| 1. |
- Arnqvist, Lisa, 1970-, et al.
(författare)
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Overexpression of CYP710A1 and CYP710A4 in transgenic Arabidopsis plants increases the level of stigmasterol at the expense of sitosterol
- 2008
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Ingår i: Planta. - : Springer Science and Business Media LLC. - 0032-0935 .- 1432-2048. ; 227:2, s. 309-317
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Tidskriftsartikel (refereegranskat)abstract
- Sitosterol and stigmasterol are major sterols in vascular plants. An altered stigmasterol:sitosterol ratio has been proposed to influence the properties of cell membranes, particularly in relation to various stresses, but biosynthesis of stigmasterol is poorly understood. Recently, however, Morikawa et al. (Plant Cell 18:1008–1022, 2006) showed in Arabidopsis thaliana that synthesis of stigmasterol and brassicasterol is catalyzed by two separate sterol C-22 desaturases, encoded by the genes CYP710A1 and CYP710A2, respectively. The proteins belong to a small cytochrome P450 subfamily having four members, denoted by CYP710A1-A4, and are related to the yeast sterol C-22 desaturase Erg5p acting in ergosterol synthesis. Here, we report on our parallel investigation of the Arabidopsis CYP710A family. To elucidate the function of CYP710A proteins, transgenic Arabidopsis plants were generated overexpressing CYP710A1 and CYP710A4. Compared to wild-type plants, both types of transformant displayed a normal phenotype, but contained increased levels of free stigmasterol and a concomitant decrease in the level of free sitosterol. CYP710A1 transformants also displayed higher levels of esterified forms of stigmasterol, cholesterol, 24-methylcholesterol and isofucosterol. The results confirm the findings of Morikawa et al. (Plant Cell 18:1008–1022, 2006) regarding the function of CYP710A1 in stigmasterol synthesis, and show that CYP710A4 also has this capacity. Furthermore, our results suggest that an increased stigmasterol level alone is sufficient to stimulate esterification of other major sterols.
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| 2. |
- Aslan, Selcuk, et al.
(författare)
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Increased production of wax esters in transgenic tobacco plants by expression of a fatty acid reductase:wax synthase gene fusion
- 2015
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Ingår i: Transgenic Research. - : Springer Science and Business Media LLC. - 0962-8819 .- 1573-9368. ; 24, s. 945-953
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Tidskriftsartikel (refereegranskat)abstract
- Wax esters are hydrophobic lipids consisting of a fatty acid moiety linked to a fatty alcohol with an ester bond. Plant-derived wax esters are today of particular concern for their potential as cost-effective and sustainable sources of lubricants. However, this aspect is hampered by the fact that the level of wax esters in plants generally is too low to allow commercial exploitation. To investigate whether wax ester biosynthesis can be increased in plants using transgenic approaches, we have here exploited a fusion between two bacterial genes together encoding a single wax ester-forming enzyme, and targeted the resulting protein to chloroplasts in stably transformed tobacco (Nicotiana benthamiana) plants. Compared to wild-type controls, transgenic plants showed both in leaves and stems a significant increase in the total level of wax esters, being eight-fold at the whole plant level. The profiles of fatty acid methyl ester and fatty alcohol in wax esters were related, and C16 and C18 molecules constituted predominant forms. Strong transformants displayed certain developmental aberrations, such as stunted growth and chlorotic leaves and stems. These negative effects were associated with an accumulation of fatty alcohols, suggesting that an adequate balance between formation and esterification of fatty alcohols is crucial for a high wax ester production. The results show that wax ester engineering in transgenic plants is feasible, and suggest that higher yields may become achieved in the near future.
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| 3. |
- Aslan, Selcuk, et al.
(författare)
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Transient silencing of the KASII genes is feasible in Nicotiana benthamiana for metabolic engineering of wax ester composition
- 2015
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- The beta-ketoacyl-ACP synthase II (KASII) is an enzyme in fatty acid biosynthesis, catalyzing the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. Mutations in KASII genes in higher plants can lead to lethality, which makes it difficult to utilize the gene for lipid metabolic engineering. We demonstrated previously that transient expression of plastid-directed fatty acyl reductases and wax ester synthases could result in different compositions of wax esters. We hypothesized that changing the ratio between C16 (palmitoyl-compounds) and C18 (stearoyl-compounds) in the plastidic acyl-ACP pool by inhibition of KASII expression would change the yield and composition of wax esters via substrate preference of the introduced enzymes. Here, we report that transient inhibition of KASII expression by three different RNAi constructs in leaves of N. benthamiana results in almost complete inhibition of KASII expression. The transient RNAi approach led to a shift of carbon flux from a pool of C18 fatty acids to C16, which significantly increased wax ester production in AtFAR6-containing combinations. The results demonstrate that transient inhibition of KASII in vegetative tissues of higher plants enables metabolic studies towards industrial production of lipids such as wax esters with specific quality and composition.
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| 4. |
- Aslan, Selcuk, et al.
(författare)
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Wax esters of different compositions produced via engineering of leaf chloroplast metabolism in Nicotiana benthamiana
- 2014
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 25, s. 103-112
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Tidskriftsartikel (refereegranskat)abstract
- In a future bin based economy, renewable sources for lipid compounds at attractive cost are needed for applications where today petrochemical derivatives are dominating. Wax esters and fatty alcohols provide diverse industrial uses, such as in lubricant and surfactant production. In this study, chloroplast metabolism was engineered to divert intermediates from de nova fatty acid biosynthesis to wax ester synthesis. To accomplish this, chloroplast targeted fatty acyl recluctases (EAR) and wax ester synthases (WS) were transiently expressed in Nic"onana benthamiuna loaves. Wax esters of different qualities and quantities were produced providing insights to the properties and interaction of the individual enzymes used. In particular, a phytyl ester synthase was found to be a premium candidate for medium chain wax ester synthesis. Catalytic activities of FAR and WS were also expressed as a fusion protein and determined functionally equivalent to the expression of individual enzymes for wax ester synthesis in chloroplasts. (C) 2014 The Authors. Published by Elsevier Inc. On behalf of International Metabolic Engineering Society.
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| 5. |
- Azadmard-Damirchi, Sodeif, et al.
(författare)
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A single step solid-phase extraction method for complete separation of sterol oxidation products in food lipids
- 2009
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1216, s. 36-42
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Tidskriftsartikel (refereegranskat)abstract
- One of the crucial steps in determination of sterol oxidation products (SOPs) in foods is their enrichment and purifications by various preparative methods for further analysis by GC and GC-MS. Among the preparative methods, SPE of various adsorbents and solvent systems, are being used most widely. At present, no single step SPE method is suitable to completely separate the SOPs. In this study, a SPE (I g silica) method, suitable for both transesterified and cold saponified oil samples. was developed to separate completely SOPs from other lipid components. This method resulted in high recovery from rapeseed oil of added 5 beta,6 beta-epoxycholestan-3 beta-ol (94-96%), cholest-5-en-3 beta-ol-7-one(94%), cholestane-3 beta,5 alpha,6 beta-triol (88-91%), cholest-5-en-3 beta,7 alpha-diol and 5 alpha,6 alpha-epoxycholestan-3 beta-ol (88-90%). The method has a high sample capacity of up to I g transesterified or cold-saponified oil sample. The method was tested and applied to different vegetable oils and to monitor the effects of refining processes on POPs in hazelnut oil. (C) 2008 Elsevier B.V. All rights reserved.
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| 6. |
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| 7. |
- Beste, Lisa, et al.
(författare)
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Synthesis of Hydroxylated Sterols in Transgenic Arabidopsis Plants Alters Growth and Steroid Metabolism
- 2011
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 157:1, s. 426-440
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Tidskriftsartikel (refereegranskat)abstract
- To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of alpha and beta forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels.
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| 8. |
- Dutta, Paresh
(författare)
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Analytical Methods for Quantification of Modified Fatty Acids and Sterols Formed as a Result of Processing
- 2009
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Ingår i: Food Analytical Methods. - : Springer Science and Business Media LLC. - 1936-9751 .- 1936-976X. ; 2, s. 30-40
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Tidskriftsartikel (refereegranskat)abstract
- Fats and oils are often submitted to technological treatments before being consumed. Some treatments like refining, hydrogenation, and frying often lead to the formation of modified fatty acids such as cyclic fatty acid monomers (CFAM), geometrical fatty acid isomers, and/or oxidized fatty acids and sterols (cholesterol and phytosterols). Both cholesterol oxidation products (COP) and phytosterol oxidation products (POP), may be present in foods. As some of the newly formed components may present some adverse effects upon consumption, methods have been developed to analyze these compounds in food products and biological samples. Gas liquid chromatography (GC) on long polar columns (100m) is a good choice to quantify trans mono- and poly-unsaturated fatty acids. In some cases a pre-fractionation step using silver nitrate thin layer chromatography (AgNO3-TLC) may be necessary to avoid GC overlapping of cis and trans isomers. Analysis of CFAM usually involves transformation of the sample in fatty acid methyl esters (FAME) which after addition of an internal standard (IS) are further hydrogenated. An enrichment step using reverse phase high performance liquid chromatography (RP-HPLC) permits to obtain a fraction which consists of a mixture of CFAM and the IS. This fraction is further analyzed by GC on a polar column. The analysis of oxidized triacylglycerol monomers (oxTG) as a group was feasible by a combination of adsorption and size-exclusion chromatography. Quantification in used frying fats and oils around the limit of rejection for human consumption (25% polar compounds) has shown that the amount of oxTG range 5.9-9.4% expressed on fat or oil weight. In foods and biological tissues, the level of oxidized sterols (SOP) is often a very small fraction of their unoxidized forms. Analysis of SOP involved extraction of lipids, saponification or transesterification, enrichment, and subsequent qualitative and quantitative determination by GC and GC-MS, or HPLC and HPLC-MS. In addition, enrichment of SOP requires complete separation from the unoxidized sterols in order to separate these compounds even by high resolution GC capillary columns.
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| 9. |
- Dutta, Paresh
(författare)
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Cholesterol and lipid oxidation in raw and pan-fried minced beef stored under aerobic packaging
- 2010
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Ingår i: Journal of the Science of Food and Agriculture. - : Wiley. - 0022-5142 .- 1097-0010. ; 90, s. 1050-1055
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The type of packaging atmosphere has been reported as a technological factor that consistently affects the quality of lipid fraction in meat. Oxidation of cholesterol and lipids was evaluated before and after pan frying in commercial refrigerated minced beef stored under aerobic atmosphere for 1 and 8 days.RESULTS: In raw beef, cholesterol and lipid oxidation developed at a slow rate. Cholesterol oxidation products (COPs) did not significantly vary (similar to 8 mu g COPs g(-1) of fat) over 8 days, while in the same period thiobarbituric acid reactive substances (TBARS) less than doubled (from 0.7 to 1.2 malondialdehyde equivalents kg(-1) of muscle). Pan frying did not influence the oxidative degree in the fresh product but consistently catalyzed cholesterol oxidation in stored beef. A significant increase was assessed in beef at the end of storage: from 8.6 to 30.0 mu g COPs g(-1) of fat in raw and cooked beef, respectively.CONCLUSION: Aerobic packaging did not appear as a pro-oxidant factor in fresh minced beef with a good oxidative quality during a short period of refrigerated storage. (C) 2010 Society of Chemical Industry
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| 10. |
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| 11. |
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| 12. |
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| 13. |
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| 14. |
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| 15. |
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| 16. |
- Kruse, Robert, et al.
(författare)
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Colloid centrifugation removes seminal plasma and cholesterol from boar spermatozoa
- 2011
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Ingår i: Reproduction, Fertility and Development. - : CSIRO Publishing. - 1031-3613 .- 1448-5990. ; 23, s. 858-865
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Tidskriftsartikel (refereegranskat)abstract
- The objective of the present study was to investigate the effect of Single-Layer Centrifugation (SLC) on boar spermatozoa, namely the effect of removal of seminal plasma proteins and cholesterol from the surface of spermatozoa. The presence of porcine seminal plasma proteins I and II (PSP-I/PSP-II) before and after SLC was studied using immunofluorescence, whereas the removal of cholesterol was shown qualitatively by thin-layer chromatography (TLC). Finally, the integrity of the sperm plasma membrane was observed by electron microscopy. It was shown that the seminal plasma proteins PSP-I and -II were removed from spermatozoa during SLC but could be restored by adding seminal plasma to the SLC-selected sperm samples. Some cholesterol was also lost from the spermatozoa during SLC but the plasma membrane itself appeared to be morphologically intact. Further studies are underway to examine the relevance of these findings to boar sperm cryopreservation and sperm fertility.
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| 17. |
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| 18. |
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| 19. |
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| 20. |
- Madawala, Samanthi, et al.
(författare)
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Novel Conjugates of 1,3-Diacylglycerol and Lipoic Acid: Synthesis, DPPH Assay, and RP-LC-MS-APCI Analysis
- 2011
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Ingår i: Journal of Lipids. - : Hindawi Limited. - 2090-3030 .- 2090-3049. ; 2011
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Tidskriftsartikel (refereegranskat)abstract
- 1,3-Diacylglycerol is known to reduce body weight and fat deposits in humans.α-Lipoic acid is a potent antioxidant and effective against many pathological conditions, including obesity and related metabolic syndromes. The present work is based on the hypothesis that the hybrid molecules of 1,3-diacylglycerol and lipoic acid possess synergistic and/or additive effects compared with the parent compounds against obesity, overweight, and related metabolic syndromes. Laboratory scale synthesis of 1,3-dioleoyl-2-lipoyl-sn-glycerol (yield 80%) and 1,3-dioleoyl-2-dihydrolipoyl-sn-glycerol (yield 70%) was performed for the first time and supported by NMR and MS data. Free radical scavenging capacity of the conjugates was assayed using DPPH test. A remarkably highin vitrofree radical scavenging capacity was demonstrated for the 1,3-dioleoyl-2-dihydrolipoyl-sn-glycerol (EC50value 0.21). RP-HPLC-MS-APCI analysis showed satisfactory separation between the conjugates (R~1). Protonated molecular ion of the conjugates atm/z809 andm/zat 811, respectively, and their characteristic fragment ions were abundant.
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| 21. |
- Madawala, Samanthi, et al.
(författare)
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Phytosterol and α-Lipoic Acid Conjugates: Synthesis, Free Radical Scavenging Capacity and RP-LC-MS-APCI Analysis
- 2012
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Ingår i: Polish Journal of Food and Nutrition Sciences. - : Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences. - 1230-0322. ; 62, s. 159-169
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Tidskriftsartikel (refereegranskat)abstract
- Plant sterols (PS) are bioactive compounds effective in reducing plasma cholesterol. Fatty acid esters of PS have improved solubility and blending properties when utilized in various food products. Naturally occurring a-lipoic acid (LA) and its reduced form dihydrolipoic acid (DHLA) are known for their antioxidant activity. In addition, they have shown an array of health beneficial properties against obesity, diabetes, cancer, cardiovascular and inflammatory diseases etc. Different LA conjugates have been reported to have increased bioactivity compared to the parent compounds. The objective of this study was to synthesize PS esters of LA (PSLA) and DHLA (PSDHLA) in order to increase their cholesterol lowering effect and reducing the risk of atherosclerosis with additional health benefits e.g. against oxidative stress.Synthesis of PSLA and PSDHLA was performed with a pure PS mixture of beta-sitosterol, stigmasterol, campesterol and brassicasterol. The free radical scavenging capacity of the conjugates was assessed by the DPPH method. Remaining percentage of DPPH free radicals was measured at the steady state for different concentrations of PSLA and PSDHLA. High free radical scavenging capacity was observed for PSDHLA compared with PSLA. Efficient concentration EC50 as a molar ratio for PSDHLA was 0.43. The derivatives were analyzed by RP-HPLC-MS-APCI. The order of the elution times of the compounds observed in HPLC-MS analysis was PS< PSDHLA< PSLA. Baseline separation was not achieved between campesterol and stigmasterol and their derivatives. These compounds could be identified by their characteristic fragment ions from the mass spectral data.
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| 22. |
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| 23. |
- Manubolu, Manjunath, et al.
(författare)
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In vitro biodegradation of cyanotoxins in the rumen fluid of cattle
- 2014
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Ingår i: BMC Veterinary Research. - : Springer Science and Business Media LLC. - 1746-6148. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background: In countries around the Baltic Sea grazing ruminants have access to and drink, surface water from lakes, rivers and in several coastal regions. The water quality of these naturally occurring reservoirs affects performance and health of livestock. In the Baltic Sea both microcystin (MC) and nodularin (NOD) occurs as cyclic peptides and have hepatotoxic effects. Although cattle obviously have died after consuming contaminated water very little information is available as to how susceptible ruminants are to the toxins produced by cyanobacteria. The critical question as to whether the rumen microflora might constitute a protective shield is unresolved. For this reason our aim is to investigate a possible degradation rate of these toxins in rumen.Results: The ability of rumen microorganisms to degrade certain important cyanotoxins (MC-LR, YR, RR and NOD) was studied in vitro by incubating with rumen fluid at three different concentrations (0.05, 0.5 and 5 mu g/mL) for 3 h. The degradation efficiencies were determined by LC-MS (ESI) positive mode. Degradation was observed in the following order MC-RR 36%, NOD 35%, MC-RR 25% and MC-LR 8.9% at lower concentrations within 3 h. However, average degradation was observed at concentration of 0.5 mu g/mL. No degradation was observed in higher concentrations for entire 3 h. The present results reveal that the degradation was both dose and time dependent.Conclusions: In conclusion the present results suggest that the rumen microbial flora may protect ruminants from being intoxicated by Cyanotoxins.
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| 24. |
- Manubolu, Manjunath, et al.
(författare)
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Protective effect of Actiniopteris radiata (Sw.) Link. against CCl4 induced oxidative stress in albino rats
- 2014
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Ingår i: Journal of Ethnopharmacology. - : Elsevier BV. - 0378-8741 .- 1872-7573. ; 153, s. 744-752
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Tidskriftsartikel (refereegranskat)abstract
- Ethnopharmacological relevance: Actiniopteris radiata is a herb with great medicinal value and is evaluated for hepatoprotective activity. To investigate the protective effect of ethanolic extract of Actiniopteris radiata (EEAR) on CCl4 induced oxidative stress in male Wistar albino rats.Materials and methods: EEAR were administered for 8 consecutive weeks to rats. Group I - control; Group II - toxin control (30% CCl4); Group III and Group IV received EEAR (250 and 500 mg/kg respectively). Antioxidant status in liver were estimated by determining the activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx); as well as by determining the levels of lipid peroxidation (LPO) and reduced glutathione (GSH). In addition, isoenzyme pattern and mRNA expression of the antioxidants were studied. Partial characterization of EEAR was performed by Liquid chromatography-mass spectrometry (LC-MS).Results: CCl4 induced oxidative stress as evidenced from increase in LPO along with reduction of SOD, CAT, GPx and GSH. Treatment with EEAR (250 and 500 mg/kg) mitigated the CCl4 induced oxidative stress. An analysis of the isozyme pattern of these antioxidant enzymes revealed variations in SOD2, CAT, GPx2 and GPx3 in CCl4 treated rats, which were normalized after EEAR treatment. Furthermore, expression of genes for the antioxidant enzymes, were down-regulated by CCl4 treatment, which were reversed by EEAR. The results of partial characterization of EEAR by LC-MS revealed the presence of rutin and other 7 unknown phenolic derivatives.Conclusions: These findings suggest the protective effect of EEAR against CCl4 induced oxidative stress might be attributed to the presence of flavonoids and phenolic compounds. (C) 2014 Elsevier Ireland Ltd. All rights reserved.
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| 25. |
- Manubolu, Manjunath, et al.
(författare)
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Variable Exposure and Responses to Cyanotoxins in Cattle Grazing on Pastures in the Coastal Zone of the Baltic Sea: A field Study
- 2014
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Ingår i: International Journal of Environmental Research. - 1735-6865. ; 8, s. 733-740
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Tidskriftsartikel (refereegranskat)abstract
- Cyanobacteria blooms are common in Baltic Sea and their intensity have been increased due to anthropogenic eutrophication. In this study we investigated the cyanotoxin levels in the water samples collected from four different locations in the Baltic Sea at three different seasons including summer 2011. Protein phosphatase 2A (PP2A) inhibition assays, Enzyme-linked immunosorbent assays (ELISA) and liquid chromatography-mass, spectrometry (LC-MS) were employed to detect cyanotoxin variants. Microcystin-LR equivalents (MCE) were detected in a number of the water samples collected at site A(0.4 to 0.64 mu g MCE/L) and at site B (0.24 to 0.44 mu g MCE/L). Cyanotoxin concentrations, as measured by ELISA, ranged between 0.98 -7.45 mu g MCE/L in samples collected at site A and between 0.12 to 0.68 mu g MCE/L, in samples collected at site B. By using LC-MS one of the molecules present in the samples from site A was determined to be nodularin (0.213 to 0.524 mu g/L) whereas samples from site B did not contain this toxin nor did they contain any of the most toxic microcystin species mentioned. The data obtained show good correlation with the MC concentration changes measured in samples and these concentrations were relatively higher during warmer months. In addition we also investigated the adsorption of toxins from water into the circulation of grazing cattle and the results show no measureable liver damage resulting from cyanotoxin poisoning.
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