Jason T. C. Tzen

Lipids, Proteins, and Structure of Seed Oil Bodies from Diverse Species

Oil bodies isolated from the mature seeds of rape (Brassica napus L.), mustard (Brassica juncea L.), cotton (Gossypium hirsutum L.), flax (Linus usitatis simum), maize (Zea mays L.), peanut (Arachis hypogaea L.), and sesame (Sesamum indicum L.) had average diameters that were different but within a narrow range (0.6–2.0 [mu]m), as measured from electron micrographs of serial sections. Their contents of triacylglycerols (TAG), phospholipids, and proteins (oleosins) were correlated with their sizes. The correlation fits a formula that describes a spherical particle surrounded by a shell of a monolayer of phospholipids embedded with oleosins. Oil bodies from the various species contained substantial amounts of the uncommon negatively charged phosphatidylserine and phosphatidylinositol, as well as small amounts of free fatty acids. These acidic lipids are assumed to interact with the basic amino acid residues of the oleosins on the surface of the phospholipid layer. Isoelectrofocusing revealed that the oil bodies from the various species had an isoelectric point of 5.7 to 6.6 and thus possessed a negatively charged surface at neutral pH. We conclude that seed oil bodies from diverse species are very similar in structure. In rapeseed during maturation, TAG and oleosins accumulated concomitantly. TAG-synthesizing acyltransferase activities appeared at an earlier stage and peaked during the active period of TAG accumulation. The concomitant accumulation of TAG and oleosins is similar to that reported earlier for maize and soybean, and the finding has an implication for the mode of oil body synthesis during seed maturation.

Protective effects of sesamin and sesamolin on hypoxic neuronal and PC12 cells

Reactive oxygen species (ROS) are important mediators of a variety of pathological processes, including inflammation and ischemic injury. The neuroprotective effects of sesame antioxidants, sesamin and sesamolin, against hypoxia or H2O2‐induced cell injury were evaluated by cell viability or lactate dehydrogenase (LDH) activity. Sesamin and sesamolin reduced LDH release of PC12 cells under hypoxia or H2O2‐stress in a dose‐dependent manner. Dichlorofluorescein (DCF)‐sensitive ROS production was induced in PC12 cells by hypoxia or H2O2‐stress but was diminished in the presence of sesamin and sesamolin. We evaluated further the role of mitogen‐activated protein kinases (MAPKs) and caspase‐3 in hypoxia‐induced PC12 cell death. Extracellular signal‐regulated protein kinase (ERK) 1, c‐jun N‐terminal kinase (JNK), and p38 MAPKs of signaling pathways were activated during hypoxia. We found that the inhibition of MAPKs and caspase‐3 by sesamin and sesamolin correlated well with the reduction in LDH release under hypoxia. Furthermore, the hypoxia‐induced apoptotic‐like cell death in cultured cortical cells as detected by a fluorescent DNA binding dye was reduced significantly by sesamin and sesamolin. Taken together, these results suggest that the protective effect of sesamin and sesamolin on hypoxic neuronal and PC12 cells might be related to suppression of ROS generation and MAPK activation.

Size and stability of reconstituted sesame oil bodies.

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.

Gene Family of Oleosin Isoforms and Their Structural Stabilization in Sesame Seed Oil Bodies

Oleosins are structural proteins sheltering the oil bodies of plant seeds. Two isoform classes termed H- and L-oleosin are present in diverse angiosperms. Two H-oleosins and one L-oleosin were identified in sesame oil bodies from the protein sequences deduced from their corresponding cDNA clones. Sequence analysis showed that the main difference between the H- and L-isoforms is an insertion of 18 residues in the C-terminal domain of H-oleosins. H-oleosin, presumably derived from L-oleosin, was duplicated independently in several species. All known oleosins can be classified as one of these two isoforms. Single copy or a low copy number was detected by Southern hybridization for each of the three oleosin genes in the sesame genome. Northern hybridization showed that the three oleosin genes were transcribed in maturing seeds where oil bodies are being assembled. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and sesame oleosin isoforms. The results indicated that reconstituted oil bodies could be stabilized by both isoforms, but L-oleosin gave slightly more structural stability than H-oleosin.

Effect of aristolochic acid on intracellular calcium concentration and its links with apoptosis in renal tubular cells

Aristolochic acid (AA) has been demonstrated to play a causal role in Chinese herbs nephropathy. However, the detailed mechanism for AA to induce apoptosis of renal tubular cells remains obscure. In this study, we show that AA evokes a rapid rise in the intracellular Ca2+ concentration of renal tubular cells through release of intracellular endoplasmic reticulum Ca2+ stores and influx of extracellular Ca2+, which in turn causes endoplasmic reticulum stress and mitochondria stress, resulting in activation of caspases and finally apoptosis. Ca2+ antagonists, including calbindin-D28k (an intracellular Ca2+ buffering protein) and BAPTA-AM (a cell-permeable Ca2+ chelator), are capable of ameliorating endoplasmic reticulum stress and mitochondria stress, and thereby enhance the resistance of the cells to AA. Moreover, we show that overexpression of the anti-apoptotic protein Bcl-2 in combination with BAPTA-AM treatment can provide renal tubular cells with almost full protection against AA-induced cytotoxicity. In conclusion, our results demonstrate an impact of AA to intracellular Ca2+ concentration and its link with AA-induced cytotoxicity.

Effect of sugar positions in ginsenosides and their inhibitory potency on Na + /K + -ATPase activity

AbstractAim:To determine whether ginsenosides with various sugar attachments may act as active components responsible for the cardiac therapeutic effects of ginseng and sanqi (the roots of Panax ginseng and Panax notoginseng) via the same molecular mechanism triggered by cardiac glycosides, such as ouabain and digoxin.Methods:The structural similarity between ginsenosides and ouabain was analyzed. The inhibitory potency of ginsenosides and ouabain on Na+/K+-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of ginsenosides to Na+/K+-ATPase.Results:Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, and possessed inhibitory potency on Na+/K+-ATPase activity. However, their inhibitory potency was significantly reduced or completely abolished when a monosaccharide was linked to the C-6 or C-20 position of the steroid-like structure; replacement of the monosaccharide with a disaccharide molecule at either of these positions caused the disappearance of the inhibitory potency. Molecular modeling and docking confirmed that the difference in Na+/K+-ATPase inhibitory potency among ginsenosides was due to the steric hindrance of sugar attachment at the C-6 and C-20 positions of the steroid-like structure.Conclusion:The cardiac therapeutic effects of ginseng and sanqi should be at least partly attributed to the effective inhibition of Na+/K+-ATPase by their metabolized ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure.

Enhanced Methionine and Cysteine Levels in Transgenic Rice Seeds by the Accumulation of Sesame 2S Albumin

A chimeric gene encoding a precursor polypeptide of sesame 2S albumin, a sulfur-rich seed storage protein, was expressed in transgenic rice plants under the control of the glutelin promoter with the aim of improving the nutritive value of rice. Rice grains harvested from the first generation of ten different transformed lines inherited the transgene, and the accumulated sesame 2S albumin was presumably processed correctly as its mature form in sesame seed. This transgene was specifically expressed in maturing rice seeds with its encoded sesame 2S albumin exclusively accumulated in the seeds. The crude protein content in rice grains from five putative homozygous lines was increased by 0.64-3.54%, and the methionine and cysteine contents of these transgenic rice grains were respectively elevated by 29-76% and 31-75% compared with those of wild-type rice grains.

Immobilization of Neocallimastix patriciarum xylanase on artificial oil bodies and statistical optimization of enzyme activity

A thermally stable and alkalophilic xylanase, XynCDBFV, from Neocallimastix patriciarum was overexpressed in Escherichia coli as a recombinant protein fused to the N-terminus of oleosin, a unique structural protein of seed oil bodies. As a result of the reconstitution of the artificial oil bodies (AOBs), the immobilization of active xylanase was accomplished. Response surface methodology (RSM) was employed for the optimization of the immobilized xylanase activity. The central composite design (CCD) and regression analysis methods were effective for determination of optimized temperature and pH conditions for the AOB-immobilized XynCDBFV. The optimal condition for the highest immobilized xylanase activity (3.93IU/mg of total protein) was observed at 59 degrees C and pH 6.0. Further, AOB-immobilized XynCDBFV retained 50% of its maximal activity after 120min at 60 degrees C, and it could be easily and simply recovered from the surface of the solution by brief centrifugation, and could be reused eight times while retaining more than 60% of its activity. These results proved it is a simple and effective method for direct immobilization of xylanases.

Magnesium lithospermate B possesses inhibitory activity on Na+,K+‐ATPase and neuroprotective effects against ischemic stroke

AbstractAim:To examine if magnesium lithospermate B (MLB) extracted from Danshen, the dried roots of Salvia miltiorrhiza, may act as an active component responsible for the cardiac therapeutic effect of this traditional Chinese herb via the same molecular mechanism triggered by cardiac glycosides, such as ouabain and digoxin. Moreover, we wanted to test if MLB may provide neuroprotection against ischemic stroke as observed for cardiac glycosides.Methods:Similarity in the chemical structure and molecular configuration between MLB and ouabain was analyzed. The inhibition potency of MLB and ouabain on Na+,K+-ATPase activity of a commercial product, as well as in purified membrane fractions from rat brain and heart tissues, was examined and compared. Neuroprotective effect of MLB against ischemic stroke was also evaluated using a cortical brain slice-based assay model.Results:Dose-dependent inhibition on the commercial Na+,K+- ATPase equivalent to that for ouabain was observed for MLB of approximately half dosage by weight. This relative potency of ouabain and MLB was also observed for their inhibition on Na+,K+-ATPase activity of plasma membrane purified from rat tissues, although these 2 inhibitors exhibited somewhat lower competence in these crude extracts. In ischemic gerbil brains, post-treatment with MLB significantly reduced the infarct size, visualized by 2,3,5-triphenyltetrazolium chloride staining, by approximately 55% when compared with the control group.Conclusion:These results evidently suggest that the cardiac therapeutic effect of Danshen should be at least partly attributed to the effective inhibition of Na+,K+- ATPase by MLB, and that MLB provides anti-ischemic neuroprotection in gerbils subjected to focal ischemia and reperfusion.

Cloning and expression of a seed-specific metallothionein-like protein from sesame

A cDNA clone, SiMT encoding an Ec type of metallothionein (MT)-like protein, was isolated from maturing seeds of sesame (Sesamum indicum L.), and its deduced protein sequence shared 47–65% similarity to other known Ec type of MT-like proteins with three highly conserved cysteine-rich segments. The transcript of SiMT was exclusively accumulated in maturing seeds from two weeks after flowering to the end of seed maturation. The results of a southern blot analysis suggested that one SiMT and one SiMT-like gene were present in the sesame genome. Recombinant SiMT fused with glutathione-S-transferase (GST) was over-expressed in Escherichia coli, and purified to homogeneity by affinity chromatography. Recombinant SiMT released from GST was harvested after factor Xa cleavage. Migration of the recombinant SiMT during SDS–PAGE was accelerated when its binding metal ions were depleted by EDTA. The metal-binding capability of recombinant SiMT was measured by inductively-coupled plasma atomic emission spectrometry. Our results show that the recombinant SiMT could trap zinc or copper ions, but not manganese ions, with a stoichiometric ratio (metal ion/SiMT) of approximately 2.