Influence of Gibberellic Acid and the Rht3 Gene on Choline and Phospholipid Metabolism in Wheat Aleurone Tissue

The effect of gibberellic acid (GA3) on phospholipid metabolismand -amylase production was studied in aleurone tissue of twonear-isogenic lines of wheat (Triticum aesuvum L.). Incubationof embryoectomized seeds from a GA-responsive line (rht3, tall)with GA₃ caused the induction of -amylase activity after a lagphase of 30 h. In the case of embryoectomized seeds from a ‘GA-insensitive’line (Rh13, dwarf), however, the lag phase was extended up to50 h. During the first 14 h following imbibition, GA₃ inhibitedcholine uptake and its subsequent incorporation into phosphatidylcholine in the Rhr3 line but not in the rht3 line. GA₃ promotedphospholipid breakdown in both the lines during this period,however. GA₃ also terminated independent turnover of the cholineN-methyl groups in phosphatidyl choline and promoted turnoverof the whole choline headgroup. These results are discussedin relation to the possibility that phosphatidyl choline turnoveris an integral part of the GA₃ signal-transduction mechanismin aleurone tissue. Key words: GA3, Rht3 gene, choline, phospholipid     

Enzymes of Intermediary Metabolism in the Aleurone Cells of Germinating Wheat

Several enzymes of the glycolysis-gluconeogenesis sequence,the pentose cycle, the tricarboxylic acid cycle, and the electrontransport chain, were studied in the aleurone tissue of germinatingTriticum vulgare. Since all the enzymes are located either inthe cytosol or in mitochondria, they acted as parameters ofthe development of metabolic capacity in these compartmentsduring germination. Each of the enzymes studied was presentin the aleurone tissue of the ungerminated grain. After 12 –18 h germination, the level of each enzyme began to increase,following a sigmoidal course which levelled off by the fourthday. This pattern of enzyme development in the aleurone tissuewas independent of the presence of the embryo, and, therefore,of hormonal control from that tissue (in contrast to the glyoxysomalenzymes). Studies using metabolic inhibitors indicated thatthe developments of the different cytosolic enzyme activitieswere dependent to different degrees upon RNA and protein synthesis.     

Using a Semi-Aqueous Medium for the Subcellular Study of Acid Phosphatase in Wheat Aleurone Tissue

Subcellular fractionation of the aleurone tissue in ethan-1,2-diol:H₂O(80:20, v/v) provided a suitable means to study acid phosphataseactivity in the protein bodies (aleurone grains). Enzyme activityin the fraction increased 7-fold during 4 d germination. Theincrease was dependent upon the presence of the embryo, butit was not regulated by gibberellic acid, indole acetic acidor glutamine when these compounds were presented either singlyor in combination. The regulatory activities of these compoundswere restricted to an acid phosphatase located in the cell wall. Key words: Triticum aestivum, aleurone tissue, acid phosphatase, plant growth regulators     

Phospholipid Metabolism in Ferrobacillus ferrooxidans

The lipid composition of the chemoautotroph Ferrobacillus ferrooxidans has been examined. Fatty acids represent 2% of the dry weight of the cells and 86% of the total are extractable with organic solvents. About 25% of the total fatty acids are associated with diacyl phospholipids. Polar carotenoids, the benzoquinone coenzyme Q-8, and most of the fatty acids are present in the neutral lipids. The phospholipids have been identified as phosphatidyl monomethylethanolamine (42%), phosphatidyl glycerol (23%), phosphatidyl ethanolamine (20%), cardiolipin (13%), phosphatidyl choline (1.5%), and phosphatidyl dimethylethanolamine (1%) by chromatography of the diacyl lipids, by chromatography in four systems of the glycerol phosphate esters derived from the lipids by mild alkaline methanolysis, and by chromatographic identification of the products of acid hydrolysis of the esters. No trace of phosphatidylserine (PS), glycerolphosphorylserine, or serine could be detected in the lipid extract or in derivatives of that extract. This casts some doubt on the postulated involvement of PS in iron metabolism. After growth in the presence of (14)C and (32)P, there was essentially no difference in the turnover of either isotope in the glycerolphosphate ester derived from each lipid in cells grown at pH 1.5 or 3.5.     

Arachidonic Acid Increases Inositol Phospholipid Metabolism and Glutamate Release in Synaptosomes Prepared from Hippocampal Tissue

We have been interested in the possibility that arachidonic acid or one of its 12-lipoxygenase metabolites may function as a retrograde messenger in long-term potentiation (LTP) in the dentate gyrus of the hippocampus. One criterion required of a retrograde messenger is that it stimulates presynaptic changes. Here, two possible presynaptic actions of arachidonic acid and its 12-lipoxygenase metabolites, 12-hydroxyeicosatetraenoic acid (HETE) and 12-hydroperoxyeicosatetraenoic acid (HPETE), are examined. We report that arachidonic acid, HETE, and HPETE significantly increase both K(+)-stimulated release of [3H]glutamate and [3H]inositol labelling of inositol phosphates in synaptosomes, whereas other biologically important fatty acids (oleic, palmitic, and stearic) failed to induce a similar response. The findings of these experiments are consistent with the hypothesis that arachidonic acid, HETE, or HPETE may play the role of a retrograde messenger in LTP.     

Control of Tissue Specificity: The Pattern of Cellular Synthetic Activities in Tissue Transformation

During transformation of the dorsal marginal iris into lenstissue after removal of the lens in the adult newt, the cellsundergo gradual changes in synthetic activities. Autoradiographicdata indicate an enhancement of RNA synthesis in the nucleusof iris cells, which is followed by enhancement of protein synthesisand onset ot DNA synthesis. After discharge of pigment granulesand a period of cellular multiplication accompanied by ribosomeproduction, the cells stop DNA synthesis and start to show detectableamounts of lens-specific proteins (-,ß-, and -crystallins).This time coincides with initiation of differentiation of primarylens fibers. In the later stages of regeneration, - and ß-crystallinsare present in the dividing cells of the lens epithelium, aswell as in the cells of the fiber area, but -crystallins aredetected only in Che cells of the fiber area. The data wereinterpreted as suggesting that the gene utilization patterntypical for the iris is not directly converted into that forthe lens, but goes through intermediate patterns before thetissue transformation is completed.     

The Role of Phosphatidylserine Decarboxylase in Brain Phospholipid Metabolism

In brain, phosphatidylethanolamine can be synthesized from free ethanolamine either by a pathway involving the formation of CDP-ethanolamine and its transfer to diglyceride, or by base-exchange of ethanolamine with existing phospholipids. Although de novo synthesis from serine has also been demonstrated, the metabolic pathway involved is not known. The enzyme phosphatidylserine decarboxylase appears to be involved in the synthesis of much of the phosphatidylethanolamine in liver, but the significance of this route in brain has been challenged. Our in vitro studies demonstrate the existence of phosphatidylserine decarboxylase activity in rat brain and characterize some of its properties. This enzyme is localized in the mitochondrial fraction, whereas the enzymes involved in base-exchange and the cytidine pathway are localized to microsomal membranes. Parallel in vivo studies showed that after the intracranial injection of L-[G-3H]serine, the specific activity of phosphatidylserine was greater in the microsomal fractions than in the mitochondrial fraction, whereas the opposite was true for phosphatidylethanolamine. When L-[U-14C]serine and [1-3H]ethanolamine were simultaneously injected, the 14C/3H ratio in mitochondrial phosphatidylethanolamine was 10 times that in microsomal phosphatidylethanolamine. The results demonstrate that serine is incorporated into the base moiety of phosphatidylethanolamine primarily through the decarboxylation of phosphatidylserine in brain mitochondria. A minimal value of 7% for the contribution of phosphatidylserine decarboxylase to whole-brain phosphatidylethanolamine synthesis can be estimated from the in vivo data.     

The Induction of Glyoxysomal Enzyme Activities in the Aleurone Cells of Germinating Wheat

The aleurone cells of quiescent Triticum vulgare grain wereobserved to contain glyoxysomes, but enzymes known to be locatedin this organelle were not detected. During germination thenumber of glyoxysomes increased, and their associated enzymeactivities appeared, increasing up to the fifth or sixth day.The appearance of ß-oxidation, isocitratase, and malatesynthetase activities were largely dependent upon the presenceof the embryo. Gibberellic acid (GA₂) was effective in replacingthe embryo in this role. It is proposed, therefore, that thedevelopment of glyoxysomal enzyme activities and probably ofthe glyoxysomes themselves, is a gibberellic acid-dependentprocess. The developments of citrate synthetase and malate dehydrogenaseactivities were only partly dependent upon gibberellic acid.Since it is known that these enzymes are located in other compartmentsbesides the glyoxysomes, it is proposed that their gibberellicacid-dependent activities are located in glyoxysomes while theirgibberellic acidindependent activities are located in the cytosoland/or the mitochondria. The developmental courses of the gibberellicacid-independent activities and the results of studies usinginhibitors of protein synthesis support this hypothesis     

Phospholipid Composition and Metabolism of Micrococcus denitrificans

The phospholipid composition of Micrococcus denitrificans was unusual in that phosphatidyl choline (PC) was a major phospholipid (30.9%). Other phospholipids were phosphatidyl glycerol (PG, 52.4%), phosphatidyl ethanolamine (PE, 5.8%), an unknown phospholipid (5.3%), cardiolipin (CL, 3.2%), phosphatidyl dimethylethanolamine (PDME, 0.9%), phosphatidyl monomethylethanolamine (PMME, 0.6%), phosphatidyl serine (PS, 0.5%), and phosphatidic acid (0.4%). Kinetics of ³²P incorporation suggested that PC was formed by the successive methylations of PE. Pulse-chase experiments with pulses of ³²P or acetate-1-¹⁴C to exponentially growing cells showed loss of isotopes from PMME, PDME, PS, and CL with biphasic kinetics suggesting the same type of multiple pools of these lipids as proposed in other bacteria. The major phospholipids, PC, PG, and PE, were metabolically stable under these conditions. The fatty acids isolated from the complex lipids were also unusual in being a simple mixture of seven fatty acids with oleic acid representing 86% of the total. Few free fatty acids and no non-extractable fatty acids associated with the cell wall or membrane were found.     

Phosphoinositides in Barley (Hordeum vulgare L.) Aleurone Tissue

[3H]Inositol labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers and analysis of phospholipids by deacylation revealed the presence of phosphatidylinositol (PtdIns), PtdIns3P, and PtdIns4P but not PtdInsP2 species. In contrast to an earlier report (P.P.N. Murthy, G. Pliska-Matyshak, L.M. Keranen, P. Lam, H.H. Mueller, N. Bhuvarahamurthy [1992] Plant Physiol 98: 1498-1501) systematic chemical degradation of PtdIns revealed no evidence of a second isomer of PtdIns. Evidence of the widespread occurrence of 3-phosphorylated PtdIns within the plant kingdom is presented.     

A Possible Role for Indoleacetic Acid, Low Temperature, and Phospholipid Metabolism in the Induction of GA(3) Responsiveness in GA(3) Insensitive (Rht3-Containing) Dwarf Wheat Aleurone

Preincubation of dwarf, Rht3-containing deembryonated seed for 4 hours in 342 nanomolar indoleacetic acid (IAA) induced maximum sensitivity to GA₃. In addition, the 4-hour IAA pretreatment caused a 2-fold increase in total phospholipids which coincided identically on a temporal basis with the induced GA₃ sensitivity. Changes in absolute levels of individual phospholipids and their acyl groups were recorded and compared with the changes observed in several Rht-containing aleurone tissues which were induced to develop GA₃ sensitivity by exposure to low temperature (5°C). Several distinct similarities between all tissues were recorded as they develop GA₃ sensitivity. One parameter, the percentage phospholipid composition, was quite similar in all tissues after they had become maximally sensitive to GA₃, suggesting that there is at least one membrane phospholipid composition which is particularly responsive to GA₃. The results indicate that (a) the basis of the GA₃ insensitivity of the Rht mutation resides in an aberrant phospholipid/fatty acid composition and/or metabolism; (b) exposure to low temperature (5°C) for 20 hours or longer, or 342 nanomolar IAA for 4 hours or longer reverses or corrects the genetic lesion, enabling the tissue to adopt a GA₃ responsive membrane composition. Finally, an hypothesis is discussed which indicates that IAA may play a controlling role in the mobilization of endospermal reserves, at least in Rht3-containing wheat aleurone.     

The Role of Membrane-Bound Enzymes in an Early Response of Aleurone Tissue to Gibberellic Acid

Treatment of aleurone layers of barley seed with gibberellicacid increases the observable phosphorylcholine glyceride transferaseactivity in a membrane fraction prepared from extracts of thealeurone cells. This gibberellic acid-dependent increase inglyceride transferase activity requires neither RNA synthesisnor protein synthesis. Membrane fractions prepared from mixturesof extracts of gibberellic acid-treated layers and control layershave a specific activity of glyceride transferase higher thanexpected on the basis of simple addition of the activities fromthe two sources. Therefore, some kind of activation is occurring.     

Metformin Decouples Phospholipid Metabolism in Breast Cancer Cells

Introduction

The antidiabetic drug metformin, currently undergoing trials for cancer treatment, modulates lipid and glucose metabolism both crucial in phospholipid synthesis. Here the effect of treatment of breast tumour cells with metformin on phosphatidylcholine (PtdCho) metabolism which plays a key role in membrane synthesis and intracellular signalling has been examined.

Methods

MDA-MB-468, BT474 and SKBr₃ breast cancer cell lines were treated with metformin and [³H-methyl]choline and [¹⁴C(U)]glucose incorporation and lipid accumulation determined in the presence and absence of lipase inhibitors. Activities of choline kinase (CK), CTP:phosphocholine cytidylyl transferase (CCT) and PtdCho-phospholipase C (PLC) were also measured. [³H] Radiolabelled metabolites were determined using thin layer chromatography.

Results

Metformin-treated cells exhibited decreased formation of [³H]phosphocholine but increased accumulation of [³H]choline by PtdCho. CK and PLC activities were decreased and CCT activity increased by metformin-treatment. [¹⁴C] incorporation into fatty acids was decreased and into glycerol was increased in breast cancer cells treated with metformin incubated with [¹⁴C(U)]glucose.

Conclusion

This is the first study to show that treatment of breast cancer cells with metformin induces profound changes in phospholipid metabolism.     

Microbial Assimilation of Hydrocarbons: Phospholipid Metabolism

An analysis of the turnover of the major phospholipids of Micrococcus cerificans growing or nongrowing cultures. The turnover rates of (14)C-PE and (14)C-PE were 61.5% of the total phospholipid, exhibited no significant rate of turnover in either growing or nongrowing cultures. The turnover rates of PE-(14)C and PE-(32)P were 3.2% per hr and 1.2% per hr, respectively. Phosphatidylglycerol (PG) exhibited a turnover rate of 11% and 7.7% per hr for (14)C and (32)P, respectively, indicating an extremely slow metabolism. PG metabolism was examined in greater detail, and the data indicated a preferential 75% incorporation of glycerol-1,3-(14)C into the unacylated portion of the PG molecule. The turnover of cardiolipin (CL) was extremely slow in growing cells whereas nongrowing cells exhibited a 30% and 36% increase per hr for (14)C-Cl and (14)C-CL, respectively. Glycerol-1,3-(14)C was not converted to phospholipid fatty acid carbon; all radioactivity appeared only in the water-soluble backbone of the phospholipids. The kinetics of assimilation of hexadecane-1-(14)C into cellular lipids is presented. Radioactivity in neutral lipid increased approximately sevenfold over the growth cycle, whereas radioactivity in phospholipid increased 50-fold during the same time period. The incorporation of radioactive fatty acids derived from the direct oxidation of hexadecane-1-(14)C demonstrated differential kinetics of assimilation into PE, PG, and CL. The results indicated a rapid turnover of phospholipid fatty acids in M. cerificans growing at the expense of hexadecane.     

The Formation of Endoplasmic Reticulum in the Aleurone Cells of Germinating Wheat: an Ultrastructural Study

In the aleurone cells of the quiescent wheat grain endoplasmicreticulum was sparse and present as short profiles. During germinationlong profiles of endoplasmic reticulum developed near to thenuclear membrane and in close association with plastids. Muchof this development occurred during the first 2 d of germination,but further development and stacking of the membranes appearedto take place after this time. The development of the long profileswas independent of the presence of the embryo and thereforeof control by gibberellic acid. On the contrary, after the secondday of germination gibberellic acid produced a decline in theamount of endoplasmic reticulum associated with vesiculationof the reticulum profiles. The results of this ultrastructuralstudy are discussed in the context of available informationon the biosynthesis of phospholipids in aleurone tissue. Someaspects of our results are at variance with those of otherswho have studied the aleurone tissue of barley. These differencesare discussed and suggestions for their resolution are made.     

Arginine-Vasopressin Stimulates Inositol Phospholipid Metabolism in Rat Hippocampus

The hippocampal vasopressin receptors have been characterised by measuring the stimulated accumulation of inositol monophosphate in the presence of 10 mM LiCl after hippocampal slices were prelabelled with [3H]inositol. Arginine-vasopressin caused a dose-dependent increase in inositol monophosphate accumulation (ED50 = 7.1 nM). The response was unchanged in the absence of Ca2+ and significantly reduced in the presence of a V1-receptor antagonist. Equimolar oxytocin was ineffective as a stimulus. This suggests that the hippocampal receptors are of the V1 type.     

Phospholipid Metabolism During Penicillinase Production in Bacillus licheniformis

During membrane-bound penicillinase production, Bacillus licheniformis forms vesicles and tubules that do not appear in the absence of penicillinase production. The major lipids of B. licheniformis were shown to be phospholipids. The proportions, metabolism, and the total phospholipid per cell were shown to be essentially the same in the uninduced control, induced and constitutive penicillinase forming cells during both the exponential and stationary growth phases. Membrane phospholipids were not secreted into the medium during penicillinase formation. In the shift from the exponential to the stationary growth phase, there was an accumulation of phosphatidyl glycerol and a marked decrease in cardiolipin. These two lipids had the most active turnover of their phospholipid phosphate of all the lipids studied.     

The Phospholipid Composition of Nuclear Subfractions from Germinating Wheat Embryos

The content of phospholipids in chromatin, nuclear matrix, and nuclear membrane from wheat (Triticum aestivum L.) embryos was studied. Subfractions of intact nuclei from dry embryos were shown to differ in the content and composition of particular phospholipids. Embryo germination resulted in the redistribution of phospholipid between nuclear subfractions. A functional role of structural changes in the nuclear membrane due to this phospholipid redistribution is discussed. It is supposed that these rearrangements change nuclear membrane permeability and its surface charge.     

Phospholipid Metabolism in Mouse Sciatic Nerve In Vivo

To probe the activities of various pathways of lipid metabolism in peripheral nerve, six phospholipid-directed precursors were individually injected into the exposed sciatic nerves of adult mice, and their incorporation into phospholipids and proteins was studied over a 2-week period. Tritiated choline, inositol, ethanolamine, serine, and glycerol were mainly used in phospholipid synthesis; in contrast, methyl-labeled methionine was primarily incorporated into protein. Phosphatidylcholine was the main lipid formed from tritiated choline, glycerol, and methionine precursors. Phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol were the main lipids formed from serine, ethanolamine, and inositol, respectively. With time there was a shift in label among phospholipids, with higher proportions of choline appearing in sphingomyelin, glycerol in phosphatidylserine, ethanolamine in phosphatidylethanolamine (plasmalogen), and inositol in polyphosphoinositides, especially phosphatidylinositol 4,5-bisphosphate. We suggest that the delay in formation of these phospholipids, which are concentrated in peripheral nerve myelin, may, at least in part, be due to their formation at a site(s) distant from the sites where the bulk of Schwann cell lipids are made. We propose that separating the synthesis of these myelin-destined lipids to near the Schwann cell's plasma membrane would facilitate their concentration in peripheral nerve myelin sheaths. At earlier labeling times, ethanolamine and glycerol were more actively incorporated into phosphatidylcholine and phosphatidylinositol, respectively, than later. The transient labeling of these phospholipids may reflect some unique role in peripheral nerve function.