9-desaturation. PFDA feeding enhanced the conversion of radioactive stearic acid into oleic acid in the liver in vivo, indicating that the induction of delta9-desaturase by PFDA functions in vivo. PFDA feeding increased the mass of octadecenoic acid (C18:1) in the liver and the proportion of C18:1 in microsomal lipid. A highly significant linear correlation existed between the microsomal desaturase activity and the proportion of C18:1 in microsomal lipid when compared using rats in five different physiological states: control, PFDA-fed, p-chlorophenoxyisobutyric acid (clofibric acid)-fed, starved and starved/refed. These results suggest that the increase in the hepatic level of C18:1 caused by feeding of PFDA to rats can be explained by the common concept of regulation, i.e. the hepatic level of C18:1 is under the control of delta9-desaturase. The dietary administration of PFDA also increased the content of cytochrome P-450 and the activity of 7-ethoxycoumarin O-de-ethylase in the liver. PMID:9230124

Omega-3 Fatty Acid Deficiency Augments Risperidone-Induced Hepatic Steatosis in Rats: Positive Association with Stearoyl-CoA Desaturase

PubMed Central

McNamara, Robert K.; Magrisso, I. Jack; Hofacer, Rylon; Jandacek, Ronald; Rider, Therese; Tso, Patrick; Benoit, Stephen C.

2012-01-01

Psychiatric patients frequently exhibit long-chain n-3 (LCn-3) fatty acid deficits and elevated triglyceride (TAG) production following chronic exposure to second generation antipsychotics (SGA). Emerging evidence suggests that SGAs and LCn-3 fatty acids have opposing effects on stearoyl-CoA desaturase-1 (SCD1), which plays a pivotal role in TAG biosynthesis. Here we evaluated whether low LCn-3 fatty acid status would augment elevations in rat liver and plasma TAG concentrations following chronic treatment with the SGA risperidone (RSP), and evaluated relationships with hepatic SCD1 expression and activity indices. In rats maintained on the n-3 fatty acid-fortified (control) diet, chronic RSP treatment significantly increased liver SCD1 mRNA and activity indices (18:1/18:0 and 16:1/16:0 ratios), and significantly increased liver, but not plasma, TAG concentrations. Rats maintained on the n-3 deficient diet exhibited significantly lower liver and erythrocyte LCn-3 fatty acid levels, and associated elevations in LCn-6/LCn-3 ratio. In n-3 deficient rats, RSP-induced elevations in liver SCD1 mRNA and activity indices (18:1/18:0 and 16:1/16:0 ratios) and liver and plasma TAG concentrations were significantly greater than those observed in RSP-treated controls. Plasma glucose levels were not altered by diet or RSP, and body weight was lower in RSP- and VEH-treated n-3 deficient rats. These preclinical data support the hypothesis that low n-3 fatty acid status exacerbates RSP-induced hepatic steatosis by augmenting SCD1 expression and activity. PMID:22750665

Influence of oilseed supplement ranging in n-6/n-3 ratio on fatty acid composition and Δ5-, Δ6-desaturase protein expression in steer muscles.

PubMed

Turner, T D; Mitchell, A; Duynisveld, J; Pickova, J; Doran, O; McNiven, M A

2012-12-01

This study investigated effects of roasted or extruded oilseed supplementation ranging in n-6/n-3 ratios from 0.3 to 5.0 on the fatty acid composition and expression of delta-5 desaturase (Δ5d) and Δ6-desaturase (Δ6d) protein in commercial steer cheek (m. masseter) and diaphragm (pars costalis diaphragmatis) muscles. In general, the n-6/n-3 ratio of the diet had a subsequent effect on the muscle n-6/n-3 ratio (P < 0.05), with muscle 18:2n-6 and 18:3n-3 content relating to proportion of dietary soya bean and linseed (P < 0.01). Compared with canola, pure linseed and soya bean diets reduced 14:1c-9 and 16:1c-9 (P < 0.05) but increased 18:1t-11 and c-9,t-11 conjugated linoleic acid (CLA) content (P < 0.01). Oilseed processing had a minor influence but extruded oilseeds increase 18:1t-11 and c-9,t-11 CLA compared with roasted (P < 0.05). Polar lipid 18:3n-3 and n-3 long-chain polyunsaturated fatty acid (LC, ⩾20 carbons PUFA) derivative content increased in relation to dietary linseed supplementation in the diaphragm (P < 0.01), whereas only 18:3n-3 was increased in the cheek (P < 0.01). Protein expression did not differ between diets; however, in each muscle the Δ5d protein expression had a stronger association with the desaturase products rather than the precursors. The relationship between Δ5d protein expression and the muscle LC n-6/n-3 ratio was negative in both muscles (P < 0.05). The relationship between Δ6d protein expression and the LC n-6/n-3 ratio was positive in the cheek (P < 0.001) and negative in the diaphragm (P < 0.05). In conclusion, diet n-6/n-3 ratio affected muscle 18:2n-6 and 18:3n-3 deposition, whereas the Δ5d and Δ6d protein expression had some influence on the polar lipid LC-PUFA profile. Results reaffirm that processed oilseeds can be used to increase the proportion of fatty acids potentially beneficial for human health, by influencing the formation of LC-PUFA and reducing the n-6/n-3 ratio.

Differences in Arachidonic Acid Levels and Fatty Acid Desaturase (FADS) Gene Variants in African Americans and European Americans with Diabetes/Metabolic Syndrome

PubMed Central

Sergeant, Susan; Hugenschmidt, Christina E.; Rudock, Megan E.; Ziegler, Julie T.; Ivester, Priscilla; Ainsworth, Hannah C.; Vaidya, Dhananjay; Case, L. Douglas; Langefeld, Carl D.; Freedman, Barry I.; Bowden, Donald W.; Mathias, Rasika A.; Chilton, Floyd H.

2012-01-01

Over the past 50 years, increases in dietary n-6 polyunsaturated fatty acids (PUFAs), such as linoleic acid, have been hypothesized to cause or exacerbate chronic inflammatory diseases. This study examines an individual’s innate capacity to synthesize n-6-long chain PUFAs (LC-PUFAs), with respect to the fatty acid desaturase (FADS) locus in Americans of African and European descent with diabetes/metabolic syndrome. Compared to European Americans (EAm), African Americans (AfAm) exhibited markedly higher serum levels of arachidonic acid (AA) (EAm 7.9±2.1; AfAm 9.8±1.9 % of total fatty acids, mean ± sd; p<2.29×10−9) and the AA to n-6-precursor fatty acid ratio, which estimates FADS1 activity (EAm 5.4±2.2, AfAm 6.9±2.2; p=1.44×10−5). Seven single nucleotide polymorphisms (SNP) mapping to the FADS locus revealed strong association with AA, eicosapentaenoic acid (EPA) and dihomogamma-linolenic acid (DGLA) in the EAm. Importantly, EAm homozygous for the minor allele (T) had significantly lower AA levels (TT: 6.3±1.0; GG: 8.5±2.1; p=3.0×10−5) and AA/DGLA ratios (TT: 3.4±0.8; GG: 6.5±2.3; p=2.2×10−7) but higher DGLA levels (TT: 1.9±0.4; GG: 1.4±0.4; p=3.3×10−7) compared to those homozygous for the major allele (GG). Allele frequency patterns suggest that the GG genotype at rs174537 (associated with higher circulating levels of AA) is much higher in AfAm (0.81) compared to EAm (0.46). Similarly, marked differences in rs174537 genotypic frequencies were observed in HapMap populations. These data suggest that there are likely important differences in the capacity of different populations to synthesize LC-PUFAs. These differences may provide a genetic mechanism contributing to health disparities between populations of African and European descent. PMID:21733300

Omega-3 fatty acid desaturase gene family from two ω-3 sources, Salvia hispanica and Perilla frutescens: Cloning, characterization and expression

PubMed Central

Xue, Yufei; Chen, Baojun; Win, Aung Naing; Fu, Chun; Lian, Jianping; Liu, Xue; Wang, Rui; Zhang, Xingcui

2018-01-01

Omega-3 fatty acid desaturase (ω-3 FAD, D15D) is a key enzyme for α-linolenic acid (ALA) biosynthesis. Both chia (Salvia hispanica) and perilla (Perilla frutescens) contain high levels of ALA in seeds. In this study, the ω-3 FAD gene family was systematically and comparatively cloned from chia and perilla. Perilla FAD3, FAD7, FAD8 and chia FAD7 are encoded by single-copy (but heterozygous) genes, while chia FAD3 is encoded by 2 distinct genes. Only 1 chia FAD8 sequence was isolated. In these genes, there are 1 to 6 transcription start sites, 1 to 8 poly(A) tailing sites, and 7 introns. The 5’UTRs of PfFAD8a/b contain 1 to 2 purine-stretches and 2 pyrimidine-stretches. An alternative splice variant of ShFAD7a/b comprises a 5’UTR intron. Their encoded proteins harbor an FA_desaturase conserved domain together with 4 trans-membrane helices and 3 histidine boxes. Phylogenetic analysis validated their identity of dicot microsomal or plastidial ω-3 FAD proteins, and revealed some important evolutionary features of plant ω-3 FAD genes such as convergent evolution across different phylums, single-copy status in algae, and duplication events in certain taxa. The qRT-PCR assay showed that the ω-3 FAD genes of two species were expressed at different levels in various organs, and they also responded to multiple stress treatments. The functionality of the ShFAD3 and PfFAD3 enzymes was confirmed by yeast expression. The systemic molecular and functional features of the ω-3 FAD gene family from chia and perilla revealed in this study will facilitate their use in future studies on genetic improvement of ALA traits in oilseed crops. PMID:29351555

Stearoyl-CoA desaturase activity is elevated by the suppression of its degradation by clofibric acid in the liver of rats.

PubMed

Toyama, Tomoaki; Kudo, Naomi; Mitsumoto, Atsushi; Hibino, Yasuhide; Tsuda, Tadashi; Kawashima, Yoichi

2007-04-01

A mechanism by which fibrates control stearoyl-CoA desaturase (SCD) in the liver was studied. Treatment of rats with 2-(4-chlorophenoxy)-2-methylpropionic acid (clofibric acid) or feeding of a fat-free diet markedly elevated hepatic activity of SCD. Both the treatment with clofibric acid and the feeding of the fat-free diet caused an increase in the steady-state level of SCD1 mRNA and enhanced transcriptional rate. The half-lives of SCD for control rats, rats treated with clofibric acid rats, and rats fed the fat-free diet were estimated to be 2.0, 3.9, and 1.9 h, respectively. Activity of palmitoyl-CoA chain elongase (PCE) was increased by both clofibric acid treatment and feeding of the fat-free diet as was observed with SCD. Steady-state level of rat fatty acid elongase 2 mRNA was increased by the treatment with clofibric acid or feeding of fat-free diet, although the transcriptional rate was not altered. Different from SCD, PCE was highly stable and its half-life was not changed by either clofibric acid or fat-free diet. These results strongly suggest that the decreased degradation of SCD is responsible for the increase in its activity in addition to increased transcription of SCD1 in the rats treated with clofibric acid.

Changes in oil content of transgenic soybeans expressing the yeast SLC1 gene.

PubMed

Rao, Suryadevara S; Hildebrand, David

2009-10-01

The wild type (Wt) and mutant form of yeast (sphingolipid compensation) genes, SLC1 and SLC1-1, have been shown to have lysophosphatidic acid acyltransferase (LPAT) activities (Nageic et al. in J Biol Chem 269:22156-22163, 1993). Expression of these LPAT genes was reported to increase oil content in transgenic Arabidopsis and Brassica napus. It is of interest to determine if the TAG content increase would also be seen in soybeans. Therefore, the wild type SLC1 was expressed in soybean somatic embryos under the control of seed specific phaseolin promoter. Some transgenic somatic embryos and in both T2 and T3 transgenic seeds showed higher oil contents. Compared to controls, the average increase in triglyceride values went up by 1.5% in transgenic somatic embryos. A maximum of 3.2% increase in seed oil content was observed in a T3 line. Expression of the yeast Wt LPAT gene did not alter the fatty acid composition of the seed oil.

Why Is Golden Rice Golden (Yellow) Instead of Red?1[w

PubMed Central

Schaub, Patrick; Al-Babili, Salim; Drake, Rachel; Beyer, Peter

2005-01-01

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of β-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, ζ-carotene desaturase, carotene cis-trans-isomerase, β-lycopene cyclase, and β-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that β-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, α/β-lycopene cyclase, β-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or ζ-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the β-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed. PMID:15821145

The food additive vanillic acid controls transgene expression in mammalian cells and mice.

PubMed

Gitzinger, Marc; Kemmer, Christian; Fluri, David A; El-Baba, Marie Daoud; Weber, Wilfried; Fussenegger, Martin

2012-03-01

Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells. When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism. Capitalizing on control components that enable Caulobacter crescentus to metabolize vanillic acid originating from lignin degradation that occurs in its oligotrophic freshwater habitat, we have designed synthetic devices that specifically adjust transgene expression in mammalian cells when exposed to vanillic acid. Even in mice transgene expression was robust, precise and tunable in response to vanillic acid. As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies.

Iron, Oxidative Stress, and Δ9 Stearoyl-CoenzymeA Desaturase Index (C16:1/C16:0): An Analysis Applying the National Health and Nutrition Examination Survey 2003–04

PubMed Central

Wu, Yue; Baylin, Ana; Colacino, Justin A

2018-01-01

Abstract Background Stearoyl-coenzyme A desaturase (SCD) is a key enzyme in fatty acid metabolism, and elevated SCD activity is associated with multiple adverse health outcomes. Diet, hormone levels, and environmental exposures are potential factors affecting SCD activity. Less is known about the relationship between micronutrients, including iron, and SCD activity. Objective The aim of this study was to investigate the association between serum ferritin level, a biomarker of circulating iron levels, and the Δ9 desaturase index (C16:1/C16:0), a biomarker of estimated SCD activity, among women in the United States. Methods The association between serum ferritin and the Δ9 desaturase index was assessed in a cross-sectional study of 447 female participants, aged 20–49 y, from NHANES 2003–2004. The multivariate analyses were performed utilizing generalized linear modeling, adjusting for potential confounders. Mediation of the relationship between serum ferritin and Δ9 desaturase index by γ-glutamyltranspeptidase (GGT), a biomarker of oxidative stress, was also assessed. Results Increased ferritin was significantly associated with a higher Δ9 desaturase index. Adjusting for waist circumference, age, race, and cotinine levels, an interquartile range increase in serum ferritin corresponded to 3.92% (95% CI: 0.88%, 7.05%) higher Δ9 desaturase index. GGT, the biomarker used to measure oxidative stress level, did not appear to mediate the association between ferritin and Δ9 desaturase index. After stratifying by pregnancy status, these associations were limited to nonpregnant individuals. Conclusions Elevated SCD activity may be associated with increased iron storage inside the human body; the association did not appear to be mediated via oxidative stress, as estimated by GGT levels.

Biosynthesis of Polyunsaturated Fatty Acids in the Razor Clam Sinonovacula constricta: Characterization of Δ5 and Δ6 Fatty Acid Desaturases.

PubMed

Ran, Zhaoshou; Xu, Jilin; Liao, Kai; Li, Shuang; Chen, Shubing; Yan, Xiaojun

2018-05-09

To investigate the endogenous long-chain polyunsaturated fatty acid (LC-PUFA) biosynthetic ability in Sinonovacula constricta, fatty acid desaturases (Fads) of this bivalve, namely, Scfad5a, Scfad5b, and Scfad6, were cloned and characterized in the current study. Meanwhile, the tissue distributions of S. constricta Fads and fatty acids (FAs) were examined. Heterologous expression in yeasts confirmed that Scfad5a and Scfad5b were both Δ5 Fads, while Scfad6 was a Δ6 Fad. However, compared with Fads in other organisms, the desaturation activities of S. constricta Fads were relatively low (especially for Scfad6), indicating an adaptation to living conditions. S. constricta Fads were expressed in all tissues examined, and particularly high expressions were found in intestine and gonad. Moreover, FAs were differently distributed among tissues, which might be correlated with their corresponding physiological roles. Taken together, the results provided an insight into LC-PUFA biosynthesis in S. constricta. Notably, Scfad6 was the first functionally characterized Δ6 Fad in marine molluscs to date.

Transgenic soybean overexpressing GmSAMT1 exhibits resistance to multiple-HG types of soybean cyst nematode Heterodera glycines

DOE PAGES

Lin, Jingyu; Mazarei, Mitra; Zhao, Nan; ...

2016-05-23

Soybean ( Glycine max (L.) Merr.) salicylic acid methyl transferase (GmSAMT1) catalyses the conversion of salicylic acid to methyl salicylate. Prior results showed that when GmSAMT1 was overexpressed in transgenic soybean hairy roots, resistance is conferred against soybean cyst nematode (SCN), Heterodera glycines Ichinohe. In this study, we produced transgenic soybean overexpressing GmSAMT1 and characterized their response to various SCN races. Transgenic plants conferred a significant reduction in the development of SCN HG type 1.2.5.7 (race 2), HG type 0 (race 3) and HG type 2.5.7 (race 5). Among transgenic lines, GmSAMT1 expression in roots was positively associated with SCNmore » resistance. In some transgenic lines, there was a significant decrease in salicylic acid titer relative to control plants. No significant seed yield differences were observed between transgenics and control soybean plants grown in one greenhouse with 22 °C day/night temperature, whereas transgenic soybean had higher yield than controls grown a warmer greenhouse (27 °C day/23 °C night) temperature. In a 1-year field experiment in Knoxville, TN, there was no significant difference in seed yield between the transgenic and nontransgenic soybean under conditions with negligible SCN infection. We hypothesize that GmSAMT1 expression affects salicylic acid biosynthesis, which, in turn, attenuates SCN development, without negative consequences to soybean yield or other morphological traits. Furthermore, we conclude that GmSAMT1 overexpression confers broad resistance to multiple SCN races, which would be potentially applicable to commercial production.« less

Transgenic soybean overexpressing GmSAMT1 exhibits resistance to multiple-HG types of soybean cyst nematode Heterodera glycines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Jingyu; Mazarei, Mitra; Zhao, Nan

Soybean ( Glycine max (L.) Merr.) salicylic acid methyl transferase (GmSAMT1) catalyses the conversion of salicylic acid to methyl salicylate. Prior results showed that when GmSAMT1 was overexpressed in transgenic soybean hairy roots, resistance is conferred against soybean cyst nematode (SCN), Heterodera glycines Ichinohe. In this study, we produced transgenic soybean overexpressing GmSAMT1 and characterized their response to various SCN races. Transgenic plants conferred a significant reduction in the development of SCN HG type 1.2.5.7 (race 2), HG type 0 (race 3) and HG type 2.5.7 (race 5). Among transgenic lines, GmSAMT1 expression in roots was positively associated with SCNmore » resistance. In some transgenic lines, there was a significant decrease in salicylic acid titer relative to control plants. No significant seed yield differences were observed between transgenics and control soybean plants grown in one greenhouse with 22 °C day/night temperature, whereas transgenic soybean had higher yield than controls grown a warmer greenhouse (27 °C day/23 °C night) temperature. In a 1-year field experiment in Knoxville, TN, there was no significant difference in seed yield between the transgenic and nontransgenic soybean under conditions with negligible SCN infection. We hypothesize that GmSAMT1 expression affects salicylic acid biosynthesis, which, in turn, attenuates SCN development, without negative consequences to soybean yield or other morphological traits. Furthermore, we conclude that GmSAMT1 overexpression confers broad resistance to multiple SCN races, which would be potentially applicable to commercial production.« less

Diversity of Δ12 Fatty Acid Desaturases in Santalaceae and Their Role in Production of Seed Oil Acetylenic Fatty Acids*

PubMed Central

Okada, Shoko; Zhou, Xue-Rong; Damcevski, Katherine; Gibb, Nerida; Wood, Craig; Hamberg, Mats; Haritos, Victoria S.

2013-01-01

Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family. PMID:24062307

Diversity of Δ12 fatty acid desaturases in santalaceae and their role in production of seed oil acetylenic fatty acids.

PubMed

Okada, Shoko; Zhou, Xue-Rong; Damcevski, Katherine; Gibb, Nerida; Wood, Craig; Hamberg, Mats; Haritos, Victoria S

2013-11-08

Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family.

Carrot Juice Administration Decreases Liver Stearoyl-CoA Desaturase 1 and Improves Docosahexaenoic Acid Levels, but Not Steatosis in High Fructose Diet-Fed Weanling Wistar Rats.

PubMed

Mahesh, Malleswarapu; Bharathi, Munugala; Reddy, Mooli Raja Gopal; Kumar, Manchiryala Sravan; Putcha, Uday Kumar; Vajreswari, Ayyalasomayajula; Jeyakumar, Shanmugam M

2016-09-01

Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent liver diseases associated with an altered lifestyle, besides genetic factors. The control and management of NAFLD mostly depend on lifestyle modifications, due to the lack of a specific therapeutic approach. In this context, we assessed the effect of carrot juice on the development of high fructose-induced hepatic steatosis. For this purpose, male weanling Wistar rats were divided into 4 groups, fed either a control (Con) or high fructose (HFr) diet of AIN93G composition, with or without carrot juice (CJ) for 8 weeks. At the end of the experimental period, plasma biochemical markers, such as triglycerides, alanine aminotransferase, and β-hydroxy butyrate levels were comparable among the 4 groups. Although, the liver injury marker, aspartate aminotransferase, levels in plasma showed a reduction, hepatic triglycerides levels were not significantly reduced by carrot juice ingestion in the HFr diet-fed rats (HFr-CJ). On the other hand, the key triglyceride synthesis pathway enzyme, hepatic stearoyl-CoA desaturase 1 (SCD1), expression at mRNA level was augmented by carrot juice ingestion, while their protein levels showed a significant reduction, which corroborated with decreased monounsaturated fatty acids (MUFA), particularly palmitoleic (C16:1) and oleic (C18:1) acids. Notably, it also improved the long chain n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA; C22:6) content of the liver in HFr-CJ. In conclusion, carrot juice ingestion decreased the SCD1-mediated production of MUFA and improved DHA levels in liver, under high fructose diet-fed conditions. However, these changes did not significantly lower the hepatic triglyceride levels.

Carrot Juice Administration Decreases Liver Stearoyl-CoA Desaturase 1 and Improves Docosahexaenoic Acid Levels, but Not Steatosis in High Fructose Diet-Fed Weanling Wistar Rats

PubMed Central

Mahesh, Malleswarapu; Bharathi, Munugala; Reddy, Mooli Raja Gopal; Kumar, Manchiryala Sravan; Putcha, Uday Kumar; Vajreswari, Ayyalasomayajula; Jeyakumar, Shanmugam M.

2016-01-01

Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent liver diseases associated with an altered lifestyle, besides genetic factors. The control and management of NAFLD mostly depend on lifestyle modifications, due to the lack of a specific therapeutic approach. In this context, we assessed the effect of carrot juice on the development of high fructose-induced hepatic steatosis. For this purpose, male weanling Wistar rats were divided into 4 groups, fed either a control (Con) or high fructose (HFr) diet of AIN93G composition, with or without carrot juice (CJ) for 8 weeks. At the end of the experimental period, plasma biochemical markers, such as triglycerides, alanine aminotransferase, and β-hydroxy butyrate levels were comparable among the 4 groups. Although, the liver injury marker, aspartate aminotransferase, levels in plasma showed a reduction, hepatic triglycerides levels were not significantly reduced by carrot juice ingestion in the HFr diet-fed rats (HFr-CJ). On the other hand, the key triglyceride synthesis pathway enzyme, hepatic stearoyl-CoA desaturase 1 (SCD1), expression at mRNA level was augmented by carrot juice ingestion, while their protein levels showed a significant reduction, which corroborated with decreased monounsaturated fatty acids (MUFA), particularly palmitoleic (C16:1) and oleic (C18:1) acids. Notably, it also improved the long chain n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA; C22:6) content of the liver in HFr-CJ. In conclusion, carrot juice ingestion decreased the SCD1-mediated production of MUFA and improved DHA levels in liver, under high fructose diet-fed conditions. However, these changes did not significantly lower the hepatic triglyceride levels. PMID:27752492

{alpha}-Lipoic acid prevents lipotoxic cardiomyopathy in acyl CoA-synthase transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Young; Department of Internal Medicine, University of Texas, Southwestern Medical Center, Dallas, TX 75390-8854; Naseem, R. Haris

2006-05-26

{alpha}-Lipoic acid ({alpha}-LA) mimics the hypothalamic actions of leptin on food intake, energy expenditure, and activation of AMP-activated protein kinase (AMPK). To determine if, like leptin, {alpha}-LA protects against cardiac lipotoxicity, {alpha}-LA was fed to transgenic mice with cardiomyocyte-specific overexpression of the acyl CoA synthase (ACS) gene. Untreated ACS-transgenic mice died prematurely with increased triacylglycerol content and dilated cardiomyopathy, impaired systolic function and myofiber disorganization, apoptosis, and interstitial fibrosis on microscopy. In {alpha}-LA-treated ACS-transgenic mice heart size, echocardiogram and TG content were normal. Plasma TG fell 50%, hepatic-activated phospho-AMPK rose 6-fold, sterol regulatory element-binding protein-1c declined 50%, and peroxisome proliferator-activatedmore » receptor-{gamma} cofactor-1{alpha} mRNA rose 4-fold. Since food restriction did not prevent lipotoxicity, we conclude that {alpha}-LA treatment, like hyperleptinemia, protects the heart of ACS-transgenic mice from lipotoxicity.« less

Transgenic expression of human neutrophil peptide-1 enhances hepatic fibrosis in mice fed a choline-deficient, L-amino acid-defined diet.

PubMed

Ibusuki, Rie; Uto, Hirofumi; Arima, Shiho; Mawatari, Seiichi; Setoguchi, Yoshiko; Iwashita, Yuji; Hashimoto, Shinichi; Maeda, Takuro; Tanoue, Shiro; Kanmura, Shuji; Oketani, Makoto; Ido, Akio; Tsubouchi, Hirohito

2013-11-01

Neutrophils infiltrate the livers of patients with nonalcoholic steatohepatitis (NASH). Human neutrophil peptides (HNPs) induce cytokine and chemokine production under inflammatory conditions, which may contribute to the progression of NASH. In this study, we focused on the effects of HNP-1 on hepatic steatosis and fibrosis in a mouse model of NASH induced by a choline-deficient, L-amino acid-defined (CDAA) diet. We generated transgenic mice expressing HNP-1 under the control of a β-actin-based promoter. HNP-1 transgenic and wild-type C57BL/6N mice were fed a CDAA diet for 16 weeks to induce hepatic steatosis and fibrosis. Serological and histological features were examined, and the effects of HNP-1 on hepatic stellate cell lines were assessed. HNP-1 transgenic and wild-type mice fed the CDAA diet showed no significant differences in serum alanine aminotransferase levels or the degree of hepatic steatosis based on Oil red O staining and hepatic triglyceride content. In contrast, Sirius Red and Azan staining showed significantly more severe hepatic fibrosis in HNP-1 transgenic mice compared with wild-type mice. In addition, significantly more α-smooth muscle actin-positive hepatic stellate cells were observed in the transgenic mice than in the wild-type mice. Finally, the proliferation of the LI90 hepatic stellate cell line increased in response to HNP-1. Our data indicate that HNP-1 enhances hepatic fibrosis in fatty liver by inducing hepatic stellate cell proliferation. Thus, neutrophil-derived HNP-1 may contribute to the progression of NASH. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Survey of the total fatty acid and triacylglycerol composition and content of 30 duckweed species and cloning of a Δ6-desaturase responsible for the production of γ-linolenic and stearidonic acids in Lemna gibba

PubMed Central

2013-01-01

Background Duckweeds, i.e., members of the Lemnoideae family, are amongst the smallest aquatic flowering plants. Their high growth rate, aquatic habit and suitability for bio-remediation make them strong candidates for biomass production. Duckweeds have been studied for their potential as feedstocks for bioethanol production; however, less is known about their ability to accumulate reduced carbon as fatty acids (FA) and oil. Results Total FA profiles of thirty duckweed species were analysed to assess the natural diversity within the Lemnoideae. Total FA content varied between 4.6% and 14.2% of dry weight whereas triacylglycerol (TAG) levels varied between 0.02% and 0.15% of dry weight. Three FA, 16:0 (palmitic), 18:2Δ9,12 (Linoleic acid, or LN) and 18:3Δ9,12,15 (α-linolenic acid, or ALA) comprise more than 80% of total duckweed FA. Seven Lemna and two Wolffiela species also accumulate polyunsaturated FA containing Δ6-double bonds, i.e., GLA and SDA. Relative to total FA, TAG is enriched in saturated FA and deficient in polyunsaturated FA, and only five Lemna species accumulate Δ6-FA in their TAG. A putative Δ6-desaturase designated LgDes, with homology to a family of front-end Δ6-FA and Δ8-spingolipid desaturases, was identified in the assembled DNA sequence of Lemna gibba. Expression of a synthetic LgDes gene in Nicotiana benthamiana resulted in the accumulation of GLA and SDA, confirming it specifies a Δ6-desaturase. Conclusions Total accumulation of FA varies three-fold across the 30 species of Lemnoideae surveyed. Nine species contain GLA and SDA which are synthesized by a Δ6 front-end desaturase, but FA composition is otherwise similar. TAG accumulates up to 0.15% of total dry weight, comparable to levels found in the leaves of terrestrial plants. Polyunsaturated FA is underrepresented in TAG, and the Δ6-FA GLA and SDA are found in the TAG of only five of the nine Lemna species that produce them. When present, GLA is enriched and SDA diminished

Enhancing freezing tolerance of Brassica napus L. by overexpression of a stearoyl-acyl carrier protein desaturase gene (SAD) from Sapium sebiferum (L.) Roxb.

PubMed

Peng, Dan; Zhou, Bo; Jiang, Yueqiao; Tan, XiaoFeng; Yuan, DeYi; Zhang, Lin

2018-07-01

Sapium sebiferum (L.) Roxb. is an important woody oil tree and traditional herbal medicine in China. Stearoyl-acyl carrier protein desaturase (SAD) is a dehydrogenase enzyme that plays a key role in the transformation of saturated fatty acids into unsaturated fatty acids in oil; these fatty acids greatly influence the freezing tolerance of plants. However, it remains unclear whether freezing tolerance can be regulated by the expression level of SsSAD in S. sebiferum L. Our research indicated that SsSAD expression in S. sebiferum L. increased under freezing stress. To further confirm this result, we constructed a pEGAD-SsSAD vector and transformed it into B. napus L. W10 by Agrobacterium tumefaciens-mediated transformation. Transgenic plants that overexpressed the SsSAD gene exhibited significantly higher linoleic (18:2) and linolenic acid (18:3) content and advanced freezing tolerance. These results suggest that SsSAD overexpression in B. napus L. can increase the content of polyunsaturated fatty acids (PUFAs) such as linoleic (18:2) and linolenic acid (18:3), which are likely pivotal in improving freezing tolerance in B. napus L. plants. Thus, SsSAD overexpression could be useful in the production of freeze-tolerant varieties of B. napus L. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of markers for Delta9-Stearoyl-ACP-Desaturase (SAD) to screen for cold acclimation

USDA-ARS?s Scientific Manuscript database

Delta 9-Stearoyl-acyl carrier protein (ACP) desaturase (SAD) is an important enzyme of fatty acid biosynthesis in higher plants. Located in the plastid stroma, SAD catalyzes the desaturation of stearoyl-ACP to oleyl-ACP. SAD plays a key role in determining the ratio of saturated fatty acids to unsat...

Stearoyl-CoA desaturase activity in bovine cumulus cells protects the oocyte against saturated fatty acid stress

PubMed Central

Aardema, Hilde; van Tol, Helena T. A.; Wubbolts, Richard W.; Brouwers, Jos F. H. M.; Gadella, Bart M.; Roelen, Bernard A. J.

2017-01-01

Abstract Metabolic rich and poor conditions are both characterized by elevated free fatty acid levels and have been associated with impaired female fertility. In particular, saturated free fatty acids have a dose-dependent negative impact on oocyte developmental competence, while monounsaturated free fatty acids appear less harmful. Cumulus cells seem to protect the oocyte against free fatty acids, and the aim of this study was to determine the mechanism behind this protection In particular, the role of the enzyme stearoyl-CoA desaturase (SCD) that converts saturated into monounsaturated fatty acids was investigated. SCD gene and protein were abundantly expressed in cumulus cells, but expression was low in oocytes. The level of SCD protein expression in cumulus cells did not change when COCs were exposed to saturated stearic acid during maturation. SCD inhibition in the presence of stearic acid significantly reduced the developmental competence of oocytes and increased the incidence of apoptosis in cumulus cells. The esterified oleic/stearic acid ratio of the neutral lipid fraction in cumulus cells decreased in the presence of SCD inhibitors when COCs were exposed to saturated free fatty acids during maturation, indicating the SCD-specific conversion of saturated fatty acids under noninhibiting conditions. The observation that cumulus cells can desaturate the potentially toxic stearic acid into oleic acid via SCD activity provides a mechanistic insight into how the cumulus cells protect the oocyte against toxicity by saturated fatty acid. PMID:28486699

Transgenic soybean overexpressing GmSAMT1 exhibits resistance to multiple-HG types of soybean cyst nematode Heterodera glycines.

PubMed

Lin, Jingyu; Mazarei, Mitra; Zhao, Nan; Hatcher, Catherine N; Wuddineh, Wegi A; Rudis, Mary; Tschaplinski, Timothy J; Pantalone, Vincent R; Arelli, Prakash R; Hewezi, Tarek; Chen, Feng; Stewart, Charles Neal

2016-11-01

Soybean (Glycine max (L.) Merr.) salicylic acid methyl transferase (GmSAMT1) catalyses the conversion of salicylic acid to methyl salicylate. Prior results showed that when GmSAMT1 was overexpressed in transgenic soybean hairy roots, resistance is conferred against soybean cyst nematode (SCN), Heterodera glycines Ichinohe. In this study, we produced transgenic soybean overexpressing GmSAMT1 and characterized their response to various SCN races. Transgenic plants conferred a significant reduction in the development of SCN HG type 1.2.5.7 (race 2), HG type 0 (race 3) and HG type 2.5.7 (race 5). Among transgenic lines, GmSAMT1 expression in roots was positively associated with SCN resistance. In some transgenic lines, there was a significant decrease in salicylic acid titer relative to control plants. No significant seed yield differences were observed between transgenics and control soybean plants grown in one greenhouse with 22 °C day/night temperature, whereas transgenic soybean had higher yield than controls grown a warmer greenhouse (27 °C day/23 °C night) temperature. In a 1-year field experiment in Knoxville, TN, there was no significant difference in seed yield between the transgenic and nontransgenic soybean under conditions with negligible SCN infection. We hypothesize that GmSAMT1 expression affects salicylic acid biosynthesis, which, in turn, attenuates SCN development, without negative consequences to soybean yield or other morphological traits. Thus, we conclude that GmSAMT1 overexpression confers broad resistance to multiple SCN races, which would be potentially applicable to commercial production. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

A New F131V Mutation in Chlamydomonas Phytoene Desaturase Locates a Cluster of Norflurazon Resistance Mutations near the FAD-Binding Site in 3D Protein Models

PubMed Central

Suarez, Julio V.; Banks, Stephen; Thomas, Paul G.; Day, Anil

2014-01-01

The green alga Chlamydomonas reinhardtii provides a tractable genetic model to study herbicide mode of action using forward genetics. The herbicide norflurazon inhibits phytoene desaturase, which is required for carotenoid synthesis. Locating amino acid substitutions in mutant phytoene desaturases conferring norflurazon resistance provides a genetic approach to map the herbicide binding site. We isolated a UV-induced mutant able to grow in very high concentrations of norflurazon (150 µM). The phytoene desaturase gene in the mutant strain contained the first resistance mutation to be localised to the dinucleotide-binding Rossmann-likedomain. A highly conserved phenylalanine amino acid at position 131 of the 564 amino acid precursor protein was changed to a valine in the mutant protein. F131, and two other amino acids whose substitution confers norflurazon resistance in homologous phytoene desaturase proteins, map to distant regions in the primary sequence of the C. reinhardtii protein (V472, L505) but in tertiary models these residues cluster together to a region close to the predicted FAD binding site. The mutant gene allowed direct 5 µM norflurazon based selection of transformants, which were tolerant to other bleaching herbicides including fluridone, flurtamone, and diflufenican but were more sensitive to beflubutamid than wild type cells. Norflurazon resistance and beflubutamid sensitivity allow either positive or negative selection against transformants expressing the mutant phytoene desaturase gene. PMID:24936791

Antiviral effects of Stichopus japonicus acid mucopolysaccharide on hepatitis B virus transgenic mice

NASA Astrophysics Data System (ADS)

Xin, Yongning; Li, Wei; Lu, Linlin; Zhou, Li; Victor, David W.; Xuan, Shiying

2016-08-01

Hepatitis B virus (HBV) is a significant global pathogen and efficient cure for HBV patients is still a challenging goal. We previously reported that acidic mucopolysaccharide from stichopus japonicus selenka (SJAMP) could inhibit HBsAg and HBeAg expression in vitro. However, the potential anti-HBV effects of SJAMP in vivo have not yet been explored. In this study, we show that SJAMP exhibits potent anti-HBV activity in HBV transgenic mice in a dose-dependent manner. Specifically, sixty HBV transgenic male BALB/c mice were randomly selected to receive the treatment of PBS, low dose SJAMP (30 mg kg-1), middle dose SJAMP (40 mg kg-1), high dose SJAMP (50 mg kg-1) and IFN (45 IU kg-1) for 30 d. SJAMP treatment suppressed serum HBV-DNA, and liver HBsAg and HBcAg levels in HBV-transgenic mice. The present study highlights the potential application of SJAMP in HBV therapy.

Handmade Cloned Transgenic Piglets Expressing the Nematode Fat-1 Gene

PubMed Central

Zhang, Peng; Zhang, Yidi; Dou, Hongwei; Yin, Jingdong; Chen, Yu; Pang, Xinzhi; Vajta, Gabor; Bolund, Lars

2012-01-01

Abstract Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been adapted worldwide, but this application is somewhat limited by its relatively low efficiency. In this study, we used handmade cloning (HMC) established previously to produce transgenic pigs that express the functional nematode fat-1 gene. Codon-optimized mfat-1 was inserted into eukaryotic expression vectors, which were transferred into primary swine donor cells. Reverse transcriptase PCR (RT-PCR), gas chromatography, and chromosome analyses were performed to select donor clones capable of converting n-6 into n-3 fatty acids. Blastocysts derived from the clones that lowered the n-6/n-3 ratio to approximately 1:1 were transferred surgically into the uteri of recipients for transgenic piglets. By HMC, 37% (n=558) of reconstructed embryos developed to the blastocyst stage after 7 days of culture in vitro, with an average cell number of 81±36 (n=14). Three recipients became pregnant after 408 day-6 blastocysts were transferred into four naturally cycling females, and a total of 14 live offspring were produced. The nematode mfat-1 effectively lowered the n-6/n-3 ratio in muscle and major organs of the transgenic pig. Our results will help to establish a reliable procedure and an efficient option in the production of transgenic animals. PMID:22686479

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

PubMed Central

Zheng, Desong; Sun, Quanxi; Liu, Jiang; Li, Yaxiao; Hua, Jinping

2016-01-01

Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of

Omega-3 fatty acid deficiency selectively up-regulates delta6-desaturase expression and activity indices in rat liver: prevention by normalization of omega-3 fatty acid status.

PubMed

Hofacer, Rylon; Jandacek, Ronald; Rider, Therese; Tso, Patrick; Magrisso, I Jack; Benoit, Stephen C; McNamara, Robert K

2011-09-01

This study investigated the effects of perinatal dietary omega-3 (n-3) fatty acid depletion and subsequent repletion on the expression of genes that regulate long-chain (LC) polyunsaturated fatty acid biosynthesis in rat liver and brain. It was hypothesized that chronic n-3 fatty acid deficiency would increase liver Fads1 and Fads2 messenger RNA (mRNA) expression/activity and that n-3 fatty acid repletion would normalize this response. Adult rats fed the n-3-free diet during perinatal development exhibited significantly lower erythrocyte, liver, and frontal cortex LCn-3 fatty acid composition and reciprocal elevations in LC omega-6 (n-6) fatty acid composition compared with controls (CONs) and repleted rats. Liver Fads2, but not Fads1, Elovl2, or Elovl5, mRNA expression was significantly greater in n-3-deficient (DEF) rats compared with CONs and was partially normalized in repleted rats. The liver 18:3n-6/18:2n-6 ratio, an index of delta6-desturase activity, was significantly greater in DEF rats compared with CON and repleted rats and was positively correlated with Fads2 mRNA expression among all rats. The liver 18:3n-6/18:2n-6 ratio, but not Fads2 mRNA expression, was also positively correlated with erythrocyte and frontal cortex LCn-6 fatty acid compositions. Neither Fads1 or Fads2 mRNA expression was altered in brain cortex of DEF rats. These results confirm previous findings that liver, but not brain, delta6-desaturase expression and activity indices are negatively regulated by dietary n-3 fatty acids. Copyright © 2011 Elsevier Inc. All rights reserved.

Bioinformatics analysis of the ς-carotene desaturase gene in cabbage (Brassica oleracea var. capitata)

NASA Astrophysics Data System (ADS)

Sun, Bo; Zheng, Aihong; Jiang, Min; Xue, Shengling; Zhang, Fen; Tang, Haoru

2018-04-01

ς-carotene desaturase (ZDS) is an important enzyme in carotenoid biosynthesis. Here, the Brassica oleracea var. capitata ZDS (BocZDS) gene sequences were obtained from Brassica database (BRAD), and preformed for bioinformatics analysis. The BocZDS gene mapped to Scaffold000363, and contains an open reading frame of 1,686 bp that encodes a 561-amino acid protein with a calculated molecular mass of 62.00 kD and an isoelectric point (pI) of 8.2. Subcellular localization predicted the BocZDS gene was in the chloroplast. The conserved domain of the BocZDS protein is PLN02487, indicating that it belongs the member of zeta-carotene desaturase. Homology analysis indicates that the ZDS protein is apparently conserved during plant evolution and is most closely related to B. oleracea var. oleracea, B. napus, and B. rapa. The findings of the present study provide a molecular basis for the elucidation of ZDS gene function in cabbage.

Impact of Unusual Fatty Acid Synthesis on Futile Cycling through β-Oxidation and on Gene Expression in Transgenic Plants1[w

PubMed Central

Moire, Laurence; Rezzonico, Enea; Goepfert, Simon; Poirier, Yves

2004-01-01

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal β-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward β-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through β-oxidation than the expression profile of genes involved in lipid metabolism. PMID:14671017

Stabilization of the yeast desaturase system by low levels of oxygen

NASA Technical Reports Server (NTRS)

Volkmann, C. M.; Klein, H. P.

1983-01-01

The stability of particulate palmitoyl-CoA desaturase preparations from anaerobically grown yeast cells was increased by exposure to low levels of oxygen. The stabilizing effect of oxygen may be based upon the increased amounts of palmitoleic acid and ergosterol that become available to the cells. These results suggest the evolutinary appearance of this system at a time when atmospheric oxygen was at a low level.

Use of radiolabeled substrates to determine the desaturase and elongase activities involved in eicosapentaenoic acid and docosahexaenoic acid biosynthesis in the marine microalga Pavlova lutheri.

PubMed

Guihéneuf, Freddy; Ulmann, Lionel; Mimouni, Virginie; Tremblin, Gérard

2013-06-01

The marine flagellate Pavlova lutheri is a microalga known to be rich in long-chain polyunsaturated fatty acids (LC-PUFAs) and able to produce large amounts of n-3 fatty acids, such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). As no previous study had attempted to measure the metabolic step of fatty acid synthesis in this alga, we used radiolabeled precursors to explore the various desaturation and elongation steps involved in LC-PUFA biosynthesis pathways. The incorporation of (14)C-labeled palmitic ([1-(14)C] 16:0) and dihomo-γ-linolenic ([1-(14)C] 20:3n-6) acids as ammonium salts within the cells was monitored during incubation periods lasting 3, 10 or 24h. Total lipids and each of the fatty acids were also monitored during these incubation periods. A decrease in the availability and/or accessibility of the radiolabeled substrates was observed over the incubation time. This decrease with incubation time observed using [1-(14)C] 16:0 and [1-(14)C] 20:3n-6 as substrates was used to monitor the conversion of (14)C-labeled arachidonic acid ([1-(14)C] 20:4n-6) into longer and more unsaturated fatty acids, such as 20:5n-3 and 22:6n-3, over shorter incubation times (1 and 3h). A metabolic relationship between the n-6 and n-3 fatty acid series was demonstrated in P. lutheri by measuring the Δ17-desaturation activity involved in the conversion of eicosatetraenoic acid to 20:5n-3. Our findings suggest that the biosynthesis pathway leading to n-3 LC-PUFA involves fatty acids of the n-6 family, which act as precursors in the biosynthesis of 20:5n-3 and 22:6n-3. This preliminary work provides a method for studying microalgal LC-PUFA biosynthesis pathways and desaturase and elongase activities in vivo using externally-radiolabeled fatty acid precursors as substrates. The use of the [1-(14)C] 20:4n-6 substrate also highlighted the relationships between the n-6 and the n-3 fatty acid series (e.g. Δ17-desaturation), and the final elongation

Accurate Analysis and Evaluation of Acidic Plant Growth Regulators in Transgenic and Nontransgenic Edible Oils with Facile Microwave-Assisted Extraction-Derivatization.

PubMed

Liu, Mengge; Chen, Guang; Guo, Hailong; Fan, Baolei; Liu, Jianjun; Fu, Qiang; Li, Xiu; Lu, Xiaomin; Zhao, Xianen; Li, Guoliang; Sun, Zhiwei; Xia, Lian; Zhu, Shuyun; Yang, Daoshan; Cao, Ziping; Wang, Hua; Suo, Yourui; You, Jinmao

2015-09-16

Determination of plant growth regulators (PGRs) in a signal transduction system (STS) is significant for transgenic food safety, but may be challenged by poor accuracy and analyte instability. In this work, a microwave-assisted extraction-derivatization (MAED) method is developed for six acidic PGRs in oil samples, allowing an efficient (<1.5 h) and facile (one step) pretreatment. Accuracies are greatly improved, particularly for gibberellin A3 (-2.72 to -0.65%) as compared with those reported (-22 to -2%). Excellent selectivity and quite low detection limits (0.37-1.36 ng mL(-1)) are enabled by fluorescence detection-mass spectrum monitoring. Results show the significant differences in acidic PGRs between transgenic and nontransgenic oils, particularly 1-naphthaleneacetic acid (1-NAA), implying the PGRs induced variations of components and genes. This study provides, for the first time, an accurate and efficient determination for labile PGRs involved in STS and a promising concept for objectively evaluating the safety of transgenic foods.

Transcriptional Activation of Two Delta-9 Palmitoyl-ACP Desaturase Genes by MYB115 and MYB118 Is Critical for Biosynthesis of Omega-7 Monounsaturated Fatty Acids in the Endosperm of Arabidopsis Seeds

PubMed Central

Troncoso-Ponce, Manuel Adrián; Barthole, Guillaume; Tremblais, Geoffrey

2016-01-01

In angiosperms, double fertilization of the embryo sac initiates the development of the embryo and the endosperm. In Arabidopsis thaliana, an exalbuminous species, the endosperm is reduced to one cell layer during seed maturation and reserves such as oil are massively deposited in the enlarging embryo. Here, we consider the strikingly different fatty acid (FA) compositions of the oils stored in the two zygotic tissues. Endosperm oil is enriched in ω-7 monounsaturated FAs, that represent more than 20 mol% of total FAs, whereas these molecular species are 10-fold less abundant in the embryo. Two closely related transcription factors, MYB118 and MYB115, are transcriptionally induced at the onset of the maturation phase in the endosperm and share a set of transcriptional targets. Interestingly, the endosperm oil of myb115 myb118 double mutants lacks ω-7 FAs. The identification of two Δ9 palmitoyl-ACP desaturases responsible for ω-7 FA biosynthesis, which are activated by MYB115 and MYB118 in the endosperm, allows us to propose a model for the transcriptional control of oil FA composition in this tissue. In addition, an initial characterization of the structure-function relationship for these desaturases reveals that their particular substrate specificity is conferred by amino acid residues lining their substrate pocket that distinguish them from the archetype Δ9 stearoyl-ACP desaturase. PMID:27681170

Overexpression of an alfalfa GDP-mannose 3, 5-epimerase gene enhances acid, drought and salt tolerance in transgenic Arabidopsis by increasing ascorbate accumulation.

PubMed

Ma, Lichao; Wang, Yanrong; Liu, Wenxian; Liu, Zhipeng

2014-11-01

GDP-mannose 3', 5'-epimerase (GME) catalyses the conversion of GDP-D-mannose to GDP-L-galactose, an important step in the ascorbic acid (ascorbic acid) biosynthetic pathway in higher plants. In this study, a novel cDNA fragment (MsGME) encoding a GME protein was isolated and characterised from alfalfa (Medicago sativa). An expression analysis confirmed that MsGME expression was induced by salinity, PEG and acidity stresses. MsGME overexpression in Arabidopsis enhanced tolerance of the transgenic plants to salt, drought and acid. Real-time PCR analysis revealed that the transcript levels of GDP-D-mannose pyrophosphorylase (GMP), L-galactose-phosphate 1-P phosphatase (GP) and GDP-L-galactose phosphorylase (GGP) were increased in transgenic Arabidopsis (T3 generation). Moreover, the ascorbate content was increased in transgenic Arabidopsis. Our results suggest that MsGME can effectively enhance tolerance of transgenic Arabidopsis to acid, drought and salt by increasing ascorbate accumulation.

Correlation of polyunsaturated fatty acids with the cold adaptation of Rhodotorula glutinis.

PubMed

He, Jing; Yang, Zhaojie; Hu, Binbin; Ji, Xiuling; Wei, Yunlin; Lin, Lianbing; Zhang, Qi

2015-11-01

This study aimed to investigate the correlation between the cold adaptation of Rhodotorula glutinis YM25079 and the membrane fluidity, content of polyunsaturated fatty acids and mRNA expression level of the Δ(12)-desaturase gene. The optimum temperature for YM25079 growth was analysed first, then the composition changes of membrane lipid in YM25079 were detected by GC-MS and membrane fluidity was evaluated by 1-anilinonaphthalene-8-sulphonate (ANS) fluorescence. Meanwhile, the encoding sequence of Δ(12)-fatty acid desaturase in YM25079 was cloned and further transformed into Saccharomyces cerevisiae INVScl for functional analysis. The mRNA expression levels of Δ(12)-fatty acid desaturase at 15°C and 25°C were analysed by real-time PCR. YM25079 could grow at 5-30°C, with the optimum temperature of 15°C. The membrane fluidity of YM25079 was not significantly reduced when the culture temperature decreased from 25°C to 15°C, but the content of polyunsaturated fatty acids (PUFAs), including linoleic acid and α-Linolenic acid increased significantly from 29.4% to 55.39%. Furthermore, a novel Δ(12)-fatty acid desaturase gene YM25079RGD12 from YM25079 was successfully identified and characterized, and the mRNA transcription level of the Δ(12)-desaturase gene was about five-fold higher in YM25079 cells grown at 15°C than that at 25°C. These results suggests that the cold adaptation of Rhodotorula glutinis YM25079 might result from higher expression of genes, especially the Δ(12)-fatty acid desaturase gene, during polyunsaturated fatty acids biosynthesis, which increased the content of PUFAs in the cell membrane and maintained the membrane fluidity at low temperature. Copyright © 2015 John Wiley & Sons, Ltd.

Bioengineering resistance to phytoene desaturase inhibitors in Arabidopsis thaliana

USDA-ARS?s Scientific Manuscript database

Three natural somatic mutations at codon 304 of the phytoene desaturase gene (pds) of Hydrilla verticillata ( L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all...

Can polymorphisms in the fatty acid desaturase (FADS) gene cluster alter the effects of fish oil supplementation on plasma and erythrocyte fatty acid profiles? An exploratory study.

PubMed

Meldrum, Suzanne J; Li, Yuchun; Zhang, Guicheng; Heaton, Alexandra E M; D'Vaz, Nina; Manz, Judith; Reischl, Eva; Koletzko, Berthold V; Prescott, Susan L; Simmer, Karen

2017-09-19

The enzymes encoded by fatty acid desaturases (FADS) genes determine the desaturation of long-chain polyunsaturated fatty acids (LCPUFA). We investigated if haplotype and single nucleotide polymorphisms (SNPs) in FADS gene cluster can influence LCPUFA status in infants who received either fish oil or placebo supplementation. Children enrolled in the Infant Fish Oil Supplementation Study (IFOS) were randomly allocated to receive either fish oil or placebo from birth to 6 months of age. Blood was collected at 6 months of age for the measurement of fatty acids and for DNA extraction. A total of 276 participant DNA samples underwent genotyping, and 126 erythrocyte and 133 plasma fatty acid measurements were available for analysis. Twenty-two FADS SNPs were selected on the basis of literature and linkage disequilibrium patterns identified from the HapMap data. Haplotype construction was completed using PHASE. For participants allocated to the fish oil group who had two copies of the FADS1 haplotype consisting of SNP minor alleles, DHA levels were significantly higher compared to other haplotypes. This finding was not observed for the placebo group. Furthermore, for members of the fish oil group only, the minor homozygous carriers of all the FADS1 SNPs investigated had significantly higher DHA than other genotypes (rs174545, rs174546, rs174548, rs174553, rs174556, rs174537, rs174448, and rs174455). Overall results of this preliminary study suggest that supplementation with fish oil may only significantly increase DHA in minor allele carriers of FADS1 SNPs. Further research is required to confirm this novel finding.

Stearoyl-Acyl Carrier Protein Desaturase Mutations Uncover an Impact of Stearic Acid in Leaf and Nodule Structure.

PubMed

Lakhssassi, Naoufal; Colantonio, Vincent; Flowers, Nicholas D; Zhou, Zhou; Henry, Jason; Liu, Shiming; Meksem, Khalid

2017-07-01

Stearoyl-acyl carrier protein desaturase (SACPD-C) has been reported to control the accumulation of seed stearic acid; however, no study has previously reported its involvement in leaf stearic acid content and impact on leaf structure and morphology. A subset of an ethyl methanesulfonate mutagenized population of soybean ( Glycine max ) 'Forrest' was screened to identify mutants within the GmSACPD-C gene. Using a forward genetics approach, one nonsense and four missense Gmsacpd-c mutants were identified to have high levels of seed, nodule, and leaf stearic acid content. Homology modeling and in silico analysis of the GmSACPD-C enzyme revealed that most of these mutations were localized near or at conserved residues essential for diiron ion coordination. Soybeans carrying Gmsacpd-c mutations at conserved residues showed the highest stearic acid content, and these mutations were found to have deleterious effects on nodule development and function. Interestingly, mutations at nonconserved residues show an increase in stearic acid content yet retain healthy nodules. Thus, random mutagenesis and mutational analysis allows for the achievement of high seed stearic acid content with no associated negative agronomic characteristics. Additionally, expression analysis demonstrates that nodule leghemoglobin transcripts were significantly more abundant in soybeans with deleterious mutations at conserved residues of GmSACPD-C. Finally, we report that Gmsacpd-c mutations cause an increase in leaf stearic acid content and an alteration of leaf structure and morphology in addition to differences in nitrogen-fixing nodule structure. © 2017 American Society of Plant Biologists. All Rights Reserved.

Hepatic delta 6-desaturase activity in lean and genetically obese ob/ob mice.

PubMed Central

Hughes, S; York, D A

1985-01-01

Hepatic delta 6-desaturase activity is primarily located in the mitochondrial fraction in mice. Both delta 6- and delta 5-desaturase activities are increased in the liver of young (6-week-old) obese mice. The increase in hepatic delta 6-desaturase activity in obese mice does not occur until weaning. Neither restriction of food intake nor hyperinsulinaemia normalize hepatic delta 6-desaturase activity of obese mice. Both cold acclimation and tri-iodothyronine (30 micrograms/day per kg) decreased hepatic delta 6-desaturase activity of obese mice to levels observed in lean mice, whereas the increase in activity in obese mice was still maintained after the induction of hypothyroidism. PMID:3977836

Total fatty acid content of the plasma membrane of Saccharomyces cerevisiae is more responsible for ethanol tolerance than the degree of unsaturation.

PubMed

Kim, Hyun-Soo; Kim, Na-Rae; Choi, Wonja

2011-03-01

The effect of change in unsaturated fatty acid composition on ethanol tolerance in Saccharomyces cerevisiae overexpressing ScOLE1 (∆9 fatty acid desaturase gene of S. cerevisiae), CaFAD2 (∆12 fatty acid desaturase gene of Candida albicans), or CaFAD3 (ω3 fatty acid desaturase gene of C. albicans) was examined. ScOLE1 over-expression increased the total unsaturated fatty acid content and enhanced ethanol tolerance, compared with a control strain. In contrast, overexpression of CaFAD2 and CaFAD3, which led to production of linoleic acid (18:2) and α-linolenic acid (18:3), respectively, neither changed total unsaturated fatty acids nor enhanced ethanol tolerance. The total unsaturated fatty acid content rather than the degree of unsaturation is thus an important factor for ethanol tolerance.

An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis, and expression in Arabidopsis.

PubMed

Meesapyodsuk, Dauenpen; Qiu, Xiao

2008-07-01

Claviceps purpurea, a fungal pathogen responsible for ergot diseases in many agriculturally important cereal crops, produces high levels of ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. It has been believed for many years that the biosynthesis of this fatty acid in C. purpurea involves a hydration process with linoleic acid as the substrate. Using degenerate polymerase chain reaction, we cloned a gene from the sclerotia encoding an enzyme (CpFAH) that has high sequence similarity to the C. purpurea oleate desaturase, but only low similarity to plant oleate hydroxylases. Functional analysis of CpFAH in yeast (Saccharomyces cerevisiae) indicated it acted predominantly as a hydroxylase, introducing hydroxyl groups at the 12-position of oleic acid and palmitoleic acid. As well, it showed Delta(12) desaturase activities on 16C and 18C monounsaturated fatty acids and, to a much lesser extent, omega(3) desaturase activities on ricinoleic acid. Heterologous expression of CpFAH under the guidance of a seed-specific promoter in Arabidopsis (Arabidopsis thaliana) wild-type and mutant (fad2/fae1) plants resulted in the accumulation of relatively higher levels of hydroxyl fatty acids in seeds. These data indicate that the biosynthesis of ricinoleic acid in C. purpurea is catalyzed by the fungal desaturase-like hydroxylase, and CpFAH, the first Delta(12) oleate hydroxylase of nonplant origin, is a good candidate for the transgenic production of hydroxyl fatty acids in oilseed crops.

Transgenic Arabidopsis Plants Expressing the Type 1 Inositol 5-Phosphatase Exhibit Increased Drought Tolerance and Altered Abscisic Acid Signaling[W

PubMed Central

Perera, Imara Y.; Hung, Chiu-Yueh; Moore, Candace D.; Stevenson-Paulik, Jill; Boss, Wendy F.

2008-01-01

The phosphoinositide pathway and inositol-1,4,5-trisphosphate (InsP3) are implicated in plant responses to stress. To determine the downstream consequences of altered InsP3-mediated signaling, we generated transgenic Arabidopsis thaliana plants expressing the mammalian type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), which specifically hydrolyzes soluble inositol phosphates and terminates the signal. Rapid transient Ca2+ responses to a cold or salt stimulus were reduced by ∼30% in these transgenic plants. Drought stress studies revealed, surprisingly, that the InsP 5-ptase plants lost less water and exhibited increased drought tolerance. The onset of the drought stress was delayed in the transgenic plants, and abscisic acid (ABA) levels increased less than in the wild-type plants. Stomatal bioassays showed that transgenic guard cells were less responsive to the inhibition of opening by ABA but showed an increased sensitivity to ABA-induced closure. Transcript profiling revealed that the drought-inducible ABA-independent transcription factor DREB2A and a subset of DREB2A-regulated genes were basally upregulated in the InsP 5-ptase plants, suggesting that InsP3 is a negative regulator of these DREB2A-regulated genes. These results indicate that the drought tolerance of the InsP 5-ptase plants is mediated in part via a DREB2A-dependent pathway and that constitutive dampening of the InsP3 signal reveals unanticipated interconnections between signaling pathways. PMID:18849493

RNAi-Mediated Knockdown of IKK1 in Transgenic Mice Using a Transgenic Construct Containing the Human H1 Promoter

PubMed Central

Moreno-Maldonado, Rodolfo; Murillas, Rodolfo; Page, Angustias; Suarez-Cabrera, Cristian; Alameda, Josefa P.; Bravo, Ana; Casanova, M. Llanos

2014-01-01

Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models. PMID:24523631

Omega-3 Fatty Acid Deficiency Increases Stearoyl-CoA Desaturase Expression and Activity Indices in Rat Liver: Positive Association with Non-Fasting Plasma Triglyceride Levels

PubMed Central

Hofacer, Rylon; Magrisso, I. Jack; Jandacek, Ronald; Rider, Therese; Tso, Patrick; Benoit, Stephen C.; McNamara, Robert K.

2011-01-01

Although omega-3 (n-3) fatty acids negatively regulate triglyceride biosynthesis, the mechanisms mediating this effect are poorly understood, and emerging evidence suggests that stearoyl-CoA desaturase (Scd1) is required for de novo triglyceride biosynthesis. To investigate this mechanism, we determined the effects of perinatal n-3 deficiency and postnatal repletion on rat liver Scd1 mRNA expression and activity indices (liver 16:1/16:0 & 18:1/18:0 ratios), and determined relationships with postprandial (non-fasting) plasma triglyceride levels. Rats were fed conventional diets with or without the n-3 fatty acid precursor α-linolenic acid (ALA, 18:3n-3) during perinatal development (E0-P100), and a subset of rats fed the ALA− diet were switched to the ALA+ diet post-weaning (P21-P100, repletion). Compared with controls, rats fed the ALA− diet exhibited significantly lower liver long-chain n-3 fatty acid compositions and elevations in monounsaturated fatty acid composition, both of which were normalized in repleted rats. Liver Scd1 mRNA expression and activity indices (16:1/16:0 & 18:1/18:0 ratios) were significantly greater in n-3 deficient rats compared with controls and repleted rats. Among all rats, liver Scd1 mRNA expression was positively correlated with liver 18:1/18:0 and 16:1/16:0 ratios. Plasma triglyceride levels, but not glucose or insulin levels, were significantly greater in n-3 deficient rats compared with controls and repleted rats. Liver Scd1 mRNA expression and activity indices were positively correlated with plasma triglyceride levels. These preclinical findings demonstrate that n-3 fatty acid status is an important determinant of liver Scd1 mRNA expression and activity, and suggest that down-regulation of Scd1 is a mechanism by which n-3 fatty acids repress constitutive triglyceride biosynthesis. PMID:22047910

Simultaneous determination of shikimic acid, salicylic acid and jasmonic acid in wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses.

PubMed

Scalabrin, Elisa; Radaelli, Marta; Capodaglio, Gabriele

2016-06-01

The presence and relative concentration of phytohormones may be regarded as a good indicator of an organism's physiological state. The integration of the rolC gene from Agrobacterium rhizogenes and of the rat glucocorticoid receptor (gr) in Nicotiana langsdorffii Weinmann plants has shown to determine various physiological and metabolic effects. The analysis of wild and transgenic N. langsdorffii plants, exposed to different abiotic stresses (high temperature, water deficit, and high chromium concentrations) was conducted, in order to investigate the metabolic effects of the inserted genes in response to the applied stresses. The development of a new analytical procedure was necessary, in order to assure the simultaneous determination of analytes and to obtain an adequately low limit of quantification. For the first time, a sensitive HPLC-HRMS quantitative method for the simultaneous determination of salicylic acid, jasmonic acid and shikimic acid was developed and validated. The method was applied to 80 plant samples, permitting the evaluation of plant stress responses and highlighting some metabolic mechanisms. Salicylic, jasmonic and shikimic acids proved to be suitable for the comprehension of plant stress responses. Chemical and heat stresses showed to induce the highest changes in plant hormonal status, differently affecting plant response. The potential of each genetic modification toward the applied stresses was marked and particularly the resistance of the gr modified plants was evidenced. This work provides new information in the study of N. langsdorffii and transgenic organisms, which could be useful for the further application of these transgenes. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

cDNA nucleotide sequence coding for stearoyl-CoA desaturase and its expression in the zebrafish (Danio rerio) embryo.

PubMed

Hsieh, S L; Liu, R W; Wu, C H; Cheng, W T; Kuo, Ching-Ming

2003-12-01

A cDNA sequence of stearoyl-CoA desaturase (SCD) was determined from zebrafish (Danio rerio) and compared to the corresponding genes in several teleosts. Zebrafish SCD cDNA has a size of 1,061 bp, encodes a polypeptide of 325 amino acids, and shares 88, 85, 84, and 83% similarities with tilapia (Oreochromis mossambicus), grass carp (Ctenopharyngodon idella), common carp (Cyprinus carpio), and milkfish (Chanos chanos), respectively. This 1,061 bp sequence specifies a protein that, in common with other fatty acid desaturases, contains three histidine boxes, believed to be involved in catalysis. These observations suggested that SCD genes are highly conserved. In addition, an oligonucleotide probe complementary to zebrafish SCD mRNA was hybridized to mRNA of approximately 396 bases with Northern blot analysis. The Northern blot and RT-PCR analyses showed that the SCD mRNA was expressed predominantly in the liver, intestine, gill, and muscle, while a lower level was found in the brain. Furthermore, we utilized whole-mount in situ hybridization and real-time quantitative RT-PCR to identify expression of the zebrafish SCD gene at five different stages of development. This revealed that very high levels of transcripts were found in zebrafish at all stages during embryogenesis and early development. Copyright 2003 Wiley-Liss, Inc.

Conserved Function of ACYL-ACYL CARRIER PROTEIN DESATURASE 5 on Seed Oil and Oleic Acid Biosynthesis between Arabidopsis thaliana and Brassica napus.

PubMed

Jin, Changyu; Li, Dong; Gao, Chenhao; Liu, Kaige; Qi, Shuanghui; Duan, Shaowei; Li, Zixiong; Gong, Jingyun; Wang, Jianjun; Hai, Jiangbo; Chen, Mingxun

2017-01-01

Previous studies have shown that several ACYL-ACYL CARRIER PROTEIN DESATURASE (AtAAD) members in Arabidopsis thaliana are responsible for oleic acid (C18:1) biosynthesis. Limited research has been conducted on another member, AtAAD5, and its paralog BnAAD5 in the closely related and commercially important plant, Brassica napus . Here, we found that AtAAD5 was predominantly and exclusively expressed in developing embryos at the whole seed developmental stages. The aad5 mutation caused a significant decrease in the amounts of oil and C18:1, and a considerable increase in the content of stearic acid (C18:0) in mature seeds, suggesting that AtAAD5 functioned as an important facilitator of seed oil biosynthesis. We also cloned the full-length coding sequence of BnAAD5-1 from the A3 subgenome of the B. napus inbred line L111. We showed that ectopic expression of BnAAD5-1 in the A. thaliana aad5-2 mutant fully complemented the phenotypes of the mutant, such as lower oil content and altered contents of C18:0 and C18:1. These results help us to better understand the functions of AAD members in A. thaliana and B. napus and provide a promising target for genetic manipulation of B. napus .

Constitutive ω-3 fatty acid production in fat-1 transgenic mice and docosahexaenoic acid administration to wild type mice protect against 2,4,6-trinitrobenzene sulfonic acid-induced colitis.

PubMed

Yum, Hye-Won; Kang, Jing X; Hahm, Ki Baik; Surh, Young-Joon

2017-06-10

Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) are known to have strong anti-inflammatory effects. In the present study, we investigated the protective effects of ω-3 PUFAs on experimentally induced murine colitis. Intrarectal administration of 2.5% 2,4,6-trinitrobenzene sulfonic acid (TNBS) caused inflammation in the colon of wild type mice, but this was less severe in fat-1 transgenic mice that constitutively produce ω-3 PUFAs from ω-6 PUFAs. The intraperitoneal administration of docosahexaenoic acid (DHA), a representative ω-3 PUFA, was also protective against TNBS-induced murine colitis. In addition, endogenously formed and exogenously introduced ω-3 PUFAs attenuated the production of malondialdehyde and 4-hydroxynonenal in the colon of TNBS-treated mice. The effective protection against inflammatory and oxidative colonic tissue damages in fat-1 and DHA-treated mice was associated with suppression of NF-κB activation and cyclooxygenase-2 expression and with elevated activation of Nrf2 and upregulation of its target gene, heme oxygenase-1. Taken together, these results provide mechanistic basis of protective action of ω-3 fatty PUFAs against experimental colitis. Copyright © 2017. Published by Elsevier Inc.

Elucidation of the sex-pheromone biosynthesis producing 5,7-dodecadienes in Dendrolimus punctatus (Lepidoptera: Lasiocampidae) reveals Delta 11- and Delta 9-desaturases with unusual catalytic properties.

PubMed

Liénard, Marjorie A; Lassance, Jean-Marc; Wang, Hong-Lei; Zhao, Cheng-Hua; Piskur, Jure; Johansson, Tomas; Löfstedt, Christer

2010-06-01

Sex pheromones produced by female moths of the Lasiocampidae family include conjugated 5,7-dodecadiene components with various oxygenated terminal groups. Here we describe the molecular cloning, heterologous expression and functional characterization of desaturases associated with the biosynthesis of these unusual chemicals. By homology-based PCR screening we characterized five cDNAs from the female moth pheromone gland that were related to other moth desaturases, and investigated their role in the production of the (Z)-5-dodecenol and (Z5,E7)-dodecadienol, major pheromone constituents of the pine caterpillar moth, Dendrolimus punctatus. Functional expression of two desaturase cDNAs belonging to the Delta 11-subfamily, Dpu-Delta 11(1)-APSQ and Dpu-Delta 11(2)-LPAE, showed that they catalysed the formation of unsaturated fatty acyls (UFAs) that can be chain-shortened by beta-oxidation and subsequently reduced to the alcohol components. A first (Z)-11-desaturation step is performed by Dpu-Delta 11(2)-LPAE on stearic acid that leads to (Z)-11-octadecenoic acyl, which is subsequently chain shortened to the (Z)-5-dodecenoic acyl precursor. The Dpu-Delta 11(1)-APSQ desaturase had the unusual property of producing Delta 8 mono-UFA of various chain lengths, but not when transformed yeast were grown in presence of (Z)-9-hexadecenoic acyl, in which case the biosynthetic intermediate (Z9,E11)-hexadecadienoic UFA was produced. In addition to a typical Z9 activity, a third transcript, Dpu-Delta 9-KPSE produced E9 mono-UFAs of various chain lengths. When provided with the (Z)-7-tetradecenoic acyl, it formed the (Z7,E9)-tetradecadienoic UFA, another biosynthetic intermediate that can be chain-shortened to (Z5,E7)-dodecadienoic acyl. Both Dpu-Delta 11(1)-APSQ and Dpu-Delta 9-KPSE thus exhibited desaturase activities consistent with the biosynthesis of the dienoic precursor. The combined action of three desaturases in generating a dienoic sex-pheromone component emphasizes the

Transgenic plants expressing GLK1 and CCA1 having increased nitrogen assimilation capacity

DOEpatents

Coruzzi, Gloria [New York, NY; Gutierrez, Rodrigo A [Santiago, CL; Nero, Damion C [Woodside, NY

2012-04-10

Provided herein are compositions and methods for producing transgenic plants. In specific embodiments, transgenic plants comprise a construct comprising a polynucleotide encoding CCA1, GLK1 or bZIP1, operably linked to a plant-specific promote, wherein the CCA1, GLK1 or bZIP1 is ectopically overexpressed in the transgenic plants, and wherein the promoter is optionally a constitutive or inducible promoter. In other embodiments, transgenic plants in which express a lower level of CCA1, GLK1 or bZIP1 are provided. Also provided herein are commercial products (e.g., pulp, paper, paper products, or lumber) derived from the transgenic plants (e.g., transgenic trees) produced using the methods provided herein.

Tauroursodeoxycholic acid, a bile acid, is neuroprotective in a transgenic animal model of Huntington's disease

PubMed Central

Keene, C. Dirk; Rodrigues, Cecilia M. P.; Eich, Tacjana; Chhabra, Manik S.; Steer, Clifford J.; Low, Walter C.

2002-01-01

Huntington's disease (HD) is an untreatable neurological disorder caused by selective and progressive degeneration of the caudate nucleus and putamen of the basal ganglia. Although the etiology of HD pathology is not fully understood, the observed loss of neuronal cells is thought to occur primarily through apoptosis. Furthermore, there is evidence in HD that cell death is mediated through mitochondrial pathways, and mitochondrial deficits are commonly associated with HD. We have previously reported that treatment with tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, prevented neuropathology and associated behavioral deficits in the 3-nitropropionic acid rat model of HD. We therefore examined whether TUDCA would also be neuroprotective in a genetic mouse model of HD. Our results showed that systemically administered TUDCA led to a significant reduction in striatal neuropathology of the R6/2 transgenic HD mouse. Specifically, R6/2 mice began receiving TUDCA at 6 weeks of age and exhibited reduced striatal atrophy, decreased striatal apoptosis, as well as fewer and smaller size ubiquitinated neuronal intranuclear huntingtin inclusions. Moreover, locomotor and sensorimotor deficits were significantly improved in the TUDCA-treated mice. In conclusion, TUDCA is a nontoxic, endogenously produced hydrophilic bile acid that is neuroprotective in a transgenic mouse model of HD and, therefore, may provide a novel and effective treatment in patients with HD. PMID:12149470

Local sources of retinoic acid coincide with retinoid-mediated transgene activity during embryonic development.

PubMed Central

Colbert, M C; Linney, E; LaMantia, A S

1993-01-01

We have assessed whether retinoic acid (RA) comes from local sources or is available widely to activate gene expression in embryos. We used an RA-responsive indicator cell line, L-C2A5, to localize RA sources. In these cells, an RA-sensitive promoter/lacZ reporter construct used previously by us to produce indicator transgenic mice is induced globally by RA in medium or locally by RA released at physiological concentrations (1 nM) from AG-1X2 resin beads. Furthermore, the cells are differentially responsive to the 9-cis and all-trans isomers of RA at low concentrations. Indicator transgenic mice with the same promoter/reporter construct were used to identify regions of RA-mediated gene activation. There are distinct domains of lacZ expression in the cervical and lumbar spinal cords of embryonic indicator mice. This pattern might reflect localized RA sources or restricted spatial and temporal expression of RA receptors, binding proteins, or other factors. To resolve this issue we compared the pattern of transgene activation in indicator cell monolayers cocultured with normal embryonic spinal cords with that in transgenic spinal cords. The explants induced reporter gene expression in L-C2A5 monolayers in a pattern identical to that in transgenic mice: alar regions of the cervical and lumbar cord were positive whereas those in the thoracic and sacral regions were not. We conclude that restricted sources of RA in the developing spinal cord mediate the local activation of RA-inducible genes. Thus, region-specific gene activation in embryos can be mediated by precisely localized sources of inductive molecules like RA. Images Fig. 1 Fig. 2 Fig. 3 PMID:8341670

Glutamic acid decarboxylase isoform distribution in transgenic mouse septum: an anti-GFP immunofluorescence study.

PubMed

Verimli, Ural; Sehirli, Umit S

2016-09-01

The septum is a basal forebrain region located between the lateral ventricles in rodents. It consists of lateral and medial divisions. Medial septal projections regulate hippocampal theta rhythm whereas lateral septal projections are involved in processes such as affective functions, memory formation, and behavioral responses. Gamma-aminobutyric acidergic neurons of the septal region possess the 65 and 67 isoforms of the enzyme glutamic acid decarboxylase. Although data on the glutamic acid decarboxylase isoform distribution in the septal region generally appears to indicate glutamic acid decarboxylase 67 dominance, different studies have given inconsistent results in this regard. The aim of this study was therefore to obtain information on the distributions of both of these glutamic acid decarboxylase isoforms in the septal region in transgenic mice. Two animal groups of glutamic acid decarboxylase-green fluorescent protein knock-in transgenic mice were utilized in the experiment. Brain sections from the region were taken for anti-green fluorescent protein immunohistochemistry in order to obtain estimated quantitative data on the number of gamma-aminobutyric acidergic neurons. Following the immunohistochemical procedures, the mean numbers of labeled cells in the lateral and medial septal nuclei were obtained for the two isoform groups. Statistical analysis yielded significant results which indicated that the 65 isoform of glutamic acid decarboxylase predominates in both lateral and medial septal nuclei (unpaired two-tailed t-test p < 0.0001 for LS, p < 0.01 for MS). This study is the first to reveal the dominance of glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice.

Association between polymorphisms in the fatty acid desaturase gene cluster and the plasma triacylglycerol response to an n-3 PUFA supplementation.

PubMed

Cormier, Hubert; Rudkowska, Iwona; Paradis, Ann-Marie; Thifault, Elisabeth; Garneau, Véronique; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

2012-08-01

Eicosapentaenoic and docosahexaenoic acids have been reported to have a variety of beneficial effects on cardiovascular disease risk factors. However, a large inter-individual variability in the plasma lipid response to an omega-3 (n-3) polyunsaturated fatty acid (PUFA) supplementation is observed in different studies. Genetic variations may influence plasma lipid responsiveness. The aim of the present study was to examine the effects of a supplementation with n-3 PUFA on the plasma lipid profile in relation to the presence of single-nucleotide polymorphisms (SNPs) in the fatty acid desaturase (FADS) gene cluster. A total of 208 subjects from Quebec City area were supplemented with 3 g/day of n-3 PUFA, during six weeks. In a statistical model including the effect of the genotype, the supplementation and the genotype by supplementation interaction, SNP rs174546 was significantly associated (p = 0.02) with plasma triglyceride (TG) levels, pre- and post-supplementation. The n-3 supplementation had an independent effect on plasma TG levels and no significant genotype by supplementation interaction effects were observed. In summary, our data support the notion that the FADS gene cluster is a major determinant of plasma TG levels. SNP rs174546 may be an important SNP associated with plasma TG levels and FADS1 gene expression independently of a nutritional intervention with n-3 PUFA.

Association between Polymorphisms in the Fatty Acid Desaturase Gene Cluster and the Plasma Triacylglycerol Response to an n-3 PUFA Supplementation

PubMed Central

Cormier, Hubert; Rudkowska, Iwona; Paradis, Ann-Marie; Thifault, Elisabeth; Garneau, Véronique; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

2012-01-01

Eicosapentaenoic and docosahexaenoic acids have been reported to have a variety of beneficial effects on cardiovascular disease risk factors. However, a large inter-individual variability in the plasma lipid response to an omega-3 (n-3) polyunsaturated fatty acid (PUFA) supplementation is observed in different studies. Genetic variations may influence plasma lipid responsiveness. The aim of the present study was to examine the effects of a supplementation with n-3 PUFA on the plasma lipid profile in relation to the presence of single-nucleotide polymorphisms (SNPs) in the fatty acid desaturase (FADS) gene cluster. A total of 208 subjects from Quebec City area were supplemented with 3 g/day of n-3 PUFA, during six weeks. In a statistical model including the effect of the genotype, the supplementation and the genotype by supplementation interaction, SNP rs174546 was significantly associated (p = 0.02) with plasma triglyceride (TG) levels, pre- and post-supplementation. The n-3 supplementation had an independent effect on plasma TG levels and no significant genotype by supplementation interaction effects were observed. In summary, our data support the notion that the FADS gene cluster is a major determinant of plasma TG levels. SNP rs174546 may be an important SNP associated with plasma TG levels and FADS1 gene expression independently of a nutritional intervention with n-3 PUFA. PMID:23016130

High Serum Phospholipid Dihomo-γ-Linoleic Acid Concentration and Low Δ5-Desaturase Activity Are Associated with Increased Risk of Type 2 Diabetes among Japanese Adults in the Hitachi Health Study.

PubMed

Akter, Shamima; Kurotani, Kayo; Sato, Masao; Hayashi, Takuya; Kuwahara, Keisuke; Matsushita, Yumi; Nakagawa, Tohru; Konishi, Maki; Honda, Toru; Yamamoto, Shuichiro; Hayashi, Takeshi; Noda, Mitsuhiko; Mizoue, Tetsuya

2017-08-01

Background: The association between the circulating fatty acid (FA) composition and type 2 diabetes (T2D) has been reported in Western populations, but evidence is scarce among Asian populations, including Japanese, who consume large amounts of fish. Objective: The objective of the present study was to prospectively examine the association between circulating concentrations of individual FAs and T2D incidence among Japanese adults. Methods: We conducted a nested case-control study in a cohort of 4754 employees, aged 34-69 y, who attended a comprehensive health checkup in 2008-2009 and donated blood samples for the Hitachi Health Study. During 5 y of follow-up, diabetes was identified on the basis of plasma glucose, glycated hemoglobin, and self-report. Two controls matched to each case by sex, age, and date of checkup were randomly chosen by using density sampling, resulting in 336 cases and 678 controls with FA measurements. GC was used to measure the FA composition in serum phospholipids. Cox proportional hazards regression was used to estimate the HRs and 95% CIs after adjusting for potential confounders. We examined the association of T2D risk with 25 different individual and combinations of FAs. Results: T2D risk was positively associated with serum dihomo-γ-linoleic acid concentration (highest compared with the lowest quartile-HR: 1.49; 95% CI: 1.04, 2.11; P- trend = 0.02) and inversely associated with Δ5-desaturase activity (highest compared with the lowest quartile-HR: 0.72; 95% CI: 0.52, 0.99; P- trend = 0.02), independent of body mass index (BMI). There were also inverse associations between T2D risk with serum total n-6 (ω-6) polyunsaturated fatty acids (PUFAs), linoleic acid, and cis -vaccenic acid, but these were attenuated and became nonsignificant after adjustment for BMI. Serum n-3 (ω-3) PUFAs and saturated fatty acids (SFAs) were not associated with T2D risk. Conclusions: T2D risk was associated with circulating concentrations of the n-6 PUFA

Cellular and Molecular Responses of Dunaliella tertiolecta by Expression of a Plant Medium Chain Length Fatty Acid Specific Acyl-ACP Thioesterase

PubMed Central

Lin, Huixin; Shen, Hui; Lee, Yuan K.

2018-01-01

Metabolic engineering of microalgae to accumulate high levels of medium chain length fatty acids (MCFAs) has met with limited success. Traditional approaches employ single introduction of MCFA specific acyl-ACP thioesterases (TEs), but our current research in transgenic Dunaliella tertiolecta line has highlighted that, there is no single rate-limiting approach that can effectively increase MCFA levels. Here, we explore the accumulation of MCFAs in D. tertiolecta after transgenic expression of myristic acid biased TE (C14TE). We observe that the MCFA levels were negatively correlated to the fatty acid (FA) synthesis genes, ketoacyl-ACP synthase II (KASII), stearoyl-CoA-9-desaturase (Δ9D), and oleoyl-CoA-12-desaturase (Δ12D). To further examine the molecular mechanism of MCFA accumulation in microalgae, we investigate the transcriptomic dynamics of the MCFA producing strain of D. tertiolecta. At the transcript level, enhanced MCFA accumulation primarily involved up-regulation of photosynthetic genes and down-regulation of genes from central carbon metabolic processes, resulting in an overall decrease in carbon precursors for FA synthesis. We additionally observe that MCFA specific peroxisomal β-oxidation gene (ACX3) was greatly enhanced to prevent excessive build-up of unusual MCFA levels. Besides, long chain acyl-CoA synthetase gene (LACS) was down-regulated, likely in attempt to control fatty acyl supply flux to FA synthesis cycle. This article provides a spatial regulation model of unusual FA accumulation in microalgae and a platform for additional metabolic engineering targeting pathways from FA synthesis, FA transport, and peroxisomal β-oxidation to achieve microalgae oils with higher levels of MCFAs. PMID:29670594

Transgenes expressing the Wnt-1 and int-2 proto-oncogenes cooperate during mammary carcinogenesis in doubly transgenic mice.

PubMed Central

Kwan, H; Pecenka, V; Tsukamoto, A; Parslow, T G; Guzman, R; Lin, T P; Muller, W J; Lee, F S; Leder, P; Varmus, H E

1992-01-01

The Wnt-1 and int-2 proto-oncogenes are transcriptionally activated by mouse mammary tumor virus insertion mutations in virus-induced tumors and encode secretory glycoproteins. To determine whether these two genes can cooperate during carcinogenesis, we have crossed two previously characterized lines of transgenic mice to obtain bitransgenic animals carrying both Wnt-1 and int-2 transgenes under the control of the mouse mammary tumor virus long terminal repeat. Mammary carcinomas appear earlier and with higher frequency in the bitransgenic animals, especially the males, than in either parental line. Nearly all bitransgenic males develop mammary neoplasms within 8 months of birth, whereas only 15% of Wnt-1 transgenic males and none of the int-2 transgenic males have tumors. In virgin bitransgenic females, tumors occur approximately 2 months earlier than in their Wnt-1 transgenic siblings; int-2 transgenic females rarely exhibit tumors. Preneoplastic glands from the bitransgenic animals of either sex demonstrate pronounced epithelial hyperplasia similar to that seen in Wnt-1 transgenic virgin females and males, and both transgenes are expressed in the hyperplastic glands and mammary tumors. RNA from the int-2 transgene is more abundant in mammary glands from bitransgenic animals than from int-2 transgenic animals; the increase is associated with high levels of RNA specific for keratin genes 14 and 18, suggesting that Wnt-1-induced epithelial hyperplasia is responsible for the observed increase in expression of the int-2 transgene. Images PMID:1530875

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm.

PubMed

Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig; Nunan, Kylie J; Madrid, Susan M; Brinch-Pedersen, Henrik; Holm, Preben B; Scheller, Henrik V

2010-04-01

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm-specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu) KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and threefold relative to wild type. The grains were shrivelled and had a 25%-33% decrease in mass. Extensive analysis of the cell walls showed a 10%-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water-extractable arabinoxylan, and a shift in the MW of the water-extractable arabinoxylan from being mainly larger than 85 kD to being between 2 and 85 kD. Ferulic acid esterase-expressing grains were also shrivelled, and the seed weight was decreased by 20%-50%. No ferulic acid esterase activity could be detected in wild-type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15%-40% increase in water-unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13% and 34%. In all the plants, the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig

2009-12-08

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and three fold relative to wild type. The grains were shriveled and had a 25-33% decrease in mass. Extensive analysis of the cell walls showed a 10-15% increase in arabinose to xylosemore » ratio, a 50% increase in the proportion of water extractable arabinoxylan, and a shift in the MW of the water extractable arabinoxylan from being mainly larger than 85 kD to being between 2 kD and 85 kD. Ferulic acid esterase expressing grains were also shriveled and the seed weight was decreased by 20-50%. No ferulic acid esterase activity could be detected in wild type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15-40% increase in water unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13 and 34%. In all the plants the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.« less

Biosynthesis and metabolic engineering of palmitoleate production, an important contributor to human health and sustainable industry.

PubMed

Wu, Yongmei; Li, Runzhi; Hildebrand, David F

2012-10-01

Palmitoleate (cis-Δ9-16:1) shows numerous health benefits such as increased cell membrane fluidity, reduced inflammation, protection of the cardiovascular system, and inhibition of oncogenesis. Plant oils containing this unusual fatty acid can also be sustainable feedstocks for producing industrially important and high-demand 1-octene. Vegetable oils rich in palmitoleate are the ideal candidates for biodiesel production. Several wild plants are known that can synthesize high levels of palmitoleate in seeds. However, low yields and poor agronomic characteristics of these plants limit their commercialization. Metabolic engineering has been developed to create oilseed crops that accumulate high levels of palmitoleate or other unusual fatty acids, and significant advances have been made recently in this field, particularly using the model plant Arabidopsis as the host. The engineered targets for enhancing palmitoleate synthesis include overexpression of Δ9 desaturase from mammals, yeast, fungi, and plants, down-regulating KASII, coexpression of an ACP-Δ9 desaturase in plastids and CoA-Δ9 desaturase in endoplasmic reticulum (ER), and optimizing the metabolic flux into triacylglycerols (TAGs). This review will mainly describe the recent progress towards producing palmitoleate in transgenic plants by metabolic engineering along with our current understanding of palmitoleate biosynthesis and its regulation, as well as highlighting the bottlenecks that require additional investigation by combining lipidomics, transgenics and other "-omics" tools. A brief review of reported health benefits and non-food uses of palmitoleate will also be covered. Copyright © 2012. Published by Elsevier Ltd.

Combined therapy of dietary fish oil and stearoyl-CoA desaturase 1 inhibition prevents the metabolic syndrome and atherosclerosis.

PubMed

Brown, J Mark; Chung, Soonkyu; Sawyer, Janet K; Degirolamo, Chiara; Alger, Heather M; Nguyen, Tam M; Zhu, Xuewei; Duong, My-Ngan; Brown, Amanda L; Lord, Caleb; Shah, Ramesh; Davis, Matthew A; Kelley, Kathryn; Wilson, Martha D; Madenspacher, Jennifer; Fessler, Michael B; Parks, John S; Rudel, Lawrence L

2010-01-01

Stearoyl-CoA desaturase 1 (SCD1) is a critical regulator of energy metabolism and inflammation. We have previously reported that inhibition of SCD1 in hyperlipidemic mice fed a saturated fatty acid (SFA)-enriched diet prevented development of the metabolic syndrome, yet surprisingly promoted severe atherosclerosis. In this study we tested whether dietary fish oil supplementation could prevent the accelerated atherosclerosis caused by SCD1 inhibition. LDLr(-/-), ApoB(100/100) mice were fed diets enriched in saturated fat or fish oil in conjunction with antisense oligonucleotide (ASO) treatment to inhibit SCD1. As previously reported, in SFA-fed mice, SCD1 inhibition dramatically protected against development of the metabolic syndrome, yet promoted atherosclerosis. In contrast, in mice fed fish oil, SCD1 inhibition did not result in augmented macrophage inflammatory response or severe atherosclerosis. In fact, the combined therapy of dietary fish oil and SCD1 ASO treatment effectively prevented both the metabolic syndrome and atherosclerosis. SCD1 ASO treatment in conjunction with dietary fish oil supplementation is an effective combination therapy to comprehensively combat the metabolic syndrome and atherosclerosis in mice.

Site-Specific Fat-1 Knock-In Enables Significant Decrease of n-6PUFAs/n-3PUFAs Ratio in Pigs

PubMed Central

Li, Mengjing; Ouyang, Hongsheng; Yuan, Hongming; Li, Jianing; Xie, Zicong; Wang, Kankan; Yu, Tingting; Liu, Minghao; Chen, Xue; Tang, Xiaochun; Jiao, Huping; Pang, Daxin

2018-01-01

The fat-1 gene from Caenorhabditis elegans encodes a fatty acid desaturase which was widely studied due to its beneficial function of converting n-6 polyunsaturated fatty acids (n-6PUFAs) to n-3 polyunsaturated fatty acids (n-3PUFAs). To date, many fat-1 transgenic animals have been generated to study disease pathogenesis or improve meat quality. However, all of them were generated using a random integration method with variable transgene expression levels and the introduction of selectable marker genes often raise biosafety concern. To this end, we aimed to generate marker-free fat-1 transgenic pigs in a site-specific manner. The Rosa26 locus, first found in mouse embryonic stem cells, has become one of the most common sites for inserting transgenes due to its safe and ubiquitous expression. In our study, the fat-1 gene was inserted into porcine Rosa 26 (pRosa26) locus via Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. The Southern blot analysis of our knock-in pigs indicated a single copy of the fat-1 gene at the pRosa26 locus. Furthermore, this single-copy fat-1 gene supported satisfactory expression in a variety of tissues in F1 generation pigs. Importantly, the gas chromatography analysis indicated that these fat-1 knock-in pigs exhibited a significant increase in the level of n-3PUFAs, leading to an obvious decrease in the n-6PUFAs/n-3PUFAs ratio from 9.36 to 2.12 (***P < 0.0001). Altogether, our fat-1 knock-in pigs hold great promise for improving the nutritional value of pork and serving as an animal model to investigate therapeutic effects of n-3PUFAs on various diseases. PMID:29563188

Site-Specific Fat-1 Knock-In Enables Significant Decrease of n-6PUFAs/n-3PUFAs Ratio in Pigs.

PubMed

Li, Mengjing; Ouyang, Hongsheng; Yuan, Hongming; Li, Jianing; Xie, Zicong; Wang, Kankan; Yu, Tingting; Liu, Minghao; Chen, Xue; Tang, Xiaochun; Jiao, Huping; Pang, Daxin

2018-05-04

The fat-1 gene from Caenorhabditis elegans encodes a fatty acid desaturase which was widely studied due to its beneficial function of converting n-6 polyunsaturated fatty acids (n-6PUFAs) to n-3 polyunsaturated fatty acids (n-3PUFAs). To date, many fat-1 transgenic animals have been generated to study disease pathogenesis or improve meat quality. However, all of them were generated using a random integration method with variable transgene expression levels and the introduction of selectable marker genes often raise biosafety concern. To this end, we aimed to generate marker-free fat-1 transgenic pigs in a site-specific manner. The Rosa26 locus, first found in mouse embryonic stem cells, has become one of the most common sites for inserting transgenes due to its safe and ubiquitous expression. In our study, the fat-1 gene was inserted into porcine Rosa 26 (pRosa26) locus via Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. The Southern blot analysis of our knock-in pigs indicated a single copy of the fat-1 gene at the pRosa26 locus. Furthermore, this single-copy fat-1 gene supported satisfactory expression in a variety of tissues in F1 generation pigs. Importantly, the gas chromatography analysis indicated that these fat-1 knock-in pigs exhibited a significant increase in the level of n-3PUFAs, leading to an obvious decrease in the n-6PUFAs/n-3PUFAs ratio from 9.36 to 2.12 (*** P < 0.0001). Altogether, our fat-1 knock-in pigs hold great promise for improving the nutritional value of pork and serving as an animal model to investigate therapeutic effects of n-3PUFAs on various diseases. Copyright © 2018 Li et al.

Exploring the potential of the bacterial carotene desaturase CrtI to increase the beta-carotene content in Golden Rice.

PubMed

Al-Babili, Salim; Hoa, Tran Thi Cuc; Schaub, Patrick

2006-01-01

To increase the beta-carotene (provitamin A) content and thus the nutritional value of Golden Rice, the optimization of the enzymes employed, phytoene synthase (PSY) and the Erwinia uredovora carotene desaturase (CrtI), must be considered. CrtI was chosen for this study because this bacterial enzyme, unlike phytoene synthase, was expressed at barely detectable levels in the endosperm of the Golden Rice events investigated. The low protein amounts observed may be caused by either weak cauliflower mosaic virus 35S promoter activity in the endosperm or by inappropriate codon usage. The protein level of CrtI was increased to explore its potential for enhancing the flux of metabolites through the pathway. For this purpose, a synthetic CrtI gene with a codon usage matching that of rice storage proteins was generated. Rice plants were transformed to express the synthetic gene under the control of the endosperm-specific glutelin B1 promoter. In addition, transgenic plants expressing the original bacterial gene were generated, but the endosperm-specific glutelin B1 promoter was employed instead of the cauliflower mosaic virus 35S promoter. Independent of codon optimization, the use of the endosperm-specific promoter resulted in a large increase in bacterial desaturase production in the T(1) rice grains. However, this did not lead to a significant increase in the carotenoid content, suggesting that the bacterial enzyme is sufficiently active in rice endosperm even at very low levels and is not rate-limiting. The endosperm-specific expression of CrtI did not affect the carotenoid pattern in the leaves, which was observed upon its constitutive expression. Therefore, tissue-specific expression of CrtI represents the better option.

Production of transgenic pigs over-expressing the antiviral gene Mx1.

PubMed

Yan, Quanmei; Yang, Huaqiang; Yang, Dongshan; Zhao, Bentian; Ouyang, Zhen; Liu, Zhaoming; Fan, Nana; Ouyang, Hongsheng; Gu, Weiwang; Lai, Liangxue

2014-01-01

The myxovirus resistance gene (Mx1) has a broad spectrum of antiviral activities. It is therefore an interesting candidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic cell nuclear transfer (SCNT) to produce transgenic pigs over-expressing the Mx1 gene. These transgenic pigs express approximately 15-25 times more Mx1 mRNA than non-transgenic pigs, and the protein level of Mx1 was also markedly enhanced. We challenged fibroblast cells isolated from the ear skin of transgenic and control pigs with influenza A virus and classical swine fever virus (CFSV). Indirect immunofluorescence assay (IFA) revealed a profound decrease of influenza A proliferation in Mx1 transgenic cells. Growth kinetics showed an approximately 10-fold reduction of viral copies in the transgenic cells compared to non-transgenic controls. Additionally, we found that the Mx1 transgenic cells were more resistant to CSFV infection in comparison to non-transgenic cells. These results demonstrate that the Mx1 transgene can protect against viral infection in cells of transgenic pigs and indicate that the Mx1 transgene can be harnessed to develop disease-resistant pigs.

Elevated Stearoyl-CoA Desaturase in Brains of Patients with Alzheimer's Disease

PubMed Central

Astarita, Giuseppe; Jung, Kwang-Mook; Vasilevko, Vitaly; DiPatrizio, Nicholas V.; Martin, Sarah K.; Cribbs, David H.; Head, Elizabeth; Cotman, Carl W.; Piomelli, Daniele

2011-01-01

The molecular bases of Alzheimer's disease (AD) remain unclear. We used a lipidomic approach to identify lipid abnormalities in the brains of subjects with AD (N = 37) compared to age-matched controls (N = 17). The analyses revealed statistically detectable elevations in levels of non-esterified monounsaturated fatty acids (MUFAs) and mead acid (20:3n-9) in mid-frontal cortex, temporal cortex and hippocampus of AD patients. Further studies showed that brain mRNAs encoding for isoforms of the rate-limiting enzyme in MUFAs biosynthesis, stearoyl-CoA desaturase (SCD-1, SCD-5a and SCD-5b), were elevated in subjects with AD. The monounsaturated/saturated fatty acid ratio (‘desaturation index’) – displayed a strong negative correlation with measures of cognition: the Mini Mental State Examination test (r = −0.80; P = 0.0001) and the Boston Naming test (r = −0.57; P = 0.0071). Our results reveal a previously unrecognized role for the lipogenic enzyme SCD in AD. PMID:22046234

Auxin Synthesis-Encoding Transgene Enhances Grape Fecundity1[OA

PubMed Central

Costantini, Elisa; Landi, Lucia; Silvestroni, Oriana; Pandolfini, Tiziana; Spena, Angelo; Mezzetti, Bruno

2007-01-01

Grape (Vitis vinifera) yield is largely dependent on the fecundity of the cultivar. The average number of inflorescences per shoot (i.e. shoot fruitfulness) is a trait related to fecundity of each grapevine. Berry number and weight per bunch are other features affecting grape yield. An ovule-specific auxin-synthesizing (DefH9-iaaM) transgene that increases the indole-3-acetic acid content of grape transgenic berries was transformed into cultivars Silcora and Thompson Seedless, which differ in the average number of inflorescences per shoots. Thompson Seedless naturally has very low shoot fruitfulness, whereas Silcora has medium shoot fruitfulness. The average number of inflorescences per shoot in DefH9-iaaM Thompson Seedless was doubled compared to its wild-type control. Berry number per bunch was increased in both transgenic cultivars. The quality and nutritional value of transgenic berries were substantially equivalent to their control fruits. The data presented indicate that auxin enhances fecundity in grapes, thus enabling to increase yield with lower production costs. PMID:17337528

Comparison of the structures of triacylglycerols from native and transgenic medium-chain fatty acid-enriched rape seed oil by liquid chromatography--atmospheric pressure chemical ionization ion-trap mass spectrometry (LC-APCI-ITMS).

PubMed

Beermann, Christopher; Winterling, Nadine; Green, Angelika; Möbius, Michael; Schmitt, Joachim J; Boehm, Günther

2007-04-01

The sn position of fatty acids in seed oil lipids affects physiological function in pharmaceutical and dietary applications. In this study the composition of acyl-chain substituents in the sn positions of glycerol backbones in triacylglycerols (TAG) have been compared. TAG from native and transgenic medium-chain fatty acid-enriched rape seed oil were analyzed by reversed-phase high performance liquid chromatography coupled with online atmospheric-pressure chemical ionization ion-trap mass spectrometry. The transformation of summer rape with thioesterase and 3-ketoacyl-[ACP]-synthase genes of Cuphea lanceolata led to increased expression of 1.5% (w/w) caprylic acid (8:0), 6.7% (w/w) capric acid (10:0), 0.9% (w/w) lauric acid (12:0), and 0.2% (w/w) myristic acid (14:0). In contrast, linoleic (18:2n6) and alpha-linolenic acid (18:3n3) levels decreased compared with the original seed oil. The TAG sn position distribution of fatty acids was also modified. The original oil included eleven unique TAG species whereas the transgenic oil contained sixty. Twenty species were common to both oils. The transgenic oil included trioctadecenoyl-glycerol (18:1/18:1/18:1) and trioctadecatrienoyl-glycerol (18:3/18:3/18:3) whereas the native oil included only the latter. The transgenic TAG were dominated by combinations of caprylic, capric, lauric, myrisitic, palmitic (16:0), stearic (18:0), oleic (18:1n9), linoleic, arachidic (20:0), behenic (22:0), and lignoceric acids (24:0), which accounted for 52% of the total fat. In the original TAG palmitic, stearic, oleic, and linoleic acids accounted for 50% of the total fat. Medium-chain triacylglycerols with capric and lauric acids combined with stearic, oleic, linoleic, alpha-linolenic, arachidic, and gondoic acids (20:1n9) accounted for 25% of the transgenic oil. The medium-chain fatty acids were mainly integrated into the sn-1/3 position combined with the essential linoleic and alpha-linolenic acids at the sn-2 position. Eight species

The growth performance of F1 transgenic mutiara catfish

NASA Astrophysics Data System (ADS)

Iskandar; Buwono, I. D.; Agung, M. U. K.

2018-04-01

The growth of catfish (African or Sangkuriang strain) these days is tend to decreased. One of the solutions due to this problem is to improve the genetics of growth using transgenesis technology, toward more profitable. The specific objective of the research is to detect the transmission of exogenous GH (African catfish GH inserts) inside the F1 transgenic Mutiara catfish using PCR (Polymerase Chain Reaction) method and to evaluate the growth performance of transgenic Mutiara catfish made using the parameters of feed conversion (FCR = Feed Conversion Ratio). Transgenic catfish (strain mutiara) F0 and F1 carried African catfish GH (600 bp) can be produced. Superiority characters of transgenic catfish represented heritability (h2 ) and heterosis (H), indicating that the offspring of hybrid F1 transgenic mutiara catfish had phenotypes rapid growth (h2 = 17.55 % and H = 42.83 %) compared to non-transgenic catfish (h 2 = 10.07 % and H = 18.56 %). Evaluation of the efficiency of feed use parameters feed conversion ratio, shows that F1 transgenic mutiara catfish (FCR = 0.85) more efficient in converting feed into meat.

Stereochemistry of a bifunctional dihydroceramide delta 4-desaturase/hydroxylase from Candida albicans; a key enzyme of sphingolipid metabolism.

PubMed

Beckmann, Christoph; Rattke, Janine; Sperling, Petra; Heinz, Ernst; Boland, Wilhelm

2003-07-21

The stereochemical course of the dihydroceramide delta 4-(E)-desaturase from Candida albicans, cloned and expressed in the yeast Saccharomyces cerevisiae strain sur2 delta, was determined using stereospecifically labelled (2R,3S)-[2,3,4,4-2H4]-palmitic acid as a metabolic probe. Mass spectrometric analysis of the dinitrophenyl-derivatives of the labelled long-chain bases revealed elimination of a single deuterium atom from C(4) (corresponding to the C(4)-HR) along with a hydrogen atom from C(5) (corresponding to the C(5)-HS). This finding is consistent with an overall syn-elimination of the two vicinal hydrogen atoms. Besides the desaturation product sphingosine (93%) minor amounts of a 4-hydroxylated product (phytosphinganine, 7%) were identified that classify the Candida enzyme as a bifunctional desaturase/hydroxylase. Both processes, desaturation and hydroxylation proceed with loss of C(4)-HR from the chiral precursor. This finding is in agreement with a two-step process involving activation of the substrate by removal of the C(4)-HR to give a C-centred radical or radicaloid followed by either disproportionation into an olefin, water and a reduced diiron complex, or to recombination of the primary reactive intermediate with an active site-bound oxygen to yield a secondary alcohol. This result demonstrates the close mechanistic relationship between desaturation and hydroxylation as two different reaction pathways of a single enzyme and strengthens the mechanistic relationship of desaturases from fatty acid metabolism and sphingolipids.

Inhibitory effects of fenretinide metabolites N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR) on fenretinide molecular targets β-carotene oxygenase 1, stearoyl-CoA desaturase 1 and dihydroceramide Δ4-desaturase 1

PubMed Central

Poliakov, Eugenia; Samuel, William; Duncan, Todd; Gutierrez, Danielle B.; Mata, Nathan L.; Redmond, T. Michael

2017-01-01

The therapeutic capacity of fenretinide (N-[4-hydroxyphenyl] retinamide; 4-HPR) has been demonstrated for several conditions, including cancer, obesity, diabetes, and ocular disease. Yet, the mechanisms of action for its pleiotropic effects are still undefined. We hypothesized that investigation of two of the major physiological metabolites of fenretinide, N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR), might begin to resolve the multifaceted effects of this synthetic retinoid. We analyzed the effects of fenretinide, MPR, 3-keto-HPR, and the non-retinoid RBP4 ligand A1120, on the activity of known targets of fenretinide, stearoyl-CoA desaturase 1 (SCD1) and dihydroceramide Δ4-desaturase 1 (DES1) in ARPE-19 cells, and purified recombinant mouse beta-carotene oxygenase 1 (BCO1) in vitro. Lipids and retinoids were extracted and quantified by liquid chromatography-mass spectrometry and reversed phase HPLC, respectively. The data demonstrate that while fenretinide is an inhibitor of the activities of these three enzymes, that 3-keto-HPR is a more potent inhibitor of all three enzymes, potentially mediating most of the in vivo beneficial effects of fenretinide. However, while MPR does not affect SCD1 and DES1 activity, it is a potent specific inhibitor of BCO1. We conclude that a deeper understanding of the mechanisms of action of fenretinide and its metabolites provides new avenues for therapeutic specificity. For example, administration of 3-keto-HPR instead of fenretinide may be preferential if inhibition of SCD1 or DES1 activity is the goal (cancer), while MPR may be better for BCO1 modulation (carotenoid metabolism). Continued investigation of fenretinide metabolites in the context of fenretinide’s various therapeutic uses will begin to resolve the pleotropic nature of this compound. PMID:28448568

Compensatory increases in tear volume and mucin levels associated with meibomian gland dysfunction caused by stearoyl-CoA desaturase-1 deficiency.

PubMed

Inaba, Takaaki; Tanaka, Yasuhisa; Tamaki, Shusaku; Ito, Tomotaka; Ntambi, James M; Tsubota, Kazuo

2018-02-20

The stearoyl-CoA desaturase (SCD) family of enzymes catalyzes monounsaturated fatty acid synthesis by inserting a cis double bond at the Δ9 position of saturated fatty acids. Disruption of these enzymes has been reported to induce a severe dry skin phenotype. Since lipid abnormalities in the meibomian glands have been associated with dry eye, we analyzed selected eye tissues contributing to tear volume and composition in genetically SCD-1-deficient mice (SCD-1 KO), including the lacrimal glands and conjunctiva. Previous histopathological analysis had revealed atrophy and loss of meibomian glands; taken together with the increased goblet cell and MUC5AC expression in the conjunctiva reported here, these findings suggest that the tear volume and mucin levels secreted are enhanced in the absence of lipid secretion as a compensatory mechanism. The expression of lipid metabolism genes in lacrimal glands was decreased in SCD1 KO mice. Thus, these results provide new pathophysiological mechanisms to pursue with regard to meibomian gland dysfunction. In addition, lack of SCD-1 causes a compensatory increase in the tear volume and mucin levels associated with changes in expression of lipid metabolism genes. These results may be useful as a new concept for dry eye treatment strategies.

Arabidopsis AtPAP1 transcription factor induces anthocyanin production in transgenic Taraxacum brevicorniculatum.

PubMed

Qiu, Jian; Sun, Shuquan; Luo, Shiqiao; Zhang, Jichuan; Xiao, Xianzhou; Zhang, Liqun; Wang, Feng; Liu, Shizhong

2014-04-01

This study developed a new purple coloured Taraxacum brevicorniculatum plant through genetic transformation using the Arabidopsis AtPAP1 gene, which overproduced anthocyanins in its vegetative tissues. Rubber-producing Taraxacum plants synthesise high-quality natural rubber (NR) in their roots and so are a promising alternative global source of this raw material. A major factor in its commercialization is the need for multipurpose exploitation of the whole plant. To add value to the aerial tissues, red/purple plants of the rubber-producing Taraxacum brevicorniculatum species were developed through heterologous expression of the production of anthocyanin pigment 1 (AtPAP1) transcription factor from Arabidopsis thaliana. The vegetative tissue of the transgenic plants showed an average of a 48-fold increase in total anthocyanin content over control levels, but with the exception of pigmentation, the transgenic plants were phenotypically comparable to controls and displayed similar growth vigor. Southern blot analysis confirmed that the AtPAP1 gene had been integrated into the genome of the high anthocyanin Taraxacum plants. The AtPAP1 expression levels were estimated by quantitative real-time PCR and were highly correlated with the levels of total anthocyanins in five independent transgenic lines. High levels of three cyanidin glycosides found in the purple plants were characterized by high performance liquid chromatography-mass spectrum analysis. The presence of NR was verified by NMR and infrared spectroscopy, and confirmed that NR biosynthesis had not been affected in the transgenic Taraxacum lines. In addition, other major phenylpropanoid products such as chlorogenic acid and quercetin glycosides were also enhanced in the transgenic Taraxacum. The red/purple transgenic Taraxacum lines described in this study would increase the future application of the species as a rubber-producing crop due to its additional health benefits.

Genome-wide analysis of the omega-3 fatty acid desaturase gene family in Gossypium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yurchenko, Olga P.; Park, Sunjung; Ilut, Daniel C.

The majority of commercial cotton varieties planted worldwide are derived from Gossypium hirsutum, which is a naturally occurring allotetraploid produced by interspecific hybridization of A- and D-genome diploid progenitor species. While most cotton species are adapted to warm, semi-arid tropical and subtropical regions, and thus perform well in these geographical areas, cotton seedlings are sensitive to cold temperature, which can significantly reduce crop yields. One of the common biochemical responses of plants to cold temperatures is an increase in omega-3 fatty acids, which protects cellular function by maintaining membrane integrity. The purpose of our study was to identify and characterizemore » the omega-3 fatty acid desaturase (FAD) gene family in G. hirsutum, with an emphasis on identifying omega-3 FADs involved in cold temperature adaptation. Results: Eleven omega-3 FAD genes were identified in G. hirsutum, and characterization of the gene family in extant A and D diploid species ( G. herbaceum and G. raimondii, respectively) allowed for unambiguous genome assignment of all homoeologs in tetraploid G. hirsutum. The omega-3 FAD family of cotton includes five distinct genes, two of which encode endoplasmic reticulum-type enzymes ( FAD3-1 and FAD3-2) and three that encode chloroplast-type enzymes ( FAD7/8-1, FAD7/8-2, and FAD7/8-3). The FAD3-2 gene was duplicated in the A genome progenitor species after the evolutionary split from the D progenitor, but before the interspecific hybridization event that gave rise to modern tetraploid cotton. RNA-seq analysis revealed conserved, gene-specific expression patterns in various organs and cell types and semi-quantitative RT-PCR further revealed that FAD7/8-1 was specifically induced during cold temperature treatment of G. hirsutum seedlings. Conclusions: The omega-3 FAD gene family in cotton was characterized at the genome-wide level in three species, showing relatively ancient establishment of the gene family

Genome-wide analysis of the omega-3 fatty acid desaturase gene family in Gossypium

DOE PAGES

Yurchenko, Olga P.; Park, Sunjung; Ilut, Daniel C.; ...

2014-11-18

The majority of commercial cotton varieties planted worldwide are derived from Gossypium hirsutum, which is a naturally occurring allotetraploid produced by interspecific hybridization of A- and D-genome diploid progenitor species. While most cotton species are adapted to warm, semi-arid tropical and subtropical regions, and thus perform well in these geographical areas, cotton seedlings are sensitive to cold temperature, which can significantly reduce crop yields. One of the common biochemical responses of plants to cold temperatures is an increase in omega-3 fatty acids, which protects cellular function by maintaining membrane integrity. The purpose of our study was to identify and characterizemore » the omega-3 fatty acid desaturase (FAD) gene family in G. hirsutum, with an emphasis on identifying omega-3 FADs involved in cold temperature adaptation. Results: Eleven omega-3 FAD genes were identified in G. hirsutum, and characterization of the gene family in extant A and D diploid species ( G. herbaceum and G. raimondii, respectively) allowed for unambiguous genome assignment of all homoeologs in tetraploid G. hirsutum. The omega-3 FAD family of cotton includes five distinct genes, two of which encode endoplasmic reticulum-type enzymes ( FAD3-1 and FAD3-2) and three that encode chloroplast-type enzymes ( FAD7/8-1, FAD7/8-2, and FAD7/8-3). The FAD3-2 gene was duplicated in the A genome progenitor species after the evolutionary split from the D progenitor, but before the interspecific hybridization event that gave rise to modern tetraploid cotton. RNA-seq analysis revealed conserved, gene-specific expression patterns in various organs and cell types and semi-quantitative RT-PCR further revealed that FAD7/8-1 was specifically induced during cold temperature treatment of G. hirsutum seedlings. Conclusions: The omega-3 FAD gene family in cotton was characterized at the genome-wide level in three species, showing relatively ancient establishment of the gene family

Temperature and metal exposure affect membrane fatty acid composition and transcription of desaturases and elongases in fathead minnow muscle and brain.

PubMed

Fadhlaoui, Mariem; Pierron, Fabien; Couture, Patrice

2018-02-01

In this study, we tested the hypothesis that metal exposure affected the normal thermal response of cell membrane FA composition and of elongase and desaturase gene transcription levels. To this end, muscle and brain membrane FA composition and FA desaturase (fads2, degs2 and scd2) and elongase (elovl2, elovl5 and elovl6) gene transcription levels were analyzed in fathead minnows (Pimephales promelas) acclimated for eight weeks to 15, 25 or 30°C exposed or not to cadmium (Cd, 6μg/l) or nickel (Ni, 450 6μg/l). The response of membrane FA composition to temperature variations or metal exposure differed between muscle and brain. In muscle, an increase of temperature induced a decrease of polyunsaturated FA (PUFA) and an increase of saturated FA (SFA) in agreement with the current paradigm. Although a similar response was observed in brain between 15 and 25°C, at 30°C, brain membrane unsaturation was higher than predicted. In both tissues, metal exposure affected the normal thermal response of membrane FA composition. The transcription of desaturases and elongases was higher in the brain and varied with acclimation temperature and metal exposure but these variations did not generally reflect changes in membrane FA composition. The mismatch between gene transcription and membrane composition highlights that several levels of control other than gene transcription are involved in adjusting membrane FA composition, including post-transcriptional regulation of elongases and desaturases and de novo phospholipid biosynthesis. Our study also reveals that metal exposure affects the mechanisms involved in adjusting cell membrane FA composition in ectotherms. Copyright © 2017 Elsevier Inc. All rights reserved.

Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene

PubMed Central

Mihálik, Daniel; Klčová, Lenka; Ondreičková, Katarína; Hudcovicová, Martina; Gubišová, Marcela; Klempová, Tatiana; Čertík, Milan; Pauk, János; Kraic, Ján

2015-01-01

The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%–0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%–1.40% (v/v) and 0%–1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat. PMID:26694368

Transgenic soybean overexpressing GmSamT1 exhibits resistance to multiple-HG types of soybean cyst nematode Heterodera glycines

USDA-ARS?s Scientific Manuscript database

Soybean (Glycine max (L.) Merr.) salicylic acid methyl transferase (GmSAMT1) catalyzes the conversion of salicylic acid to methyl salicylate. Prior results showed that when GmSAMT1 was overexpressed in transgenic soybean hairy roots, resistance is conferred against soybean cyst nematode (SCN), Heter...

Omega-3 fatty acids: new insights into the pharmacology and biology of docosahexaenoic acid, docosapentaenoic acid, and eicosapentaenoic acid.

PubMed

Davidson, Michael H

2013-12-01

Fish oil contains a complex mixture of omega-3 fatty acids, which are predominantly eicosapentaenoic acid (EPA), docosapentaenoic acid, and docosahexaenoic acid (DHA). Each of these omega-3 fatty acids has distinct biological effects that may have variable clinical effects. In addition, plasma levels of omega-3 fatty acids are affected not only by dietary intake, but also by the polymorphisms of coding genes fatty acid desaturase 1-3 for the desaturase enzymes that convert short-chain polyunsaturated fatty acids to long-chain polyunsaturated fatty acids. The clinical significance of this new understanding regarding the complexity of omega-3 fatty acid biology is the purpose of this review. FADS polymorphisms that result in either lower levels of long-chain omega-3 fatty acids or higher levels of long-chain omega-6 polyunsaturated fatty acids, such as arachidonic acid, are associated with dyslipidemia and other cardiovascular risk factors. EPA and DHA have differences in their effects on lipoprotein metabolism, in which EPA, with a more potent peroxisome proliferator-activated receptor-alpha effect, decreases hepatic lipogenesis, whereas DHA not only enhances VLDL lipolysis, resulting in greater conversion to LDL, but also increases HDL cholesterol and larger, more buoyant LDL particles. Overall, these results emphasize that blood concentrations of individual long-chain polyunsaturated fatty acids, which reflect both dietary intake and metabolic influences, may have independent, but also complementary- biological effects and reinforce the need to potentially provide a complex mixture of omega-3 fatty acids to maximize cardiovascular risk reduction.

Genome-Wide Survey and Characterization of Fatty Acid Desaturase Gene Family in Brassica napus and Its Parental Species.

PubMed

Xue, Yufei; Chen, Baojun; Wang, Rui; Win, Aung Naing; Li, Jiana; Chai, Yourong

2018-02-01

Rapeseed (Brassica napus) is an important oilseed crop worldwide, and fatty acid (FA) compositions determine the nutritional and economic value of its seed oil. Fatty acid desaturases (FADs) play a pivotal role in regulating FA compositions, but to date, no comprehensive genome-wide analysis of FAD gene family in rapeseed and its parent species has been reported. In this study, using homology searches, 84, 45, and 44 FAD genes were identified in rapeseed, Brassica rapa, and Brassica oleracea genomes, respectively. These FAD genes were unevenly located in 17 chromosomes and 2 scaffolds of rapeseed, 9 chromosomes and 1 scaffold of B. rapa, and all the chromosomes of B. oleracea. Phylogenetic analysis showed that the soluble and membrane-bound FADs in the three Brassica species were divided into four and six subfamilies, respectively. Generally, the soluble FADs contained two conserved histidine boxes, while three highly conserved histidine boxes were harbored in membrane-bound FADs. Exon-intron structure, intron phase, and motif composition and position were highly conserved in each FAD subfamily. Putative subcellular locations of FAD proteins in three Brassica species were consistent with those of corresponding known FADs. In total, 25 of simple sequence repeat (SSR) loci were found in FAD genes of the three Brassica species. Transcripts of selected FAD genes in the three species were examined in various organs/tissues or stress treatments from NCBI expressed sequence tag (EST) database. This study provides a critical molecular basis for quality improvement of rapeseed oil and facilitates our understanding of key roles of FAD genes in plant growth and development and stress response.

Alpha linolenic acid in maternal diet halts the lipid disarray due to saturated fatty acids in the liver of mice offspring at weaning.

PubMed

Shomonov-Wagner, Limor; Raz, Amiram; Leikin-Frenkel, Alicia

2015-02-26

Alpha linolenic acid (ALA, 18:3) in maternal diets has been shown to attenuate obesity associated insulin resistance (IR) in adult offspring in mice. The objective in the present study was to detect the early effects of maternal dietary saturated fatty acids (SFA) and their partial substitution with ω-3 ALA, docosa hexenoic acid (DHA,22:6) and eicosapentenoic acid 20:5 (EPA,20:5) on the HOMA index, liver lipids and fatty acid desaturases in the offspring at weaning. 3 month old C57Bl6/J female mice were fed diets containing normal amount of calories but rich in SFA alone or partially replaced with ALA, DHA or EPA before mating, during pregnancy and lactation. Pregnant mice fed SFA produced offspring with the highest HOMA index, liver lipids and desaturase activities. ALA prevented SFA induced lipid increase but DHA and EPA only reduced it by 42% and 31% respectively. ALA, DHA and EPA decreased HOMA index by 84%, 75% and 83% respectively. ALA, DHA and EPA decreased Δ6 and SCD1 desaturase activities about 30%. SFA feeding to mothers predisposes their offspring to develop IR and liver lipid accumulation already at weaning. ω3 fatty acids reduce IR, ALA halts lipid accumulation whereas DHA and EPA only blunt it.ALA and DHA restore the increased SCD1 to normal. These studies suggest that ω-3 fatty acids have different potencies to preclude lipid accumulation in the offspring partially by affecting pathways associated to SCD1 modulation.

Arabidopsis Enhanced Drought Tolerance1/HOMEODOMAIN GLABROUS11 Confers Drought Tolerance in Transgenic Rice without Yield Penalty1[W][OA

PubMed Central

Yu, Linhui; Chen, Xi; Wang, Zhen; Wang, Shimei; Wang, Yuping; Zhu, Qisheng; Li, Shigui; Xiang, Chengbin

2013-01-01

Enhancing drought tolerance without yield decrease has been a great challenge in crop improvement. Here, we report the Arabidopsis (Arabidopsis thaliana) homodomain-leucine zipper transcription factor Enhanced Drought Tolerance/HOMEODOMAIN GLABROUS11 (EDT1/HDG11) was able to confer drought tolerance and increase grain yield in transgenic rice (Oryza sativa) plants. The improved drought tolerance was associated with a more extensive root system, reduced stomatal density, and higher water use efficiency. The transgenic rice plants also had higher levels of abscisic acid, proline, soluble sugar, and reactive oxygen species-scavenging enzyme activities during stress treatments. The increased grain yield of the transgenic rice was contributed by improved seed setting, larger panicle, and more tillers as well as increased photosynthetic capacity. Digital gene expression analysis indicated that AtEDT1/HDG11 had a significant influence on gene expression profile in rice, which was consistent with the observed phenotypes of transgenic rice plants. Our study shows that AtEDT1/HDG11 can improve both stress tolerance and grain yield in rice, demonstrating the efficacy of AtEDT1/HDG11 in crop improvement. PMID:23735506

Heavy ion mutagenesis combined with triclosan screening provides a new strategy for improving the arachidonic acid yield in Mortierella alpina.

PubMed

Zhang, Huidan; Lu, Dong; Li, Xin; Feng, Yingang; Cui, Qiu; Song, Xiaojin

2018-05-02

Arachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment. The obtained mutant strain F-23 ultimately achieved an ARA yield of 5.26 g L - 1 , which is 3.24 times higher than that of the wild-type strain. In addition, quantitative real-time PCR confirmed that the expression levels of fatty acid synthase (FAS), Δ5-desaturase, Δ6-desaturase, and Δ9-desaturase were all significantly up-regulated in the mutant F-23 strain, especially Δ6- and Δ9-desaturase, which were up-regulated 3- and 2-fold, respectively. This study confirmed a feasible mutagenesis breeding strategy for improving ARA production and provided a mutant of Mortierella alpina with high ARA yield.

Hydrostatic pressure decreases membrane fluidity and lipid desaturase expression in chondrocyte progenitor cells.

PubMed

Montagne, Kevin; Uchiyama, Hiroki; Furukawa, Katsuko S; Ushida, Takashi

2014-01-22

Membrane biomechanical properties are critical in modulating nutrient and metabolite exchange as well as signal transduction. Biological membranes are predominantly composed of lipids, cholesterol and proteins, and their fluidity is tightly regulated by cholesterol and lipid desaturases. To determine whether such membrane fluidity regulation occurred in mammalian cells under pressure, we investigated the effects of pressure on membrane lipid order of mouse chondrogenic ATDC5 cells and desaturase gene expression. Hydrostatic pressure linearly increased membrane lipid packing and simultaneously repressed lipid desaturase gene expression. We also showed that cholesterol mimicked and cholesterol depletion reversed those effects, suggesting that desaturase gene expression was controlled by the membrane physical state itself. This study demonstrates a new effect of hydrostatic pressure on mammalian cells and may help to identify the molecular mechanisms involved in hydrostatic pressure sensing in chondrocytes. © 2013 Elsevier Ltd. All rights reserved.

Flavonoid production in transgenic hop (Humulus lupulus L.) altered by PAP1/MYB75 from Arabidopsis thaliana L.

PubMed

Gatica-Arias, A; Farag, M A; Stanke, M; Matoušek, J; Wessjohann, L; Weber, G

2012-01-01

Hop is an important source of secondary metabolites, such as flavonoids. Some of these are pharmacologically active. Nevertheless, the concentration of some classes as flavonoids in wild-type plants is rather low. To enhance the production in hop, it would be interesting to modify the regulation of genes in the flavonoid biosynthetic pathway. For this purpose, the regulatory factor PAP1/AtMYB75 from Arabidopsis thaliana L. was introduced into hop plants cv. Tettnanger by Agrobacterium-mediated genetic transformation. Twenty kanamycin-resistant transgenic plants were obtained. It was shown that PAP1/AtMYB75 was stably incorporated and expressed in the hop genome. In comparison to the wild-type plants, the color of female flowers and cones of transgenic plants was reddish to pink. Chemical analysis revealed higher levels of anthocyanins, rutin, isoquercitin, kaempferol-glucoside, kaempferol-glucoside-malonate, desmethylxanthohumol, xanthohumol, α-acids and β-acids in transgenic plants compared to wild-type plants.

The GmFAD7 gene family from soybean: identification of novel genes and tissue-specific conformations of the FAD7 enzyme involved in desaturase activity.

PubMed

Andreu, Vanesa; Lagunas, Beatriz; Collados, Raquel; Picorel, Rafael; Alfonso, Miguel

2010-07-01

The FAD7 gene encodes a omega3 fatty acid desaturase which catalyses the production of trienoic fatty acids (TAs) in plant chloroplasts. A novel GmFAD7 gene (named GmFAD7-2) has been identified in soybean, with high homology to the previously annotated GmFAD7 gene. Genomic sequencing analysis together with searches at the soybean genome database further confirmed that both GmFAD7 genes were located in two different loci within the soybean genome, suggesting that the soybean omega3 plastidial desaturase FAD7 is encoded by two different paralogous genes. Both GmFAD7-1 and GmFAD7-2 genes were expressed in all soybean tissues examined, displaying their highest mRNA accumulation in leaves. This expression profile contrasted with GmFAD3A and GmFAD3B mRNA accumulation, which was very low in this tissue. These results suggested a concerted control of plastidial and reticular omega3 desaturase gene expression in soybean mature leaves. Analysis of GmFAD7 protein distribution in different soybean tissues showed that, in mature leaves, two bands were detected, coincident with the higher expression level of both GmFAD7 genes and the highest 18:3 fatty acid accumulation. By contrast, in seeds, where FAD7 activity is low, specific GmFAD7 protein conformations were observed. These GmFAD7 protein conformations were affected in vitro by changes in the redox conditions of thiol groups and iron availability. These results suggest the existence of tissue-specific post-translational regulatory mechanisms affecting the distribution and conformation of the FAD7 enzymes related with the control of its activity.

Characterization of a Maize Wip1 Promoter in Transgenic Plants

PubMed Central

Zhang, Shengxue; Lian, Yun; Liu, Yan; Wang, Xiaoqing; Liu, Yunjun; Wang, Guoying

2013-01-01

The Maize Wip1 gene encodes a wound-induced Bowman-Birk inhibitor (BBI) protein which is a type of serine protease inhibitor, and its expression is induced by wounding or infection, conferring resistance against pathogens and pests. In this study, the maize Wip1 promoter was isolated and its function was analyzed. Different truncated Wip1 promoters were fused upstream of the GUS reporter gene and transformed into Arabidopsis, tobacco and rice plants. We found that (1) several truncated maize Wip1 promoters led to strong GUS activities in both transgenic Arabidopsis and tobacco leaves, whereas low GUS activity was detected in transgenic rice leaves; (2) the Wip1 promoter was not wound-induced in transgenic tobacco leaves, but was induced by wounding in transgenic rice leaves; (3) the truncated Wip1 promoter had different activity in different organs of transgenic tobacco plants; (4) the transgenic plant leaves containing different truncated Wip1 promoters had low GUS transcripts, even though high GUS protein level and GUS activities were observed; (5) there was one transcription start site of Wip1 gene in maize and two transcription start sites of GUS in Wip1::GUS transgenic lines; (6) the adjacent 35S promoter which is present in the transformation vectors enhanced the activity of the truncated Wip1 promoters in transgenic tobacco leaves, but did not influence the disability of truncated Wip1231 promoter to respond to wounding signals. We speculate that an ACAAAA hexamer, several CAA trimers and several elements similar to ACAATTAC octamer in the 5′-untranslated region might contribute to the strong GUS activity in Wip1231 transgenic lines, meanwhile, compared to the 5′-untranslated region from Wip1231 transgenic lines, the additional upstream open reading frames (uORFs) in the 5′-untranslated region from Wip1737 transgenic lines might contribute to the lower level of GUS transcript and GUS activity. PMID:24322445

Evolution of the Insect Desaturase Gene Family with an Emphasis on Social Hymenoptera

PubMed Central

Helmkampf, Martin; Cash, Elizabeth; Gadau, Jürgen

2015-01-01

Desaturase genes are essential for biological processes, including lipid metabolism, cell signaling, and membrane fluidity regulation. Insect desaturases are particularly interesting for their role in chemical communication, and potential contribution to speciation, symbioses, and sociality. Here, we describe the acyl-CoA desaturase gene families of 15 insects, with a focus on social Hymenoptera. Phylogenetic reconstruction revealed that the insect desaturases represent an ancient gene family characterized by eight subfamilies that differ strongly in their degree of conservation and frequency of gene gain and loss. Analyses of genomic organization showed that five of these subfamilies are represented in a highly microsyntenic region conserved across holometabolous insect taxa, indicating an ancestral expansion during early insect evolution. In three subfamilies, ants exhibit particularly large expansions of genes. Despite these expansions, however, selection analyses showed that desaturase genes in all insect lineages are predominantly undergoing strong purifying selection. Finally, for three expanded subfamilies, we show that ants exhibit variation in gene expression between species, and more importantly, between sexes and castes within species. This suggests functional differentiation of these genes and a role in the regulation of reproductive division of labor in ants. The dynamic pattern of gene gain and loss of acyl-CoA desaturases in ants may reflect changes in response to ecological diversification and an increased demand for chemical signal variability. This may provide an example of how gene family expansions can contribute to lineage-specific adaptations through structural and regulatory changes acting in concert to produce new adaptive phenotypes. PMID:25425561

Bleaching herbicide norflurazon inhibits phytoene desaturase by competition with the cofactors.

PubMed

Breitenbach, J; Zhu, C; Sandmann, G

2001-11-01

Cofactor requirement was determined for the heterologous expressed phytoene desaturases from the cyanobacterium Synechococcus and the higher plant Gentiana lutea. The cyanobacterial enzyme is dependent on either NAD(P) or plastoquinone, whereas only quinones such as plastoquinone can function as a cofactor for the phytoene desaturase from G. lutea. Enzyme kinetic studies were carried out to determine a possible competition between the cofactors and the bleaching herbicide norflurazon. For the Synechococcus enzyme, competition between norflurazon and NADP, as well as plastoquinone, could be demonstrated. The K(m) values for these cofactors were 6.6 mM and 0.23 microM, respectively. Inhibition of the phytoene desaturase from G. lutea by norflurazon was also competitive with respect to plastoquinone. The K(m) values of both enzymes for plastoquinone were very close.

Generation and Characterization of Human Heme Oxygenase-1 Transgenic Pigs

PubMed Central

Yang, Jaeseok; Cho, Bumrae; Hwang, Jong-Ik; Park, Sol Ji; Hurh, Sunghoon; Kim, Hwajung; Lee, Eun Mi; Ro, Han; Kang, Jung Taek; Kim, Su Jin; Won, Jae-Kyung; O'Connell, Philip J.; Kim, Hyunil; Surh, Charles D.; Lee, Byeong-Chun; Ahn, Curie

2012-01-01

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation. PMID:23071605

Generation and characterization of human heme oxygenase-1 transgenic pigs.

PubMed

Yeom, Hye-Jung; Koo, Ok Jae; Yang, Jaeseok; Cho, Bumrae; Hwang, Jong-Ik; Park, Sol Ji; Hurh, Sunghoon; Kim, Hwajung; Lee, Eun Mi; Ro, Han; Kang, Jung Taek; Kim, Su Jin; Won, Jae-Kyung; O'Connell, Philip J; Kim, Hyunil; Surh, Charles D; Lee, Byeong-Chun; Ahn, Curie

2012-01-01

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.

Effects of Astaxanthin and Docosahexaenoic-Acid-Acylated Astaxanthin on Alzheimer's Disease in APP/PS1 Double-Transgenic Mice.

PubMed

Che, Hongxia; Li, Qian; Zhang, Tiantian; Wang, Dandan; Yang, Lu; Xu, Jie; Yanagita, Teruyoshi; Xue, Changhu; Chang, Yaoguang; Wang, Yuming

2018-05-16

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with the characteristics of senile plaques, neuroinflammation, neurofibrillary tangles, and destruction of synapse structure stability. Previous studies have verified the protective effects of astaxanthin (AST). However, whether synthesized docosahexaenoic-acid-acylated AST diesters (AST-DHA) could delay AD pathogenesis remains unclear. In the present study, APP/PSEN1 (APP/PS1) double-transgenic mice were administrated with AST and AST-DHA for 2 months. The results of radial 8-arm maze and Morris water maze tests showed that AST-DHA exerted more significant effects than AST in enhancing learning and memory levels of APP/PS1 mice. Further mechanical studies suggested that AST-DHA was superior to AST in regulating the parameters of oxidative stress, reducing tau hyperphosphorylation, suppressing neuroinflammation, and regulating inflammasome expression and activation in APP/PS1 mice. The findings suggested that AST-DHA attenuated cognitive disorders by reducing pathological features in APP/PS1 mice, suggesting that AST-DHA might be a potential therapeutic agent for AD.

Maize ARGOS1 (ZAR1) transgenic alleles increase hybrid maize yield.

PubMed

Guo, Mei; Rupe, Mary A; Wei, Jun; Winkler, Chris; Goncalves-Butruille, Marymar; Weers, Ben P; Cerwick, Sharon F; Dieter, Jo Ann; Duncan, Keith E; Howard, Richard J; Hou, Zhenglin; Löffler, Carlos M; Cooper, Mark; Simmons, Carl R

2014-01-01

Crop improvement for yield and drought tolerance is challenging due to the complex genetic nature of these traits and environmental dependencies. This study reports that transgenic over-expression of Zea mays AR GOS1 (ZAR1) enhanced maize organ growth, grain yield, and drought-stress tolerance. The ZAR1 transgene exhibited environmental interactions, with yield increase under Temperate Dry and yield reduction under Temperate Humid or High Latitude environments. Native ZAR1 allele variation associated with drought-stress tolerance. Two founder alleles identified in the mid-maturity germplasm of North America now predominate in Pioneer's modern breeding programme, and have distinct proteins, promoters and expression patterns. These two major alleles show heterotic group partitioning, with one predominant in Pioneer's female and the other in the male heterotic groups, respectively. These two alleles also associate with favourable crop performance when heterozygous. Allele-specific transgene testing showed that, of the two alleles discussed here, each allele differed in their impact on yield and environmental interactions. Moreover, when transgenically stacked together the allelic pair showed yield and environmental performance advantages over either single allele, resembling heterosis effects. This work demonstrates differences in transgenic efficacy of native alleles and the differences reflect their association with hybrid breeding performance.

Maize ARGOS1 (ZAR1) transgenic alleles increase hybrid maize yield

PubMed Central

Guo, Mei

2014-01-01

Crop improvement for yield and drought tolerance is challenging due to the complex genetic nature of these traits and environmental dependencies. This study reports that transgenic over-expression of Zea mays ARGOS1 (ZAR1) enhanced maize organ growth, grain yield, and drought-stress tolerance. The ZAR1 transgene exhibited environmental interactions, with yield increase under Temperate Dry and yield reduction under Temperate Humid or High Latitude environments. Native ZAR1 allele variation associated with drought-stress tolerance. Two founder alleles identified in the mid-maturity germplasm of North America now predominate in Pioneer’s modern breeding programme, and have distinct proteins, promoters and expression patterns. These two major alleles show heterotic group partitioning, with one predominant in Pioneer’s female and the other in the male heterotic groups, respectively. These two alleles also associate with favourable crop performance when heterozygous. Allele-specific transgene testing showed that, of the two alleles discussed here, each allele differed in their impact on yield and environmental interactions. Moreover, when transgenically stacked together the allelic pair showed yield and environmental performance advantages over either single allele, resembling heterosis effects. This work demonstrates differences in transgenic efficacy of native alleles and the differences reflect their association with hybrid breeding performance. PMID:24218327

The Crystal Structure of the Ivy delta4-16:0-ACP Desaturase Reveals Structural Details of the Oxidized Active Site and Potential Determinants of Regioselectivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guy,J.; Whittle, E.; Kumaran, D.

2007-01-01

The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the {Delta}{sup 4} desaturation of 16:0-ACP and the{Delta}{sup 9} desaturation of 18:0-ACP and further desaturates{Delta}{sup 9}-16:1 or {Delta}{sup 9}-18:1 to the corresponding {Delta}{sup 4,9} dienes. The crystal structure of the enzyme has been solved to 1.95{angstrom} resolution, and both the iron-iron distance of 3.2{angstrom} and the presence of a {mu}-oxo bridge reveal this to be the only reported structure of a desaturase in the oxidized FeIII-FeIII form. Significant differences are seen between the oxidized active site and the reduced active site of the Ricinus communis (castor) desaturase;more » His{sup 227} coordination to Fe2 is lost, and the side chain of Glu{sup 224}, which bridges the two iron ions in the reduced structure, does not interact with either iron. Although carboxylate shifts have been observed on oxidation of other diiron proteins, this is the first example of the residue moving beyond the coordination range of both iron ions. Comparison of the ivy and castor structures reveal surface amino acids close to the annulus of the substrate-binding cavity and others lining the lower portion of the cavity that are potential determinants of their distinct substrate specificities. We propose a hypothesis that differences in side chain packing explains the apparent paradox that several residues lining the lower portion of the cavity in the ivy desaturase are bulkier than their equivalents in the castor enzyme despite the necessity for the ivy enzyme to accommodate three more carbons beyond the diiron site.« less

High levels of retinal membrane docosahexaenoic acid increase susceptibility to stress-induced degenerations⃞

PubMed Central

Tanito, Masaki; Brush, Richard S.; Elliott, Michael H.; Wicker, Lea D.; Henry, Kimberly R.; Anderson, Robert E.

2009-01-01

The fat-1 gene cloned from C. elegans encodes an n-3 fatty acid desaturase that converts n-6 to n-3 PUFA. Mice carrying the fat-1 transgene and wild-type controls were fed an n-3-deficient/n-6-enriched diet [fat-1- safflower oil (SFO) and wt-SFO, respectively]. Fatty acid profiles of rod outer segments (ROS), cerebellum, plasma, and liver demonstrated significantly lower n-6/n-3 ratios and higher docosahexaenoic acid (DHA) levels in fat-1-SFO compared with wt-SFO. When mice were exposed to light stress: 1) the outer nuclear layer (ONL) thickness was reduced; 2) amplitudes of the electroretinogram (ERG) were lower; 3) the number of apoptotic photoreceptor cells was greater; and 4) modification of retinal proteins by 4-hydroxyhexenal (4-HHE), an end-product of n-3 PUFA oxidation was increased in both fat-1-SFO and wt mice fed a regular lab chow diet compared with wt-SFO. The results indicate a positive correlation between the level of DHA, the degree of n-3 PUFA lipid peroxidation, and the vulnerability of the retina to photooxidative stress. In mice not exposed to intense light, the reduction in DHA resulted in reduced efficacy in phototransduction gain steps, while no differences in the retinal morphology or retinal biochemistry. These results highlight the dual roles of DHA in cellular physiology and pathology. PMID:19023138

Tissue-specific, nutritional, and developmental regulation of rat fatty acid elongases

PubMed Central

Wang, Yun; Botolin, Daniela; Christian, Barbara; Busik, Julia; Xu, Jinghua; Jump, Donald B.

2008-01-01

Of the six fatty acid elongase (Elovl) subtypes expressed in mammals, adult rat liver expresses four subtypes: Elovl-5 > Elovl-1 = Elovl-2 = Elovl-6. Overnight starvation and fish oil-enriched diets repressed hepatic elongase activity in livers of adult male rats. Diet-induced changes in elongase activity correlate with Elovl-5 and Elovl-6 mRNA abundance. Adult rats fed the peroxisome proliferator-activated receptor α (PPARα) agonist WY14,643 have increased hepatic elongase activity, Elovl-1, Elovl-5, Elovl-6, Δ5, Δ6, and Δ9 desaturase mRNA abundance, and mead acid (20:3,n-9) content. PPARα agonists affect both fatty acid elongation and desaturation pathways leading to changes in hepatic lipid composition. Elovl activity is low in fetal liver but increases significantly after birth. Developmental changes in hepatic elongase activity paralleled the postnatal induction of Elovl-5 mRNA and mRNAs encoding the PPARα-regulated transcripts, Δ5 and Δ6 desaturase, and cytochrome P450 4A. In contrast, Elovl-6, Δ9 desaturase, and FAS mRNA abundance paralleled changes in hepatic sterol regulatory element binding protein 1c (SREBP-1c) nuclear content. SREBP-1c is present in fetal liver nuclei, absent from nuclei immediately after birth, and reappears in nuclei at weaning, 21 days postpartum. In conclusion, changes in Elovl-5 expression may account for much of the nutritional and developmental control of fatty acid elongation activity in the rat liver. PMID:15654130

Sex Pheromone Evolution Is Associated with Differential Regulation of the Same Desaturase Gene in Two Genera of Leafroller Moths

PubMed Central

Albre, Jérôme; Liénard, Marjorie A.; Sirey, Tamara M.; Schmidt, Silvia; Tooman, Leah K.; Carraher, Colm; Greenwood, David R.; Löfstedt, Christer; Newcomb, Richard D.

2012-01-01

Chemical signals are prevalent in sexual communication systems. Mate recognition has been extensively studied within the Lepidoptera, where the production and recognition of species-specific sex pheromone signals are typically the defining character. While the specific blend of compounds that makes up the sex pheromones of many species has been characterized, the molecular mechanisms underpinning the evolution of pheromone-based mate recognition systems remain largely unknown. We have focused on two sets of sibling species within the leafroller moth genera Ctenopseustis and Planotortrix that have rapidly evolved the use of distinct sex pheromone blends. The compounds within these blends differ almost exclusively in the relative position of double bonds that are introduced by desaturase enzymes. Of the six desaturase orthologs isolated from all four species, functional analyses in yeast and gene expression in pheromone glands implicate three in pheromone biosynthesis, two Δ9-desaturases, and a Δ10-desaturase, while the remaining three desaturases include a Δ6-desaturase, a terminal desaturase, and a non-functional desaturase. Comparative quantitative real-time PCR reveals that the Δ10-desaturase is differentially expressed in the pheromone glands of the two sets of sibling species, consistent with differences in the pheromone blend in both species pairs. In the pheromone glands of species that utilize (Z)-8-tetradecenyl acetate as sex pheromone component (Ctenopseustis obliquana and Planotortrix octo), the expression levels of the Δ10-desaturase are significantly higher than in the pheromone glands of their respective sibling species (C. herana and P. excessana). Our results demonstrate that interspecific sex pheromone differences are associated with differential regulation of the same desaturase gene in two genera of moths. We suggest that differential gene regulation among members of a multigene family may be an important mechanism of molecular innovation in

Acyl CoA profiles of transgenic plants that accumulate medium-chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds.

PubMed

Larson, Tony R; Edgell, Teresa; Byrne, James; Dehesh, Katayoon; Graham, Ian A

2002-11-01

Several Brassica napus lines transformed with genes responsible for the synthesis of medium- or long-chain fatty acids were examined to determine limiting factor(s) for the subsequent accumulation of these fatty acids in seed lipids. Examination of a decanoic acid (10:0) accumulating line revealed a disproportionately high concentration of 10:0 CoA during seed development compared to long-chain acyl CoAs isolated from the same tissues, suggesting that poor incorporation of 10:0 CoA into seed lipids limits 10:0 fatty acid accumulation. This relationship was also seen for dodecanoyl (12:0) CoA and fatty acid in a high 12:0 line, but not for octadecanoic (18:0) CoA and fatty acid in a high 18:0 line. Comparison of 10:0 CoA and fatty acid proportions from seeds at different developmental stages for transgenic B. napus and Cuphea hookeriana, the source plant for the medium-chain thioesterase and 3-ketoacyl-ACP synthase transgenes, revealed that C. hookeriana incorporates 10:0 CoA into seed lipids more efficiently than transgenic B. napus. Furthermore, beta-oxidation and glyoxylate cycle activities were not increased above wild type levels during seed development in the 8:0/10:0 line, suggesting that lipid catabolism was not being induced in response to the elevated 10:0 CoA concentrations. Taken together, these data suggest that transgenic plants that are engineered to synthesize medium-chain fatty acids may lack the necessary mechanisms, such as specific acyltransferases, to incorporate these fatty acids efficiently into seed lipids.

Antibody-induced albuminuria and accelerated focal glomerulosclerosis in the Thy-1.1 transgenic mouse.

PubMed

Assmann, Karel J M; van Son, Jacco P H F; Dïjkman, Henry B P M; Mentzel, Stef; Wetzels, Jack F M

2002-07-01

Podocytes play an important role in the development of proteinuria and focal glomerulosclerosis. Previously we have demonstrated that a combination of two monoclonal antibodies (mAb) against aminopeptidase A (APA), an enzyme present on podocytes, induces a massive acute albuminuria in mice. The present study examined the relationship between the acute antibody-induced albuminuria and the development of focal glomerulosclerosis in the Thy-1.1 transgenic mouse. This mouse expresses a hybrid human-mouse Thy-1.1 antigen on the podocytes, and slowly but spontaneously develops albuminuria and focal glomerulosclerosis. Five-week-old non-albuminuric Thy-1.1 transgenic and non-transgenic control mice were injected with anti-APA and anti-Thy-1.1 mAb or saline. Albuminuria was measured at days 1, 7, 14 and 21. At day 21 kidneys were processed for light microscopy, immunofluorescence, and electron microscopy. Injection of anti-APA and anti-Thy1.1 mAb in Thy-1.1 transgenic mice induced an albuminuria at day 1 that persisted at day 21. The acute albuminuria after injection of anti-APA mAb was more prominent but transient in non-transgenic mice. In non-trangenic mice no albuminuria could be induced with anti-Thy 1.1 mAb. Light microscopy revealed normal glomeruli at day 1 in all transgenic mice, however, at day 21 advanced glomerulosclerotic lesions were seen in mice injected with either anti-APA mAb (37+/-19% of glomeruli affected) or anti-Thy-1.1 mAb (71+/-5%). Non-transgenic mice did not reveal sclerotic lesions at any time investigated. In the transgenic mice the percentage of focal glomerulosclerosis at day 21 did not correlate with albuminuria at day 21. However, we found a highly significant correlation between percentage of focal glomerulosclerosis and the time-averaged albuminuria over the three-week study period (P < 0.001). Injection of a combination of anti-APA or anti-Thy-1.1 mAb into one mo old, non-albuminuric Thy-1.1 transgenic mice induces an acute albuminuria at

Cardiac phenotype induced by a dysfunctional α 1C transgene: a general problem for the transgenic approach.

PubMed

Asemu, Girma; Fishbein, Kenneth; Lao, Qi Zong; Ravindran, Arippa; Herbert, Ron; Canuto, Holly C; Spencer, Richard G; Soldatov, Nikolai M

2011-01-01

Based on stable integration of recombinant DNA into a host genome, transgenic technology has become an important genetic engineering methodology. An organism whose genetic characteristics have been altered by the insertion of foreign DNA is supposed to exhibit a new phenotype associated with the function of the transgene. However, successful insertion may not be sufficient to achieve specific modification of function. In this study we describe a strain of transgenic mouse, G7-882, generated by incorporation into the mouse genome of human CaV 1.2 α(1C) cDNA deprived of 3'-UTR to exclude transcription. We found that, in response to chronic infusion of isoproterenol, G7-882 develops dilated cardiomyopathy, a misleading "transgenic artifact" compatible with the expected function of the incorporated "correct" transgene. Specifically, using magnetic resonance imaging (MRI), we found that chronic β-adrenergic stimulation of G7-882 mice caused left ventricular hypertrophy and aggravated development of dilated cardiomyopathy, although no significant changes in the kinetics, density and voltage dependence of the calcium current were observed in G7-882 cardiomyocytes as compared to cells from wild type mice. This result illustrates the possibility that even when a functional transgene is expressed, an observed change in phenotype may be due to the artifact of "incidental incorporation" leading to misleading conclusions. To exclude this possibility and thus provide a robust tool for exploring biological function, the new transgenic phenotype must be replicated in several independently generated transgenic strains.

Maternal single nucleotide polymorphisms in the fatty acid desaturase 1 and 2 coding regions modify the impact of prenatal supplementation with DHA on birth weight.

PubMed

Gonzalez-Casanova, Ines; Rzehak, Peter; Stein, Aryeh D; Garcia Feregrino, Raquel; Rivera Dommarco, Juan A; Barraza-Villarreal, Albino; Demmelmair, Hans; Romieu, Isabelle; Villalpando, Salvador; Martorell, Reynaldo; Koletzko, Berthold; Ramakrishnan, Usha

2016-04-01

Specific single nucleotide polymorphisms (SNPs) in the fatty acid desaturase (FADS) gene affect the activity and efficiency of enzymes that are responsible for the conversion of polyunsaturated fatty acids (PUFAs) into their long-chain active form. A high prevalence of SNPs that are associated with slow PUFA conversion has been described in Hispanic populations. We assessed the heterogeneity of the effect of prenatal supplementation with docosahexaenoic acid (DHA) on birth weight across selected FADS SNPs in a sample of Mexican women and their offspring. We obtained information on the maternal genotype from stored blood samples of 654 women who received supplementation with 400 mg DHA/d or a placebo from weeks 18 to 22 of gestation through delivery as part of a randomized controlled trial conducted in Cuernavaca, Mexico. We selected 4 tag SNPs (rs174455, rs174556, rs174602, and rs498793) in the FADS region for analysis. We used an ANOVA to test for the heterogeneity of the effect on birth weight across each of the 4 SNPs. The mean ± SD birth weight was 3210 ± 470 g, and the weight-for-age z score (WAZ) was -0.24 ± 1.00. There were no intention-to-treat differences in birth weights. We showed significant heterogeneity by SNP rs174602 (P= 0.02); offspring of carriers of alleles TT and TC in the intervention group were heavier than those in the placebo group (WAZ: -0.13 ± 0.14 and -0.20 ± 0.08 compared with -0.55 ± 0.15 and -0.39 ± 0.09, respectively); there were no significant differences in offspring of rs174602 CC homozygotes (WAZ: -0.26 ± 0.09 in the intervention group compared with -0.04 ± 0.09 in the placebo group). We showed no significant heterogeneity across the other 3 FADS SNPs. Differential responses to prenatal DHA supplementation on the basis of the genetic makeup of target populations could explain the mixed evidence of the impact of DHA supplementation on birth weight. This trial was registered at clinicaltrials.gov as NCT00646360. © 2016

Biofortification of safflower: an oil seed crop engineered for ALA-targeting better sustainability and plant based omega-3 fatty acids.

PubMed

Rani, Arti; Panwar, Asha; Sathe, Manjary; Chandrashekhara, Karunakara Alageri; Kush, Anil

2018-05-11

Alpha-linolenic acid (ALA) deficiency and a skewed n6:n3 fatty acid ratio in the diet is a major explanation for the prevalence of cardiovascular diseases and inflammatory/autoimmune diseases. There is mounting evidence of the health benefits associated with omega-3 long chain polyunsaturated fatty acids (LC PUFA's). Although present in abundance in fish, a number of factors limit our consumption of fish based omega-3 PUFA's. To name a few, overexploitation of wild fish stocks has reduced their sustainability due to increased demand of aquaculture for fish oil and meal; the pollution of marine food webs has raised concerns over the ingestion of toxic substances such as heavy metals and dioxins; vegetarians do not consider fish-based sources for supplemental nutrition. Thus alternative sources are being sought and one approach to the sustainable supply of LC-PUFAs is the metabolic engineering of transgenic plants with the capacity to synthesize n3 LC-PUFAs. The present investigation was carried out with the goal of developing transgenic safflower capable of producing pharmaceutically important alpha-linolenic acid (ALA, C18:3, n3). This crop was selected as the seeds accumulate ~ 78% of the total fatty acids as linoleic acid (LA, C18:2, n6), the immediate precursor of ALA. In the present work, ALA production was achieved successfully in safflower seeds by transforming safflower hypocotyls with Arabidopsis specific delta 15 desaturase (FAD3) driven by truncated seed specific promoter. Transgenic safflower fortified with ALA is not only potentially valuable nutritional superior novel oil but also has reduced ratio of LA to ALA which is required for good health.

Cold affects the transcription of fatty acid desaturases and oil quality in the fruit of Olea europaea L. genotypes with different cold hardiness

PubMed Central

Matteucci, M.; D'Angeli, S.; Errico, S.; Lamanna, R.; Perrotta, G.; Altamura, M. M.

2011-01-01

The olive tree lacks dormancy and is low temperature sensitive, with differences in cold tolerance and oil quality among genotypes. The oil is produced in the drupe, and the unsaturated fatty acids contribute to its quality. The aim of the present research was to investigate the relationship among development, cold response, expression of fatty acid desaturase (FAD) genes, and unsaturated fatty acid composition in drupes belonging to genotypes differing in leaf cold tolerance, but producing good oil (i.e. the non-hardy Moraiolo, the semi-hardy Frantoio, and the hardy Canino). In all genotypes, cold sensitivity, evaluated by cold-induced transient increases in cytosolic calcium, was high in the epi-mesocarp cells before oil body formation, and decreased during oil biogenesis. However, genotype-dependent differences in cold sensitivity appeared at the end of oil production. Genotype-dependent differences in FAD2.1, FAD2.2, FAD6, and FAD7 expression levels occurred in the epi-mesocarp cells during the oleogenic period. However, FAD2.1 and FAD7 were always the highest in the first part of this period. FAD2.2 and FAD7 increased after cold applications during oleogenesis, independently of the genotype. Unsaturated fatty acids increased in the drupes of the non-hardy genotype, but not in those of the hardy one, after cold exposure at the time of the highest FAD transcription. The results show a direct relationship between FAD expression and lipid desaturation in the drupes of the cold-sensitive genotype, and an inverse relationship in those of the cold-resistant genotype, suggesting that drupe cold acclimation requires a fine FAD post-transcriptional regulation. Hypotheses relating FAD desaturation to storage and membrane lipids, and genotype cold hardiness are discussed. PMID:21357772

Gene Expression of Desaturase (FADS1 and FADS2) and Elongase (ELOVL5) Enzymes in Peripheral Blood: Association with Polyunsaturated Fatty Acid Levels and Atopic Eczema in 4-Year-Old Children

PubMed Central

Chisaguano, Aida Maribel; Montes, Rosa; Pérez-Berezo, Teresa; Castellote, Ana Isabel; Guerendiain, Marcela; Bustamante, Mariona; Morales, Eva; García-Esteban, Raquel; Sunyer, Jordi; Franch, Àngels; López-Sabater, M. Carmen

2013-01-01

Abstract Background It is unknown if changes in the gene expression of the desaturase and elongase enzymes are associated with abnormal n-6 long chain polyunsaturated fatty acid (LC-PUFA) levels in children with atopic eczema (AE). We analyzed whether mRNA-expression of genes encoding key enzymes of LC-PUFA synthesis (FADS1, FADS2 and ELOVL5) is associated with circulating LC-PUFA levels and risk of AE in 4-year-old children. Methods AE (n=20) and non-AE (n=104) children participating in the Sabadell cohort within the INfancia y Medio Ambiente (INMA) Project were included in the present study. RT-PCR with TaqMan Low-Density Array cards was used to measure the mRNA-expression of FADS1, FADS2 and ELOVL5. LC-PUFA levels were measured by fast gas chromatography in plasma phospholipids. The relationship of gene expression with LC-PUFA levels and enzyme activities was evaluated by Pearson’s rank correlation coefficient, and logistic regression models were used to study its association with risk of developing AE. Results Children with AE had lower levels of several n-6 PUFA members, dihomo-γ-linolenic (DGLA) and arachidonic (AA) acids. mRNA-expression levels of FADS1 and 2 strongly correlated with DGLA levels and with D6D activity. FADS2 and ELOVL5 mRNA-expression levels were significantly lower in AE than in non-AE children (-40.30% and -20.36%; respectively), but no differences were found for FADS1. Conclusions and Significance Changes in the mRNA-expression levels of FADS1 and 2 directly affect blood DGLA levels and D6D activity. This study suggests that lower mRNA-expressions of FADS2 and ELOVL5 are associated with higher risk of atopic eczema in young children. PMID:24167612

Gene expression of desaturase (FADS1 and FADS2) and Elongase (ELOVL5) enzymes in peripheral blood: association with polyunsaturated fatty acid levels and atopic eczema in 4-year-old children.

PubMed

Chisaguano, Aida Maribel; Montes, Rosa; Pérez-Berezo, Teresa; Castellote, Ana Isabel; Guerendiain, Marcela; Bustamante, Mariona; Morales, Eva; García-Esteban, Raquel; Sunyer, Jordi; Franch, Angels; López-Sabater, M Carmen

2013-01-01

It is unknown if changes in the gene expression of the desaturase and elongase enzymes are associated with abnormal n-6 long chain polyunsaturated fatty acid (LC-PUFA) levels in children with atopic eczema (AE). We analyzed whether mRNA-expression of genes encoding key enzymes of LC-PUFA synthesis (FADS1, FADS2 and ELOVL5) is associated with circulating LC-PUFA levels and risk of AE in 4-year-old children. AE (n=20) and non-AE (n=104) children participating in the Sabadell cohort within the INfancia y Medio Ambiente (INMA) Project were included in the present study. RT-PCR with TaqMan Low-Density Array cards was used to measure the mRNA-expression of FADS1, FADS2 and ELOVL5. LC-PUFA levels were measured by fast gas chromatography in plasma phospholipids. The relationship of gene expression with LC-PUFA levels and enzyme activities was evaluated by Pearson's rank correlation coefficient, and logistic regression models were used to study its association with risk of developing AE. Children with AE had lower levels of several n-6 PUFA members, dihomo-γ-linolenic (DGLA) and arachidonic (AA) acids. mRNA-expression levels of FADS1 and 2 strongly correlated with DGLA levels and with D6D activity. FADS2 and ELOVL5 mRNA-expression levels were significantly lower in AE than in non-AE children (-40.30% and -20.36%; respectively), but no differences were found for FADS1. Changes in the mRNA-expression levels of FADS1 and 2 directly affect blood DGLA levels and D6D activity. This study suggests that lower mRNA-expressions of FADS2 and ELOVL5 are associated with higher risk of atopic eczema in young children.

Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

PubMed Central

Jhappan, C; Geiser, A G; Kordon, E C; Bagheri, D; Hennighausen, L; Roberts, A B; Smith, G H; Merlino, G

1993-01-01

Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland. Images PMID:8491177

Induction of 1-acylglycerophosphocholine acyltransferase genes by fibrates in the liver of rats.

PubMed

Yamazaki, Tohru; Wakabayashi, Michiko; Ikeda, Erika; Tanaka, Shizuyo; Sakamoto, Takeshi; Mitsumoto, Atsushi; Kudo, Naomi; Kawashima, Yoichi

2012-01-01

The effect of fibrates (clofibric acid, bezafibrate and fenofibrate) on the gene expression and activity of 1-acylglycerophosphocholine acyltransferase (LPCAT) was investigated. The administration of 0.1% (w/w) clofibric acid, bezafibrate or fenofibrate in diet for 14 d to rats induced LPCAT activity in hepatic microsomes in the following order: fenofibrate>bezafibrate>clofibric acid. The LPCAT induced by fenofibrate preferred to arachidonoyl-CoA and linoleoyl-CoA to a greater extent than did LPCAT in control microsomes. The treatment with the fibrates resulted in upregulation of the relative expression of mRNAs encoding LPCAT3 and LPCAT4 in the following order: fenofibrate>bezafibrate>clofibric acid. The administration of fibrates did not change the expression of genes encoding either LPCAT1 or LPCAT2. The treatment with fibrates elevated relative levels of both mRNAs encoding Δ6 desaturase (Fads2) and Δ5 desaturase (Fads1) in the order of fenofibrate>bezafibrate>clofibric acid, and the extent of the increase in the level of Δ6 desaturase mRNA was greater than that of Δ5 desaturase. Fatty acid profile in hepatic phosphatidylcholine (PC) was significantly changed by the treatments with fibrates. These results suggest (i) that fibrates induce LPCAT activity in hepatic microsomes by elevating the expression of genes encoding LPCAT3 and LPCAT4, (ii) that the changes in fatty acid profile of hepatic PC are, in part, due to the elevated expression of two isoforms, LPCAT3 and LPCAT4, and (iii) that the ability of fibrates to induce these changes are in the order of fenofibrate>bezafibrate>clofibric acid.

Overexpression of Poplar Pyrabactin Resistance-Like Abscisic Acid Receptors Promotes Abscisic Acid Sensitivity and Drought Resistance in Transgenic Arabidopsis.

PubMed

Yu, Jingling; Yang, Lei; Liu, Xiaobing; Tang, Renjie; Wang, Yuan; Ge, Haiman; Wu, Mengting; Zhang, Jiang; Zhao, Fugeng; Luan, Sheng; Lan, Wenzhi

2016-01-01

Drought stress is an important environmental factor limiting productivity of plants, especially fast growing species with high water consumption like poplar. Abscisic acid (ABA) is a phytohormone that positively regulates seed dormancy and drought resistance. The PYR1 (Pyrabactin Resistance 1)/ PYRL (PYR-Like)/ RCAR (Regulatory Component of ABA Receptor) (PYR/PYL/RCAR) ABA receptor family has been identified and widely characterized in Arabidopsis thaliana. However, their functions in poplars remain unknown. Here, we report that 2 of 14 PYR/PYL/RCAR orthologues in poplar (Populus trichocarpa) (PtPYRLs) function as a positive regulator of the ABA signal transduction pathway. The Arabidopsis transient expression and yeast two-hybrid assays showed the interaction among PtPYRL1 and PtPYRL5, a clade A protein phosphatase 2C, and a SnRK2, suggesting that a core signalling complex for ABA signaling pathway exists in poplars. Phenotypic analysis of PtPYRL1 and PtPYRL5 transgenic Arabidopsis showed that these two genes positively regulated the ABA responses during the seed germination. More importantly, the overexpression of PtPYRL1 and PtPYRL5 substantially improved ABA sensitivity and drought stress tolerance in transgenic plants. In summary, we comprehensively uncovered the properties of PtPYRL1 and PtPYRL5, which might be good target genes to genetically engineer drought-Resistant plants.

Chronic sucrose intake decreases concentrations of n6 fatty acids, but not docosahexaenoic acid in the rat brain phospholipids.

PubMed

Mašek, Tomislav; Starčević, Kristina

2017-07-13

We investigated the influence of high sucrose intake, administered in drinking water, on the lipid profile of the brain and on the expression of SREBP1c and Δ-desaturase genes. Adult male rats received 30% sucrose solution for 20 weeks (Sucrose group), or plain water (Control group). After the 20th week of sucrose treatment, the Sucrose group showed permanent hyperglycemia. Sucrose treatment also increased the amount of total lipids and fatty acids in the brain. The brain fatty acid profile of total lipids as well as phosphatidylethanolamine, phosphatidylcholine and cardiolipin of the Sucrose group was extensively changed. The most interesting change was a significant decrease in n6 fatty acids, including the important arachidonic acid, whereas the content of oleic and docosahexaenoic acid remained unchanged. RT-qPCR revealed an increase in Δ-5-desaturase and SREBP1c gene expression. In conclusion, high sucrose intake via drinking water extensively changes rat brain fatty acid profile by decreasing n6 fatty acids, including arachidonic acid. In contrast, the content of docosahexaenoic acid remains constant in the brain total lipids as well as in phospholipids. Changes in the brain fatty acid profile reflect changes in the lipid metabolism of the rat lipogenic tissues and concentrations in the circulation. Copyright © 2017 Elsevier B.V. All rights reserved.

Depressed expression of FAE1 and FAD2 genes modifies fatty acid profiles and storage compounds accumulation in Brassica napus seeds.

PubMed

Shi, Jianghua; Lang, Chunxiu; Wang, Fulin; Wu, Xuelong; Liu, Renhu; Zheng, Tao; Zhang, Dongqing; Chen, Jinqing; Wu, Guanting

2017-10-01

In plants, the enzymes fatty acid dehydrogenase 2 (FAD2) and fatty acid elongase 1 (FAE1) have been shown in previous studies to play important roles in the de novo biosynthesis of fatty acids. However, the effects of depressed expression of FAD2 and FAE1 on seed storage compounds accumulation remains to be elucidated. In this study, we produced RNA interfering transgenic rapeseeds lines, BnFAD2-Ri, BnFAE1-Ri and BnFAD2/BnFAE1-Ri, which exhibited depressed expression of the BnFAD2 and BnFAE1 genes under the control of seed-specific napin A promoter. These transgenic rapeseeds showed normal growth and development as compared with the wild type (CY2). Depressed expression of BnFAD2 and BnFAE1 genes modified fatty acid profiles, leading to increased oleic acid and decreased erucic acid contents in transgenic seeds. Consistent with these results, the ratios of C18:1/C18:2 and C18:1/C18:3 in C18 unsaturated fatty acids were greatly increased due to increased oleic acid content in transgenic seeds. Moreover, depressed expression of BnFAD2 and BnFAE1 genes resulted in slightly decreased oil contents and increased protein contents in transgenic seeds. Our results demonstrated that depressed expression of BnFAD2 and BnFAE1 greatly improves seed nutritional quality by modulating the fatty acid metabolism and storage products accumulation and that BnFAD2 and BnFAE1 are reliable targets for genetic improvement of rapeseed in seed nutritional quality. Copyright © 2017 Elsevier B.V. All rights reserved.

Genetic variation in polyunsaturated fatty acid metabolism and its potential relevance for human development and health.

PubMed

Glaser, Claudia; Lattka, Eva; Rzehak, Peter; Steer, Colin; Koletzko, Berthold

2011-04-01

Blood and tissue contents of polyunsaturated fatty acid (PUFA) and long-chain PUFA (LC-PUFA) are related to numerous health outcomes including cardiovascular health, allergies, mental health and cognitive development. Evidence has accumulated to show that in addition to diet, common polymorphisms in the fatty acid desaturase (FADS) gene cluster have very marked effects on human PUFA and LC-PUFA status. Recent results suggest that in addition to fatty acid desaturase 1 and fatty acid desaturase 2, the gene product of fatty acid desaturase 3 is associated with desaturating activity. New data have become available to show that FADS single nucleotide polymorphisms (SNPs) also modulate docosahexaenoic acid status in pregnancy as well as LC-PUFA levels in children and in human milk. There are indications that FADS SNPs modulate the risk for allergic disorders and eczema, and the effect of breastfeeding on later cognitive development. Mechanisms by which FADS SNPs modulate PUFA levels in blood, breast milk and tissues should be explored further. More studies are required to explore the effects of FADS gene variants in populations with different ethnic backgrounds, lifestyles and dietary habits, and to investigate in greater depth the interaction of gene variants, diet and clinical end points, including immune response and developmental outcomes. Analyses of FADS gene variants should be included into all sizeable cohort and intervention studies addressing biological effects of PUFA and LC-PUFA in order to consider these important confounders, and to enhance study sensitivity and precision. © 2011 Blackwell Publishing Ltd.

Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis

USDA-ARS?s Scientific Manuscript database

Producing unusual fatty acids (FAs) in crop plants has been a long-standing goal of green chemistry. However, expression of the enzymes that catalyze the primary synthesis of these unusual FAs in transgenic plants typically results in low levels of the desired FA. For example, seed-specific expressi...

Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy1

PubMed Central

Gao, Xiaoqing; Zhou, Jie; Li, Jun; Zou, Xiaowei; Zhao, Jianhua; Li, Qingliang; Xia, Ran; Yang, Ruifang; Wang, Dekai; Zuo, Zhaoxue; Tu, Jumin; Tao, Yuezhi; Chen, Xiaoyun; Xie, Qi; Zhu, Zengrong

2015-01-01

Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops. PMID:25371551

Molecular and functional characterization of cry1Ac transgenic pea lines.

PubMed

Teressa Negawo, Alemayehu; Baranek, Linda; Jacobsen, Hans-Jörg; Hassan, Fathi

2016-10-01

Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses.

Molecular and functional characterization of cry1Ac transgenic pea lines

PubMed Central

Teressa Negawo, Alemayehu; Baranek, Linda; Jacobsen, Hans-Jörg; Hassan, Fathi

2016-01-01

ABSTRACT Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses. PMID:27764552

Maternal single nucleotide polymorphisms in the fatty acid desaturase 1 and 2 coding regions modify the impact of prenatal supplementation with DHA on birth weight12

PubMed Central

Gonzalez-Casanova, Ines; Rzehak, Peter; Stein, Aryeh D; Garcia Feregrino, Raquel; Dommarco, Juan A Rivera; Barraza-Villarreal, Albino; Demmelmair, Hans; Romieu, Isabelle; Villalpando, Salvador; Martorell, Reynaldo; Koletzko, Berthold; Ramakrishnan, Usha

2016-01-01

Background: Specific single nucleotide polymorphisms (SNPs) in the fatty acid desaturase (FADS) gene affect the activity and efficiency of enzymes that are responsible for the conversion of polyunsaturated fatty acids (PUFAs) into their long-chain active form. A high prevalence of SNPs that are associated with slow PUFA conversion has been described in Hispanic populations. Objective: We assessed the heterogeneity of the effect of prenatal supplementation with docosahexaenoic acid (DHA) on birth weight across selected FADS SNPs in a sample of Mexican women and their offspring. Design: We obtained information on the maternal genotype from stored blood samples of 654 women who received supplementation with 400 mg DHA/d or a placebo from weeks 18 to 22 of gestation through delivery as part of a randomized controlled trial conducted in Cuernavaca, Mexico. We selected 4 tag SNPs (rs174455, rs174556, rs174602, and rs498793) in the FADS region for analysis. We used an ANOVA to test for the heterogeneity of the effect on birth weight across each of the 4 SNPs. Results: The mean ± SD birth weight was 3210 ± 470 g, and the weight-for-age z score (WAZ) was −0.24 ± 1.00. There were no intention-to-treat differences in birth weights. We showed significant heterogeneity by SNP rs174602 (P = 0.02); offspring of carriers of alleles TT and TC in the intervention group were heavier than those in the placebo group (WAZ: −0.13 ± 0.14 and −0.20 ± 0.08 compared with −0.55 ± 0.15 and −0.39 ± 0.09, respectively); there were no significant differences in offspring of rs174602 CC homozygotes (WAZ: −0.26 ± 0.09 in the intervention group compared with −0.04 ± 0.09 in the placebo group). We showed no significant heterogeneity across the other 3 FADS SNPs. Conclusion: Differential responses to prenatal DHA supplementation on the basis of the genetic makeup of target populations could explain the mixed evidence of the impact of DHA supplementation on birth weight. This

WRINKLED1 Rescues Feedback Inhibition of Fatty Acid Synthesis in Hydroxylase-Expressing Seeds1[OPEN

PubMed Central

Browse, John

2016-01-01

Previous attempts at engineering Arabidopsis (Arabidopsis thaliana) to produce seed oils containing hydroxy fatty acids (HFA) have resulted in low yields of HFA compared with the native castor (Ricinus communis) plant and caused undesirable effects, including reduced total oil content. Recent studies have led to an understanding of problems involved in the accumulation of HFA in oils of transgenic plants, which include metabolic bottlenecks and a decrease in the rate of fatty acid synthesis. Focusing on engineering the triacylglycerol assembly mechanisms led to modest increases in the HFA content of seed oil, but much room for improvement still remains. We hypothesized that engineering fatty acid synthesis in the plastids to increase flux would facilitate enhanced total incorporation of fatty acids, including HFA, into seed oil. The transcription factor WRINKLED1 (WRI1) positively regulates the expression of genes involved in fatty acid synthesis and controls seed oil levels. We overexpressed Arabidopsis WRI1 in seeds of a transgenic line expressing the castor fatty acid hydroxylase. The proportion of HFA in the oil, the total HFA per seed, and the total oil content of seeds increased to an average of 20.9%, 1.26 µg, and 32.2%, respectively, across five independent lines, compared with 17.6%, 0.83 µg, and 27.9%, respectively, for isogenic segregants. WRI1 and WRI1-regulated genes involved in fatty acid synthesis were up-regulated, providing for a corresponding increase in the rate of fatty acid synthesis. PMID:27208047

RNAi trigger fragment truncation attenuates soybean FAD2-1 transcript suppression and yields intermediate oil phenotypes.

PubMed

Wagner, Nicholas; Mroczka, Andrew; Roberts, Peter D; Schreckengost, William; Voelker, Toni

2011-09-01

Suppression of the microsomal ω6 oleate desaturase during the seed development of soybean (Glycine max) with the 420-bp soybean FAD2-1A intron as RNAi trigger shifts the conventional fatty acid composition of soybean oil from 20% oleic and 60% polyunsaturates to one containing greater than 80% oleic acid and less than 10% polyunsaturates. To determine whether RNAi could be attenuated by reducing the trigger fragment length, transgenic plants were generated to express successively shorter 5' or 3' deletion derivatives of the FAD2-1A intron. We observed a gradual reduction in transcript suppression with shorter trigger fragments. Fatty acid composition was less affected with shorter triggers, and triggers less than 60 bp had no phenotypic effect. No trigger sequences conferring significantly higher or lower suppression efficiencies were found, and the primary determinant of suppression effect was sequence length. The observed relationship of transcript suppression with the induced fatty acid phenotype indicates that RNAi is a saturation process and not a step change between suppressed and nonsuppressed states and intermediate suppression states can be achieved. © 2010 Monsanto. Plant Biotechnology Journal © 2010 Society for Experimental Biology and Blackwell Publishing Ltd.

Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants.

PubMed

Moire, Laurence; Rezzonico, Enea; Goepfert, Simon; Poirier, Yves

2004-01-01

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal beta-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward beta-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through beta-oxidation than the expression profile of genes involved in lipid metabolism.

Metabolomic screening of regional brain alterations in the APP/PS1 transgenic model of Alzheimer's disease by direct infusion mass spectrometry.

PubMed

González-Domínguez, Raúl; García-Barrera, Tamara; Vitorica, Javier; Gómez-Ariza, José Luis

2015-01-01

The identification of pathological mechanisms underlying to Alzheimer's disease is of great importance for the discovery of potential markers for diagnosis and disease monitoring. In this study, we investigated regional metabolic alterations in brain from the APP/PS1 mice, a transgenic model that reproduces well some of the neuropathological and cognitive deficits observed in human Alzheimer's disease. For this purpose, hippocampus, cortex, cerebellum and olfactory bulbs were analyzed using a high-throughput metabolomic approach based on direct infusion mass spectrometry. Metabolic fingerprints showed significant differences between transgenic and wild-type mice in all brain tissues, being hippocampus and cortex the most affected regions. Alterations in numerous metabolites were detected including phospholipids, fatty acids, purine and pyrimidine metabolites, acylcarnitines, sterols and amino acids, among others. Furthermore, metabolic pathway analysis revealed important alterations in homeostasis of lipids, energy management, and metabolism of amino acids and nucleotides. Therefore, these findings demonstrate the potential of metabolomic screening and the use of transgenic models for understanding pathogenesis of Alzheimer's disease. Copyright © 2014 Elsevier B.V. All rights reserved.

Fatty acid utilization by young Wistar rats fed a cafeteria diet.

PubMed

Esteve, M; Rafecas, I; Fernández-López, J A; Remesar, X; Alemany, M

1992-12-02

The content and accretion of fatty acids in 30, 45 and 60-day old Wistar rats fed either reference chow or a cafeteria diet has been studied, together with their actual fatty acid intake during that period. Diet had a small overall effect on the pattern of deposition of fatty acids, but the deposition of fat was much higher in cafeteria rats. The fat-rich cafeteria diet allowed the direct incorporation of most fatty acids into lipid storage, whilst chow-feeding activated lipogenesis and the deposition of a shorter chain and more saturated type of fatty acids. During the second month of the rat's life, the elongation pathway as well as delta 9-desaturase became functional, thus helping to shape the pattern of fatty acids actually accrued. The 60-day rats showed a relative impairment in the operation of delta 5-desaturase, since their lipids had a higher C20:4/C20:3 ratio than those of the diet ingested. Cafeteria-diet feeding minimized this effect since the large supply of dietary polyunsaturated fatty acids made the operation of the elongation-desaturase pathways practically unnecessary.

Endogenous ω-3 Polyunsaturated Fatty Acid Production Confers Resistance to Obesity, Dyslipidemia, and Diabetes in Mice

PubMed Central

Li, Jie; Li, Fanghong R.; Wei, Dong; Jia, Wei; Kang, Jing X.; Stefanovic-Racic, Maja

2014-01-01

Despite the well-documented health benefits of ω-3 polyunsaturated fatty acids (PUFAs), their use in clinical management of hyperglycemia and obesity has shown little success. To better define the mechanisms of ω-3 PUFAs in regulating energy balance and insulin sensitivity, we deployed a transgenic mouse model capable of endogenously producing ω-3 PUFAs while reducing ω-6 PUFAs owing to the expression of a Caenorhabditis elegans fat-1 gene encoding an ω-3 fatty acid desaturase. When challenged with high-fat diets, fat-1 mice strongly resisted obesity, diabetes, hypercholesterolemia, and hepatic steatosis. Endogenous elevation of ω-3 PUFAs and reduction of ω-6 PUFAs did not alter the amount of food intake but led to increased energy expenditure in the fat-1 mice. The requirements for the levels of ω-3 PUFAs as well as the ω-6/ω-3 ratios in controlling blood glucose and obesity are much more stringent than those in lipid metabolism. These metabolic phenotypes were accompanied by attenuation of the inflammatory state because tissue levels of prostaglandin E2, leukotriene B4, monocyte chemoattractant protein-1, and TNF-α were significantly decreased. TNF-α–induced nuclear factor-κB signaling was almost completely abolished. Consistent with the reduction in chronic inflammation and a significant increase in peroxisome proliferator–activated receptor-γ activity in the fat-1 liver tissue, hepatic insulin signaling was sharply elevated. The activities of prolipogenic regulators, such as liver X receptor, stearoyl-CoA desaturase-1, and sterol regulatory element binding protein-1 were sharply decreased, whereas the activity of peroxisome proliferator–activated receptor-α, a nuclear receptor that facilitates lipid β-oxidation, was markedly increased. Thus, endogenous conversion of ω-6 to ω-3 PUFAs via fat-1 strongly protects against obesity, diabetes, inflammation, and dyslipidemia and may represent a novel therapeutic modality to treat these prevalent

A Palmitic Acid Elongase Affects Eicosapentaenoic Acid and Plastidial Monogalactosyldiacylglycerol Levels in Nannochloropsis1

PubMed Central

Dolch, Lina-Juana; Rak, Camille; Broughton, Richard; Leterrier, Marina; Tellier, Frédérique; Faure, Jean-Denis; Falconet, Denis; Jouhet, Juliette

2017-01-01

Nannochloropsis species are oleaginous eukaryotes containing a plastid limited by four membranes, deriving from a secondary endosymbiosis. In Nannochloropsis, thylakoid lipids, including monogalactosyldiacylglycerol (MGDG), are enriched in eicosapentaenoic acid (EPA). The need for EPA in MGDG is not understood. Fatty acids are de novo synthesized in the stroma, then converted into very-long-chain polyunsaturated fatty acids (FAs) at the endoplasmic reticulum (ER). The production of MGDG relies therefore on an EPA supply from the ER to the plastid, following an unknown process. We identified seven elongases and five desaturases possibly involved in EPA production in Nannochloropsis gaditana. Among the six heterokont-specific saturated FA elongases possibly acting upstream in this pathway, we characterized the highly expressed isoform Δ0-ELO1. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that NgΔ0-ELO1 could elongate palmitic acid. Nannochloropsis Δ0-elo1 mutants exhibited a reduced EPA level and a specific decrease in MGDG. In NgΔ0-elo1 lines, the impairment of photosynthesis is consistent with a role of EPA-rich MGDG in nonphotochemical quenching control, possibly providing an appropriate MGDG platform for the xanthophyll cycle. Concomitantly with MGDG decrease, the level of triacylglycerol (TAG) containing medium chain FAs increased. In Nannochloropsis, part of EPA used for MGDG production is therefore biosynthesized by a channeled process initiated at the elongation step of palmitic acid by Δ0-ELO1, thus acting as a committing enzyme for galactolipid production. Based on the MGDG/TAG balance controlled by Δ0-ELO1, this study also provides novel prospects for the engineering of oleaginous microalgae for biotechnological applications. PMID:27895203

Identification of Secretory Odontoblasts Using DMP1-GFP Transgenic Mice

PubMed Central

Balic, Anamaria; Mina, Mina

2011-01-01

Terminal differentiation of odontoblasts from dental papilla is a long process involving several intermediate steps and changes in the transcriptional profile and expression of proteins secreted by cells in the odontoblast lineage. Transgenic mouse lines in which GFP expression is under the control of tissue-and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stages of differentiation along a lineage. Our previous studies showed utilization of pOBCol3.6GFP and pOBCol2.3GFP animals for identification of odontoblasts at early and late stages of polarization respectively. In the present study we used the DMP1-GFP transgenic animal as an experimental model to examine its expression during the differentiation of odontoblasts from progenitor cells in vivo and in vitro. Our observations showed that DMP1-GFP transgene is first activated in secretory/functional odontoblasts engaged in secretion of predentin and then transiently expressed at high levels in newly differentiated odontoblasts. Expression of DMP1-GFP was down-regulated in highly differentiated odontoblasts. The temporal and spatial pattern of expression of DMP1-GFP transgene closely mimics the expression of endogenous DMP1. This transgenic animal will facilitate studies of gene expression and biological functions in secretory/functional odontoblasts. PMID:21172466

Flanking sequence determination and specific PCR identification of transgenic wheat B102-1-2.

PubMed

Cao, Jijuan; Xu, Junyi; Zhao, Tongtong; Cao, Dongmei; Huang, Xin; Zhang, Piqiao; Luan, Fengxia

2014-01-01

The exogenous fragment sequence and flanking sequence between the exogenous fragment and recombinant chromosome of transgenic wheat B102-1-2 were successfully acquired using genome walking technology. The newly acquired exogenous fragment encoded the full-length sequence of transformed genes with transformed plasmid and corresponding functional genes including ubi, vector pBANF-bar, vector pUbiGUSPlus, vector HSP, reporter vector pUbiGUSPlus, promoter ubiquitin, and coli DH1. A specific polymerase chain reaction (PCR) identification method for transgenic wheat B102-1-2 was established on the basis of designed primers according to flanking sequence. This established specific PCR strategy was validated by using transgenic wheat, transgenic corn, transgenic soybean, transgenic rice, and non-transgenic wheat. A specifically amplified target band was observed only in transgenic wheat B102-1-2. Therefore, this method is characterized by high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of transgenic wheat B102-1-2.

Dietary fish oil replacement by linseed oil: Effect on growth, nutrient utilization, tissue fatty acid composition and desaturase gene expression in silver barb (Puntius gonionotus) fingerlings.

PubMed

Nayak, Madhusmita; Saha, Ashis; Pradhan, Avinash; Samanta, Mrinal; Giri, Shiba Shankar

2017-03-01

Silver barb (Puntius gonionotus) is considered a promising medium carp species for freshwater aquaculture in Asia. This study in silver barb was carried out to evaluate the effects of total or partial substitution of dietary fish oil (FO) with linseed oil (LO) on growth, nutrient utilization, whole-body composition, muscle and liver fatty acid composition. Fish (12.1±0.4g of initial body weight) were fed for 60days with five experimental iso-proteinous, iso-lipidic and iso-caloric diets in which FO (control diet) was replaced by 33.3%, 50%, 66.7% and 100% LO. Final weight, weight gain, percent weight gain, SGR decreased linearly (p<0.001) with increasing LO levels in the diets. Dietary LO substitution levels did not significantly (p>0.05) affect the feed conversion ratio (FCR), protein efficiency ratio (PER) and whole body proximate composition. Furthermore, enhanced level of LO increased α-linolenic acid (ALA; 18:3n3) and linoleic acid (LA; 18:2n6) and decreased eicosapentaenoic acid (EPA; 20:5n3) and docosahexaenoic acid (DHA; 22:6n3) in muscle and liver. To understand the molecular mechanism of long chain-polyunsaturated fatty acid (LC-PUFA) biosynthesis, we cloned and characterized the fatty acyl Δ6 desaturase (Δ6 fad) cDNA and investigated its expression in various organs/tissues following replacement of FO with LO in the diet. The full-length Δ6 fad cDNA was 2056bp encoding 444 amino acids and was widely expressed in various organs/tissues. Replacement of FO with LO increased the expression of Δ6 fad mRNA in liver, muscle and intestine but no significant difference was found in the brain. Copyright © 2016 Elsevier Inc. All rights reserved.

A study of associations between early DHA status and fatty acid desaturase (FADS) SNP and developmental outcomes in children of obese mothers.

PubMed

Andersen, Karina R; Harsløf, Laurine B S; Schnurr, Theresia M; Hansen, Torben; Hellgren, Lars I; Michaelsen, Kim F; Lauritzen, Lotte

2017-01-01

DHA from diet or endogenous synthesis has been proposed to affect infant development, however, results are inconclusive. In this study, we aim to verify previously observed fatty acid desaturase gene cluster (FADS) SNP-specific associations with erythrocyte DHA status in 9-month-old children and sex-specific association with developmental outcomes. The study was performed in 166 children (55 % boys) of obese mothers. Erythrocyte fatty acid composition was analysed in blood-samples obtained at 9 months of age, and developmental outcomes assessed by the Ages and Stages Questionnaire at 3 years. Erythrocyte DHA level ranged from 4·4 to 9·9 % of fatty acids, but did not show any association with FADS SNP or other potential determinants. Regression analysis showed associations between erythrocyte DHA and scores for personal-social skills (β 1·8 (95 % CI 0·3, 3·3), P=0·019) and problem solving (β 3·4 (95 % CI 1·2, 5·6), P=0·003). A tendency was observed for an association in opposite direction between minor alleles (G-variant) of rs1535 and rs174575 and personal-social skills (P=0·062 and 0·068, respectively), which became significant when the SNP were combined based on their previously observed effect on erythrocyte DHA at 9 months of age (β 2·6 (95 % CI 0·01, 5·1), P=0·011). Sex-SNP interaction was indicated for rs174575 genotype on fine motor scores (P=0·016), due to higher scores among minor allele carrying girls (P=0·043), whereas no effect was seen among boys. In conclusion, DHA-increasing FADS SNP and erythrocyte DHA status were consistently associated with improved personal-social skills in this small cohort of children of obese mothers irrespective of sex, but the sample was too small to verify potential sex-specific effects.

Induction of Lipid and Oleosin Biosynthesis by (+)-Abscisic Acid and Its Metabolites in Microspore-Derived Embryos of Brassica napus L.cv Reston (Biological Responses in the Presence of 8[prime]-Hydroxyabscisic Acid).

PubMed Central

Zou, J.; Abrams, G. D.; Barton, D. L.; Taylor, D. C.; Pomeroy, M. K.; Abrams, S. R.

1995-01-01

Microspore-derived (MD) embryos of Brassica napus L. cv Reston were used to test the effects of (+)-abscisic acid ([(+)-ABA]) and its metabolites, 8[prime]-hydroxyabscisic acid (8[prime]-OH ABA) and (-)-phaseic acid (PA), on the accumulation of very long-chain monounsaturated fatty acids (VLCMFAs) and induction of genes encoding a 19-kD oleosin protein and a [delta]15 desaturase during embryogenesis. Developing early to mid-cotyledonary MD embryos at 16 to 19 d in culture were treated with 10 [mu]M hormone/metabolite for 4 d. At various times during incubation, embryos and medium were analyzed to determine levels of hormone/metabolite, VLCMFAs, and oleosin or [delta]15 desaturase transcripts. The VLCMFAs, 20:1 and 22:1, primarily in triacylglycerols, increased by 200% after 72 h in the presence of (+)-ABA and 8[prime]-OH ABA relative to the control. In contrast, treatment with PA for 72 h had little effect (20% increase) on the level of VLCMFAs. The first 24 to 72 h of (+)-ABA treatment were critical in the induction of VLCMFA biosynthesis, with 8[prime]-OH ABA lagging slightly behind (+)-ABA in promoting this response. The accumulation of VLCMFAs was positively correlated with an increase in elongase activity. (+)-ABA and its 8[prime]-OH ABA metabolite induced the accumulation of a 19-kD oleosin transcript within 2 to 4 h in culture. In addition, both (+)-ABA and 8[prime]-OH ABA induced the same level of [delta]15 desaturase transcript by 8 h. PA had no effect on the induction of either oleosin or [delta]15 desaturase transcripts. To our knowledge, this is the first report of the biological activity of 8[prime]-OH ABA and of stimulatory effects of (+)-ABA and 8[prime]-OH ABA on lipid and oleosin biosynthesis. PMID:12228493

Cardiac phenotype induced by a dysfunctional α1C transgene

PubMed Central

Lao, Qi Zong; Ravindran, Arippa; Herbert, Ron; Canuto, Holly C

2011-01-01

Based on stable integration of recombinant DNA into a host genome, transgenic technology has become an important genetic engineering methodology. An organism whose genetic characteristics have been altered by the insertion of foreign DNA is supposed to exhibit a new phenotype associated with the function of the transgene. However, successful insertion may not be sufficient to achieve specific modification of function. In this study we describe a strain of transgenic mouse, G7-882, generated by incorporation into the mouse genome of human Cav1.2 α1C cDNA deprived of 3′-UTR to exclude transcription. We found that, in response to chronic infusion of isoproterenol, G7-882 develops dilated cardiomyopathy, a misleading “transgenic artifact” compatible with the expected function of the incorporated “correct” transgene. Specifically, using magnetic resonance imaging (MRI), we found that chronic β-adrenergic stimulation of G7-882 mice caused left ventricular hypertrophy and aggravated development of dilated cardiomyopathy, although no significant changes in the kinetics, density and voltage dependence of the calcium current were observed in G7-882 cardiomyocytes as compared to cells from wild type mice. This result illustrates the possibility that even when a functional transgene is expressed, an observed change in phenotype may be due to the artifact of “incidental incorporation” leading to misleading conclusions. To exclude this possibility and thus provide a robust tool for exploring biological function, the new transgenic phenotype must be replicated in several independently generated transgenic strains. PMID:21224729

Alterations by peroxisome proliferators of acyl composition of hepatic phosphatidylcholine in rats, mice and guinea-pigs. Role of stearoyl-CoA desaturase.

PubMed Central

Kawashima, Y; Hirose, A; Kozuka, H

1986-01-01

Rats, mice and guinea-pigs were administered p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). The treatments of rats and mice with either clofibric acid or tiadenol increased markedly the activities of stearoyl-CoA desaturase, palmitoyl-CoA chain elongation, 1-acylglycerophosphate (1-acyl-GP) acyltransferase and 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase, but not 2-acylglycerophosphocholine (2-acyl-GPC) acyltransferase in liver microsomes. The treatment of guinea-pigs with clofibric acid did not cause any change in the activities of these enzymes. The treatment of guinea-pigs with tiadenol caused a slight, but significant, increase in the activities of 1-acyl-GP acyltransferase and 1-acyl-GPC acyltransferase. The treatment of rats and mice with either clofibric acid or tiadenol increased markedly the proportion of 18:1 and decreased greatly the proportion of 18:0 in liver microsomal phosphatidylcholine. However, there is a considerable difference in the effects of the two peroxisome proliferators on the composition of polyunsaturated fatty acids in phosphatidylcholine between rats and mice. The treatment of guinea-pigs with either of the two peroxisome proliferators caused no change in acyl composition of phosphatidylcholine. The possible role of stearoyl-CoA desaturation in the regulation of acyl composition of phosphatidylcholine was discussed. PMID:2874791

Characterization of a higher plant herbicide-resistant phytoene desaturase and its use as a selectable marker

USDA-ARS?s Scientific Manuscript database

Three natural somatic mutations at codon 304 of the phytoene desaturase gene (pds) of Hydrilla verticillata ( L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all...

Systemic Down-Regulation of Delta-9 Desaturase Promotes Muscle Oxidative Metabolism and Accelerates Muscle Function Recovery following Nerve Injury

PubMed Central

Henriques, Alexandre; Lequeu, Thiebault; Rene, Frederique; Bindler, Françoise; Dirrig-Grosch, Sylvie; Oudart, Hugues; Palamiuc, Lavinia; Metz-Boutigue, Marie-Helene; Dupuis, Luc; Marchioni, Eric; Gonzalez De Aguilar, Jose-Luis; Loeffler, Jean-Philippe

2013-01-01

The progressive deterioration of the neuromuscular axis is typically observed in degenerative conditions of the lower motor neurons, such as amyotrophic lateral sclerosis (ALS). Neurodegeneration in this disease is associated with systemic metabolic perturbations, including hypermetabolism and dyslipidemia. Our previous gene profiling studies on ALS muscle revealed down-regulation of delta-9 desaturase, or SCD1, which is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids. Interestingly, knocking out SCD1 gene is known to induce hypermetabolism and stimulate fatty acid beta-oxidation. Here we investigated whether SCD1 deficiency can affect muscle function and its restoration in response to injury. The genetic ablation of SCD1 was not detrimental per se to muscle function. On the contrary, muscles in SCD1 knockout mice shifted toward a more oxidative metabolism, and enhanced the expression of synaptic genes. Repressing SCD1 expression or reducing SCD-dependent enzymatic activity accelerated the recovery of muscle function after inducing sciatic nerve crush. Overall, these findings provide evidence for a new role of SCD1 in modulating the restorative potential of skeletal muscles. PMID:23785402

Salicylic acid deficiency in NahG transgenic lines and sid2 mutants increases seed yield in the annual plant Arabidopsis thaliana

PubMed Central

Abreu, Maria Elizabeth; Munné-Bosch, Sergi

2009-01-01

Salicylic acid-deficient NahG transgenic lines and sid2 mutants were used to evaluate the role of this compound in the development of the short-lived, annual plant Arabidopsis thaliana, with a particular focus on the interplay between salicylic acid and other phytohormones. Low salicylic acid levels led to increased growth, as well as to smaller abscisic acid levels and reduced damage to PSII (as indicated by Fv/Fm ratios) during the reproductive stages in rosette leaves of NahG transgenic lines and sid2 mutants, compared with wild-type plants. Furthermore, salicylic acid deficiency highly influenced seed yield and composition. Seed production increased by 4.4-fold and 3.5-fold in NahG transgenic lines and sid2 mutants, respectively, compared to the wild type. Salicylic acid deficiency also improved seed composition in terms of antioxidant vitamin concentrations, seeds of salicylic acid-deficient plants showing higher levels of α- and γ-tocopherol (vitamin E) and β-carotene (pro-vitamin A) than seeds of wild-type plants. Seeds of salicylic acid-deficient plants also showed higher nitrogen concentrations than seeds of wild-type plants. It is concluded that (i) the sid2 gene, which encodes for isochorismate synthase, plays a central role in salicylic acid biosynthesis during plant development in A. thaliana, (ii) salicylic acid plays a role in the regulation of growth, senescence, and seed production, (iii) there is a cross-talk between salicylic acid and other phytohormones during plant development, and (iv) the concentrations of antioxidant vitamins in seeds may be influenced by the endogenous levels of salicylic acid in plants. PMID:19188277

[Reproduction,genotype identification and evaluation of APP/PS1 transgenic mice].

PubMed

Tan, Long; Li, Hai-Qiang; Li, Yi-Bo; Liu, Wei; Pang, Wei; Jiang, Yu-Gang

2018-02-08

To identify the genotype of (APP/PS1) transgenic mice and evaluate the changing of cognitive and behavioral fu nctions, provide an effective animal model for the Alzheimer's disease (AD) research. Male APP/PS1 transgenic mice mated with female APP/PS1 transgenic mice, and the genotype of their filial mice was identified by PCR. The APP +/PS1 + mice were assigned into AD model group (AD group, n =8), and the APP/PS1 mice were assigned into control group (CT group, n =8). The Morris water maze test was carried out to detect the capacity of learning and memory of mice. After that, the mice were sacrificed and the brain tissues were sampled and stained by HE and congo red for the pathological examination. ①A APP/PS1 genome DNA about 360 bp size was detected. The methods of feeding and breeding were successful to attain APP/PS1 transgenic mice.②Statistical significance was found in the differences of the capacity of learning and memory between 7-month-old APP/PS1 positive mice and negative mice ( P <0.05).③The results of HE stain showed that the structure and cellular morphology of hippocampus of AD mice were obviously abnormal. The results of congo red stain showed that positive amyloid plaque was observed in brains of AD mice. APP/PS1 transgenic mice present typical symptoms and behaviors of Alzheimer's disease. The transgenic mouse is an effective tool for the research and prevention of AD.

Podocyte changes upon induction of albuminuria in Thy-1.1 transgenic mice.

PubMed

Smeets, Bart; Dijkman, Henry B P M; te Loeke, Nathalie A J M; van Son, Jacco P H F; Steenbergen, Eric J; Assmann, Karel J M; Wetzels, Jack F M; Groenen, Patricia J T A

2003-12-01

Thy-1.1 transgenic mice, characterized by ectopic expression of the Thy-1.1 protein on podocytes, spontaneously develop proteinuria and focal glomerulosclerosis (FGS). Injection of a monoclonal antibody (mAb) directed against the Thy-1.1 protein in young transgenic mice induces a massive albuminuria that is followed by an accelerated FGS within 3 weeks. This albuminuria is complement and leukocyte independent. The time course of proteinuria, the pathogenesis of the acute proteinuria and the dose dependency of FGS are unknown. Albuminuria was measured in Thy-1.1 transgenic mice after injection of different doses of anti-Thy-1.1 mAb and at different time points within the first 24 h after injection. Podocytic foot processes and slit pore diameter were quantitated by electron microscopy. Changes in expression of slit pore constituents (podocin, CD2AP, nephrin and ZO-1), cytoskeleton-associated proteins (actin, alpha-actinin, ezrin and synaptopodin), the GDH-podocyte adhesion molecules alpha(3)-integrin, and heparan sulfate were studied by immunofluorescence. FGS was scored by light microscopy at 3 weeks after induction of albuminuria. Albuminuria in Thy-1.1 transgenic mice was observed within 10 min after anti-Thy-1.1 mAb injection. This rapid development of albuminuria was accompanied by a reduction in number of podocytic foot processes from 20.0 +/- 0.7/10 microm glomerular basement membrane (GBM) in saline-treated transgenic mice to 8.0 +/- 0.5 and 2.2 +/- 0.2 in anti-Thy-1.1-treated mice, at 10 min and 8 h after treatment, respectively. In addition, we observed a significant decrease in width of remaining slit pores, from 32.7 +/- 1.1 to 26.8 +/- 1.4 nm at 10 min after mAb injection. By immunofluorescence, we did not observe major changes in the expression pattern of any of the proteins studied. There was no correlation between the injected dose of the anti-Thy-1.1 mAb and the acute albuminuria. In contrast, the percentage of FGS at 3 weeks correlated with the

Effect of two intermediate electron donors, NADPH and FADH(2), on Spirulina Delta (6)-desaturase co-expressed with two different immediate electron donors, cytochrome b (5) and ferredoxin, in Escherichia coli.

PubMed

Kurdrid, Pavinee; Subudhi, Sanjukta; Cheevadhanarak, Supapon; Tanticharoen, Morakot; Hongsthong, Apiradee

2007-12-01

When the gene desD encoding Spirulina Delta(6)-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron donor, i.e. the cytochrome b (5) domain from Mucor rouxii, the results showed the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6)-desaturase. The results revealed that in E. coli cells, where cytochrome b (5) is absent and ferredoxin, a natural electron donor of Delta(6)-desaturase, is present at a very low level, the cytochrome b (5) domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Delta(6)-desaturase in E. coli. In comparison to the co-expression of cytochrome b ( 5 ) with the Delta(6)-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Delta(6)-desaturase co-expressed with cytochrome b (5) and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and FADH(2) (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately 50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will be affected.

Comparison of two fluorescent antibody techniques (FATS) for detection and quantification of Renibacterium salmoninarum in coelomic fluid of spawning chinook salmon Oncorhynchus tshawytscha

USGS Publications Warehouse

Elliott, D.G.; McKibben, C.L.

1997-01-01

Two versions of the fluorescent antibody technique (FAT) were compared for detection and quantification of Renibacterium salmoninarum in coelomic fluid samples from naturally infected spawning chinook salmon Oncorhynchus tshawytscha. For the membrane filtration-FAT (MF-FAT), trypsin-treated samples were passed through 0.2 ??m polycarbonate filters to concentrate bacteria for direct enumeration by immunofluorescence microscopy. For the smear-FAT (S-FAT), samples were centrifuged at 8800 x g for 10 min and the pelleted material was smeared on slides for immunofluorescence staining Detected prevalences of Renibacterium salmoninarum were 1.8 to 3.4 times higher by the MF-FAT than by the S-FAT: differences were significant at p ??? 0.0002. The S-FAT consistently detected R. salmoninarum only in samples with calculated bacterial concentrations ??? 2.4 x 103 cells ml-1 by MF-FAT testing. Increasing the area examined on a filter or slide from 50 to 100 microscope fields at 1000x magnification resulted in the detection of a maximum of 4% additional positive samples by the MF-FAT and 7% additional positive samples by the S-FAT. In individual samples for which bacterial counts were obtained by both the MF-FAT and the S-FAT, the counts averaged from 47 times (??30 SD) to 175 times (??165 SD) higher by the MF-FAT. Centrifugation of samples at 10000 x g for 10 min resulted in a 4-fold increase in mean bacterial counts by the S-FAT compared with a 10-min centrifugation at 2000 x g, but the highest calculated bacterial concentration obtained by S-FAT testing was more than 6-fold lower than that obtained for the same sample by MF-FAT testing. Because of its greater sensitivity, the MF-FAT is preferable to the S-FAT for use in critical situations requiring the detection of low numbers of R. salmoninarum.

Identification of a Δ5-like fatty acyl desaturase from the cephalopod Octopus vulgaris (Cuvier 1797) involved in the biosynthesis of essential fatty acids.

PubMed

Monroig, Oscar; Navarro, Juan C; Dick, James R; Alemany, Frederic; Tocher, Douglas R

2012-08-01

Long-chain polyunsaturated fatty acids (LC-PUFA) have been identified as essential compounds for common octopus (Octopus vulgaris), but precise dietary requirements have not been determined due, in part, to the inherent difficulties of performing feeding trials on paralarvae. Our objective is to establish the essential fatty acid (EFA) requirements for paralarval stages of the common octopus through characterisation of the enzymes of endogenous LC-PUFA biosynthetic pathways. In this study, we isolated a cDNA with high homology to fatty acyl desaturases (Fad). Functional characterisation in recombinant yeast showed that the octopus Fad exhibited Δ5-desaturation activity towards saturated and polyunsaturated fatty acyl substrates. Thus, it efficiently converted the yeast's endogenous 16:0 and 18:0 to 16:1n-11 and 18:1n-13, respectively, and desaturated exogenously added PUFA substrates 20:4n-3 and 20:3n-6 to 20:5n-3 (EPA) and 20:4n-6 (ARA), respectively. Although the Δ5 Fad enables common octopus to produce EPA and ARA, the low availability of its adequate substrates 20:4n-3 and 20:3n-6, either in the diet or by limited endogenous synthesis from C(18) PUFA, might indicate that EPA and ARA are indeed EFA for this species. Interestingly, the octopus Δ5 Fad can also participate in the biosynthesis of non-methylene-interrupted FA, PUFA that are generally uncommon in vertebrates but have been found previously in marine invertebrates, including molluscs, and now also confirmed to be present in specific tissues of common octopus.

Erythrocyte polyunsaturated fatty acid composition is associated with depression and FADS genotype in Caucasians.

PubMed

Cribb, Lachlan; Murphy, Jenifer; Froud, Amy; Oliver, Georgina; Bousman, Chad A; Ng, Chee H; Sarris, Jerome

2017-05-29

Polyunsaturated fatty acids (PUFAs) play an important role in the pathophysiology of major depressive disorder (MDD), related, in part, to their role in inflammatory systems. The enzymes δ-5 and δ-6 desaturase are the rate-limiting steps in the metabolism of PUFAs and are encoded in the genes fatty acid desaturase (FADS) 1 and 2, respectively. Single nucleotide polymorphisms (SNPs) and haplotypes within the FADS gene cluster have been shown to influence PUFA composition. The objective of this study was to determine whether key omega-3 (n-3) and omega-6 (n-6) fatty acids may be associated with depression, and to explore the role of FADS genotype in PUFA variation. Four erythrocyte long chain (LC) fatty acids (linoleic acid [LA], α-linolenic acid [ALA], arachidonic acid [AA] and Eicosapentaenoic acid [EPA]), as well as six SNPs (rs174537, rs174547, rs174570, rs174575, rs498793 and rs3834458) within the FADS gene cluster were measured in a sample of 207 participants (154 with MDD versus 53 non-depressed controls). The precursor LC-PUFAs LA and ALA appeared to be negatively associated with depression (P < 0.001 and P < 0.01, respectively), while AA:LA (surrogate measure of desaturase activity) was positively associated with depression (P < 0.01). No significant differences were noted in erythrocyte EPA, AA or AA:EPA between groups. Minor alleles of each SNP (excluding rs498793) were associated with variation in desaturase activity and LA. Both rs174537 and rs174547 were associated with ALA. No genotype was associated with EPA or AA. Minor alleles of rs174537 and rs174547 were significantly associated with lower odds of MDD (although significance was lost after correction for multiple comparisons). Precursor LC-PUFAs, LA and ALA, appear to be associated with MDD and potentially modulated by genetic variation in the FADS gene cluster. These results provide support for the consideration of PUFA composition, diet and FADS genetic variation in the

Synthesis of docosahexaenoic acid from eicosapentaenoic acid in retina neurons protects photoreceptors from oxidative stress.

PubMed

Simón, María Victoria; Agnolazza, Daniela L; German, Olga Lorena; Garelli, Andrés; Politi, Luis E; Agbaga, Martin-Paul; Anderson, Robert E; Rotstein, Nora P

2016-03-01

Oxidative stress is involved in activating photoreceptor death in several retinal degenerations. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, protects cultured retina photoreceptors from apoptosis induced by oxidative stress and promotes photoreceptor differentiation. Here, we investigated whether eicosapentaenoic acid (EPA), a metabolic precursor to DHA, had similar effects and whether retinal neurons could metabolize EPA to DHA. Adding EPA to rat retina neuronal cultures increased opsin expression and protected photoreceptors from apoptosis induced by the oxidants paraquat and hydrogen peroxide (H2 O2 ). Palmitic, oleic, and arachidonic acids had no protective effect, showing the specificity for DHA. We found that EPA supplementation significantly increased DHA percentage in retinal neurons, but not EPA percentage. Photoreceptors and glial cells expressed Δ6 desaturase (FADS2), which introduces the last double bond in DHA biosynthetic pathway. Pre-treatment of neuronal cultures with CP-24879 hydrochloride, a Δ5/Δ6 desaturase inhibitor, prevented EPA-induced increase in DHA percentage and completely blocked EPA protection and its effect on photoreceptor differentiation. These results suggest that EPA promoted photoreceptor differentiation and rescued photoreceptors from oxidative stress-induced apoptosis through its elongation and desaturation to DHA. Our data show, for the first time, that isolated retinal neurons can synthesize DHA in culture. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in retina photoreceptors, and its precursor, eicosapentaenoic acid (EPA) have multiple beneficial effects. Here, we show that retina neurons in vitro express the desaturase FADS2 and can synthesize DHA from EPA. Moreover, addition of EPA to these cultures protects photoreceptors from oxidative stress and promotes their differentiation through its metabolization to DHA. © 2015 International Society for Neurochemistry.

Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins.

PubMed Central

Lindqvist, Y; Huang, W; Schneider, G; Shanklin, J

1996-01-01

The three-dimensional structure of recombinant homodimeric delta9 stearoyl-acyl carrier protein desaturase, the archetype of the soluble plant fatty acid desaturases that convert saturated to unsaturated fatty acids, has been determined by protein crystallographic methods to a resolution of 2.4 angstroms. The structure was solved by a combination of single isomorphous replacement, anomalous contribution from the iron atoms to the native diffraction data and 6-fold non-crystallographic symmetry averaging. The 363 amino acid monomer consists of a single domain of 11 alpha-helices. Nine of these form an antiparallel helix bundle. The enzyme subunit contains a di-iron centre, with ligands from four of the alpha-helices in the helix bundle. The iron ions are bound in a highly symmetric environment, with one of the irons forming interactions with the side chains of E196 and H232 and the second iron with the side chains of E105 and H146. Two additional glutamic acid side chains, from E143 and E229, are within coordination distance to both iron ions. A water molecule is found within the second coordination sphere from the iron atoms. The lack of electron density corresponding to a mu-oxo bridge, and the long (4.2 angstroms) distance between the iron ions suggests that this probably represents the diferrous form of the enzyme. A deep channel which probably binds the fatty acid extends from the surface into the interior of the enzyme. Modelling of the substrate, stearic acid, into this channel places the delta9 carbon atom in the vicinity of one of the iron ions. Images PMID:8861937

Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins.

PubMed

Lindqvist, Y; Huang, W; Schneider, G; Shanklin, J

1996-08-15

The three-dimensional structure of recombinant homodimeric delta9 stearoyl-acyl carrier protein desaturase, the archetype of the soluble plant fatty acid desaturases that convert saturated to unsaturated fatty acids, has been determined by protein crystallographic methods to a resolution of 2.4 angstroms. The structure was solved by a combination of single isomorphous replacement, anomalous contribution from the iron atoms to the native diffraction data and 6-fold non-crystallographic symmetry averaging. The 363 amino acid monomer consists of a single domain of 11 alpha-helices. Nine of these form an antiparallel helix bundle. The enzyme subunit contains a di-iron centre, with ligands from four of the alpha-helices in the helix bundle. The iron ions are bound in a highly symmetric environment, with one of the irons forming interactions with the side chains of E196 and H232 and the second iron with the side chains of E105 and H146. Two additional glutamic acid side chains, from E143 and E229, are within coordination distance to both iron ions. A water molecule is found within the second coordination sphere from the iron atoms. The lack of electron density corresponding to a mu-oxo bridge, and the long (4.2 angstroms) distance between the iron ions suggests that this probably represents the diferrous form of the enzyme. A deep channel which probably binds the fatty acid extends from the surface into the interior of the enzyme. Modelling of the substrate, stearic acid, into this channel places the delta9 carbon atom in the vicinity of one of the iron ions.

Non-destructive Determination of Shikimic Acid Concentration in Transgenic Maize Exhibiting Glyphosate Tolerance Using Chlorophyll Fluorescence and Hyperspectral Imaging

PubMed Central

Feng, Xuping; Yu, Chenliang; Chen, Yue; Peng, Jiyun; Ye, Lanhan; Shen, Tingting; Wen, Haiyong; He, Yong

2018-01-01

The development of transgenic glyphosate-tolerant crops has revolutionized weed control in crops in many regions of the world. The early, non-destructive identification of superior plant phenotypes is an important stage in plant breeding programs. Here, glyphosate-tolerant transgenic maize and its parental wild-type control were studied at 2, 4, 6, and 8 days after glyphosate treatment. Visible and near-infrared hyperspectral imaging and chlorophyll fluorescence imaging techniques were applied to monitor the performance of plants. In our research, transgenic maize, which was highly tolerant to glyphosate, was phenotyped using these high-throughput non-destructive methods to validate low levels of shikimic acid accumulation and high photochemical efficiency of photosystem II as reflected by maximum quantum yield and non-photochemical quenching in response to glyphosate. For hyperspectral imaging analysis, the combination of spectroscopy and chemometric methods was used to predict shikimic acid concentration. Our results indicated that a partial least-squares regression model, built on optimal wavelengths, effectively predicted shikimic acid concentrations, with a coefficient of determination value of 0.79 for the calibration set, and 0.82 for the prediction set. Moreover, shikimic acid concentration estimates from hyperspectral images were visualized on the prediction maps by spectral features, which could help in developing a simple multispectral imaging instrument for non-destructive phenotyping. Specific physiological effects of glyphosate affected the photochemical processes of maize, which induced substantial changes in chlorophyll fluorescence characteristics. A new data-driven method, combining mean fluorescence parameters and featuring a screening approach, provided a satisfactory relationship between fluorescence parameters and shikimic acid content. The glyphosate-tolerant transgenic plants can be identified with the developed discrimination model

Non-destructive Determination of Shikimic Acid Concentration in Transgenic Maize Exhibiting Glyphosate Tolerance Using Chlorophyll Fluorescence and Hyperspectral Imaging.

PubMed

Feng, Xuping; Yu, Chenliang; Chen, Yue; Peng, Jiyun; Ye, Lanhan; Shen, Tingting; Wen, Haiyong; He, Yong

2018-01-01

The development of transgenic glyphosate-tolerant crops has revolutionized weed control in crops in many regions of the world. The early, non-destructive identification of superior plant phenotypes is an important stage in plant breeding programs. Here, glyphosate-tolerant transgenic maize and its parental wild-type control were studied at 2, 4, 6, and 8 days after glyphosate treatment. Visible and near-infrared hyperspectral imaging and chlorophyll fluorescence imaging techniques were applied to monitor the performance of plants. In our research, transgenic maize, which was highly tolerant to glyphosate, was phenotyped using these high-throughput non-destructive methods to validate low levels of shikimic acid accumulation and high photochemical efficiency of photosystem II as reflected by maximum quantum yield and non-photochemical quenching in response to glyphosate. For hyperspectral imaging analysis, the combination of spectroscopy and chemometric methods was used to predict shikimic acid concentration. Our results indicated that a partial least-squares regression model, built on optimal wavelengths, effectively predicted shikimic acid concentrations, with a coefficient of determination value of 0.79 for the calibration set, and 0.82 for the prediction set. Moreover, shikimic acid concentration estimates from hyperspectral images were visualized on the prediction maps by spectral features, which could help in developing a simple multispectral imaging instrument for non-destructive phenotyping. Specific physiological effects of glyphosate affected the photochemical processes of maize, which induced substantial changes in chlorophyll fluorescence characteristics. A new data-driven method, combining mean fluorescence parameters and featuring a screening approach, provided a satisfactory relationship between fluorescence parameters and shikimic acid content. The glyphosate-tolerant transgenic plants can be identified with the developed discrimination model

Comparative proteomic analysis of rd29A:RdreB1BI transgenic and non-transgenic strawberries exposed to low temperature.

PubMed

Gu, Xianbin; Gao, Zhihong; Zhuang, Weibing; Qiao, Yushan; Wang, Xiuyun; Mi, Lin; Zhang, Zhen; Lin, Zhilin

2013-05-01

Low-temperature stress is one of the major abiotic stresses in plants worldwide, and the dehydration responsive element binding protein (DREB) transcription factor induces expression of genes involved in environmental stress tolerance in plants. A proteomic approach based on two-dimensional gel electrophoresis (2-DE) and subsequent mass spectrometric identification was used to study the changes in the leaf proteome profiles of rd29A:RdreB1BI transgenic and non-transgenic strawberries exposed to low-temperature conditions. By comparing the proteomic profiles, we located 21 protein spots that were reproducibly up- or down-regulated by more than twofold between transgenic and non-transgenic strawberries. Eight identified proteins function in energy and metabolism, four in biosynthetic processes, four were stress and defense related, three spots were identified as cold-stress related expressed sequence tags (ESTs), and two were unknown proteins. The change patterns of low-temperature tolerance proteins, including photosynthetic proteins (RuBisCO large subunit and RuBisCO activase), cytoplasmic Cu/Zn-superoxide dismutase (Cu/Zn-SOD), late embryogenesis abundant protein 14-A (Lea14-A), eukaryotic translation initiation factor 5A (eIF5A), and cold-stress related ESTs, were differentially regulated between non-transgenic and rd29A:RdreB1BI transgenic strawberries. They are likely important gene products in the regulatory network of the RdreB1BI gene. Consequently, this study provides the first characterization of the transgenic strawberry proteome and the predicted target proteins of the RdreB1BI gene by using proteomic approaches. Copyright © 2013 Elsevier GmbH. All rights reserved.

Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation.

PubMed

Banik, Mitali; Duguid, Scott; Cloutier, Sylvie

2011-06-01

Three genes encoding fatty acid desaturase 3 (fad3a, fad3b, and a novel fad3c) were cloned from four flax genotypes varying in linolenic acid content. Real-time PCR was used to quantify expression levels of the three fad3 genes during seed development. High amounts of both fad3a and fad3b transcripts were observed and reached their peak levels at 20 days after anthesis, except for fad3a from SP2047 where only low level expression was observed throughout seed development. Transcript accumulation of the novel fad3c gene was at similar background levels. The fatty acid composition was analysed for all genotypes and stages of development and compared with the fad3 gene expression patterns. α-Linolenic acid gradually accumulated during seed development, while linoleic acid was transient and decreased in M5791, UGG5-5, and AC McDuff. In contrast, the linolenic acid present in the early stages of development nearly completely disappeared in SP2047, while linoleic acid steadily accumulated. fad3a of the low linolenic acid line SP2047 encoded a truncated protein caused by a premature stop codon resulting from a single point mutation, and the low level of transcript accumulation in this genotype is likely due to nonsense-mediated mRNA decay caused by the premature termination of translation as a result of this early stop codon. Although substantial amounts of transcript accumulation occurred with fad3b of SP2047 genotype, cloning of the gene revealed a mutation in the first histidine box causing an amino acid change. Heterologous expression in yeast of the SP2047 and UGG5-5 fad3b genes showed that the mutation in the histidine box in SP2047 caused the enzyme inactivity. Taken together, these results showed that fad3a and fad3b are responsible for linolenic acid accumulation in flax seeds but did not support a major role for the novel fad3c. These observations were further supported by phenotypic and genotypic assessment of a doubled haploid population. Expression patterns

Umbilical cord PUFA are determined by maternal and child fatty acid desaturase (FADS) genetic variants in the Avon Longitudinal Study of Parents and Children (ALSPAC)

PubMed Central

Lattka, Eva; Koletzko, Berthold; Zeilinger, Sonja; Hibbeln, Joseph R.; Klopp, Norman; Ring, Susan M.; Steer, Colin D.

2012-01-01

Fetal supply with long-chain PUFA (LC-PUFA) during pregnancy is important for brain growth and visual and cognitive development and is provided by materno–fetal placental transfer. We recently showed that maternal fatty acid desaturase (FADS) genotypes modulate the amounts of LC-PUFA in maternal blood. Whether FADS genotypes influence the amounts of umbilical cord fatty acids has not been investigated until now. The aim of the present study was to investigate the influence of maternal and child FADS genotypes on the amounts of LC-PUFA in umbilical cord venous plasma as an indicator of fetal fatty acid supply during pregnancy. A total of eleven cord plasma n-6 and n-3 fatty acids were analysed for association with seventeen FADS gene cluster SNP in over 2000 mothers and children from the Avon Longitudinal Study of Parents and Children. In a multivariable analysis, the maternal genotype effect was adjusted for the child genotype and vice versa to estimate which of the two has the stronger influence on cord plasma fatty acids. Both maternal and child FADS genotypes and haplotypes influenced amounts of cord plasma LC-PUFA and fatty acid ratios. Specifically, most analysed maternal SNP were associated with cord plasma levels of the precursor n-6 PUFA, whereas the child genotypes were mainly associated with more highly desaturated n-6 LC-PUFA. This first study on FADS genotypes and cord fatty acids suggests that fetal LC-PUFA status is determined to some extent by fetal fatty acid conversion. Associations of particular haplotypes suggest specific effects of SNP rs498793 and rs968567 on fatty acid metabolism. PMID:22877655

Umbilical cord PUFA are determined by maternal and child fatty acid desaturase (FADS) genetic variants in the Avon Longitudinal Study of Parents and Children (ALSPAC).

PubMed

Lattka, Eva; Koletzko, Berthold; Zeilinger, Sonja; Hibbeln, Joseph R; Klopp, Norman; Ring, Susan M; Steer, Colin D

2013-04-14

Fetal supply with long-chain PUFA (LC-PUFA) during pregnancy is important for brain growth and visual and cognitive development and is provided by materno-fetal placental transfer. We recently showed that maternal fatty acid desaturase (FADS) genotypes modulate the amounts of LC-PUFA in maternal blood. Whether FADS genotypes influence the amounts of umbilical cord fatty acids has not been investigated until now. The aim of the present study was to investigate the influence of maternal and child FADS genotypes on the amounts of LC-PUFA in umbilical cord venous plasma as an indicator of fetal fatty acid supply during pregnancy. A total of eleven cord plasma n-6 and n-3 fatty acids were analysed for association with seventeen FADS gene cluster SNP in over 2000 mothers and children from the Avon Longitudinal Study of Parents and Children. In a multivariable analysis, the maternal genotype effect was adjusted for the child genotype and vice versa to estimate which of the two has the stronger influence on cord plasma fatty acids. Both maternal and child FADS genotypes and haplotypes influenced amounts of cord plasma LC-PUFA and fatty acid ratios. Specifically, most analysed maternal SNP were associated with cord plasma levels of the precursor n-6 PUFA, whereas the child genotypes were mainly associated with more highly desaturated n-6 LC-PUFA. This first study on FADS genotypes and cord fatty acids suggests that fetal LC-PUFA status is determined to some extent by fetal fatty acid conversion. Associations of particular haplotypes suggest specific effects of SNP rs498793 and rs968567 on fatty acid metabolism.

Transgenic cells with increased plastoquinone levels and methods of use

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sayre, Richard T.; Subramanian, Sowmya; Cahoon, Edgar

Disclosed herein are transgenic cells expressing a heterologous nucleic acid encoding a prephenate dehydrogenase (PDH) protein, a heterologous nucleic acid encoding a homogentisate solanesyl transferase (HST) protein, a heterologous nucleic acid encoding a deoxyxylulose phosphate synthase (DXS) protein, or a combination of two or more thereof. In particular examples, the disclosed transgenic cells have increased plastoquinone levels. Also disclosed are methods of increasing cell growth rates or production of biomass by cultivating transgenic cells expressing a heterologous nucleic acid encoding a PDH protein, a heterologous nucleic acid encoding an HST protein, a heterologous nucleic acid encoding a DXS protein, ormore » a combination of two or more thereof under conditions sufficient to produce cell growth or biomass.« less

Caudal dysgenesis in islet-1 transgenic mice

PubMed Central

Muller, Yunhua Li; Yueh, Yir Gloria; Yaworsky, Paul J.; Salbaum, J. Michael; Kappen, Claudia

2014-01-01

Maternal diabetes during pregnancy is responsible for the occurrence of diabetic embryopathy, a spectrum of birth defects that includes heart abnormalities, neural tube defects, and caudal dysgenesis syndromes. Here, we report that mice transgenic for the homeodomain transcription factor Isl-1 develop profound caudal growth defects that resemble human sacral/caudal agenesis. Isl-1 is normally expressed in the pancreas and is required for pancreas development and endocrine cell differentiation. Aberrant regulation of this pancreatic transcription factor causes increased mesodermal cell death, and the severity of defects is dependent on transgene dosage. Together with the finding that mutation of the pancreatic transcription factor HLXB9 causes sacral agenesis, our results implicate pancreatic transcription factors in the pathogenesis of birth defects associated with diabetes. PMID:12738808

Rice mutants deficient in ω-3 fatty acid desaturase (FAD8) fail to acclimate to cold temperatures.

PubMed

Tovuu, Altanzaya; Zulfugarov, Ismayil S; Wu, Guangxi; Kang, In Soon; Kim, Choongrak; Moon, Byoung Yong; An, Gynheung; Lee, Choon-Hwan

2016-12-01

To investigate the role of ω-3 fatty acid (FA) desaturase (FAD8) during cold acclimation in higher plants, we characterized three independent T-DNA insertional knock-out mutants of OsFAD8 from rice (Oryza sativa L.). At room temperature (28 °C), osfad8 plants exhibited significant alterations in fatty acid (FA) unsaturation for all four investigated plastidic lipid classes. During a 5-d acclimation period at 4 °C, further changes in FA unsaturation in both wild-type (WT) and mutant plants varied according to the type of lipid. We also monitored the fluidity of the thylakoid membrane using a threshold temperature to represent the change in fluorescence. The values were altered significantly by both FAD8 mutation and cold acclimation, suggesting that factors other than FAD8 are involved in C18 FA unsaturation and fluctuations in membrane fluidity. Similarly, significant changes were noted for both the mutant and WT samples in terms of their FA compositions as well as activities related to photosystem (PS) I, PSII, and photoprotection. This included the development of non-photochemical quenching and increased zeaxanthin accumulation. Despite the relatively small changes in FA composition during cold acclimation, cold-inducible FAD8 knock-out mutants displayed strong differences in photoprotective activities and a further drop in membrane fluidity. The mutants were more sensitive than WT to short-term low-temperature stress that resulted in increased production of reactive oxygen species after 5 d of chilling. Taken together, our findings suggest that FA unsaturation by OsFAD8 is crucial for the acclimation of higher plants to low-temperature stress. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Why is golden rice golden (yellow) instead of red?

PubMed

Schaub, Patrick; Al-Babili, Salim; Drake, Rachel; Beyer, Peter

2005-05-01

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of beta-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, zeta-carotene desaturase, carotene cis-trans-isomerase, beta-lycopene cyclase, and beta-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that beta-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, alpha/beta-lycopene cyclase, beta-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or zeta-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the beta-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are

[Cloning and functional characterization of phytoene desaturase in Andrographis paniculata].

PubMed

Shen, Qin-qin; Li, Li-xia; Zhan, Peng-lin; Wang, Qiang

2015-10-01

A full-length cDNA of phytoene desaturase (PDS) gene from Andrographis paniculata was obtained through RACE-PCR. The cDNA sequence consists of 2 224 bp with an intact ORF of 1 752 bp (GeneBank: KP982892), encoding a ploypeptide of 584 amino acids. Homology analysis showed that the deduced protein has extensive sequence similarities to PDS from other plants, and contains a conserved NAD ( H) -binding domain of plant dehydrase cofactor binding-domain in N-terminal. Phylogenetic analysis demonstrated that ApPDS was more related to PDS of Sesamum indicum and Pogostemon cablin. The semi-quantitative RT-PCR analysis revealed that ApPDS expressed in whole aboveground tissues with the highest expression in leaves. Virus induced gene silencing (VIGS) was performed to characterize the functional of ApPDS in planta. Significant photobleaching was not observed in infiltrated leaves, while the PDS gene has been down-regulated significantly at the yellowish area. To the best of our knowledge, this represents the first report of PDS gene cloning and functional characterization from A. paniculata, which lays the foundation for further investigation of new genes, especially that correlative to andrographolide biosynthetic pathway.

Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice

PubMed Central

Khare, Sanjay D.; Sarosi, Ildiko; Xia, Xing-Zhong; McCabe, Susan; Miner, Kent; Solovyev, Irina; Hawkins, Nessa; Kelley, Michael; Chang, David; Van, Gwyneth; Ross, Larry; Delaney, John; Wang, Ling; Lacey, David; Boyle, William J.; Hsu, Hailing

2000-01-01

TALL-1/Blys/BAFF is a member of the tumor necrosis factor (TNF) ligand superfamily that is functionally involved in B cell proliferation. Here, we describe B cell hyperplasia and autoimmune lupus-like changes in transgenic mice expressing TALL-1 under the control of a β-actin promoter. The TALL-1 transgenic mice showed severe enlargement of spleen, lymph nodes, and Peyer's patches because of an increased number of B220+ cells. The transgenic mice also had hypergammaglobulinemia contributed by elevations of serum IgM, IgG, IgA, and IgE. In addition, a phenotype similar to autoimmune lupus-like disease was also seen in TALL-1 transgenic mice, characterized by the presence of autoantibodies to nuclear antigens and immune complex deposits in the kidney. Prolonged survival and hyperactivity of transgenic B cells may contribute to the autoimmune lupus-like phenotype in these animals. Our studies further confirm TALL-1 as a stimulator of B cells that affect Ig production. Thus, TALL-1 may be a primary mediator in B cell-associated autoimmune diseases. PMID:10716715

A myo-inositol-1-phosphate synthase gene, IbMIPS1, enhances salt and drought tolerance and stem nematode resistance in transgenic sweet potato.

PubMed

Zhai, Hong; Wang, Feibing; Si, Zengzhi; Huo, Jinxi; Xing, Lei; An, Yanyan; He, Shaozhen; Liu, Qingchang

2016-02-01

Myo-inositol-1-phosphate synthase (MIPS) is a key rate limiting enzyme in myo-inositol biosynthesis. The MIPS gene has been shown to improve tolerance to abiotic stresses in several plant species. However, its role in resistance to biotic stresses has not been reported. In this study, we found that expression of the sweet potato IbMIPS1 gene was induced by NaCl, polyethylene glycol (PEG), abscisic acid (ABA) and stem nematodes. Its overexpression significantly enhanced stem nematode resistance as well as salt and drought tolerance in transgenic sweet potato under field conditions. Transcriptome and real-time quantitative PCR analyses showed that overexpression of IbMIPS1 up-regulated the genes involved in inositol biosynthesis, phosphatidylinositol (PI) and ABA signalling pathways, stress responses, photosynthesis and ROS-scavenging system under salt, drought and stem nematode stresses. Inositol, inositol-1,4,5-trisphosphate (IP3 ), phosphatidic acid (PA), Ca(2+) , ABA, K(+) , proline and trehalose content was significantly increased, whereas malonaldehyde (MDA), Na(+) and H2 O2 content was significantly decreased in the transgenic plants under salt and drought stresses. After stem nematode infection, the significant increase of inositol, IP3 , PA, Ca(2+) , ABA, callose and lignin content and significant reduction of MDA content were found, and a rapid increase of H2 O2 levels was observed, peaked at 1 to 2 days and thereafter declined in the transgenic plants. This study indicates that the IbMIPS1 gene has the potential to be used to improve the resistance to biotic and abiotic stresses in plants. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Effect of dietary n-3 fatty acids supplementation on fatty acid metabolism in atorvastatin-administered SHR.Cg-Leprcp/NDmcr rats, a metabolic syndrome model.

PubMed

Al Mamun, Abdullah; Hashimoto, Michio; Katakura, Masanori; Tanabe, Yoko; Tsuchikura, Satoru; Hossain, Shahdat; Shido, Osamu

2017-01-01

The effects of cholesterol-lowering statins, which substantially benefit future cardiovascular events, on fatty acid metabolism have remained largely obscured. In this study, we investigated the effects of atorvastatin on fatty acid metabolism together with the effects of TAK-085 containing highly purified eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ethyl ester on atorvastatin-induced n-3 polyunsaturated fatty acid lowering in SHR.Cg-Lepr cp /NDmcr (SHRcp) rats, as a metabolic syndrome model. Supplementation with 10mg/kg body weight/day of atorvastatin for 17 weeks significantly decreased plasma total cholesterol and very low density lipoprotein cholesterol. Atorvastatin alone caused a subtle change in fatty acid composition particularly of EPA and DHA in the plasma, liver or erythrocyte membranes. However, the TAK-085 consistently increased both the levels of EPA and DHA in the plasma, liver and erythrocyte membranes. After confirming the reduction of plasma total cholesterol, 300mg/kg body weight/day of TAK-085 was continuously administered for another 6 weeks. Supplementation with TAK-085 did not decrease plasma total cholesterol but significantly increased the EPA and DHA levels in both the plasma and liver compared with rats administered atorvastatin only. Supplementation with atorvastatin alone significantly decreased sterol regulatory element-binding protein-1c, Δ5- and Δ6-desaturases, elongase-5, and stearoyl-coenzyme A (CoA) desaturase-2 levels and increased 3-hydroxy-3-methylglutaryl-CoA reductase mRNA expression in the liver compared with control rats. TAK-085 supplementation significantly increased stearoyl-CoA desaturase-2 mRNA expression. These results suggest that long-term supplementation with atorvastatin decreases the EPA and DHA levels by inhibiting the desaturation and elongation of n-3 fatty acid metabolism, while TAK-085 supplementation effectively replenishes this effect in SHRcp rat liver. Copyright © 2016 Elsevier Masson

Overexpression of OsRAA1 Causes Pleiotropic Phenotypes in Transgenic Rice Plants, including Altered Leaf, Flower, and Root Development and Root Response to Gravity1

PubMed Central

Ge, Lei; Chen, Hui; Jiang, Jia-Fu; Zhao, Yuan; Xu, Ming-Li; Xu, Yun-Yuan; Tan, Ke-hui; Xu, Zhi-Hong; Chong, Kang

2004-01-01

There are very few root genes that have been described in rice as a monocotyledonous model plant so far. Here, the OsRAA1 (Oryza sativa Root Architecture Associated 1) gene has been characterized molecularly. OsRAA1 encodes a 12.0-kD protein that has 58% homology to the AtFPF1 (Flowering Promoting Factor 1) in Arabidopsis, which has not been reported as modulating root development yet. Data of in situ hybridization and OsRAA1∷GUS transgenic plant showed that OsRAA1 expressed specifically in the apical meristem, the elongation zone of root tip, steles of the branch zone, and the young lateral root. Constitutive expression of OsRAA1 under the control of maize (Zea mays) ubiquitin promoter resulted in phenotypes of reduced growth of primary root, increased number of adventitious roots and helix primary root, and delayed gravitropic response of roots in seedlings of rice (Oryza sativa), which are similar to the phenotypes of the wild-type plant treated with auxin. With overexpression of OsRAA1, initiation and growth of adventitious root were more sensitive to treatment of auxin than those of the control plants, while their responses to 9-hydroxyfluorene-9-carboxylic acid in both transgenic line and wild type showed similar results. OsRAA1 constitutive expression also caused longer leaves and sterile florets at the last stage of plant development. Analysis of northern blot and GUS activity staining of OsRAA1∷GUS transgenic plants demonstrated that the OsRAA1 expression was induced by auxin. At the same time, overexpression of OsRAA1 also caused endogenous indole-3-acetic acid to increase. These data suggested that OsRAA1 as a new gene functions in the development of rice root systems, which are mediated by auxin. A positive feedback regulation mechanism of OsRAA1 to indole-3-acetic acid metabolism may be involved in rice root development in nature. PMID:15247372

Epistatic effects between pairs of the growth hormone secretagogue receptor 1a, growth hormone, growth hormone receptor, non-SMC condensin I complex, subunit G and stearoyl-CoA desaturase genes on carcass, price-related and fatty acid composition traits in Japanese Black cattle.

PubMed

Komatsu, Masanori; Nishino, Kagetomo; Fujimori, Yuki; Haga, Yasutoshi; Iwama, Nagako; Arakawa, Aisaku; Aihara, Yoshito; Takeda, Hisato; Takahashi, Hideaki

2018-02-01

Growth hormone secretagogue receptor 1a (GHSR1a), growth hormone (GH), growth hormone receptor (GHR), non-SMC condensin I complex, subunit G (NCAPG) and stearoyl-CoA desaturase (SCD), are known to play important roles in growth and lipid metabolisms. Single and epistatic effects of the five genes on carcass, price-related and fatty acid (FA) composition traits were analyzed in a commercial Japanese Black cattle population of Ibaraki Prefecture. A total of 650 steers and 116 heifers for carcass and price-related traits, and 158 steers for FA composition traits were used in this study. Epistatic effects between pairs of the five genes were found in several traits. Alleles showing strain-specific differences in the five genes had significant single and epistatic effects in some traits. The data suggest that a TG-repeat polymorphism of the GHSR1a.5'UTR-(TG) n locus plays a central role in gene-gene epistatic interaction of FA composition traits in the adipose tissue of Japanese Black cattle. © 2017 Japanese Society of Animal Science.

The Yeast HAL1 Gene Improves Salt Tolerance of Transgenic Tomato1

PubMed Central

Gisbert, Carmina; Rus, Ana M.; Bolarín, M. Carmen; López-Coronado, J. Miguel; Arrillaga, Isabel; Montesinos, Consuelo; Caro, Manuel; Serrano, Ramon; Moreno, Vicente

2000-01-01

Overexpression of the HAL1 gene in yeast has a positive effect on salt tolerance by maintaining a high internal K+ concentration and decreasing intracellular Na+ during salt stress. In the present work, the yeast gene HAL1 was introduced into tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens-mediated transformation. A sample of primary transformants was self-pollinated, and progeny from both transformed and non-transformed plants (controls) were evaluated for salt tolerance in vitro and in vivo. Results from different tests indicated a higher level of salt tolerance in the progeny of two different transgenic plants bearing four copies or one copy of the HAL1 gene. In addition, measurement of the intracellular K+ to Na+ ratios showed that transgenic lines were able to retain more K+ than the control under salt stress. Although plants and yeast cannot be compared in an absolute sense, these results indicate that the mechanism controlling the positive effect of the HAL1 gene on salt tolerance may be similar in transgenic plants and yeast. PMID:10806256

Repeated intraperitoneal injections of liposomes containing phosphatidic acid and cardiolipin reduce amyloid-β levels in APP/PS1 transgenic mice.

PubMed

Ordóñez-Gutiérrez, Lara; Re, Francesca; Bereczki, Erika; Ioja, Eniko; Gregori, Maria; Andersen, Alina J; Antón, Marta; Moghimi, S Moein; Pei, Jin-Jing; Masserini, Massimo; Wandosell, Francisco

2015-02-01

The accumulation of extracellular amyloid-beta (Aβ) peptide and intracellular neurofibrillary tangles in the brain are two major neuropathological hallmarks of Alzheimer's disease (AD). It is thought that an equilibrium exists between Aβ in the brain and in the peripheral blood and thus, it was hypothesized that shifting this equilibrium towards the blood by enhancing peripheral clearance might reduce Aβ levels in the brain: the 'sink effect'. We tested this hypothesis by intraperitoneally injecting APP/PS1 transgenic mice with small unilamellar vesicles containing either phosphatidic acid or cardiolipin over 3weeks. This treatment reduced significantly the amount of Aβ in the plasma and the brain levels of Aβ were lighter affected. Nevertheless, this dosing regimen did modulate tau phosphorylation and glycogen synthase kinase 3 activities in the brain, suggesting that the targeting of circulating Aβ may be therapeutically relevant in AD. Intraperitoneal injection of small unilamellar vesicles containing phosphatidic acid or cardiolipin significantly reduced the amount of amyloid-beta (Aß) peptide in the plasma in a rodent model. Brain levels of Aß were also affected - although to a lesser extent - suggesting that targeting of circulating Aß may be therapeutically relevant of Alzheimer's disease. Copyright © 2015 Elsevier Inc. All rights reserved.

NKX3.1 Genotype and IGF-1 Interact in Prostate Cancer Risk

DTIC Science & Technology

2009-05-01

Steadman DJ, Giuffrida D, Gelmann EP. DNA-binding sequence of the human prostate-specific homeodomain protein NKX3.1. Nucleic Acids Res 2000;28...Gelmann EP. DNA-binding sequence of the human prostate-specific homeodomain protein NKX3.1. Nucleic Acids Res 2000;28:2389–95. 20. Wu X, Senechal K...3212836 /UG=Hs.21765 fatty acid desaturase 3 204733_at 5.74 gb:NM_002774.1 /DEF=Homo sapiens kallikrein 6 (neurosin, zyme) (KLK6), mRNA. /FEA=mRNA /GEN

Eicosapentenoic Acid Attenuates Allograft Rejection in an HLA-B27/EGFP Transgenic Rat Cardiac Transplantation Model.

PubMed

Liu, Zhong; Hatayama, Naoyuki; Xie, Lin; Kato, Ken; Zhu, Ping; Ochiya, Takahiro; Nagahara, Yukitoshi; Hu, Xiang; Li, Xiao-Kang

2012-01-01

The development of an animal model bearing definite antigens is important to facilitate the evaluation and modulation of specific allo-antigen responses after transplantation. In the present study, heterotopic cardiac transplantation was performed from F344/EGFPTg and F344/HLA-B27Tg rats to F344 rats. The F344 recipients accepted the F344/EGFPTg transplants, whereas they rejected the cardiac tissue from the F344/HLA-B27Tg rats by 39.4 ± 6.5 days, due to high production of anti-HLA-B27 IgM- and IgG-specific antibodies. In addition, immunization of F344 rats with skin grafts from F344/HLA-B27Tg rats resulted in robust production of anti- HLA-B27 IgM and IgG antibodies and accelerated the rejection of a secondary cardiac allograft (7.4 ± 1.9 days). Of interest, the F344 recipients rejected cardiac grafts from double transgenic F344/HLA-B27&EGFPTg rats within 9.0 ± 3.2 days, and this was associated with a significant increase in the infiltration of lymphocytes by day 7, suggesting a role for cellular immune rejection. Eicosapentenoic acid (EPA), one of the ω-3 polyunsaturated fatty acids in fish oil, could attenuate the production of anti-HLA IgG antibodies and B-cell proliferation, significantly prolonging double transgenic F344HLA-B27&EGFPTg to F344 rat cardiac allograft survival (36.1 ± 13.6 days). Moreover, the mRNA expression in the grafts was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), revealing an increase in the expression of the HO-1, IL-10, TGF-β, IDO, and Foxp3 genes in the EPA-treated group. Hence, our data indicate that HLA-B27 and/or GFP transgenic proteins are useful for establishing a unique animal transplantation model to clarify the mechanism underlying the allogeneic cellular and humoral immune response, in which the transplant antigens are specifically presented. Furthermore, we also demonstrated that EPA was effective in the treatment of rat cardiac allograft rejection and may allow the development of

Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz).

PubMed

Beltrán, J; Prías, M; Al-Babili, S; Ladino, Y; López, D; Beyer, P; Chavarriaga, P; Tohme, J

2010-05-01

A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava's starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.

Erythrocyte and plasma fatty acid patterns in dogs with atopic dermatitis and healthy dogs in the same household

PubMed Central

Zimmermann, Annett; Gück, Thomas; Oechtering, Gerhard

2006-01-01

Abstract Recent studies have indicated that dogs with canine atopic dermatitis (CAD) may have a disorder of fatty acid metabolism: possibly low or absent activity of Δ6-desaturase or Δ5-desaturase, or both. To clarify this possibility, we examined the erythrocyte and plasma fatty acid patterns of 8 dogs with CAD and their 8 healthy housemates. Atopic dermatitis was diagnosed according to the criteria proposed by Willemse; other causes of dermatitis were excluded clinically and by appropriate tests. Erythrocyte ghosts were prepared from blood samples. Membrane lipids were extracted and separated by thin-layer chromatography. From plasma and lipid fractions, fatty acid content was determined by gas chromatography. In erythrocytes, but not in plasma, we observed significant differences in the fatty acid pattern that suggested a reduction in the n6 fatty acid products of the Δ6- and Δ5-desaturases in dogs with atopic dermatitis when compared with healthy housemates. PMID:16850941

Transgenic barley (Hordeum vulgare L.) expressing the wheat aluminium resistance gene (TaALMT1) shows enhanced phosphorus nutrition and grain production when grown on an acid soil.

PubMed

Delhaize, Emmanuel; Taylor, Phillip; Hocking, Peter J; Simpson, Richard J; Ryan, Peter R; Richardson, Alan E

2009-06-01

Barley (Hordeum vulgare L.), genetically modified with the Al(3+) resistance gene of wheat (TaALMT1), was compared with a non-transformed sibling line when grown on an acidic and highly phosphate-fixing ferrosol supplied with a range of phosphorus concentrations. In short-term pot trials (26 days), transgenic barley expressing TaALMT1 (GP-ALMT1) was more efficient than a non-transformed sibling line (GP) at taking up phosphorus on acid soil, but the genotypes did not differ when the soil was limed. Differences in phosphorus uptake efficiency on acid soil could be attributed not only to the differential effects of aluminium toxicity on root growth between the genotypes, but also to differences in phosphorus uptake per unit root length. Although GP-ALMT1 out-performed GP on acid soil, it was still not as efficient at taking up phosphorus as plants grown on limed soil. GP-ALMT1 plants grown in acid soil possessed substantially smaller rhizosheaths than those grown in limed soil, suggesting that root hairs were shorter. This is a probable reason for the lower phosphorus uptake efficiency. When grown to maturity in large pots, GP-ALMT1 plants produced more than twice the grain as GP plants grown on acid soil and 80% of the grain produced by limed controls. Expression of TaALMT1 in barley was not associated with a penalty in either total shoot or grain production in the absence of Al(3+), with both genotypes showing equivalent yields in limed soil. These findings demonstrate that an important crop species can be genetically engineered to successfully increase grain production on an acid soil.

Effect of the feeding system on the fatty acid composition, expression of the Δ9-desaturase, Peroxisome Proliferator-Activated Receptor Alpha, Gamma, and Sterol Regulatory Element Binding Protein 1 genes in the semitendinous muscle of light lambs of the Rasa Aragonesa breed

PubMed Central

2010-01-01

Background Conjugated linoleic acids (CLAs) are receiving increasing attention because of their beneficial effects on human health, with milk and meat products derived from ruminants as important sources of CLA in the human diet. SCD gene is responsible for some of the variation in CLA concentration in adipose tissues, and PPARγ, PPARα and SREBP1 genes are regulator of SCD gene. The aim of this work was to evaluate the effect of the feeding system on fatty acid composition, CLA content and relative gene expression of Δ9-desaturase (SCD), Peroxisome Proliferator-Activated Receptor Gamma (PPARγ), Peroxisome Proliferator-Activated Receptor Alpha, (PPARα) and Sterol Regulatory Element Binding Protein (SREBP1) in Rasa Aragonesa light lambs in semitendinous muscle. Forty-four single-born male lambs were used to evaluate the effect of the feeding system, varying on an intensity gradient according to the use of concentrates: 1. grazing alfalfa, 2. grazing alfalfa with a supplement for lambs, 3. indoor lambs with grazing ewes and 4. drylot. Results Both grazing systems resulted in a higher concentration of vaccenic acid (VA), CLA, CLA/VA acid ratio, and a lower oleic content, oleic acid (C18:1)/stearic acid (C18:0) ratio, PUFA n-6/n-3 ratio and SCD expression compared to other diets. In addition feeding system affected the fatty acid composition and SCD expression, possibly due to CLA concentration or the PUFA n-6/n-3 ratio. Both expression of the SCD gene and the feeding system were important factors affecting CLA concentration in the animal's semitendinous muscle. PPARγ, PPARα and SREBP1 expression seemed to be unaffected by the feeding system. Although no significant results were found, PPARγ, PPARα and SREBP1 showed similar expression pattern as SCD. Moreover, the correlation results between SCD expression and PPARγ (p < 0.01), as well as SREBP1 (p < 0.01) expression, may suggest that these genes were affecting SCD expression in a different way. Conclusions

Catalytic properties of an expressed and purified higher plant type zeta-carotene desaturase from Capsicum annuum.

PubMed

Breitenbach, J; Kuntz, M; Takaichi, S; Sandmann, G

1999-10-01

The zeta-carotene desaturase from Capsicum annuum (EC 1.14.99.-) was expressed in Escherichia coli, purified and characterized biochemically. The enzyme acts as a monomer with lipophilic quinones as cofactors. Km values for the substrate zeta-carotene or the intermediate neurosporene in the two-step desaturation reaction are almost identical. Product analysis showed that different lycopene isomers are formed, including substantial amounts of the all-trans form, together with 7,7',9,9'-tetracis prolycopene via the corresponding neurosporene isomers. The application of different geometric isomers as substrates revealed that the zeta-carotene desaturase has no preference for certain isomers and that the nature of the isomers formed during catalysis depends strictly on the isomeric composition of the substrate.

Enriched endogenous n-3 polyunsaturated fatty acids alleviate cognitive and behavioral deficits in a mice model of Alzheimer's disease.

PubMed

Wu, Kefeng; Gao, Xiang; Shi, Baoyan; Chen, Shiyu; Zhou, Xin; Li, Zhidong; Gan, Yuhong; Cui, Liao; Kang, Jing Xuan; Li, Wende; Huang, Ren

2016-10-01

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that accompanied by memory deficits and neuropsychiatric dysfunction. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have seemly therapeutic potential in AD, but the benefit of n-3 PUFAs is still in debates. Here, we employed a transgenic mice carry fat-1 gene to encode n-3 desaturase from Caenorhabditis elegans, which increase endogenous n-3 PUFAs by converting n-6 PUFAs to n-3 PUFAs crossed with amyloid precursor protein (APP) Tg mice to evaluate the protective effects of endogenous n-3 PUFAs on cognitive and behavioral deficits of APP Tg mice. We fed APP, APP/fat-1 and fat-1 mice with n-6 PUFAs rich diet. Brain tissues were collected at 3, 9 and 12 months for fatty acid and gene expression analysis, histology and protein assays. Morris Water Maze Test, open field test and elevated plus maze test were performed to measure the behavior capability. From the results, the expression of fat-1 transgene increased cortical n-3: n-6 PUFAs ratio and n-3 PUFAs concentrations, and sensorimotor dysfunction and cognitive deficits in AD were significantly less severe in APP/fat-1 mice with endogenous n-3 PUFAs than in APP mice controls. The protection against disturbance of spontaneous motor activity and cognitive deficits in AD was strongly correlated with increased n-3: n-6 PUFAs ratio and endogenous n-3 PUFAs, reduced APP generation, inhibited amyloid β peptide aggregation, suppressed nuclear factor-kappa B and astroglia activation, and reduced death of neurons in the cortex of APP/fat-1 mice compared with APP mice controls. In conclusion, our study demonstrates that an available medication with the maintenance of enriched n-3 PUFAs in the brain could slow down cognitive decline and prevent neuropsychological disorder in AD. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Functional analysis of multiple carotenogenic genes from Lycium barbarum and Gentiana lutea L. for their effects on beta-carotene production in transgenic tobacco.

PubMed

Ji, Jing; Wang, Gang; Wang, Jiehua; Wang, Ping

2009-02-01

Carotenoids are red, yellow and orange pigments, which are widely distributed in nature and are especially abundant in yellow-orange fruits and vegetables and dark green leafy vegetables. Carotenoids are essential for photosynthesis and photoprotection in plant life and also have different beneficial effects in humans and animals (van den Berg et al. 2000). For example, beta-carotene plays an essential role as the main dietary source of vitamin A. To obtain further insight into beta-carotene biosynthesis in two important economic plant species, Lycium barbarum and Gentiana lutea L., and to investigate and prioritize potential genetic engineering targets in the pathway, the effects of five carotenogenic genes from these two species, encoding proteins including geranylgeranyl diphosphate synthase, phytoene synthase and delta-carotene desaturase gene, lycopene beta-cyclase, lycopene epsilon-cyclase were functionally analyzed in transgenic tobacco (Nicotiana tabacum) plants. All transgenic tobacco plants constitutively expressing these genes showed enhanced beta-carotene contents in their leaves and flowers to different extents. The addictive effects of co-ordinate expression of double transgenes have also been investigated.

Bleaching Herbicide Flurtamone Interferes with Phytoene Desaturase

PubMed Central

Sandmann, Gerhard; Ward, Carl E.; Lo, William C.; Nagy, Jon O.; Böger, Peter

1990-01-01

The mode of action of the furanone herbicide flurtamone and derivatives was investigated with cress seedlings and with the unicellular cyanobacterium Anacystis. Either in the light or in the dark these compounds inhibited the formation of α- and β-carotene and all of the xanthophylls in the seedlings. Instead, phytoene, a precursor of colored carotenoids, was accumulated. In illuminated seedlings photooxidative destruction of chlorophyll was observed. The I50 value of flurtamone inhibition of carotenoid biosynthesis in intact Anacystis cells and the K1 value for interaction of flurtamone with phytoene desaturase with Anacystis thylakoids were 30 and 18 nanomoles, respectively. Concentrations of flurtamone which strongly inhibited carotenoid synthesis had no direct peroxidative activities and did not inhibit photosynthetic electron transport. PMID:16667736

EPA, DHA, and Lipoic Acid Differentially Modulate the n-3 Fatty Acid Biosynthetic Pathway in Atlantic Salmon Hepatocytes.

PubMed

Bou, Marta; Østbye, Tone-Kari; Berge, Gerd M; Ruyter, Bente

2017-03-01

The aim of the present study was to investigate how EPA, DHA, and lipoic acid (LA) influence the different metabolic steps in the n-3 fatty acid (FA) biosynthetic pathway in hepatocytes from Atlantic salmon fed four dietary levels (0, 0.5, 1.0 and 2.0%) of EPA, DHA or a 1:1 mixture of these FA. The hepatocytes were incubated with [1- 14 C] 18:3n-3 in the presence or absence of LA (0.2 mM). Increased endogenous levels of EPA and/or DHA and LA exposure both led to similar responses in cells with reduced desaturation and elongation of [1- 14 C] 18:3n-3 to 18:4n-3, 20:4n-3, and EPA, in agreement with reduced expression of the Δ6 desaturase gene involved in the first step of conversion. DHA production, on the other hand, was maintained even in groups with high endogenous levels of DHA, possibly due to a more complex regulation of this last step in the n-3 metabolic pathway. Inhibition of the Δ6 desaturase pathway led to increased direct elongation to 20:3n-3 by both DHA and LA. Possibly the route by 20:3n-3 and then Δ8 desaturation to 20:4n-3, bypassing the first Δ6 desaturase step, can partly explain the maintained or even increased levels of DHA production. LA increased DHA production in the phospholipid fraction of hepatocytes isolated from fish fed 0 and 0.5% EPA and/or DHA, indicating that LA has the potential to further increase the production of this health-beneficial FA in fish fed diets with low levels of EPA and/or DHA.

Degeneration of oxidative muscle fibers in HTLV-1 tax transgenic mice.

PubMed

Nerenberg, M I; Wiley, C A

1989-12-01

The HTLV-1 tax gene under control of the HTLV-1 long terminal repeat (LTR) was introduced into transgenic mice. Previously tax protein expression in the muscle and peripheral nerves of three independent mouse lines was reported. Here the localization of this transgenic protein at a cellular and subcellular level is described. Tax protein was expressed in oxidative muscle fibers that developed severe progressive atrophy. It localized to the cytoplasma where it was associated with structures resembling degenerating Z bands. This pattern of muscle fiber involvement is similar to that observed in human retroviral associated myopathy. This transgenic mouse model suggests that preferential expression of the HTLV-1 viral promoter in oxidative muscle fibers may explain the productive infection of these fibers in HTLV-1 myopathy.

Identification and Analysis of a Gene from Calendula officinalis Encoding a Fatty Acid Conjugase

PubMed Central

Qiu, Xiao; Reed, Darwin W.; Hong, Haiping; MacKenzie, Samuel L.; Covello, Patrick S.

2001-01-01

Two homologous cDNAs, CoFad2 and CoFac2, were isolated from a Calendula officinalis developing seed by a polymerase chain reaction-based cloning strategy. Both sequences share similarity to FAD2 desaturases and FAD2-related enzymes. In C. officinalis plants CoFad2 was expressed in all tissues tested, whereas CoFac2 expression was specific to developing seeds. Expression of CoFad2 cDNA in yeast (Saccharomyces cerevisiae) indicated it encodes a Δ12 desaturase that introduces a double bond at the 12 position of 16:1(9Z) and 18:1(9Z). Expression of CoFac2 in yeast revealed that the encoded enzyme acts as a fatty acid conjugase converting 18:2(9Z, 12Z) to calendic acid 18:3(8E, 10E, 12Z). The enzyme also has weak activity on the mono-unsaturates 16:1(9Z) and 18:1(9Z) producing compounds with the properties of 8,10 conjugated dienes. PMID:11161042

Drought stress-induced compositional changes in tolerant transgenic rice and its wild type.

PubMed

Nam, Kyong-Hee; Kim, Do-Young; Shin, Hee Jae; Nam, Ki Jung; An, Joo Hee; Pack, In-Soon; Park, Jung-Ho; Jeong, Soon-Chun; Kim, Ho Bang; Kim, Chang-Gi

2014-06-15

Comparing well-watered versus deficit conditions, we evaluated the chemical composition of grains harvested from wild-type (WT) and drought-tolerant, transgenic rice (Oryza sativa L.). The latter had been developed by inserting AtCYP78A7, which encodes a cytochrome P450 protein. Two transgenic Lines, '10B-5' and '18A-4', and the 'Hwayoung' WT were grown under a rainout shelter. After the harvested grains were polished, their levels of key components, including proximates, amino acids, fatty acids, minerals and vitamins were analysed to determine the effect of watering system and genotype. Drought treatment significantly influenced the levels of some nutritional components in both transgenic and WT grains. In particular, the amounts of lignoceric acid and copper in the WT decreased by 12.6% and 39.5%, respectively, by drought stress, whereas those of copper and potassium in the transgenics rose by 88.1-113.3% and 10.4-11.9%, respectively, under water-deficit conditions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Overexpression of a Novel Apple NAC Transcription Factor Gene, MdNAC1, Confers the Dwarf Phenotype in Transgenic Apple (Malus domestica)

PubMed Central

Jia, Dongfeng; Gong, Xiaoqing; Li, Mingjun; Li, Chao; Sun, Tingting

2018-01-01

Plant height is an important trait for fruit trees. The dwarf characteristic is commonly associated with highly efficient fruit production, a major objective when breeding for apple (Malus domestica). We studied the function of MdNAC1, a novel NAC transcription factor (TF) gene in apple related to plant dwarfing. Localized primarily to the nucleus, MdNAC1 has transcriptional activity in yeast cells. Overexpression of the gene results in a dwarf phenotype in transgenic apple plants. Their reduction in size is manifested by shorter, thinner stems and roots, and a smaller leaf area. The transgenics also have shorter internodes and fewer cells in the stems. Levels of endogenous abscisic acid (ABA) and brassinosteroid (BR) are lower in the transgenic plants, and expression is decreased for genes involved in the biosynthesis of those phytohormones. All of these findings demonstrate that MdNAC1 has a role in plants dwarfism, probably by regulating ABA and BR production. PMID:29702625

Misregulated progesterone secretion and impaired pregnancy in Cyp11a1 transgenic mice.

PubMed

Chien, Yu; Cheng, Wei-Cheng; Wu, Menq-Rong; Jiang, Si-Tse; Shen, Che-Kun James; Chung, Bon-chu

2013-10-01

Normal pregnancy is supported by increased levels of progesterone (P4), which is secreted from ovarian luteal cells via enzymatic steps catalyzed by P450scc (CYP11A1) and HSD3B. The development and maintenance of corpora lutea during pregnancy, however, are less well understood. Here we used Cyp11a1 transgenic mice to delineate the steps of luteal cell differentiation during pregnancy. Cyp11a1 in a bacterial artificial chromosome was injected into mouse embryos to generate transgenic mice with transgene expression that recapitulated endogenous Cyp11a1 expression. Cyp11a1 transgenic females displayed reduced pregnancy rate, impaired implantation and placentation, and decreased litter size in utero, although they produced comparable numbers of blastocysts. The differentiation of transgenic luteal cells was delayed during early pregnancy as shown by the delayed activation of genes involved in steroidogenesis and cholesterol availability. Luteal cell mitochondria were elongated, and their numbers were reduced, with morphology and numbers similar to those observed in granulosa cells. Transgenic luteal cells accumulated lipid droplets and secreted less progesterone during early pregnancy. The progesterone level returned to normal on gestation day 9 but was not properly withdrawn at term, leading to delayed stillbirth. P4 supplementation rescued the implantation rates but not the ovarian defects. Thus, overexpression of Cyp11a1 disrupts normal development of the corpus luteum, leading to progesterone insufficiency during early pregnancy. Misregulation of the progesterone production in Cyp11a1 transgenic mice during pregnancy resulted in aberrant implantation, anomalous placentation, and delayed parturition.

Characterization and Ectopic Expression of CoWRI1, an AP2/EREBP Domain-Containing Transcription Factor from Coconut (Cocos nucifera L.) Endosperm, Changes the Seeds Oil Content in Transgenic Arabidopsis thaliana and Rice (Oryza sativa L.)

PubMed Central

Sun, RuHao; Ye, Rongjian; Gao, Lingchao; Zhang, Lin; Wang, Rui; Mao, Ting; Zheng, Yusheng; Li, Dongdong; Lin, Yongjun

2017-01-01

Coconut (Cocos nucifera L.) is a key tropical crop and a member of the monocotyledonous family Arecaceae (Palmaceae). Few genes and related metabolic processes involved in coconut endosperm development have been investigated. In this study, a new member of the WRI1 gene family was isolated from coconut endosperm and was named CoWRI1. Its transcriptional activities and interactions with the acetyl-CoA carboxylase (BCCP2) promoter of CoWRI1 were confirmed by the yeast two-hybrid and yeast one-hybrid approaches, respectively. Functional characterization was carried out through seed-specific expression in Arabidopsis and endosperm-specific expression in rice. In transgenic Arabidopsis, high over-expressions of CoWRI1 in seven independent T2 lines were detected by quantitative real-time PCR. The relative mRNA accumulation of genes encoding enzymes involved in either fatty acid biosynthesis or triacylglycerols assembly (BCCP2, KASI, MAT, ENR, FATA, and GPDH) were also assayed in mature seeds. Furthermore, lipid and fatty acids C16:0 and C18:0 significantly increased. In two homozygous T2 transgenic rice lines (G5 and G2), different CoWRI1 expression levels were detected, but no CoWRI1 transcripts were detected in the wild type. Analyses of the seed oil content, starch content, and total protein content indicated that the two T2 transgenic lines showed a significant increase (P < 0.05) in seed oil content. The transgenic lines also showed a significant increase in starch content, whereas total protein content decreased significantly. Further analysis of the fatty acid composition revealed that palmitic acid (C16:0) and linolenic acid (C18:3) increased significantly in the seeds of the transgenic rice lines, but oleic acid (C18:1) levels significantly declined. PMID:28179911

Characterization and Ectopic Expression of CoWRI1, an AP2/EREBP Domain-Containing Transcription Factor from Coconut (Cocos nucifera L.) Endosperm, Changes the Seeds Oil Content in Transgenic Arabidopsis thaliana and Rice (Oryza sativa L.).

PubMed

Sun, RuHao; Ye, Rongjian; Gao, Lingchao; Zhang, Lin; Wang, Rui; Mao, Ting; Zheng, Yusheng; Li, Dongdong; Lin, Yongjun

2017-01-01

Coconut ( Cocos nucifera L.) is a key tropical crop and a member of the monocotyledonous family Arecaceae ( Palmaceae ). Few genes and related metabolic processes involved in coconut endosperm development have been investigated. In this study, a new member of the WRI1 gene family was isolated from coconut endosperm and was named CoWRI1 . Its transcriptional activities and interactions with the acetyl-CoA carboxylase ( BCCP2 ) promoter of CoWRI1 were confirmed by the yeast two-hybrid and yeast one-hybrid approaches, respectively. Functional characterization was carried out through seed-specific expression in Arabidopsis and endosperm-specific expression in rice. In transgenic Arabidopsis , high over-expressions of CoWRI1 in seven independent T2 lines were detected by quantitative real-time PCR. The relative mRNA accumulation of genes encoding enzymes involved in either fatty acid biosynthesis or triacylglycerols assembly (BCCP2, KASI, MAT, ENR, FATA, and GPDH) were also assayed in mature seeds. Furthermore, lipid and fatty acids C16:0 and C18:0 significantly increased. In two homozygous T2 transgenic rice lines (G5 and G2), different CoWRI1 expression levels were detected, but no CoWRI1 transcripts were detected in the wild type. Analyses of the seed oil content, starch content, and total protein content indicated that the two T2 transgenic lines showed a significant increase ( P < 0.05) in seed oil content. The transgenic lines also showed a significant increase in starch content, whereas total protein content decreased significantly. Further analysis of the fatty acid composition revealed that palmitic acid (C16:0) and linolenic acid (C18:3) increased significantly in the seeds of the transgenic rice lines, but oleic acid (C18:1) levels significantly declined.

Comparative analysis of nutritional compositions of transgenic RNAi-mediated virus-resistant bean (event EMB-PV051-1) with its non-transgenic counterpart.

PubMed

Carvalho, José L V; de Oliveira Santos, Juliana; Conte, Carmine; Pacheco, Sidney; Nogueira, Elsa O P L; Souza, Thiago L P O; Faria, Josias C; Aragão, Francisco J L

2015-10-01

Golden mosaic is among the most economically important diseases that severely reduce bean production in Latin America. In 2011, a transgenic bean event named Embrapa 5.1 (EMB-PV051-1), resistant to bean golden mosaic virus, was approved for commercial release in Brazil. The aim of this study was to measure and evaluate the nutritional components of the beans, as well as the anti-nutrient levels in the primary transgenic line and its derived near-isogenic lines after crosses and backcrosses with two commercial cultivars. Nutritional assessment of transgenic crops used for human consumption is an important aspect of safety evaluations. Results demonstrated that the transgenic bean event, cultivated under field conditions, was substantially equivalent to that of the non-transgenic bean plants. In addition, the amounts of the nutritional components are within the range of values observed for several bean commercial varieties grown across a range of environments and seasons.