Macular vascularisation changes analysed using OCT angiography after successful rhegmatogenous retinal detachment repair.

AIM: To analyse the macular vascularisation changes analysed using optical coherence tomography angiography (OCTA) after successful rhegmatogenous retinal detachment (RRD) repair by comparing gas vs silicone oil and macula-on vs macula-off. METHODS: This retrospective data collection included 77 eyes with RRD that underwent pars plana vitrectomy (PPV) and gas or silicone oil tamponade. We performed an OCTA during the postoperative control between 6 and 24mo after the last surgery and evaluated the main parameters measured by OCTA: foveal avascular zone (FAZ) and parafoveolar vascular density (PVD) in the superficial capillary plexus. The patients were divided into four groups: RRD with macular involvement treated with gas tamponade, RRD without macular involvement treated with gas tamponade, RRD with macular involvement treated with silicone oil tamponade and RRD without macular involvement treated with silicone oil tamponade. A one-way ANOVA test combined with post hoc Bonferroni corrections compared FAZ sizes and PVD in all four groups. RESULTS: The FAZ size was statistically significantly larger in eyes with RRD involving the macula than in those not involving it (P=0.005). There was no statistically significant difference in the FAZ sizes of the eyes treated with silicone oil tamponade compared to those treated with gas tamponade (P=0.54). There was no statistically significant difference in the PVD comparing all four groups. CONCLUSION: Despite the known risks associated with silicone oil, our findings suggest that the type of tamponade used during PPV to treat an RRD has no significant effect on the future integrity of the PVD or the size of the FAZ in the superficial capillary plexus as measured by OCTA.

Nonsterol Triterpenoids as Major Constituents of Olea europaea.

Plant triterpenoids represent a large and structurally diverse class of natural products. A growing interest has been focused on triterpenoids over the past decade due to their beneficial effects on human health. We show here that these bioactive compounds are major constituents of several aerial parts (floral bud, leaf bud, stem, and leaf) of olive tree, a crop exploited so far almost exclusively for its fruit and oil. O. europaea callus cultures were analyzed as well. Twenty sterols and twenty-nine nonsteroidal tetra- and pentacyclic triterpenoids belonging to seven types of carbon skeletons (oleanane, ursane, lupane, taraxerane, taraxastane, euphane, and lanostane) were identified and quantified by GC and GC-MS as free and esterified compounds. The oleanane-type compounds, oleanolic acid and maslinic acid, were largely predominant in all the organs tested, whereas they are practically absent in olive oil. In floral buds, they represented as much as 2.7% of dry matter. In callus cultures, lanostane-type compounds were the most abundant triterpenoids. In all the tissues analyzed, free and esterified triterpene alcohols exhibited different distribution patterns of their carbon skeletons. Taken together, these data provide new insights into largely unknown triterpene secondary metabolism of Olea europaea.

The plastidial 2-C-methyl-D-erythritol 4-phosphate pathway provides the isoprenyl moiety for protein geranylgeranylation in tobacco BY-2 cells.

Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-trans-geranylgeraniol (GGol). Furthermore, 1-deoxy-d-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-d-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-d-erythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation.

Sterol-dependent endocytosis mediates post-cytokinetic acquisition of PIN2 auxin efflux carrier polarity.

The polarization of yeast and animal cells relies on membrane sterols for polar targeting of proteins to the plasma membrane, their polar endocytic recycling and restricted lateral diffusion. However, little is known about sterol function in plant-cell polarity. Directional root growth along the gravity vector requires polar transport of the plant hormone auxin. In Arabidopsis, asymmetric plasma membrane localization of the PIN-FORMED2 (PIN2) auxin transporter directs root gravitropism. Although the composition of membrane sterols influences gravitropism and localization of two other PIN proteins, it remains unknown how sterols contribute mechanistically to PIN polarity. Here, we show that correct membrane sterol composition is essential for the acquisition of PIN2 polarity. Polar PIN2 localization is defective in the sterol-biosynthesis mutant cyclopropylsterol isomerase1-1 (cpi1-1) which displays altered sterol composition, PIN2 endocytosis, and root gravitropism. At the end of cytokinesis, PIN2 localizes initially to both newly formed membranes but subsequently disappears from one. By contrast, PIN2 frequently remains at both daughter membranes in endocytosis-defective cpi1-1 cells. Hence, sterol composition affects post-cytokinetic acquisition of PIN2 polarity by endocytosis, suggesting a mechanism for sterol action on establishment of asymmetric protein localization.

Characterization of lipid rafts from Medicago truncatula root plasma membranes: a proteomic study reveals the presence of a raft-associated redox system.

Several studies have provided new insights into the role of sphingolipid/sterol-rich domains so-called lipid rafts of the plasma membrane (PM) from mammalian cells, and more recently from leaves, cell cultures, and seedlings of higher plants. Here we show that lipid raft domains, defined as Triton X-100-insoluble membranes, can also be prepared from Medicago truncatula root PMs. These domains have been extensively characterized by ultrastructural studies as well as by analysis of their content in lipids and proteins. M. truncatula lipid domains are shown to be enriched in sphingolipids and Delta(7)-sterols, with spinasterol as the major compound, but also in steryl glycosides and acyl-steryl glycosides. A large number of proteins (i.e. 270) have been identified. Among them, receptor kinases and proteins related to signaling, cellular trafficking, and cell wall functioning were well represented whereas those involved in transport and metabolism were poorly represented. Evidence is also given for the presence of a complete PM redox system in the lipid rafts.

Formation of triterpenoids throughout Olea europaea fruit ontogeny.

Drupes were handpicked from olive (Olea europaea L.) trees, cv chemlali, at 13 distinct stages of fruit development, referred to as weeks after flowering (WAF), and analyzed for their free and esterified sterols and triterpenoids content. These two classes of compounds are synthesized via the acetate/mevalonate pathway and share common precursors up to oxidosqualene (OS). Cyclization of OS in either cycloartenol or beta-amyrin constitutes a branch point between primary (sterol pathway) and secondary (triterpenoid pathway) metabolisms. At the onset of fruit development, i.e., between 12 and 18 WAF, drupes were found to contain high amounts of alpha- and beta-amyrins as well as more-oxygenated compounds such as triterpenic diols (erythrodiol and uvaol) and acids (oleanolic, ursolic and maslinic acids). Concomitantly, sterol precursors were barely detectable. From 21 WAF, when the olive fruit reached its final size and began to turn from green to purple, alpha- and beta-amyrins were no longer present, while 4,4-dimethyl- and 4alpha-methylsterols started to be formed, indicating a redirection of the carbon flux from the triterpenoid pathway towards the sterol pathway. Between 21 and 30 WAF, sterol end products, mainly represented by sitosterol, progressively accumulated and triterpenic diols were replaced by triterpenic acids, essentially maslinic acid. Interestingly, the developing olive fruit was found to accumulate significant amounts of parkeol as an ester conjugate. Whatever the stage of development, triterpenoids represent the major triterpenic compounds of the olive fruit.

Insights into the role of specific lipids in the formation and delivery of lipid microdomains to the plasma membrane of plant cells.

The existence of sphingolipid- and sterol-enriched microdomains, known as lipid rafts, in the plasma membrane (PM) of eukaryotic cells is well documented. To obtain more insight into the lipid molecular species required for the formation of microdomains in plants, we have isolated detergent (Triton X-100)-resistant membranes (DRMs) from the PM of Arabidopsis (Arabidopsis thaliana) and leek (Allium porrum) seedlings as well as from Arabidopsis cell cultures. Here, we show that all DRM preparations are enriched in sterols, sterylglucosides, and glucosylceramides (GluCer) and depleted in glycerophospholipids. The GluCer of DRMs from leek seedlings contain hydroxypalmitic acid. We investigated the role of sterols in DRM formation along the secretory pathway in leek seedlings. We present evidence for the presence of DRMs in both the PM and the Golgi apparatus but not in the endoplasmic reticulum. In leek seedlings treated with fenpropimorph, a sterol biosynthesis inhibitor, the usual Delta(5)-sterols are replaced by 9beta,19-cyclopropylsterols. In these plants, sterols and hydroxypalmitic acid-containing GluCer do not reach the PM, and most DRMs are recovered from the Golgi apparatus, indicating that Delta(5)-sterols and GluCer play a crucial role in lipid microdomain formation and delivery to the PM. In addition, DRM formation in Arabidopsis cells is shown to depend on the unsaturation degree of fatty acyl chains as evidenced by the dramatic decrease in the amount of DRMs prepared from the Arabidopsis mutants, fad2 and Fad3+, affected in their fatty acid desaturases.

Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane.

A large body of evidence from the past decade supports the existence of functional microdomains in membranes of animal and yeast cells, which play important roles in protein sorting, signal transduction, or infection by pathogens. They are based on the dynamic clustering of sphingolipids and cholesterol or ergosterol and are characterized by their insolubility, at low temperature, in nonionic detergents. Here we show that similar microdomains also exist in plant plasma membrane isolated from both tobacco leaves and BY2 cells. Tobacco lipid rafts were found to be greatly enriched in a sphingolipid, identified as glycosylceramide, as well as in a mixture of stigmasterol, sitosterol, 24-methylcholesterol, and cholesterol. Phospho- and glycoglycerolipids of the plasma membrane were largely excluded from lipid rafts. Membrane proteins were separated by one- and two-dimensional gel electrophoresis and identified by tandem mass spectrometry or use of specific antibody. The data clearly indicate that tobacco microdomains are able to recruit a specific set of the plasma membrane proteins and exclude others. We demonstrate the recruitment of the NADPH oxidase after elicitation by cryptogein and the presence of the small G protein NtRac5, a negative regulator of NADPH oxidase, in lipid rafts.

A review of tobacco BY-2 cells as an excellent system to study the synthesis and function of sterols and other isoprenoids.

In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate (IPP), the universal precursor for isoprenoid biosynthesis. In this paper we review findings and observations made primarily with tobacco BY-2 cells (TBY-2), which have proven to be an excellent system in which to study the two biosynthetic pathways. A major advantage of these cells as an experimental system is their ability to readily take up specific inhibitors and stably- and/or radiolabeled precursors. This permits the functional elucidation of the role of isoprenoid end products and intermediates. Because TBY-2 cells undergo rapid cell division and can be synchronized within the cell cycle, they constitute a highly suitable test system for determination of those isoprenoids and intermediates that act as cell cycle inhibitors, thus giving an indication of which branches of the isoprenoid pathway are essential. Through chemical complementation; and use of precursors, intracellular compartmentation can be elucidated, as well as the extent to which the plastidial and cytosolic pathways contribute to the syntheses of specific groups of isoprenoids (e.g., sterols) via exchange of intermediates across membranes. These topics are discussed in the context of the pertinent literature.

Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase.

To get some insight into the regulatory mechanisms controlling the sterol branch of the mevalonate pathway, tobacco (Nicotiana tabacum cv Bright Yellow-2) cell suspensions were treated with squalestatin-1 and terbinafine, two specific inhibitors of squalene synthase (SQS) and squalene epoxidase, respectively. These two enzymes catalyze the first two steps involved in sterol biosynthesis. In highly dividing cells, SQS was actively expressed concomitantly with 3-hydroxy-3-methylglutaryl coenzyme A reductase and both sterol methyltransferases. At nanomolar concentrations, squalestatin was found to inhibit efficiently sterol biosynthesis as attested by the rapid decrease in SQS activity and [(14)C]radioactivity from acetate incorporated into sterols. A parallel dose-dependent accumulation of farnesol, the dephosphorylated form of the SQS substrate, was observed without affecting farnesyl diphosphate synthase steady-state mRNA levels. Treatment of tobacco cells with terbinafine is also shown to inhibit sterol synthesis. In addition, this inhibitor induced an impressive accumulation of squalene and a dose-dependent stimulation of the triacylglycerol content and synthesis, suggesting the occurrence of regulatory relationships between sterol and triacylglycerol biosynthetic pathways. We demonstrate that squalene was stored in cytosolic lipid particles, but could be redirected toward sterol synthesis if required. Inhibition of either SQS or squalene epoxidase was found to trigger a severalfold increase in enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, giving first evidence for a positive feedback regulation of this key enzyme in response to a selective depletion of endogenous sterols. At the same time, no compensatory responses mediated by SQS were observed, in sharp contrast to the situation in mammalian cells.

Inhibition of the sterol pathway in leek seedlings impairs phosphatidylserine and glucosylceramide synthesis but triggers an accumulation of triacylglycerols.

Like most higher plants, leek seedlings (Allium porrum L.) contain a mixture of Delta(5)-sterols in which sitosterol largely predominates. As previously reported (Plant Physiol., 117 (1998) 931), these compounds, which are synthesized at the endoplasmic reticulum level, were shown to be actively transported to the plasma membrane via a membrane-mediated process, together with phosphatidylserine (PS). In the present work, leek seedlings were allowed to germinate for 7 days in the presence of fenpropimorph, a sterol biosynthesis inhibitor. Such a treatment was found to trigger an almost complete replacement of the usual sterols by 9beta,19-cyclopropylsterols (mainly cycloeucalenol and 29-norcycloartenol). Extensive lipid analyses and labeling experiments with sodium [14C]acetate were performed to examine potential changes in the content and the rate of synthesis of the other lipid molecular species. The results indicate that the inhibition of the sterol pathway was accompanied by a severe decrease in PS and glucosylceramide synthesis as well as by a redirection of fatty acids toward the storage triacylglycerol pathway. Triacyglycerols are shown to accumulate concomitantly with a significant increase in intracellular lipid droplets in both aerial parts and roots of leek seedlings. Taken together, the present data emphasize that a coordinated regulation of the biosynthetic pathways of sterols and some specific lipid molecular species could take place during plant membrane biogenesis.