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Therapeutic potential of Gymnema sylvestre on diverse cell types is predominantly due to a variety of terpenoids and their derivatives. However, their bioavailability becomes limited due to poor solubility and lower lipophilic properties, provoking the search for novel membranotropic terpenoids and their mechanism of action. A terpenoid fraction purified from Gymnema sylvestre exhibited broad spectrum antimicrobial activity against both Gram positive and Gram negative bacteria with IC 50 ˂ 0.1 mg/ml. Evaluation of its membranotropic effect in vitro on reconstituted model membrane revealed that the fraction induced flip-flop of fluorescent phospholipid analogs across the lipid bilayer. The terpenoid-induced lipid flipping was biphasic with a fast linear phase (rate constant (k 1 ) = 3 to 5 S -1 ) and a second slow exponential phase (rate constant (k 2 ) = (4 to 9) × 10 -3  S -1 ). The lipid-flippase activity of the terpenoid fraction showed concentration and incubation-dependent cooperativity, indicating their lipophilic nature and membrane-destabilizing activity that facilitated lipid translocation. For the first time, our study reveals the flippase activity of a terpenoid fraction of Gymnema sylvestre that could be further explored for their membrane-mediated pharmacological properties. Graphical Abstract.

Phospholipid translocation (flip-flop) in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum of eukaryotic cells (rat liver) and bacterial cytoplasmic membranes is a fundamental step in membrane biogenesis. It is known that flip-flop in these membranes occurs without a metabolic energy requirement, bidirectionally with no specificity for phospholipid headgroup. In this study, we demonstrate for the first time ATP-independent flippase activity in endoplasmic reticulum membranes of plants using spinach as a model system. For this, we generated proteoliposomes from a Triton X-100 extract of endoplasmic reticulum membranes of spinach and assayed them for flippase activity using fluorescently labeled phospholipids. The half-time for flipping was found to be 0.7-1.0 min. We also show that (a) proteoliposomes can flip fluorescently labeled analogues of phosphatidylcholine and phosphatidylethanolamine, (b) flipping activity is protein-mediated, (c) more than one class of lipid translocator (flippase) is present in spinach membranes, based on the sensitivity to protease and protein-modifying reagents, and (d) translocation of PC and PE is affected differently upon treatment with protease and protein-modifying reagents. Ca (2+)-dependent scrambling activity was not observed in the vesicles reconstituted from plant ER membranes, ruling out the possibility of the involvement of scramblase in translocation of phospholipids. These results suggest the existence of biogenic membrane flippases in plants and that the mechanism of membrane biogenesis is similar to that found in animals.

A novel method for electroformation of liposomes and phytosomes using copper electrode is described. Liposomes made at 2 V and 10 Hz AC field from L-α-egg-phosphatidylcholine (egg-PC), K. pneumoniae phosphatidylethanolamine, K. pneumoniae polar lipids and E. coli polar lipids on copper electrode were (777.9 ± 118.4), (370.2 ± 100.5), (825.3 ± 21.54), and (281.3 ± 42.3) nm in diameter, respectively. Giant vesicles were formed at 30 V and 10 Hz AC field from polar lipids of K. pneumoniae and E. coli were (106 ± 29.7) and (86 ± 24.3) µm in diameter, respectively. All liposomes were unilamellar as indicated by their unilamellar indices of 50 ± 2, had surface charge comparable to vesicles made from lipid(s) with similar composition and exhibited only 1–2 mol% of oxidized lipids. Cu concentration in the liposomal samples was <1.5 ppm for large unilamellar vesicles (LUVs) and ˂5 ppm for giant unilamellar vesicles (GUVs). The vesicles were stable for >15 d without loss of their size, charge, or unilamellarity. The method was successfully applied to prepare phytosomes from egg-PC and a phytochemical fraction of Dimorphocalyx glabellus, a medicinal plant with anti-diuretic properties. Phytosomes formed were 1000–1500 nm in diameter and exhibited altered fluorescence and absorbance properties compared to the unencapsulated phytochemical. Phytosomes with phytochemical: egg-PC ratio from 0.15 to 1.5 had encapsulation efficiency ranging 90–30%, respectively, and was stable for 1 month. Our method is easy, inexpensive and convenient that will prove to be useful for preparation of liposomes and phytosomes. [ABSTRACT FROM AUTHOR]

Human phospholipid scramblase 1(hPLSCR1), when expressed in E. coli (BL-21 DE3), forms inclusion bodies that are functionally inactive. We studied the effects of various stress inducing agents and chaperones on soluble expression of hPLSCR1 in E. coli (BL-21 DE3). Addition of 3% (v/v) ethanol before induction and decreasing the post-induction temperature to 15°C increased the solubility of hPLSCR1 to ~10 and ~15% respectively. Presence of groES-groEL complex solubilized the hPLSCR1 to ~30% of the total hPLSCR1. Absence of groES-groEL did not improve the solubility of hPLSCR1 suggesting that groES and groEL are the essential chaperones for the correct folding of hPLSCR1 when over-expressed in E. coli. [ABSTRACT FROM AUTHOR]

Abstract: Background: Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca2+ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca2+/Mg2+ binding to human scramblases and conformational changes taking place in them remains unknown. Methods: In the present study, we analyzed the Ca2+ and Mg2+ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry. Results: The results in this study show that (i) affinities of the peptides are in the order hPLSCR1 >hPLSCR3>hPLSCR2>hPLSCR4 for Ca2+ and in the order hPLSCR1>hPLSCR2>hPLSCR3>hPLSCR4 for Mg2+, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca2+ and Mg2+ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families. Conclusions: Based on the above results, we hypothesize that the Ca2+ binding motif of hPLSCR1 is a novel type of Ca2+ binding motif. General significance: Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function. [Copyright &y& Elsevier]

Abstract: Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic organisms. They are believed to be involved in destroying plasma membrane phospholipid asymmetry at critical cellular events like cell activation, injury and apoptosis. However, a detailed mechanism of phospholipid scrambling still awaits a proper understanding. The most studied member of this family, phospholipid scramblase 1 (PLSCR1) (a 37kDa protein), is involved in rapid Ca2+ dependent transbilayer redistribution of plasma membrane phospholipids. Recently the function of PLSCR1 as a phospholipids translocator has been challenged and evidences suggest that PLSCR1 acts as signaling molecule. It has been shown to be involved in protein phosphorylation and as a potential activator of genes in response to interferon and other cytokines. Interferon induced rapid biosynthesis of PLSCR1 targets some of the protein into the nucleus, where it binds to the promoter region of inositol 1,4,5-triphosphate (IP3) receptor type 1 (IP3R1) gene and induces its expression. Palmitoylation of PLSCR1 acts as a switch, controlling its localization either to the PM or inside the nucleus. In the present review, we discuss the current understanding of PLSCR1 in relation to its trafficking, localization and signaling functions. [Copyright &y& Elsevier]