On the Compartmentation of Triacylglycerol Synthesis in Developing Seeds of Brassica napus

Compartmentation of storage lipid biosynthesis in developing erucate-rich rapeseeds during the period of rapid triacylglycerol accumulation has been investigated by labelling acyl residues and the glycerol backbone in endomembrane lipids of isolated embryos with radioactive precursors, either before (“in vivo”) or after (“in vitro”) subcellular fractionation. In contrast to the low light environment within the pod under normal environmental conditions, the photosynthetic and lipid synthesizing capacities of the embryos were significantly stimulated by their illumination in the isolated state. Both ways of demonstrating “de novo” synthesis of triacylglycerols and erucic acid in endomembrane vesicles show their significantly higher accumulation in oil bodies than in microsomal fractions, where membrane lipids predominate. The increased diacylglycerol acylation in erucate-rich rape embryos appears to be coupled to an alternative elongation mechanism for oleic acid, with another immediate acyl donor than 18:1-CoA. The present results are interpreted as a spatial separation of triacylglycerol formation, with very long-chain fatty acids obtained from residual lipid synthesis and fatty acid elongating capacity located on the endoplasmic reticulum.     

植物中从二酰甘油到三酰甘油的两条合成新途径

过去认为植物中只有一条从二酰甘油到三酰甘油的合成途径。近年来,在一些植物体内又发现了从二酰甘油到三酰甘油合成的两条新途径。该文介绍这两条新途径及其意义。     

Triacylglycerol Lipase in Developing Rat Sciatic Nerve

Triacylglycerol lipase activity, with a pH optimum of 7.5, was demonstrated in cell-free homogenates of rat sciatic endoneurium. 1,2-Diacylglycerol was the major product of triacylglycerol hydrolysis. A rapid decline in lipase activity was found in rats up to 2 months of age. After this time, the decrease continued, but at a much slower rate. Such developmental changes in triacylglycerol lipase activity may, at least in part, account for the slower metabolic turnover of endoneurial triacylglycerol in adult rat sciatic nerve.     

Glycerol Affects Root Development through Regulation of Multiple Pathways in Arabidopsis

Glycerol metabolism has been well studied biochemically. However, the means by which glycerol functions in plant development is not well understood. This study aimed to investigate the mechanism underlying the effects of glycerol on root development in Arabidopsis thaliana. Exogenous glycerol inhibited primary root growth and altered lateral root development in wild-type plants. These phenotypes appeared concurrently with increased endogenous glycerol-3-phosphate (G3P) and H₂O₂ contents in seedlings, and decreased phosphate levels in roots. Upon glycerol treatment, G3P level and root development did not change in glycerol kinase mutant gli1, but G3P level increased in gpdhc1 and fad-gpdh mutants, which resulted in more severely impaired root development. Overexpression of the FAD-GPDH gene attenuated the alterations in G3P, phosphate and H₂O₂ levels, leading to increased tolerance to exogenous glycerol, which suggested that FAD-GPDH plays an important role in modulating this response. Free indole-3-acetic acid (IAA) content increased by 46%, and DR5pro::GUS staining increased in the stele cells of the root meristem under glycerol treatment, suggesting that glycerol likely alters normal auxin distribution. Decreases in PIN1 and PIN7 expression, β-glucuronidase (GUS) staining in plants expressing PIN7pro::GUS and green fluorescent protein (GFP) fluorescence in plants expressing PIN7pro::PIN7-GFP were observed, indicating that polar auxin transport in the root was downregulated under glycerol treatment. Analyses with auxin-related mutants showed that TIR1 and ARF7 were involved in regulating root growth under glycerol treatment. Glycerol-treated plants showed significant reductions in root meristem size and cell number as revealed by CYCB1;1pro::GUS staining. Furthermore, the expression of CDKA and CYCB1 decreased significantly in treated plants compared with control plants, implying possible alterations in cell cycle progression. Our data demonstrated that glycerol treatment altered endogenous levels of G3P, phosphate and ROS, affected auxin distribution and cell division in the root meristem, and eventually resulted in modifications of root development.     

Triacylglycerol Bioassembly in Microspore-Derived Embryos of Brassica napus L. cv Reston

Erucic acid (22:1) was chosen as a marker to study triacylglycerol (TAG) biosynthesis in a Brassica napus L. cv Reston microspore-derived (MD) embryo culture system. TAGs accumulating during embryo development exhibited changes in acyl composition similar to those observed in developing zygotic embryos of the same cv, particularly with respect to erucic and eicosenoic acids. However, MD embryos showed a much higher rate of incorporation of ¹⁴C-erucoyl moieties into TAGs in vitro than zygotic embryos. Homogenates of early-late cotyledonary stage MD embryos (14-29 days in culture) were assessed for the ability to incorporate 22:1 and 18:1 (oleoyl) moieties into glycerolipids. In the presence of [1-¹⁴C]22:1-coenzyme A (CoA) and various acyl acceptors, including glycerol-3-phosphate (G-3-P), radiolabeled erucoyl moieties were rapidly incorporated into the TAG fraction, but virtually excluded from other Kennedy Pathway intermediates as well as complex polar lipids. This pattern of erucoyl incorporation was unchanged during time course experiments or upon incubation of homogenates with chemicals known to inhibit Kennedy Pathway enzymes. In marked contrast, parallel experiments conducted using [1-¹⁴C]18:1-CoA and G-3-P indicated that ¹⁴C oleoyl moieties were incorporated into lyso-phosphatidic acids, phosphatidic acids, diacylglycerols, and TAGs of the Kennedy Pathway, as well as other complex polar lipids, such as phosphatidylcholines and phosphatidylethanolamines. When supplied with l-[2-³H(N)]G-3-P and [1-¹⁴C]22:1-CoA, the radiolabeled TAG pool contained both isotopes, indicating G-3-P to be a true acceptor of erucoyl moieties. Radio-high-performance liquid chromatography, argentation thin-layer chromatography/gas chromatography-mass spectrometry, and stereospecific analyses of radiolabeled TAGs indicated that 22:1 was selectively incorporated into the sn-3 position by a highly active diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), while oleoyl moieties were inserted into the sn-1 and sn-2 positions. In the presence of sn-1,2-dierucin and [1-¹⁴C]22:1-CoA, homogenates and microsomal preparations were able to produce radiolabeled trierucin, a TAG not found endogenously in this species. A 105,000g pellet fraction contained 22:1-CoA:DGAT exhibiting the highest specific activity. The rate of 22:1-CoA:DGAT activity in vitro could more than account for the maximal rate of TAG biosynthesis observed in vivo during embryo development. In double label experiments, G-3-P was shown to stimulate the conversion of [³H]phosphatidylcholines to [³H]diacylglycerols, which subsequently acted as acceptors for ¹⁴C erucoyl moieties. In vitro, 22:1 moieties did not enter the sn-1 position of TAGs by a postsynthetic modification or transacylation of preformed TAGs.     

Zinc Levels Modulate Lifespan through Multiple Longevity Pathways in Caenorhabditis elegans

Zinc is an essential trace metal that has integral roles in numerous biological processes, including enzymatic function, protein structure, and cell signaling pathways. Both excess and deficiency of zinc can lead to detrimental effects on development and metabolism, resulting in abnormalities and disease. We altered the zinc balance within Caenorhabditis elegans to examine how changes in zinc burden affect longevity and healthspan in an invertebrate animal model. We found that increasing zinc levels in vivo with excess dietary zinc supplementation decreased the mean and maximum lifespan, whereas reducing zinc levels in vivo with a zinc-selective chelator increased the mean and maximum lifespan in C. elegans. We determined that the lifespan shortening effects of excess zinc required expression of DAF-16, HSF-1 and SKN-1 proteins, whereas the lifespan lengthening effects of the reduced zinc may be partially dependent upon this set of proteins. Furthermore, reducing zinc levels led to greater nuclear localization of DAF-16 and enhanced dauer formation compared to controls, suggesting that the lifespan effects of zinc are mediated in part by the insulin/IGF-1 pathway. Additionally, zinc status correlated with several markers of healthspan in worms, including proteostasis, locomotion and thermotolerance, with reduced zinc levels always associated with improvements in function. Taken together, these data support a role for zinc in regulating both development and lifespan in C. elegans, and that suggest that regulation of zinc homeostasis in the worm may be an example of antagonistic pleiotropy.     

Proteomic Analysis of Developing Somatic Embryos of Coffea arabica

Differential protein profiles of three stages of somatic embryogenesis, including globular, torpedo, and cotyledonary somatic embryos, of Coffea arabica cv. Catuaí Vermelho were analyzed in an attempt to better understand somatic embryogenesis in coffee plants. Somatic embryos at these different stages of development were collected from in vitro-grown cultures, and then macerated in liquid nitrogen. Proteins were extracted with phenol and further quantified using the Bradford method. The bidimensional electrophoresis analysis revealed a wide range of proteins ranging between 10 and 160?kDa and of pH values ranging from 3 to 10. Several differentially expressed proteins were identified by mass spectrometry, and some were found to be specific to these different stages of somatic embryogenesis in coffee. The enolase and 11S storage globulin proteins, for example, could be used as molecular markers for somatic embryo development stages and for embryogenic and non-embryogenic genotype differentiation, respectively.     

Modulation of the Surface Proteome through Multiple Ubiquitylation Pathways in African Trypanosomes

Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes.     

Control of Lipid Synthesis during Soybean Seed Development: Enzymic and Immunochemical Assay of Acyl Carrier Protein

During soybean seed (Glycine max, var Am Soy 71) development, the rate of lipid biosynthesis per seed increases greatly. As the seed reaches maturity, lipid synthesis declines. To study the controls over the oil synthesis and storage process, we have chosen acyl carrier protein (ACP) as a representative marker for the fatty acid synthetase pathway. We have quantitated soybean ACP levels by both enzymic and immunochemical methods. Escherichia coli acyl-ACP synthetase was used as an assay for enzymically active ACP. Total ACP protein was determined by immunoassay using antibodies prepared in rabbits against spinach ACP. These antibody preparations also bind ACP isolated from soybeans, allowing development of a radioimmunoassay based on competition with [³H]palmitoyl-ACP. The enzymic and immunochemical measurement of ACP at various stages of seed development have indicated that ACP activity and ACP antigen increase markedly in correlation with the in vivo increase in lipid synthesis. These results indicate that a major control over the increase in lipid synthesis arises through regulation of the levels of the fatty acid biosynthetic proteins. However, as the seed reaches maturity and lipid biosynthesis declines, ACP per seed remains relatively high. In the mature seed, we found that more than 95% of the ACP is localized in the cotyledons, less than 5% is in the axis, and less than 1% is in the seed coat.     

Protein and RNA Content and Synthesis in Embryos and Endosperms from Developing Triticum duram Seeds

Protein and RNA contents and synthesis were evaluated in the course of wheat grain (T. durum cv. Cappelli) development. Embryos and endosperms were considered separately during five phases from the 20th day after anthests until full ripenes was reached. No clean-cut changes were observed in the pattern of soluble proteins of the embryos. In the endosperms protein synthesis continues till the later phases and appears to be due to the albumin + globulin component. Screening of bands of endosperm proteins from electrophoresis indicates that the gliadins are synthesized early, with the exception of a Ω - gliadin. Glutelins with high relative molecular mass also appear to be synthesized when the grain approaches full ripeness. The RNA content of the embryo and the endosperm is high in the early stages, when high cell proliferation occurs, and declines later on. The synthesis of RNA during in vitro imbibition is, however, higher in the later phases of ripening. Most of RNA synthesized in the embryos was ribosomal. To whom correspondence should be sent.     

The Glycolytic Inhibitor 2-Deoxyglucose Activates Multiple Prosurvival Pathways through IGF1R

Recent molecular studies indicate that aerobic glycolysis plays an important role in tumorigenesis and is a valid target for cancer therapy. Although 2-deoxyglucose (2-DG) is well characterized as a glycolytic inhibitor, we recently discovered that it activates a prosurvival oncoprotein, AKT, through PI3K. In this study, we discovered that 2-DG treatments disrupted the binding between insulin-like growth factor 1 (IGF-1) and IGF-binding protein 3 (IGFBP3) so that the free form of IGF-1 could be released from the IGF-1·IGFBP3 complex to activate IGF-1 receptor (IGF1R) signaling. Because IGF1R signaling is involved, PI3K/AKT constitutes only one of the prosurvival pathways that are activated by 2-DG treatment; we validated that MEK-ERK signaling was also induced in an IGF1R-dependent manner in some cancer cell lines. Furthermore, our phospho-specific antibody microarray analysis indicated that 2-DG up-regulated the phosphorylation of 64 sites within various signaling pathways in H460 cells. Chemical inhibition of IGF1R reduced 57 of these up-regulations. These data suggest that 2-DG-induced activation of many survival pathways can be jointly attenuated through IGF1R inhibition. Our in vitro analysis demonstrated that treatment with a combination of subtoxic doses of 2-DG and the IGF1R inhibitor II reduced cancer cell proliferation 90% and promoted significant apoptosis.Cancer cells display high rates of aerobic glycolysis in comparison with their nontransformed counterparts (i.e. the Warburg effect (1)). Whether increased aerobic glycolysis drives tumor formation or merely represents a byproduct of oncogenic transformation has been a subject of controversy. Two recent studies demonstrated that the Warburg effect can be reversed in some cancer cells by either the depletion of lactate dehydrogenenase A or switching pyruvate kinase expression from M2 to M1 isoform (2, 3). Interestingly, the reversal of the Warburg effect correlates with a reduction in the ability of the isogenic cancer cells to form tumors in nude mouse xenografts. Viewed in combination, these observations appeared to indicate that tumor cells preferentially use glucose for purposes other than oxidative phosphorylation and that aerobic glycolysis is a valid target for cancer therapeutics.Targeting glycolysis for cancer treatment has been explored previously as a therapeutic approach (4, 5). Of all the glycolysis inhibitors that were evaluated, 2-deoxyglucose (2-DG)³ is the one that has been best characterized in animal model studies and human clinical trials (6–8). It is converted by hexokinase to phosphorylated 2-DG, which becomes trapped inside the cell and inhibits hexokinase (9). As a direct consequence of 2-DG treatment, intracellular ATP is depleted (10, 11), which ultimately suppresses cell proliferation in vitro (12, 13). Nonetheless, the implementation of 2-DG as an anticancer agent in vivo has been a disappointment. Whereas 2-DG suppresses cell growth in vitro, studies using xenografts indicate that 2-DG treatment, when provided as a single agent, does not inhibit tumor growth (6).Because 2-DG is a small molecule, we suspected that it activates other signaling pathways and decided to evaluate its off-target effects. Our initial findings indicated that 2-DG activates AKT function through phosphatidylinositol 3-kinase (PI3K) and is independent of glycolysis or mTOR inhibition. Thus, the inhibitory effect on growth produced by 2-DG-mediated glycolysis inhibition may be partial offset by the fact there is also 2-DG-induced AKT activation (14). In the current study, we used a phospho-specific antibody array to identify IGF1R as the upstream receptor tyrosine kinase that is responsible for the activation of AKT signaling. Using recombinant IGF-1 and IGFBP3 proteins, we discovered that the inhibition of IGF-1 by IGFBP3 is disrupted in the presence of 2-DG. As 2-DG treatment activates IGF-1 signaling, we evaluated other prosurvival signaling pathways such as ERK signaling, which was also activated by 2-DG treatment in some cancer cell lines. Lastly, we tested to see whether an inhibitor of IGF1R would interfere with the prosurvival pathways and increase apoptosis if given in combination with 2-DG.     

beta-Conglycinins in Developing Soybean Seeds

The temporal sequence of development of the major proteins of seeds of soybean (Merr.) has been studied during development of cotyledons from flowering to maturity. A well-defined difference occurred in the times of appearance and the periods of maximum accumulation of α, α′-, and β-subunits of betaconglycinin. Whereas α- and α′-subunits appeared 15 to 17 days after flowering, accumulation of β-subunit did not commence until 22 days after flowering. Such alterations in subunit composition infer that changes also occurred in the amino acid composition of betaconglycinin during maturation, particularly in the content of methionine which is low in the β-subunit.     

Digoxin Suppresses Tumor Malignancy through Inhibiting Multiple Src-Related Signaling Pathways in Non-Small Cell Lung Cancer

Non-small cell lung cancer is the predominant type of lung cancer, resulting in high mortality worldwide. Digoxin, a cardiac glycoside, has recently been suggested to be a novel chemotherapeutic agent. Src is an oncogene that plays an important role in cancer progression and is therefore a potential target for cancer therapy. Here, we investigated whether digoxin could suppress lung cancer progression through the inhibition of Src activity. The effects of digoxin on lung cancer cell functions were investigated using colony formation, migration and invasion assays. Western blotting and qPCR assays were used to analyze the mRNA and protein expression levels of Src and its downstream proteins, and a cell viability assay was used to measure cellular cytotoxicity effects. The results of the cell function assays revealed that digoxin inhibited the proliferation, invasion, migration, and colony formation of A549 lung cancer cells. Similar effects of digoxin were also observed in other lung cancer cell lines. Furthermore, we found that digoxin significantly suppressed Src activity and its protein expression in a dose- and time-dependent manner as well as reduced EGFR and STAT3 activity. Our data suggest that digoxin is a potential anticancer agent that may suppress lung cancer progression through inhibiting Src and the activity of related proteins.     

Dynamics of Ubiquitin Pools in Developing Sea Urchin Embryos

The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid- and solution-phase immunochemical assays, employing anti-ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin ( ca . 8 × 10⁸ molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13-fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later-stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus . The results of pulse-labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12-fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin-encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.     

Abscisic Acid in Developing Zygotic Embryos of Theobroma cacao

Abscisic acid (ABA) levels were measured by enzyme-linked immunosorbent assay in developing zygotic embryos of Theobroma cacao. ABA was detected in all embryos tested, with a peak of ABA at levels of 1 to 3 micrograms per gram fresh weight during early maturation. This corresponded to embryos of 10 to 30% dry weight and to early stages of anthocyanin and lipid accumulation.