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Satish C Bhatla - One of the best experts on this subject based on the ideXlab platform.
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sodium chloride stress induces nitric oxide accumulation in root tips and Oil Body surface accompanying slower oleosin degradation in sunflower seedlings
Physiologia Plantarum, 2010Co-Authors: Anisha David, Sunita Yadav, Satish C BhatlaAbstract:Present work highlights the involvement of endogenous nitric oxide (NO) in sodium chloride (NaCl)-induced biochemical regulation of seedling growth in sunflower (Helianthus annuus L., cv. Morden). The growth response is dependent on NaCl concentration to which seedlings are exposed, they being tolerant to 40 mM NaCl and showing a reduction in extension growth at 120 mM NaCl. NaCl sensitivity of sunflower seedlings accompanies a fourfold increase in Na(+) /K(+) ratio in roots (as compared to that in cotyledons) and rapid transport of Na(+) to the cotyledons, thereby enhancing Na(+) /K(+) ratio in cotyledons as well. A transient increase in endogenous NO content, primarily contributed by putative NOS activity in roots of 4-day-old seedlings subjected to NaCl stress and the relative reduction in Na(+) /K(+) ratio after 4 days, indicates that NO regulates Na(+) accumulation, probably by affecting the associated transporter proteins. Root tips exhibit an early and transient enhanced expression of 4,5-diaminofluorescein diacetate (DAF-2DA) positive NO signal in the presence of 120 mM NaCl. Oil bodies from 2-day-old seedling cotyledons exhibit enhanced localization of NO signal in response to 120 mM NaCl treatment, coinciding with a greater retention of the principal Oil Body membrane proteins, i.e. oleosins. Abolition of DAF positive fluorescence by the application of specific NO scavenger [2-phenyl-4,4,5,5-tetramethyllimidazoline-1-oxyl-3-oxide (PTIO)] authenticates the presence of endogenous NO. These novel findings provide evidence for a possible protective role of NO during proteolytic degradation of oleosins prior to/accompanying lipolysis.
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temporal and spatial analysis of lipid accumulation oleosin expression and fatty acid partitioning during seed development in sunflower helianthus annuus l
Acta Physiologiae Plantarum, 2010Co-Authors: Vibha Kaushik, Mukesh K Yadav, Satish C BhatlaAbstract:Biochemical and fluorescence microscopic imaging approach has been adopted to investigate the accumulation of Oil bodies at specific stages of seed development in Helianthus annuus L. cv. Morden. Seed filling in sunflower is marked with a rapid accumulation of proteins and lipids upto 30 DAA, after which protein accumulation declines whereas lipids continue to accumulate. Earliest signs of lipid accumulation are evident as early as during globular stage of embryo development. Spatially, a developing seed exhibits enhanced lipid deposition in peripheral cells. Oil Body biogenesis is observed as early as 10 DAA, as is evident from the fluorescence microscopic detection of Nile red-positive entities in the protoplasts. To begin with, expression of one of the oleosin (the principal Oil Body membrane proteins) isoforms (16 kDa), is slower than the other two (17.5 and 20 kDa). Fatty acid composition of Oil Body lipids is quite similar to that of total seed lipids. An enhanced accumulation of linoleic acid is evident during later stages of seed filling. The proportion of major saturated fatty acids, palmitic (16:0) and stearic (18:0), however, do not alter much during the later phases of seed development. Present findings provide new information on Oil Body development, lipid accumulation and fatty acid composition, for a better understanding of the phasing of physiological and biochemical events associated with Oilseed development.
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co localization of putative calcium channels phenylalkylamine binding sites on Oil bodies in protoplasts from dark grown sunflower seedling cotyledons
Plant Signaling & Behavior, 2009Co-Authors: Shweta Vandana, Satish C BhatlaAbstract:Oil bodies are spherical entities containing a triacylglycerol (TAG) matrix encased by a phospholipid monolayer, which is stabilized by Oil Body-specific proteins, principally oleosins. Biochemical investigations in the recent past have also demonstrated the expression of calcium-binding proteins, called caleosins, as a component of Oil Body membranes during seed germination. Using DM-Bodipy-phenylalkylamine (PAA; a fluorescent derivative of phenylalkylamine)-a fluorescent probe known to bind L-type calcium channel proteins, present investigations provide the first report on the localization and preferential accumulation of putative calcium channel proteins on/around Oil bodies during peak lipolytic phase in protoplasts derived from dark-grown sunflower (Helianthus annuus L. cv Morden) seedling cotyledons. Specificity of DM-Bodipy-PAA labeling was confirmed by using bepridil, a non-fluorescent competitor of PAA while non-specific dye accumulation has been ruled out by using Bodipy-FL as control. Co-localization of fluorescence from DM-Bodipy-PAA binding sites (ex: 504 nm; em: 511 nm) and nile red fluorescing Oil bodies (ex: 552 nm; em: 636 nm) has been undertaken by epifluorescence and confocal laser scanning microscopy (CLSM). It revealed the affinity of PAA-sensitive ion channels for the Oil Body surface. Findings from the current investigations highlight the significance of calcium and calcium channel proteins during Oil Body mobilization in sunflower.
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evidence for the probable Oil Body association of a thiol protease leading to oleosin degradation in sunflower seedling cotyledons
Plant Physiology and Biochemistry, 2006Co-Authors: S Vandana, Satish C BhatlaAbstract:The activity of a 65 kDa, cytosolic protease from sunflower seedling cotyledons coincides with the degradation of oleosins during seed germination. Further investigations carried out in this laboratory have demonstrated the probable association of a thiol-protease with Oil bodies, leading to gradual degradation of oleosins during seedling growth. Evidence to this effect have been brought out through zymographic detection of protease activity from Oil bodies, degradation of oleosins by electrophoretically eluted protease from the seedling cotyledons and inhibition of protease activity by thiol-protease inhibitor, such as N-ethylmaleimide (NEM). In addition to these biochemical evidence, visualization of thiol-protease activity has also been achieved by a novel fluorescence microscopic method and confocal imaging. It involves the uptake and binding of a fluorogenic thiol-protease inhibitor (fluorescein mercuric acetate, FMA) at the intracellular thiol-protease activity sites in protoplasts, leading to fluorescence emission at 523 nm following excitation at 499 nm. Maximum protease activity is observed in 4-d-old seedling cotyledons, coinciding with the phase of active triacylglycerol (TAGs) hydrolysis. All these observations provide evidence for the expression of the said thiol-protease activity on the Oil Body surface, leading to gradual proteolysis of oleosins during seed germination.
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Spatial and temporal changes in lipase activity sites during Oil Body mobilization in protoplasts from sunflower seedling cotyledons
Plant Growth Regulation, 2005Co-Authors: Ami Gupta, Satish C BhatlaAbstract:Hydrolysis of triacylglycerols (TAGs) catalyzed by lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) action, is the principal biochemical event during Oil Body mobilization in germinating Oilseeds. Employing a fluorescence microscopic technique developed in the author’s laboratory, a shift in the intracellular lipase activity has been demonstrated in the protoplasts of sunflower seedling cotyledons during seed germination. Lipase activity is primarily confined to protein storage vacuoles (PSVs) in 1 d old seedling cotyledons. At 2 d old stage, a relocalization of lipase activity begins and activity can be observed both on PSVs and Oil bodies. At later stages of development (3–6 d), smaller PSVs coalesce into a large vegetative vacuole devoid of lipase activity. During this phase, lipase activity is confined to Oil bodies only and maximum activity is detected in 4 d old seedlings, coinciding with maximum rate of lipolysis. Thus, present investigations on protoplasts from seedling cotyledons provide evidence for intracellular shift in lipase activity to sites of TAG hydrolysis (Oil bodies) and also show a structural and functional reorganization of PSVs.
Linbo Zhang - One of the best experts on this subject based on the ideXlab platform.
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a new nanoscale transdermal drug delivery system Oil Body linked oleosin hegf improves skin regeneration to accelerate wound healing
Journal of Nanobiotechnology, 2018Co-Authors: Weidong Qiang, Xinxin Lan, Muhammad Noman, Tingting Zhou, Xiaomei Zhang, Yongxin Guo, Jie Zheng, Hongyu Wang, Lili Guan, Linbo ZhangAbstract:Background Epidermal growth factor (EGF) can promote cell proliferation as well as migration, which is feasible in tissue wound healing. Oil bodies have been exploited as an important platform to produce exogenous proteins. The exogenous proteins were expressed in Oil bodies from plant seeds. The process can reduce purification steps, thereby significantly reducing the purification cost. Mostly, the diameter of Oil Body particle ranges between 1.0 and 1.5 µm in the safflower seeds, however, it reduces to 700–1000 nm in the transgenic safflower seeds. The significant reduction of particle size in transgenic seeds is extremely beneficial to skin absorption.
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Oil Body bound oleosin rhfgf9 fusion protein expressed in safflower carthamus tinctorius l stimulates hair growth and wound healing in mice
BMC Biotechnology, 2018Co-Authors: Jingbo Cai, Ruicheng Wen, Xiuran Wang, Haishan Tian, Linbo Zhang, Chao JiangAbstract:Fibroblast growth factor 9 (FGF9) is a heparin-binding growth factor, secreted by both mesothelial and epithelial cells, which participates in hair follicle regeneration, wound healing, and bone development. A suitable source of recombinant human FGF9 (rhFGF9) is needed for research into potential clinical applications. We present that expression of oleosin-rhFGF9 fusion protein in safflower (Carthamus tinctorius L.) seeds stimulates hair growth and wound healing. The oleosin-rhFGF9 expressed in safflower seeds, in which it localizes to the surface of Oil bodies. The expression of oleosin-rhFGF9 was confirmed by polyacrylamide gel electrophoresis and western blotting. According to BCA and Enzyme-linked immunosorbent assay (ELISA) assay, the results show that the expression level of oleosin-rhFGF9 was 0.14% of Oil Body protein. The Oil Body bound oleosin-rhFGF9 showed mitogenic activity towards NIH3T3 cells in a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The efficacy of Oil Body bound oleosin-rhFGF9 in promoting hair growth and wound healing was investigated in C57BL/6 mice. In a hair regeneration experiment, 50 μg/μl Oil Body bound oleosin-rhFGF9 was applied to the dorsal skin of mice in the resting phase of the hair growth cycle. After 15 days, thicker hair and increased number of new hairs were seen compared with controls. Furthermore, the number of new hairs was greater compared with rhFGF9-treated mice. The hair follicles of mice treated with Oil Body bound oleosin-rhFGF9 expressed β-catenin more abundantly. In a wound healing experiment, dorsal skin wounds were topically treated with 50 μg/μl Oil Body bound oleosin-rhFGF9. Wound healing was quicker compared with mice treated with rhFGF9 and controls, especially in the earlier stages of healing. The Oil Body bound oleosin-rhFGF9 promotes both hair growth and wound healing. It appears to promote hair growth, at least in part, by up-regulating β-catenin expression. The potential of Oil Body bound oleosin-rhFGF9 as an external drug can treat the alopecia and wounds or use in further clinical application.
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Oil Body bound oleosin-rhFGF9 fusion protein expressed in safflower (Carthamus tinctorius L.) stimulates hair growth and wound healing in mice
BMC, 2018Co-Authors: Jingbo Cai, Ruicheng Wen, Xiuran Wang, Haishan Tian, Linbo Zhang, Chao JiangAbstract:Abstract Background Fibroblast growth factor 9 (FGF9) is a heparin-binding growth factor, secreted by both mesothelial and epithelial cells, which participates in hair follicle regeneration, wound healing, and bone development. A suitable source of recombinant human FGF9 (rhFGF9) is needed for research into potential clinical applications. We present that expression of oleosin-rhFGF9 fusion protein in safflower (Carthamus tinctorius L.) seeds stimulates hair growth and wound healing. Results The oleosin-rhFGF9 expressed in safflower seeds, in which it localizes to the surface of Oil bodies. The expression of oleosin-rhFGF9 was confirmed by polyacrylamide gel electrophoresis and western blotting. According to BCA and Enzyme-linked immunosorbent assay (ELISA) assay, the results show that the expression level of oleosin-rhFGF9 was 0.14% of Oil Body protein. The Oil Body bound oleosin-rhFGF9 showed mitogenic activity towards NIH3T3 cells in a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The efficacy of Oil Body bound oleosin-rhFGF9 in promoting hair growth and wound healing was investigated in C57BL/6 mice. In a hair regeneration experiment, 50 μg/μl Oil Body bound oleosin-rhFGF9 was applied to the dorsal skin of mice in the resting phase of the hair growth cycle. After 15 days, thicker hair and increased number of new hairs were seen compared with controls. Furthermore, the number of new hairs was greater compared with rhFGF9-treated mice. The hair follicles of mice treated with Oil Body bound oleosin-rhFGF9 expressed β-catenin more abundantly. In a wound healing experiment, dorsal skin wounds were topically treated with 50 μg/μl Oil Body bound oleosin-rhFGF9. Wound healing was quicker compared with mice treated with rhFGF9 and controls, especially in the earlier stages of healing. Conclusions The Oil Body bound oleosin-rhFGF9 promotes both hair growth and wound healing. It appears to promote hair growth, at least in part, by up-regulating β-catenin expression. The potential of Oil Body bound oleosin-rhFGF9 as an external drug can treat the alopecia and wounds or use in further clinical application
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Oil Body bound oleosin rhfgf 10 a novel drug delivery system that improves skin penetration to accelerate wound healing and hair growth in mice
International Journal of Molecular Sciences, 2017Co-Authors: Jing Yang, Weidong Qiang, Jingbo Cai, Haishan Tian, Linbo Zhang, Hongyu Wang, Jian Huang, Chao JiangAbstract:Recombinant human fibroblast growth factor 10 (rhFGF-10) is frequently used to treat patients with skin injuries. It can also promote hair growth. However, the effective application of rhFGF-10 is limited because of its poor stability and transdermal absorption. In this study, polymerase chain reaction (PCR) and Southern blotting were used to identify transgenic safflowers carrying a gene encoding an oleosin-rhFGF-10 fusion protein. The size and structural integrity of oleosin-rhFGF-10 in Oil bodies extracted from transgenic safflower seeds was characterized by polyacrylamide gel electrophoresis and western blotting. Oil Body extracts containing oleosin-rhFGF-10 were topically applied to mouse skin. The absorption of oleosin-rhFGF-10 was studied by immunohistochemistry. Its efficiency in promoting wound healing and hair regeneration were evaluated in full thickness wounds and hair growth assays. We identified a safflower line that carried the transgene and expressed a 45 kDa oleosin-rhFGF-10 protein. Oil Body-bound oleosin-rhFGF-10 was absorbed by the skin with higher efficiency and speed compared with prokaryotically-expressed rhFGF-10. Oleosin-rhFGF-10 also enhanced wound closure and promoted hair growth better than rhFGF-10. The application of oleosin-rhFGF-10 in Oil bodies promoted its delivery through the skin, providing a basis for improved therapeutic effects in enhancing wound healing and hair growth.
David A. Gray - One of the best experts on this subject based on the ideXlab platform.
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physical and oxidative stability of pre emulsified Oil bodies extracted from soybeans
Food Chemistry, 2012Co-Authors: Bingcan Chen, David A. Gray, David Julian Mcclements, Eric A DeckerAbstract:Abstract Soybeans contain Oil bodies that are coated by a layer of oleosin proteins. In nature, this protein coating protects the Oil bodies from environmental stresses and might be utilised by food manufacturers for the same purpose. In this study, an aqueous extraction method was developed to increase the yield of Oil bodies extracted from soybean. This method involved a two-step procedure: (i) blending, dispersion, and filtration of soybeans; (ii) homogenisation, suspension, and centrifugation of the filter cake. Using this extraction method about 65% of the Oil bodies could be obtained. The mean particle diameter ( d 43 ) and sedimentation of the resulting Oil bodies increased during storage, suggesting they were prone to aggregation. Heat treatment (90 °C, 30 min) of the Oil Body suspensions immediately after extraction improved their storage stability, which was attributed to deactivation of endogenous enzymes such as lipase and lipoxygenase. Heat treatment did not adversely affect the oxidative stability of the Oil Body suspensions at pH 3 or 7 during storage at 37 °C. These results suggest that this aqueous extraction method can be used to prepare Oil Body suspensions with improved long-term stability.
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Oxidative stability of sunflower Oil bodies
European Journal of Lipid Science and Technology, 2008Co-Authors: Ian D. Fisk, Daniel A. White, Mitaben Dhirajlal Lad, David A. GrayAbstract:This study investigates the oxidative stability of sunflower Oil Body suspensions (10 wt-% lipid). Two washed suspensions of Oil bodies were evaluated over 8 days at three temperatures (5, 25 and 45 °C) against three comparable sunflower Oil emulsions stabilized with dodecyltrimethylammonium bromide (DTAB), polyoxyethylene-sorbitan monolaurate (Tween 20) and sodium dodecyl sulfate (SDS) (17 mM). The development of oxidation was monitored by measuring the presence of lipid hydroperoxides and the formation of hexanal. Lipid hydroperoxide concentrations in the DTAB, SDS and Tween 20 emulsions were consistently higher than in the Oil Body suspensions; furthermore, hexanal formation was not detected in the Oil Body emulsions, whereas hexanal was present in the headspace of the formulated emulsions. The reasons for the extended resistance to oxidation of the Oil Body suspensions are hypothesized to be due to the presence of residual seed proteins in the continuous phase and the presence of a strongly stabilized lipid-water interface.
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sunflower seed Oil Body emulsions rheology and stability assessment of a natural emulsion
Food Hydrocolloids, 2008Co-Authors: Daniel A. White, Ian D. Fisk, J R Mitchell, Bettina Wolf, Sandra E Hill, David A. GrayAbstract:Abstract The viscoelastic characteristics of a purified Oil Body cream (67.6±0.7% lipid, 5.4±0.7% protein and 25.2±0.1% moisture) recovered from sunflower seeds ( Helianthus annuus L.) have been determined. Moreover, the effects of pH (2–7) and CaCl 2 concentration (0–150 mM) on rheology and physical stability of Oil Body emulsions have been studied. Oscillatory measurements showed that the purified Oil Body cream exhibited weak gel-like behaviour. Diluted Oil Body emulsions (⩽20 wt% Oil) showed extensive creaming (creaming index=56–59%) at pH 5–6 resulting in significant ( P D (3,2)=11–13 μm compared with 0.3 μm at pH 7) and viscosity (0.025–0.035 Pa s at shear rate 10 s −1 compared with 0.008 Pa s at pH 7). Microscopic examination revealed that the emulsion droplets aggregated at pH 5–6 but did not coalesce. The influence of CaCl 2 was investigated at pH above (pH 7) and below (pH 3) the isoelectric point (IEP) of the intrinsic oleosin proteins (pH 5⩽IEP⩽pH 6) associated with the surface of the Oil bodies. At pH 7, Oil bodies were stable up to a CaCl 2 concentration of 1.5 mM; at 5–150 mM CaCl 2 creaming occurred (66–70%), and significant ( P D (3,2)=10–13 μm) and viscosity (0.015 Pa s at shear rate 10 s −1 ) were observed. At pH 3 there was no significant ( P >0.05) influence of CaCl 2 on emulsion stability or rheology. These findings demonstrate that rheology and stability of Oil Body emulsions are, like processed emulsions, affected by pH and by ionic concentration when the pH is above the IEP of the surface components of the Oil Body. The novelty in this work therefore lies in the source of the emulsion, a natural, pre-formed Oil-in-water emulsion derived from seed tissue.
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tocopherol an intrinsic component of sunflower seed Oil bodies
Journal of the American Oil Chemists' Society, 2006Co-Authors: Ian D. Fisk, Daniel A. White, Andre Carvalho, David A. GrayAbstract:Oil bodies were removed from mature sunflower through wet grinding followed by filtration then centrifugation and recovered as the buoyant fraction. Washing this fraction with buffer (water-washed Oil bodies, WWOB) or 9 M urea (urea-washed Oil bodies, UWOB) resulted in the removal of extraneous proteins. SDS-PAGE of the proteins still associated with the Oil Body fraction after washing indicated that this effect was particularly dramatic with urea washing. Thirty-eight percent of the total seed tocopherol was recovered in WWOB after only one cycle of Oil Body recovery. The total phenolic content (TPC) of differentially washed sunflower seed Oil bodies was used as a marker for the nonspecific association of phenolic compounds to Oil bodies. This value decreased with increased removal of proteins from Oil bodies, whereas the converse was true for tocopherol values, which increased from 214 mg total tocopherol kg−1 WWOB [dry wt basis (dwb)] to 392 mg total tocopherol kg−1 UWOB (dwb). The ratio of the four tocopherol isomers remained constant in the seed and Oil Body preparations (α:β:γ:δ approximately 94∶5∶0.5∶0.5). This work provides evidence that an intrinsic population of tocopherol molecules exists in the Oil bodies of mature sunflower seeds.
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Tocopherol—An Intrinsic Component of Sunflower Seed Oil Bodies
Journal of the American Oil Chemists' Society, 2006Co-Authors: Ian D. Fisk, Daniel A. White, Andre Carvalho, David A. GrayAbstract:Oil bodies were removed from mature sunflower through wet grinding followed by filtration then centrifugation and recovered as the buoyant fraction. Washing this fraction with buffer (water-washed Oil bodies, WWOB) or 9 M urea (urea-washed Oil bodies, UWOB) resulted in the removal of extraneous proteins. SDS-PAGE of the proteins still associated with the Oil Body fraction after washing indicated that this effect was particularly dramatic with urea washing. Thirty-eight percent of the total seed tocopherol was recovered in WWOB after only one cycle of Oil Body recovery. The total phenolic content (TPC) of differentially washed sunflower seed Oil bodies was used as a marker for the nonspecific association of phenolic compounds to Oil bodies. This value decreased with increased removal of proteins from Oil bodies, whereas the converse was true for tocopherol values, which increased from 214 mg total tocopherol kg−1 WWOB [dry wt basis (dwb)] to 392 mg total tocopherol kg−1 UWOB (dwb). The ratio of the four tocopherol isomers remained constant in the seed and Oil Body preparations (α:β:γ:δ approximately 94∶5∶0.5∶0.5). This work provides evidence that an intrinsic population of tocopherol molecules exists in the Oil bodies of mature sunflower seeds.
Jason T C Tzen - One of the best experts on this subject based on the ideXlab platform.
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A unique caleosin in Oil bodies of lily pollen.
Plant & cell physiology, 2008Co-Authors: Pei-luen Jiang, Guang-yuh Jauh, Co-shing Wang, Jason T C TzenAbstract:In view of the recent isolation of stable Oil bodies as well as a unique oleosin from lily pollen, this study examined whether other minor proteins were present in this lipid-storage organelle. Immunological cross-recognition using antibodies against three minor Oil-Body proteins from sesame suggested that a putative caleosin was specifically detected in the Oil-Body fraction of pollen extract. A cDNA fragment encoding this putative pollen caleosin, obtained by PCR cloning, was confirmed by immunodetection and MALDI-MS analyses of the recombinant protein over-expressed in Escherichia coli and the native form. Caleosin in lily pollen Oil bodies seemed to be a unique isoform distinct from that in lily seed Oil bodies.
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Determination and analyses of the N-termini of Oil-Body proteins, steroleosin, caleosin and oleosin.
Plant physiology and biochemistry : PPB, 2005Co-Authors: Li Jen Lin, Pao-chi Liao, Hsueh Hui Yang, Jason T C TzenAbstract:Seed Oil bodies comprise a triacylglycerol matrix shielded by a monolayer of phospholipids and proteins. These surface proteins include an abundant structural protein, oleosin, and at least two minor protein classes termed caleosin and steroleosin. Two steroleosin isoforms (41 and 39 kDa), one caleosin (27 kDa), and two oleosin isoforms (17 and 15 kDa) have been identified in Oil bodies isolated from sesame seeds. The signal peptides responsible for targeting of these proteins to Oil bodies have not been experimentally determined. Hydropathy analyses indicate that the hydrophobic domain putatively responsible for Oil-Body anchoring is located in the N-terminal region of steroleosin, but in the central region of caleosin or oleosin. Direct amino acid sequencing showed that both steroleosin isoforms possessed a free methionine residue at their N-termini while caleosin and oleosin isoforms were N-terminally blocked. Mass spectrometry analyses revealed that N-termini of both caleosin and 17 kDa oleosin were acetylated after the removal of the first methionine. In addition, deamidation was observed at a glutamine residue in the N-terminal region of 17 kDa oleosin.
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Size and stability of reconstituted sesame Oil bodies.
Biotechnology progress, 2003Co-Authors: Chichung Peng, I-ping Lin, Ching‐kuan Lin, Jason T C TzenAbstract:Oil bodies of sesame seeds comprise a triacylglycerol matrix, which is surrounded by a monolayer of phospholipids embedded with unique proteins, mainly structural proteins termed oleosins. Artificial Oil bodies were successfully reconstituted with various compositions of triacylglycerols, phospholipids, and Oil-Body proteins. The sizes of reconstituted Oil bodies displayed a normal distribution with an average size proportional to the ratio of triacylglycerols to Oil-Body proteins. Both thermostability and structural stability of reconstituted Oil bodies decreased as their sizes increased, and vice versa. Proteinase K digestion indicated that oleosins anchored both native and reconstituted Oil bodies via their central hydrophobic domains. The stability of reconstituted Oil bodies, as well as the purified ones from sesame seeds, could be substantially enhanced after their surface proteins were cross-linked by glutaraldehyde or genipin.
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A new method for seed Oil Body purification and examination of Oil Body integrity following germination.
Journal of biochemistry, 1997Co-Authors: Jason T C Tzen, Chichung Peng, Dor-jih Cheng, Emily C.f. Chen, Joyce M.h. ChiuAbstract:Plant seeds store triacylglycerols as energy sources for germination and postgerminative growth of seedlings. The triacylglycerols are preserved in small, discrete, intracellular organelles called Oil bodies. A new method was developed to purify seed Oil bodies. The method included extraction, flotation by centrifugation, detergent washing, ionic elution, treatment with a chaotropic agent, and integrity testing by use of hexane. These processes subsequently removed non-specifically associated or trapped proteins within the Oil bodies. Oil bodies purified by this method maintained their integrity and displayed electrostatic repulsion and steric hindrance on their surface. Compared with the previous procedure, this method allowed higher purification of Oil bodies, as demonstrated by SDS-PAGE using five species of Oilseeds. Oil bodies purified from sesame were further analyzed by two-dimensional gel electrophoresis and revealed two potential oleosin isoforms. The integrity of Oil bodies in germinating sesame seedlings was examined by hexane extraction. Our results indicated that consumption of triacylglycerols reduced gradually the total amount of Oil bodies in seedlings, whereas no alteration was observed in the integrity of remaining Oil bodies. This observation implies that Oil bodies in germinating seeds are not degraded simultaneously. It is suggested that glyoxisomes, with the assistance of mitochondria, fuse and digest Oil bodies one at a time, while the remaining Oil bodies are preserved intact during the whole period of germination.
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surface structure and properties of plant seed Oil bodies
Journal of Cell Biology, 1992Co-Authors: Jason T C Tzen, A H C HuangAbstract:Storage triacylglycerols (TAG) in plant seeds are present in small discrete intracellular organelles called Oil bodies. An Oil Body has a matrix of TAG, which is surrounded by phospholipids (PL) and alkaline proteins, termed oleosins. Oil bodies isolated from mature maize (Zea mays) embryos maintained their discreteness, but coalesced after treatment with trypsin but not with phospholipase A2 or C. Phospholipase A2 or C exerted its activity on Oil bodies only after the exposed portion of oleosins had been removed by trypsin. Attempts were made to reconstitute Oil bodies from their constituents. TAG, either extracted from Oil bodies or of a 1:2 molar mixture of triolein and trilinolein, in a dilute buffer were sonicated to produce droplets of sizes similar to those of Oil bodies; these droplets were unstable and coalesced rapidly. Addition of Oil Body PL or dioleoyl phosphatidylcholine, with or without charged stearylamine/stearic acid, or oleosins, to the medium before sonication provided limited stabilization effects to the TAG droplets. High stability was achieved only when the TAG were sonicated with both Oil Body PL (or dioleoyl phosphatidylcholine) and oleosins of proportions similar to or higher than those in the native Oil bodies. These stabilized droplets were similar to the isolated Oil bodies in chemical properties, and can be considered as reconstituted Oil bodies. Reconstituted Oil bodies were also produced from TAG of a 1:2 molar mixture of triolein and trilinolein, dioleoyl phosphatidylcholine, and oleosins from rice (Oryza sativa), wheat (Triticum aestivum), rapeseed (Brassica napus), soybean (Glycine max), or jojoba (Simmondsia chinensis). It is concluded that both oleosins and PL are required to stabilize the Oil bodies and that oleosins prevent Oil bodies from coalescing by providing steric hindrance. A structural model of an Oil Body is presented. The current findings on seed Oil bodies could be extended to the intracellular storage lipid particles present in diverse organisms.
Eliot M. Herman - One of the best experts on this subject based on the ideXlab platform.
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suppression of soybean oleosin produces micro Oil bodies that aggregate into Oil Body er complexes
Molecular Plant, 2008Co-Authors: Monica A. Schmidt, Eliot M. HermanAbstract:Using RNAi, the seed Oil Body protein 24-kDa oleosin has been suppressed in transgenic soybeans. The endoplasmic reticulum (ER) forms micro-Oil bodies about 50 nm in diameter that coalesce with adjacent Oil bodies forming a hierarchy of Oil Body sizes. The Oil bodies in the oleosin knockdown form large Oil Body-ER complexes with the interior dominated by micro-Oil bodies and intermediate-sized Oil bodies, while the peripheral areas of the complex are dominated by large Oil bodies. The complex merges to form giant Oil bodies with onset of seed dormancy that disrupts cell structure. The transcriptome of the oleosin knockdown shows few changes compared to wild-type. Proteomic analysis of the isolated Oil bodies of the 24-kDa oleosin knockdown shows the absence of the 24-kDa oleosin and the presence of abundant caleosin and lipoxygenase. The formation of the micro-Oil bodies in the oleosin knockdown is interpreted to indicate a function of the oleosin as a surfactant.
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Suppression of Soybean Oleosin Produces Micro-Oil Bodies that Aggregate into Oil Body/ER Complexes
Molecular plant, 2008Co-Authors: Monica A. Schmidt, Eliot M. HermanAbstract:Using RNAi, the seed Oil Body protein 24-kDa oleosin has been suppressed in transgenic soybeans. The endoplasmic reticulum (ER) forms micro-Oil bodies about 50 nm in diameter that coalesce with adjacent Oil bodies forming a hierarchy of Oil Body sizes. The Oil bodies in the oleosin knockdown form large Oil Body-ER complexes with the interior dominated by micro-Oil bodies and intermediate-sized Oil bodies, while the peripheral areas of the complex are dominated by large Oil bodies. The complex merges to form giant Oil bodies with onset of seed dormancy that disrupts cell structure. The transcriptome of the oleosin knockdown shows few changes compared to wild-type. Proteomic analysis of the isolated Oil bodies of the 24-kDa oleosin knockdown shows the absence of the 24-kDa oleosin and the presence of abundant caleosin and lipoxygenase. The formation of the micro-Oil bodies in the oleosin knockdown is interpreted to indicate a function of the oleosin as a surfactant.
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Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of Oil-Body formation in seeds
The Plant journal : for cell and molecular biology, 1997Co-Authors: Cristina Sarmiento, Joanne H.e. Ross, Eliot M. Herman, Denis J. MurphyAbstract:Two genomic clones, encoding isoforms A and B of the 24 kDa soybean oleosin and containing 5 kbp and 1 kbp, respectively, of promoter sequence, were inserted separately into rapeseed plants. T2 seeds from five independent transgenic lines, three expressing isoform A and two expressing isoform B, each containing one or two copies of the transgene, were analysed in detail. In all five lines, the soybean transgenes exhibited the same patterns of mRNA and protein accumulation as the resident rapeseed oleosins, i.e. their expression was absolutely seed-specific and peaked at the mid-late stages of cotyledon development. The 24 kDa soybean oleosin was targeted to and stably integrated into Oil bodies, despite the absence of a soybean partner isoform. The soybean protein accumulated in young embryos mainly as a 23 kDa polypeptide, whereas a 24 kDa protein predominated later in development. The ratio of rapeseed:soybean oleosin in the transgenic plants was about 5:1 to 6:1, as determined by SDS-PAGE and densitometry. Accumulation of these relatively high levels of soybean oleosin protein did not affect the amount of endogenous rapeseed oleosin. Immunoblotting studies showed that about 95% of the recombinant soybean 24 kDa oleosin (and the endogenous 19 kDa rapeseed oleosin) was targeted to Oil bodies, with the remainder associated with the microsomal fraction. Sucrose density-gradient centrifugation showed that the oleosins were associated with a membrane fraction of buoyant density 1.10-1.14 g ml-1, which partially overlapped with several endoplasmic reticulum (ER) markers. Unlike oleosins associated with Oil bodies, none of the membrane-associated oleosins could be immunoprecipitated in the presence of protein A-Sepharose, indicating a possible conformational difference between the two pools of oleosin. Complementary electron microscopy-immunocytochemical studies of transgenic rapeseed revealed that all Oil bodies examined could be labelled with both the soybean or rapeseed anti-oleosin antibodies, indicating that each Oil Body contained a mixed population of soybean and rapeseed oleosins. A small but significant proportion of both soybean and rapeseed oleosins was located on ER membranes in the vicinity of Oil bodies, but none were detected on the bulk ER cisternae. This is the first report of apparent targeting of oleosins via ER to Oil bodies in vivo and of possible associated conformational/processing changes in the protein. Although Oil-Body formation per se can occur independently of oleosins, it is proposed that the relative net amounts of oleosin and Oil accumulated during the course of seed development are a major determinant of Oil-Body size in desiccation-tolerant seeds.
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Oleosins: Their Subcellular Targeting and Role in Oil-Body Ontogeny
Physiology Biochemistry and Molecular Biology of Plant Lipids, 1997Co-Authors: Denis J. Murphy, Joanne H.e. Ross, C. Sarmiento, Eliot M. HermanAbstract:Seed Oil bodies in plants are surrounded by a monolayer of highly unusual and abundant proteins, the oleosins, which can comprise as much as 8–15% total seed protein [1,2]. However, relatively little is known about the subcellular targeting and processing of oleosins, and their role in Oil-Body formation and maturation. We have expressed a soybean 24 kDa oleosin in transgenic rapeseed, in order to elucidate these issues. Detailed experimental data will be published elsewhere [3], but in this report, we discuss the implications of our findings for the mechanism of Oil-Body ontogeny in plants.
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Cotranslational Integration of Soybean (Glycine max) Oil Body Membrane Protein Oleosin into Microsomal Membranes
Plant physiology, 1993Co-Authors: Deborah S. Loer, Eliot M. HermanAbstract:Storage triglycerides in Oil seeds are sequestered in discrete organelles termed Oil bodies. They are bounded by a monolayer of phospholipids in which a few distinct proteins (oleosins) are embedded. Synthesis of soybean (Glycine max) 24-kD oleosin was analyzed by in vitro transcription and translation in reticulocyte lysate in the presence of canine microsomes. Our results show that 24-kD oleosin is cotranslationally integrated into microsomal membranes. We demonstrate that oleosin is integrated into a bilayer membrane in preference to the Oil Body monolayer membrane, indicating that oleosin is synthesized on the endoplasmic reticulum (ER). A new model of Oil Body assembly involving a conformational change through initial association with the ER membrane is proposed.
