scholarly journals Evidence for phospholipases from Trypanosoma cruzi active on phosphatidylinositol and inositolphosphoceramide

1999 ◽  
Vol 345 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Laura E. BERTELLO ◽  
Maria Júlia M. ALVES ◽  
Walter COLLI ◽  
Rosa M. de LEDERKREMER
Keyword(s):  
Fatty Acid ◽  
Trypanosoma Cruzi ◽  
Phospholipase C ◽  
Enzyme Activities ◽  
Neutral Lipids ◽  
Particulate Material ◽  
Phospholipase A1 ◽  
Free System ◽  
Lipid Moiety ◽  
A Cell

The lipid moiety in the glycosylphosphatidylinositol anchors of glycoproteins of Trypanosoma cruzi consists of an alkylacylglycerol, a lysoalkylglycerol or a ceramide. Previously, we showed that the inositolphosphoceramides (IPCs) are the major components in the precursor inositolphospholipids of epimastigote and trypomastigote forms. Using 3H-labelled subfractions of IPC, phosphatidylinositol (PI) and glycoinositolphospholipids (GIPLs) as substrates with a cell-free system, we now demonstrate the association of at least five enzyme activities with the trypanosomal membranous particulate material. These include: phospholipase A1 and phospholipase A2, enzymes that release free fatty acid from the PI and GIPLs; an acyltransferase responsible for the acylation of the generated monoacyl or monoalkylglycerolipids with endogenous unlabelled fatty acid; two activities of phospholipase C, one releasing ceramide from IPC and the other alkylacylglycerol, alkylglycerol or diacylglycerol from PI. The neutral lipids were also generated on incubation of the GIPLs. The phospholipase C activities were inhibited by p-chloromercuriphenylsulphonic acid, as reported for other PI phospholipases C. An IPC-fatty-acid hydrolase, releasing fatty acid from the labelled IPC, was also observed. The enzyme activities reported in the present study may be acting in remodelling reactions leading to the anchor of the mature glycoproteins of T. cruzi.

scholarly journals Stimulatory and inhibitory actions of lysophosphatidylcholine, depending on its fatty acid residue, on the phospholipase C/Ca2+ system in HL-60 leukaemia cells

1998 ◽  
Vol 336 (2) ◽  
pp. 491-500 ◽  
Author(s):  
Fumikazu OKAJIMA ◽  
Koichi SATO ◽  
Hideaki TOMURA ◽  
Atsushi KUWABARA ◽  
Hiromi NOCHI ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Phospholipase C ◽  
Inositol Phosphate ◽  
Inhibitory Effect ◽  
Fatty Acid Residue ◽  
Stimulatory Action ◽  
Intact Cells ◽  
Free System ◽  
A Cell ◽  
G Protein Coupled

We examined the mechanism of action of lysophosphatidylcholine (LPC), which is suggested to be involved in the pathogenesis of atherosclerosis and inflammatory disorders, in HL-60 leukaemia cells. Extracellular 1-palmitoyl LPC increased the intracellular Ca2+ concentration in association with production of inositol phosphate. These actions of LPC were markedly inhibited by treatment of the cells with pertussis toxin and U73122, a phospholipase C inhibitor. The lipid-induced stimulation of the phospholipase C/Ca2+ system was also attenuated in the dibutyryl cAMP-induced differentiated (neutrophil-like) cells, in which phospholipase C activation induced by NaF or formyl-Met-Leu-Phe was enhanced. In contrast with the stimulatory action of 1-palmitoyl LPC, 1-stearoyl LPC was inhibitory for the phospholipase C/Ca2+ system stimulated by NaF as well as by 1-palmitoyl LPC or other Ca2+-mobilizing agonists. In a cell-free system, only an inhibitory effect on phospholipase C activity was observed even by 1-palmitoyl LPC; 1-stearoyl LPC was more inhibitive than 1-palmitoyl LPC. Taken together, these results suggest that atherogenic and inflammatory LPC exerts both stimulatory and inhibitory actions on the phospholipase C/Ca2+ system depending on the species of fatty acid residue of the lipid; the stimulatory effect is possibly mediated through G-protein-coupled receptors; the inhibitory effect might be caused by dysfunction of the components involved in the enzyme system owing to the amphiphilic nature of the lipid. 1-Palmitoyl LPC prefers the former receptor stimulation at least in intact cells, but 1-stearoyl LPC preferentially exerts the latter inhibitory action.

scholarly journals Photosensitization to ultraviolet irradiation and selective killing of cells following uptake of pyrene-containing fatty acid

1986 ◽  
Vol 85 (1) ◽  
pp. 149-159
Author(s):  
E. Fibach ◽  
O. Morand ◽  
S. Gatt
Keyword(s):  
Fatty Acid ◽  
Ultraviolet Irradiation ◽  
Plasma Membranes ◽  
Incubation Temperature ◽  
Serum Proteins ◽  
Neutral Lipids ◽  
Cell Types ◽  
Dodecanoic Acid ◽  
A Cell ◽  
The Individual

Cells were incubated with 12-(1-pyrene)-dodecanoic acid (P12), a long-chain fatty acid to which a pyrene ring has been attached covalently. This acid was transported across the plasma membranes of cells and subsequently incorporated into their neutral lipids and phospholipids. Irradiation of these pyrene-containing cells for short periods (0.5-4 min) with ultraviolet light at 366 nm resulted in eventual cell death. Similar irradiation had no effect on cells that had not been exposed to P12. The time of the period of irradiation necessary for inducing the toxic process was related to the quantity of P12 incorporated, the latter being a function of the respective metabolic activity of the individual cell type. The degree of incorporation of P12 into a cell, and consequently its acquired sensitivity to killing by ultraviolet irradiation at 366 nm, was affected by the incubation temperature and addition of non-fluorescent fatty acid, albumin or other serum proteins. Different degrees of incorporation of P12 into various cell types were used for selective killing and elimination of cell populations by irradiation at 366 nm. The combined procedure of preincubation with P12 followed by ultraviolet irradiation thus permitted selection of cell types with a greater resistance to this procedure.

scholarly journals Formation and Remodeling of Inositolphosphoceramide during Differentiation of Trypanosoma cruzi from Trypomastigote to Amastigote

2003 ◽  
Vol 2 (4) ◽  
pp. 756-768 ◽  
Author(s):  
Maria Laura Salto ◽  
Laura E. Bertello ◽  
Mauricio Vieira ◽  
Roberto Docampo ◽  
Silvia N. J. Moreno ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Trypanosoma Cruzi ◽  
Palmitic Acid ◽  
Phospholipase C ◽  
Phospholipase A ◽  
Acidic Ph ◽  
Acid Hydrolase ◽  
Aureobasidin A

ABSTRACT Differentiation of Trypanosoma cruzi trypomastigotes to amastigotes inside myoblasts or in vitro, at low extracellular pH, in the presence of [3H]palmitic acid or [3H]inositol revealed differential labeling of inositolphosphoceramide and phosphatidylinositol, suggesting that a remodeling process takes place in both lipids. Using 3H-labeled inositolphosphoceramide and phosphatidylinositol as substrates, we demonstrated the association of at least five enzymatic activities with the membranes of amastigotes and trypomastigotes. These included phospholipase A1, phospholipase A2, inositolphosphoceramide-fatty acid hydrolase, acyltransferase, and a phospholipase C releasing either ceramide or a glycerolipid from the inositolphospholipids. These enzymes may be acting in remodeling reactions leading to the anchor of mature glycoproteins or glycoinositolphospholipids and helping in the transformation of the plasma membrane, a necessary step in the differentiation of slender trypomastigotes to round amastigotes. Synthesis of inositolphosphoceramide and particularly of glycoinositolphospholipids was inhibited by aureobasidin A, a known inhibitor of fungal inositolphosphoceramide synthases. The antibiotic impaired the differentiation of trypomastigotes at acidic pH, as indicated by an increased appearance of intermediate forms and a decreased expression of the Ssp4 glycoprotein, a characteristic marker of amastigote forms. Aureobasidin A was also toxic to differentiating trypomastigotes at acidic pH but not to trypomastigotes maintained at neutral pH. Our data suggest that inositolphosphoceramide is implicated in T. cruzi differentiation and that its metabolism could provide important targets for the development of antiparasitic therapies.

scholarly journals Cell-free acylation of rat brain myelin proteolipid protein and DM-20

1987 ◽  
Vol 246 (3) ◽  
pp. 611-617 ◽  
Author(s):  
T Yoshimura ◽  
D Agrawal ◽  
H C Agrawal
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Palmitic Acid ◽  
Rat Brain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Proteolipid Protein ◽  
Free System ◽  
Cell Free System ◽  
Selective Labelling ◽  
A Cell

Incubation of rat brain myelin with [3H]palmitic acid in the presence of ATP, CoA and MgCl2 or [14C]-palmitoyl-CoA in a cell-free system resulted in the selective labelling of ‘PLP’ [proteolipid protein; Folch & Lees (1951) J. Biol. Chem. 191, 807-817] and ‘DM-20’ [Agrawal, Burton, Fishman, Mitchell & Prensky (1972) J. Neurochem. 19, 2083-2089] which, after polyacrylamide-gel electrophoresis in SDS, were revealed by fluorography. These results provide evidence of the association of fatty acid-CoA ligase and acyltransferase in isolated myelin. Palmitic acid is covalently bound to PLP and DM-20, because 70 and 92% of the radioactivity was removed from proteolipid proteins after treatment with hydroxylamine and methanolic NaOH respectively. Incubation of myelin with [3H]palmitic acid in the absence of ATP, CoA, MgCl2, or all three, decreased incorporation of fatty acid into PLP to 3, 55, 18 and 2% respectively. The cell-free system exhibits specificity with respect to the chain length of the fatty acids, since myristic acid is incorporated into PLP at a lower rate when compared with palmitic and oleic acids. The acylation of PLP is an enzymic reaction, since (1) maximum incorporation of [3H]palmitic acid into PLP occurred at physiological temperatures and decreased with an increase in the temperature; (2) acylation of PLP with [3H]palmitic acid and [14C]palmitoyl-CoA was severely inhibited by SDS (0.05%); and (3) the incorporation of fatty acid and palmitoyl-CoA into PLP was substantially decreased by the process of freezing-thawing and freeze-drying of myelin. We have provided evidence that all of the enzymes required for acylation of PLP and DM-20 are present in isolated rat brain myelin. Acylation of PLP in a cell-free system with fatty acids and palmitoyl-CoA suggests that a presynthesized pool of non-acylated PLP and DM-20 is available for acylation.

Sperm exocytosis reconstructed in a cell-free system: evidence for the involvement of phospholipase C and actin filaments in membrane fusion

1995 ◽  
Vol 108 (6) ◽  
pp. 2525-2535 ◽  
Author(s):  
B. Spungin ◽  
I. Margalit ◽  
H. Breitbart
Keyword(s):  
Membrane Fusion ◽  
Phospholipase C ◽  
Present Report ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Intact Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Free Membrane

We used a cell-free system to study membrane fusion during sperm exocytosis (acrosome reaction). Extracted bovine sperm plasma and outer acrosomal membranes were labeled with chlorophyll a or DCY, respectively. The occurrence of membrane fusion is indicated by the ability of the probes to diffuse from one membrane species to another which is revealed by resonance energy transfer between the two probes. We have previously shown using this system that the requirement of capacitation for sperm exocytosis is retained in cell-free membrane fusion, and that the pH and calcium dependence of the cell-free fusion mimics those of exocytosis in intact cells. In the present report we further characterize the fusion of sperm membranes which we observe in our assay. Phosphoproteins and phospholipases were found to be involved in the membrane fusion step of sperm exocytosis. Protein kinases, phosphatases, and Gi-like proteins, while involved in exocytosis in intact cells, are not involved specifically in the membrane fusion step of exocytosis. The role of membrane bound F-actin in regulating membrane fusion was also studied using fluorescently labeled phalloidin. The results show that cortical F-actin has two roles in regulating sperm exocytosis. One is to form a scaffolding to hold phospholipase C at the membrane. It also functions as a physical barrier to membrane fusion which is removed by the increases in intracellular calcium and pH which precede fusion.

scholarly journals Phosphatidylinositol 4,5-Bisphosphate Regulates Two Steps of Homotypic Vacuole Fusion

2000 ◽  
Vol 11 (3) ◽  
pp. 807-817 ◽  
Author(s):  
Andreas Mayer ◽  
Dietrich Scheglmann ◽  
Stephen Dove ◽  
Alexandra Glatz ◽  
William Wickner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Phospholipase C ◽  
Daughter Cell ◽  
Free System ◽  
Cell Free System ◽  
Vacuole Fusion ◽  
A Cell ◽  
Phosphatidylinositol 4 ◽  
Phosphatidylinositol 3

Yeast vacuoles undergo cycles of fragmentation and fusion as part of their transmission to the daughter cell and in response to changes of nutrients and the environment. Vacuole fusion can be reconstituted in a cell free system. We now show that the vacuoles synthesize phosphoinositides during in vitro fusion. Of these phosphoinositides, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are important for fusion. Monoclonal antibodies to PI(4,5)P2, neomycin (a phosphoinositide ligand), and phosphatidylinositol-specific phospholipase C interfere with the reaction. Readdition of PI(4,5)P2 restores fusion in each case. Phosphatidylinositol 3-phosphate and PI(3,5)P2 synthesis are not required. PI(4,5)P2is necessary for priming, i.e., for the Sec18p (NSF)-driven release of Sec17p (α-SNAP), which activates the vacuoles for subsequent tethering and docking. Therefore, it represents the kinetically earliest requirement identified for vacuole fusion so far. Furthermore, PI(4,5)P2 is required at a step that can only occur after docking but before the BAPTA sensitive step in the latest stage of the reaction. We hence propose that PI(4,5)P2controls two steps of vacuole fusion.

Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

2019 ◽  
pp. 27-33
Author(s):  
YuE Kravchenko ◽  
SV Ivanov ◽  
DS Kravchenko ◽  
EI Frolova ◽  
SP Chumakov
Keyword(s):  
Phage Display ◽  
Selection Procedure ◽  
Free System ◽  
Phage Displays ◽  
High Affinity ◽  
Binding Domains ◽  
A Cell ◽  
Vhh Antibodies ◽  
Efficiency Of Selection ◽  
Selection Of

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

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