极长链脂肪酸的合成缺陷对酵母细胞膜的稳定性和多烯类药物敏感性的影响

【背景】脂肪酸延长酶家族参与脂肪酸代谢具有真核生物的高度保守性,且与膜脂的代谢密切相关。但细胞极长链脂肪酸(Very long-chain fatty acid,VLCFA)的合成缺陷对膜的稳定性及多烯类药物的敏感性影响并不完全明晰。【目的】探究细胞VLCFA延长酶ELO1、ELO2和ELO3的作用及功能。【方法】研究脂肪酸延长酶缺陷型elo1?、elo2?和elo3?对多烯类药物两性霉素B (Amphotericin B,AmB)、制霉菌素(Nystatin,Ny)及唑类硝酸益康唑(Econazolenitrate,Eco)的响应,检测不同酵母细胞的麦角固醇,检测其对Na+的响应及胞内钠钾离子水平。【结果】发现细胞VLCFA延长酶ELO2和ELO3缺陷后对AmB高度敏感;VLCFA延长酶缺陷突变株elo2?和elo3?对其它多烯类药物Ny及唑类药物Eco也十分敏感;细胞膜不饱和脂肪酸增加也会改变膜的稳定性,实验结果表明外源油酸(Oleic acid,OLA)增加了elo2?和elo3?突变体的AmB敏感性;相对野生型BY4741和elo1?,缺陷菌株elo2?和elo3?中麦角固醇的含量有显著下降;钠钾离子平衡是维护细胞正常生理的必要条件,也是检测细胞膜稳定性的重要参数,发现VLCFA的合成缺陷菌株对高浓度的NaCl比野生型菌株更敏感,使用ICP-AES检测不同浓度AmB胁迫下细胞内钠钾离子水平,也显示VLCFA延长酶缺陷菌株中,钠水平表现出上升趋势,并且细胞内钾含量明显降低。【结论】细胞VLCFA的合成缺陷会导致细胞膜更脆弱、稳定性下降,从而提高真菌对多烯类药物的敏感性,也表明脂肪酸延长酶是潜在的抗真菌治疗靶点。     

Amphotericin B-Induced Carbohydrate Changes on the Trypanosoma cruzi Surface Membrane

ABSTRACT Changes in the cell surface carbohydrates of Trypanosoma cruzi epimastigotes induced by Amphotericin B (AmB) were assessed by chemical methods and by agglutination assay employing a panel of highly purified lectins of various sugar specificities, Escherichia coli K12 with mannose-sensitive fimbriae was also used as an agglutination probe. Amphotericin B caused a decrease in the total carbohydrate content of all glycoconjugate fractions isolated. Exposure to AmB strongly affected the mannose/galactose ratio (1:5) in the CHCI3/methanol/H2O soluble fraction. These sugars in 1.4:1 ratio were the major hexose components of control cells. The decrease in the mannose content (48 to 15%) after AmB treatment agrees with the marked decrease in the T. cruzi cell surface receptors for fimbriated E. coli K12. Also, an increase in the galactose content (74%) as compared with control cells (34%) is in agreement with the peanut agglutinin and Euonymus europaeus lectins agglutination results. Differences in the cell surface carbohydrates induced by AmB could be associated with alterations in the membrane structure and organization.     

Amphotericin B-induced carbohydrate changes on the Trypanosoma cruzi surface membrane.

Changes in the cell surface carbohydrates of Trypanosoma cruzi epimastigotes induced by Amphotericin B (AmB) were assessed by chemical methods and by agglutination assay employing a panel of highly purified lectins of various sugar specificities. Escherichia coli K12 with mannose-sensitive fimbriae was also used as an agglutination probe. Amphotericin B caused a decrease in the total carbohydrate content of all glycoconjugate fractions isolated. Exposure to AmB strongly affected the mannose/galactose ratio (1:5) in the CHCl3/methanol/H2O soluble fraction. These sugars in 1.4:1 ratio were the major hexose components of control cells. The decrease in the mannose content (48 to 15%) after AmB treatment agrees with the marked decrease in the T. cruzi cell surface receptors for fimbriated E. coli K12. Also, an increase in the galactose content (74%) as compared with control cells (34%) is in agreement with the peanut agglutinin and Euonymus europaeus lectins agglutination results. Differences in the cell surface carbohydrates induced by AmB could be associated with alterations in the membrane structure and organization.     

Concentration dependent dual effect of the monolauryl ester of sucrose on the antifungal activity and absorption spectra of amphotericin B (Fungizone)

A mild detergent, the monolauryl ester of sucrose (LS), at concentrations which ranged from 0.008 to 0.03%, enhanced amphotericin B (AmB) toxicity against Saccharomyces cerevisiae and Cryptococcus neoformans cells. At higher concentrations, 0.06 to 2.5%, LS inhibited AmB effects on these two fungi. We analyzed changes in the absorption spectrum of AmB induced by LS at these two concentration ranges by comparing ratios (R values) of AmB absorbance at 409 nm, the wavelength characteristic of non-aggregated (monomeric) AmB, to absorbance at 328 nm, the wavelength characteristic of aggregated AmB. Low concentrations of LS caused a decrease in R, whereas the higher LS concentrations increased R. Therefore, LS had concentration-dependent dual effects on the antifungal activity of AmB which correlated with shifts in the physical states of AmB. The concentration range of LS required to inhibit the antifungal effects of AmB was about 1000-fold greater than the previously reported concentrations required to inhibit AmB toxicity to mammalian cells (Gruda, I., Gauthier, E., Elberg, S., Brajtburg, J. and Medoff, G. (1988) Biochem. Biophys. Res. Commun. 154, 954-958). This suggests that LS may be a useful agent to decrease AmB toxicity to host cells without affecting the antifungal effects. Moreover, increase in AmB toxicity induced by low concentrations of LS suggests the possibility that synergistic interaction between fatty acid esters and polyene antibiotics may have therapeutic value.     

Amphotericin B Membrane Action: Role for Two Types of Ion Channels in Eliciting Cell Survival and Lethal Effects

The formation of aqueous pores by the polyene antibiotic amphotericin B (AmB) is at the basis of its fungicidal and leishmanicidal action. However, other types of nonlethal and dose-dependent biphasic effects that have been associated with the AmB action in different cells, including a variety of survival responses, are difficult to reconcile with the formation of a unique type of ion channel by the antibiotic. In this respect, there is increasing evidence indicating that AmB forms nonaqueous (cation-selective) channels at concentrations below the threshold at which aqueous pores are formed. The main foci of this review will be (1) to provide a summary of the evidence supporting the formation of cation-selective ion channels and aqueous pores by AmB in lipid membrane models and in the membranes of eukaryotic cells; (2) to discuss the influence of membrane parameters such as thickness fluctuations, the type of sterol present and the existence of sterol-rich specialized lipid raft microdomains in the formation process of such channels; and (3) to develop a cell model that serves as a framework for understanding how the intracellular K(+) and Na(+) concentration changes induced by the cation-selective AmB channels enhance multiple survival response pathways before they are overcome by the more sustained ion fluxes, Ca(2+)-dependent apoptotic events and cell lysis effects that are associated with the formation of AmB aqueous pores.     

Effect of diet on hyperthermia-induced cell lethality and prostaglandin release.

Hyperthermia-induced cell lethality is thought to be mediated through injury to the cell membrane. Membrane perturbation results in the release of prostaglandins (PG) and leukotrienes (LT). These compounds are potent biological mediators and may modify the tumor microenvironment and therapeutic efficacy. Membrane composition and PG/LT release are influenced by the dietary fatty acids. The relationship between these variables and response to hyperthermia was examined in vitro using murine P388 leukemia cells grown as an ascites in mice provided either saturated fatty acid diet (SFA; 16% beef tallow) or unsaturated fatty acid diet (UFA; 16% safflower oil). Cells were harvested and exposed in vitro to either 37 degrees C or 43.5 degrees C for periods up to 2 hours. Hyperthermic exposure for 2 hours resulted in 40% cell lethality in SFA cells and 55% in UFA cells. The phospholipid and total cholesterol content was higher (33% and 50% respectively) in the UFA versus the SFA cells. Hyperthermia produced a six-fold increase in prostaglandin E2 PGE2 release by SFA cells and a 4.5-fold increase by UFA cells. No LTC4 was detected. Alteration of dietary fat affects cell lethality and PG release following hyperthermic treatment. The increase in phospholipid and cholesterol content of UFA cells may be a response to reduced membrane fluidity.     

Fatty acid composition in native and cultured human endothelial cells

Endothelial cells from human umbilical veins were isolated by collagenase treatment. Cells were cultured in the presence of either 20% fetal bovine serum (FBS) or 20% human serum (HS). At confluency, endothelial cell lipids were labeled with tracer concentrations of tritiated arachidonic acid, then extracted and separated into lipid subclasses by thin layer chromatography. The fatty acid composition of each lipid class was determined by glass capillary gas-liquid chromatography analysis and compared to that of cells freshly isolated from the cord (NC cells). The fatty acid compositions differed only in phospholipids. Polyunsaturated fatty acids (PFAs), arachidonic, and linoleic acids were depleted in FBS cell phospholipids and replaced by both stearic and oleic acids. No significant difference could be observed between NC cell and HS cell phospholipids. We conclude that PFAs might be decreased in FBS cells because of the relative paucity of PFAs in FBS as compared to HS. It seems therefore more convenient to cultivate endothelial cells in the presence of HS, especially in respect to their phospholipid content of arachidonic acid, which is the physiological reservoir for prostacyclin synthesis.     

Role of triglycerides in endothelial cell arachidonic acid metabolism

Arachidonic acid was incorporated into triglycerides by cultured bovine endothelial cells in a time- and concentration-dependent manner. At 75 microM or higher, more arachidonic acid was incorporated into triglycerides than into phospholipids. The triglyceride content of the cells increased as much as 5.5-fold, cytoplasmic inclusions appeared, and arachidonic acid comprised 22% of the triglyceride fatty acids. Triglyceride turnover occurred during subsequent maintenance culture; there was a 60% decrease in the radioactive arachidonic acid contained in triglycerides and a 40% decrease in triglyceride content in 6 hr. Most of the radioactivity was released into the medium as free fatty acid. The turnover of arachidonic acid, but not oleic acid in cellular triglycerides, decreased when supplemental fatty acid was added to the maintenance medium. Incorporation and turnover of radioactive arachidonic acid in triglycerides also was observed in human skin fibroblasts, 3T3-L1 cells, and MDCK cells. Other fatty acids were incorporated into triglycerides by the endothelial cells; the amounts after a 16-hr incubation with 50 microM fatty acid were 20:3 greater than 20:4 greater than 18:1 greater than 18:2 greater than 22:6 greater than 16:0 greater than 20:5. These findings indicate that triglyceride formation and turnover can play a role in the fatty acid metabolism of endothelial cells and that arachidonic acid can be stored in endothelial cell triglycerides.     

Amphotericin B both inhibits and enhances T-cell proliferation: inhibitory effect is mediated through H(2)O(2) production via cyclooxygenase pathway by macrophages

Amphotericin B (AmB) has been shown to have both immunosuppressive and -enhancing effects, making its precise nature of action enigmatic. In the present study, we found that AmB inhibited concanavalin A (Con A)-induced T cell proliferation if added within first 30 min of stimulation, after which inhibition began to diminish rapidly. However, AmB did not inhibit T-cell proliferation induced by a combination of PMA and ionomycin. AmB inhibition of Con A-induced proliferation was completely overcome by cyclooxygenase inhibitor ibuprofen ([alpha-methyl-4-(isobutyl)phenylacetic acid]) and H(2)O(2) scavenger catalase. In fact, in the presence of ibuprofen and catalase, AmB enhanced, instead of suppressing, Con A-induced proliferation in a dose-dependent way. The effect of catalase was limited to the removal of extracellular H(2)O(2) only, as the enzyme did not enter the cells. AmB stimulated H(2)O(2) production by macrophages, but not by a lymphocyte population, which was inhibited by ibuprofen. Our T-cell preparation contained about 3% macrophages, and AmB inhibition of proliferation was further pronounced by increasing the macrophage number by as little as 1%. Finally, AmB inhibition of Con A-induced T-cell proliferation was completely overcome by 2-mercaptoethanol. On the basis of these results, we suggest that AmB stimulates H(2)O(2) production by macrophages through the activation of the cyclooxygenase pathway of arachidonate metabolism. H(2)O(2) then inhibits Con A-induced T-cell proliferation by interfering with an early step of the T-cell receptor signaling pathway through the oxidative modification of some signaling proteins. Our results also show that AmB enhances T-cell proliferation, which can be seen only after blocking its inhibitory effect.     

Mechanism of Action of Amphotericin B at the Cellular Level. Its Modulation by Delivery Systems

Abstract

Amphotericin B (AmB) is the drug of choice for the treatment of systemic fungal infections, but its use is hampered by its severe side-effects. A better understanding of its mechanisms of action is needed to develop new AmB formulations with an optimal selectivity between fungal and mammalian cells. Interactions between AmB and cells depend on the concentration of the drug. Stimulatory effects, modulation of the activity of immunocompetent cells and inhibition of yeast adherence are early events that precede the actual cellular toxicity. If membrane permeability alterations are considered to be the first toxic step, cell death results not only from osmotic imbalances, but also from additional mechanisms, such as lipid peroxidation, inhibition of membrane enzymes and blockade of endocytosis. The selectivity between fungal and mammalian cells takes its origin from the difference in the nature of the membrane sterol: ergosterol in fungi, cholesterol in mammalian cells. Transmembrane pores result from different mechanisms according to the sterol: ergosterol-AmB complexes are formed from monomelic AmB in solution, which is the only form present in aqueous medium at low AmB concentrations, whereas pores in the cholesterol containing membrane result from the adsorption onto the membrane surface of aqueous self-associated AmB, that appears in medium when AmB concentration increases. The liposomes seem to sequester AmB in a manner which makes it unavailable for mammalian cells, but maintains its access to fungal cells. The transfer of AmB by progressive diffusion of free AmB through the aqueous phase could explain the enhancement of the therapeutic index of the drug by liposomes, since the induction of pore formation needs a higher threshold of drug for host cell than for fungal cell membranes. The closed structure of the vehicle is not required to enhance the selectivity of the drug: esters of sucrose or high concentration of sodium deoxycholate afford a protective effect as well. Macrophages, after phagocytosis of liposomal AmB, may be considered as a reservoir of AmB, from which the drug is progressively released. Finally, the strong binding of AmB to the delivery system reduces the amount of drug bound to serum components and thus the endocytosis of AmB through the LDL receptor, resulting in lower toxicity.     

Effect of aggregation of amphotericin B on lysophosphatidylcholine micelles as related to its complex formation with cholesterol or ergosterol

The effect of aggregation of amphotericin B (AmB), as well as the complex formation of AmB with cholesterol or ergosterol, was investigated in micelles and vesicles. AmB in lysophosphatidylcholine (LPC) micelles adopted a more favorable monomeric form than that in other drug formulations. At an LPC/AmB ratio of 200, AmB existed only in monomeric form. Such monomeric behavior is likely dependent upon the fluidity and size of the micelles. In LPC micelles composed of 90% monomeric AmB, AmB-ergosterol complex formation occurred with an increase in the sterol concentration, but the complex formation of AmB-cholesterol was slight. On the other hand, in LPC micelles composed of 40% monomeric AmB, the complex formation of AmB-cholesterol as well as AmB-ergosterol was extensive. These results suggest that the complex formation of AmB with both sterols is highly dependent upon the aggregated state of AmB. In addition, using monolayers, mixtures of AmB/LPC/ergosterol were became more stable with rising temperature, while the stability of mixtures of AmB/LPC/cholesterol remained unchanged, implying that complex formation of AmB with cholesterol is different from that of AmB with ergosterol.     

Effect of docosahexaenoic acid on membrane fluidity and function in intact cultured Y-79 retinoblastoma cells.

Considerable metabolic energy is expended in ensuring that membranes possess a characteristic fatty acid composition. The nature of the specific requirement of the retina for high levels of docosahexaenoic acid (DHA) is as yet undefined. Previous work has speculated that DHA is required to maintain the fluid nature and permeability necessary for optimal retinal function. Cultured Y-79 retinoblastoma cells were grown in serum-containing media with and without supplemental DHA. Resultant changes in membrane fluidity were assessed using fluorescent probes. No differences were observed in rotational probe mobility as assessed by fluorescence polarization despite a fourfold increase in cellular DHA content. Lateral probe mobility as assessed by pyrene eximer formation was significantly enhanced in DHA-supplemented cells. Both the DHA content and total fatty acid unsaturation index in retinoblastoma cells were directly correlated with membrane fluidity as reported by eximer formation (Pearson's rho = 0.96 and 0.92, respectively). DHA supplementation also resulted in a significant increase in cellular choline uptake. We speculate that the effect of DHA content on retinal function may be mediated by changes in membrane fluidity and associated enzyme and transport activities.     

Fatty acid composition in native and cultured human endothelial cells

Summary Endothelial cells from human umbilical veins were isolated by collagenase treatment. Cells were cultured in the presence of either 20% fetal bovine serum (FBS) or 20% human serum (HS). At confluency, endothelial cell lipids were labeled with tracer concentrations of tritiated arachidonic acid, then extracted and separated into lipid subclasses by thin layer chromatography. The fatty acid composition of each lipid class was determined by glass capillary gas-liquid chromatography analysis and compared to that of cells freshly isolated from the cord (NC cells). The fatty acid compositions differed only in phospholipids. Polyunsaturated fatty acids (PFAs), arachidonic, and linoleic acids were depleted in FBS cell phospholipids and replaced by both stearic and oleic acids. No significant difference could be observed between NC cell and HS cell phospholipids. We conclude that PFAs might be decreased in FBS cells because of the relative paucity of PFAs in FBS as compared to HS. It seems therefore more convenient to cultivate endothelial cells in the presence of HS, especially in respect to their phospholipid content of arachidonic acid, which is the physiological reservoir for prostacyclin synthesis. This work was supported by a grant from the Délégation Générale à la Recherche Scientifique et Technique, Paris, France (79.7.0091).     

Quantitative and qualitative analysis of the antifungal activity of allicin alone and in combination with antifungal drugs

The antifungal activity of allicin and its synergistic effects with the antifungal agents flucytosine and amphotericin B (AmB) were investigated in Candida albicans (C. albicans). C. albicans was treated with different conditions of compounds alone and in combination (allicin, AmB, flucytosine, allicin + AmB, allicin + flucytosine, allicin + AmB + flucytosine). After a 24-hour treatment, cells were examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM) to measure morphological and biophysical properties associated with cell death. The clearing assay was conducted to confirm the effects of allicin. The viability of C. albicans treated by allicin alone or with one antifungal drug (AmB, flucytosine) in addition was more than 40% after a 24-hr treatment, but the viability of groups treated with combinations of more than two drugs was less than 32%. When the cells were treated with allicin alone or one type of drug, the morphology of the cells did not change noticeably, but when cells were treated with combinations of drugs, there were noticeable morphological changes. In particular, cells treated with allicin + AmB had significant membrane damage (burst or collapsed membranes). Classification of cells according to their cell death phase (CDP) allowed us to determine the relationship between cell viability and treatment conditions in detail. The adhesive force was decreased by the treatment in all groups compare to the control. Cells treated with AmB + allicin had a greater adhesive force than cells treated with AmB alone because of the secretion of molecules due to collapsed membranes. All cells treated with allicin or drugs were softer than the control cells. These results suggest that allicin can reduce MIC of AmB while keeping the same efficacy.     

The growth inhibitory effect of conjugated linoleic acid on a human hepatoma cell line, HepG2, is induced by a change in fatty acid metabolism, but not the facilitation of lipid peroxidation in the cells

We investigated the growth inhibitory effect of conjugated linoleic acid (CLA) on HepG2 (human hepatoma cell line), exploring whether the inhibitory action occurs via lipid peroxidation in the cells. When the cells were incubated up to 72 h with 5-40 microM of CLA (a mixture of 9c,11t-18:2 and 10t,12c-18:2), cell proliferation was clearly inhibited in a dose and time dependent manner but such an inhibition was not confirmed with linoleic acid (LA). In order to evaluate the possible contribution of lipid peroxidation exerted by CLA to cell growth inhibition, alpha-tocopherol (5-20 microM) and BHT (1-10 microM) as potent antioxidants were added to the medium with CLA (20 microM), which did not restore cell growth at all. Furthermore, after 72 h incubation, the membranous phospholipid hydroperoxide formation in the CLA-supplemented cells was suppressed respectively to 25% and 50% of that in LA-supplemented cells and control cells. No difference was observed by a conventional lipid peroxide assay, the TBA test, between CLA-supplemented cells and LA-supplemented cells. Although the cellular lipid peroxidation was not stimulated, lipid contents (triacylglycerol, total cholesterol and free cholesterol) and fatty acid contents (palmitic acid, palmitoleic acid and stearic acid) markedly increased in CLA-supplemented cells compared with LA-supplemented and control cells. Moreover, supplementation with 20 microM LA and 20 microM arachidonic acid profoundly interfered with the inhibitory effect of CLA in HepG2. These results suggest that the growth inhibitory effect of CLA on HepG2 is due to changes in fatty acid metabolism but not to lipid peroxidation.     

Lipid Metabolism of Manganese-deficient Algae: I. Effect of Manganese Deficiency on the Greening and the Lipid Composition of Euglena Gracilis Z 1

The growth of photoautotrophic Euglena gracilis Z is strongly inhibited by manganese deficiency, whereas chlorophyll formation is not appreciably affected. The galactosyldiglyceride content of the manganese-deficient photo-autotrophic Euglena was about 40% lower on the basis of either chlorophyll content or dry weight. When dark-grown cultures of Euglena were grown photoheterotrophically in light sufficient for the greening of the cells, or photosynthesis, manganese deficiency resulted in a reduction of the cellular content of chlorophyll and galactosyldiglycerides to 40% of control values, indicating interference with chloroplast formation. The fatty acids of the photoheterotrophic manganese-deficient cells were mainly saturated, with an unusual accumulation (about 45%) of the total fatty acids) of myristic acid. In spite of this, the galactosyldiglycerides contain mainly unsaturated fatty acids. Ninety per cent of the fatty acids of the monogalactosyldiglyceride are unsaturated, including large amounts of alpha-linolenic acid. The ratio of chlorophyll to galactosyldiglyceride content of the cells was remarkably constant at all manganese deficiency levels.     

Effect of long-chain fatty acids in the culture medium on fatty acid composition of WEHI-3 and J774A.1 cells

As a first step in determining the mechanism of action of specific fatty acids on immunological function of macrophages, a comparative study of the effect of long-chain polyunsaturated fatty acids (PUFA) in the medium was conducted in two macrophage cell lines, J774A.1 and WEHI-3. The baseline fatty-acid profiles of the two cell lines differed in the % distribution of saturated (SFA) and unsaturated fatty acids (UFA). J774A.1 cells had a higher % of SFA (primarily palmitic acid) than WEHI-3 cells. Conversely, WEHI-3 cells had a higher % of UFA (primarily oleic acid) than J774A.1 cells. Neither cell line had detectable amounts of alpha-linolenic acid (ALA) or eicosapentaenoic acid (EPA). The most abundant polyunsaturated fatty acid in both cells lines was arachidonic acid (AA). The efficiency of transport of fatty acids from the medium to the macrophages by two delivery vehicles (BSA complexes and ethanolic suspensions) was compared. Overall, fatty acids were transported satisfactorily by both delivery systems. Alpha-linolenic acid and doscosahexenoic acid (DHA) were transported more efficiently by the ethanolic suspension system. Linoleic acid (LA) was taken up more completely than ALA, and DHA was taken up more completely than EPA by both cell cultures and delivery systems. A dose-response effect was demonstrated for LA, ALA, EPA and DHA in both J774A.1 and WEHI-3 cells. Addition of polyunsaturated fatty acids (PUFA) to the cell cultures modified the total lipid fatty acid composition of the cells. The presence of ALA in the culture medium resulted in a significant decrease in AA in both cell lines. The omega-3/omega-6 fatty acid ratio (omega-3/omega-6), polyunsaturated/saturated fatty acid ratio (P/S), and unsaturation index (UI) increased directly with the amount of PUFA and omega-3 fatty acid provided in the medium. The results indicate that the macrophage cell lines have similar, but not identical, fatty acid profiles that may be the result of differences in fatty acid metabolism. These distinctions could in turn produce differences in immunological function. The ethanol fatty-acid delivery system, when compared with the fatty acid-BSA complex system, is preferable for measurement of dose-response effects, because the cellular fatty acid content increased in proportion to the amount of fatty acid provided in the medium. Similar dose-response results were observed in a previous in vivo study using flaxseed, rich in ALA, as a source of PUFA.     

Increased calcium permeability is not responsible for the rapid lethal effects of amphotericin B on Leishmania sp

The mode of action of the polyene antibiotic amphotericin B (AmB), the drug of choice for the treatment of systemic fungal infections and visceral leishmaniasis, is still unclear. An increase in intracellular Ca2+ concentration [( Ca2+]i), toxic in many cases, has been postulated as a possible lethal mechanism for AmB. Cell permeabilization to ethidium bromide (EB) was used as a criterion of viability. Kinetics of the DNA-EB fluorescent complex formation was studied in ergosterol-containing Leishmania promastigotes. Intracellular Ca2+ concentration was measured using quin-2 fluorescence in parallel aliquots. It is shown in this work that AmB can act as an efficient Ca2+ ionophore. However, the rapid permeabilization effect induced by AmB on these cells was not dependent on an increase in [Ca2+]i. On the contrary, it was found that leishmanicidal effect of AmB was enhanced in the absence of external calcium. Furthermore, A23187 a Ca2+ ionophore did not provoke cell permeabilization to EB.     

Modification of the fatty acid composition of individual phospholipids and neutral lipids after infection of the simian erythrocyte by Plasmodium knowlesi

Using capillary gas-liquid chromatography, we have analyzed the alteration in the total fatty acid, phospholipid and neutral lipid compositions of the monkey erythrocyte, after infection by the malarial parasite Plasmodium knowlesi. Data based on fatty acid quantitation show that the phospholipid composition is altered, with particularly large increases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the most abundant phospholipids in normal and P. knowlesi-schizont-infected cells. Unesterified fatty acids were found to be less abundant in infected cells. The total fatty acid content of the cell is increased 6-fold during infection, and total fatty acid composition is also changed: the infected cells are richer in palmitate (+23%), oleate (+29%) and linoleate (+89%), but contained less stearate (-27%) and arachidonate (-40%). The determination of the fatty acid composition of individual phospholipids, neutral lipids and unesterified fatty acids showed that choline-containing phospholipids (PC and sphingomyelin) were not as altered in their fatty acid pattern as anionic phospholipids (PE, phosphatidylserine (PS) and phosphatidylinositol (PI) and lysophosphatidylcholine (lysoPC). Specific alterations in the fatty acid compositions of individual phospholipids were detected, whereas the rise in linoleic acid was the only change during infection that was recovered in each phospholipid (except PC), neutral lipid and unesterified fatty acids. The fatty acid composition of the neutral lipids and unesterified fatty acids was particularly modified: the only rise in arachidonic acid level was observed in these lipid classes after infection. The total plasmalogen level of the erythrocyte is decreased in infected cells (-60%), but their level is increased in PI.     

Fatty acid profiles of cells from the insect cell line iprl-21 (Spodoptera frugiperda) and of the tissue culture medium after repeated use

Summary When IPL-1 medium was used for three serial incubations of cells of the IPRL-21 insect cell line (Spodoptera frugiperda, J. E. Smith) at least 23 fatty acids were identified from the media and/or from the cells. During the first incubation only negligible changes occurred in the total fatty acid content of the medium, but after the second and third incubations the total content decreased. Seven of the 23 fatty acids (palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and arachidonic acids) comprised 92% of the total fatty acid content, but the specific concentrations varied after each 7-day incubation. During the first incubation, the concentration of the monoene fatty acids increased, and the concentration of the more highly unsaturated fatty acids decreased. During the second and third serial incubations, the specific concentrations of all fatty acids decreased, with the exception of palmitoleic acid. These changes in the total fatty acid content and in the specific concentration of individual fatty acids in the cell indicated uptake of fatty acids from the medium and/or cellular lipid biosynthesis. The fatty acid content of the cells differed during the active growth phase and the stationary phase.