Role Of Plasma Membrane Research Articles

Impedance Spectroscopic Investigation of the Bilayer Lipid Membranes Formed from the Phosphatidylserine–Ceramide Mixture

Electrochemical impedance spectroscopy was used for the study of two-component lipid membranes. Phosphatidylserine and ceramide were to be investigated because they play an important biochemical role in cell membranes. The research on biolipid interaction was focused on a quantitative description of processes that take part in a bilayer. Assumed models of interaction between amphiphilic molecules and the equilibria that take place there were described by mathematical equations for the studied system. The possibility of complex formation for a two-component system forming bilayers was assumed, which could explain the deviation from the additivity rule. The molecular area and the equilibrium constant of the complex were determined.

Physicochemical analysis of phosphatidylcholine-ceramide system in bilayer lipid membranes.

Electrochemical impedance spectroscopy was used for the study of two-component lipid membranes. Phosphatidylcholine and ceramide were to be investigated, since they play an important biochemical role in cell membranes. The research on biolipid interaction was focused on quantitative description of processes that take part in a bilayer. Assumed models of interaction between amphiphilic molecules and the equilibria that take place there were described by mathematical equations for the studied system. The possibility of complex formation for two-component system forming bilayers was assumed that could explain the deviation from additivity rule. Equilibria were described by mathematical equations that were further verified experimentally. The determined values of parameters (stability constant, molecular area of complex, capacitance and conductance of the lipid membranes formed from molecules and complexes) were used for calculation of model curves. The comparison of model curves and experimental points verified the assumed model.

PH signature for the responses of arbuscular mycorrhizal fungi to external stimuli

Environmental and developmental signals can elicit differential activation of membrane proton (H+) fluxes as one of the primary responses of plant and fungal cells. In recent work,1 we could determine that during the presymbiotic growth of arbuscular mycorrhizal (AM) fungi specific domains of H+ flux are activated by clover root factors, namely host root exudates or whole root system. Consequently, activation on hyphal growth and branching were observed and the role of plasma membrane H+-ATPase was investigated. The specific inhibitors differentially abolished most of hyphal H+ effluxes and fungal growth. As this enzyme can act in signal transduction pathways, we believe that spatial and temporal oscillations of the hyphal H+ fluxes could represent a pH signature for both early events of the AM symbiosis and fungal ontogeny.Addendum to: Ramos AC, Façanha AR, Feijó JA. Proton (H+) flux signature for the presymbiotic development of the arbuscular mycorrhizal fungi. New Phytol 2008; 178:177-88.

Multiply Methyl‐Branched Fatty Acids and Diacids in the Polar Lipids of a Microaerophilic Subsurface Microbial Community

A previously unreported series of di- and tri-methylated fatty acids, as well as saturated and monounsaturated diacids were identified in polar lipids isolated from environmental subsurface sediment samples. Mechanisms are proposed for their formation, but their origin and role in cell membranes remains unknown.

Free fatty acid transport across adipocytes is mediated by an unknown membrane protein pump

The role of cell membranes in regulating the flux of long chain free fatty acids (FFA) into and out of adipocytes is intensely debated. Four different membrane proteins including, FABPpm, CD36/FAT, caveolin-1, and FATP have been identified as facilitating FFA transport. Moreover, CD36 and caveolin-1 are also reported to mediate transport in conjunction with lipid rafts. The principal evidence for these findings is a correlation of the level of FFA uptake with the expression level of these proteins and with the integrity of lipid rafts. The 3T3-L1 and 3T3-F442A cell lines in their preadipocyte states reveal little or no expression of these proteins and correspondingly low levels of uptake. Here we have microinjected the adipocyte and preadipocyte cell lines with ADIFAB, the fluorescent indicator of FFA. The ADIFAB fluorescence allowed us to monitor the intracellular unbound FFA concentration during FFA influx and efflux. We show that these measurements of transport, in contrast to FFA uptake measurements, correlate neither with expression of these proteins nor with lipid raft integrity in preadipocytes and adipocytes. Transport characteristics, including the generation of an ATP-dependent FFA concentration gradient, are virtually identical in adipocytes and preadipocytes. We suggest that the origin of the discrepancy between uptake and our measurements is that most of the FFA transported into the cells is lost during the uptake but not in the transport protocols. We conclude that long chain fatty acid transport in adipocytes is very likely mediated by an as-yet-unidentified membrane protein pump.

Involvement of NADPH oxidase in sulfur dioxide-induced oxidative stress in plant cells

Bisulfite, a major form of SO2 in aqueous phase of apoplast, may reduce photosynthesis rate and thereby crop yield through inducing reactive oxygen species (ROS). In this study, ROS production was directly detected in a living cell of leaf of spinach (Spinacia oleracea L.) using laser scanning confocal microscopes with the assistance of the fluorescence probe dichlorofluorescin diacetate (H2DCF-DA). Results showed that, under bisulfite stress, a large quantity of ROS indicated by DCF fluorescence was produced in epidermic tissue. The role of plasma membrane (PM) NADPH oxidase in bisulfite-induced ROS production was also investigated. Treatment with bisulfite resulted in a significant increase in the content of ROS and the activity of PM NADPH oxidase in spinach leaves. The effects caused by bisulfite were inhibited pronouncedly by pretreatment with two widely used NADPH oxidase inhibitors (diphenyleneiodonium and quinacrine). Moreover, the change patterns of the bisulfite-induced increase and inhibitor-caused decrease in the two parameters were quite similar. Additionally, only a small amount of ROS could be observed on in vitro chloroplasts under bisulfite stress. Based on all the results, we conclude that ROS is involved in bisulfite-induced stress, and the bisulfite-induced enhancements in levels of ROS originate mainly from PM NADPH oxidase.

Biosynthesis of polyunsaturated fatty acids in lower eukaryotes

Polyunsaturated fatty acids have important structural roles in cell membranes. They are also intermediates in the synthesis of biologically active molecules such as eicosanoids, which mediate fever, inflammation, blood pressure and neurotransmission. Arachidonic and docosahexaenoic acids are essential components of brain tissues and, through their involvement in the development of neural and retinal functions, important dietary nutrients for neonatal babies. Lower eukaryotes are particularly rich in C20-22 polyunsaturated fatty acids. Fungi and marine microalgae are currently used to produce nutraceutic oils. Other protists and algae are being studied because of the variability in their enzymes involved in polyunsaturated fatty acid biosynthesis. Such enzymes could be used as source for the production of transgenic organisms able to synthesize designed oils for human diet or, in the case of parasitic protozoa, they might be identified as putative chemotherapeutic targets. Polyunsaturated fatty acids can be synthesized by two different pathways: an anaerobic one, by using polyketide synthase related enzymes, and an aerobic one, which involves the action of elongases and oxygen dependent desaturases. Desaturases can be classified into three main types, depending on which of the consecutive steps of polyunsaturated fatty acid synthesis they are involved with. The enzymes may be specialized to act on: saturated substrates (type I); mono- and di-unsaturated fatty acids by introducing additional double bonds at the methyl-end site of the existing double bonds (type II); or the carboxy half ('front-end') of polyunsaturated ones (type III). Type III desaturases require the alternating action of elongases. A description of the enzymes that have been isolated and functionally characterized is provided, in order to highlight the different pathways found in lower eukaryotes.

The Coexistence of the Oxidative and Reductive Systems in Roots: The Role of Plasma Membranes

Different components of the plasma membrane bound and associated redox system, which participate in the energy transfer from the predominantly reducing intercellular environment to the extracellular oxidizing environment, are reviewed. Special attention is given to plant root cells. An analysis of the plasma membrane-associated redox components, such as the cytochromes, quinones, and different types of oxidoreductases (dehydrogenases, oxidases, peroxidases, and superoxide dismutases), is made, as well as their coupling with naturally occurring extracellular substrates, such as oxygen and its reactive forms, phenols, ascorbate, nitrate, ferric ion, and organic acids. The participation of different free radical species in most of the plasma membrane-bound redox reactions is documented.

Effects of an oxidative stress on human hemoglobin: a multiwavelength visible spectrometry study

This study was carried out to investigate hemoglobin behavior and the role of cell membrane during oxidative stress of human red blood cells induced by a water-soluble radical initiator, 2,2′-azobis(amidino-propane) hydrochloride (AAPH) and compare the observed data to the one obtained with purified human haemoglobin solution. The different forms of hemoglobin were identified and quantified by multiwavelength visible spectrometry using multiple linear regression analysis. Hemolysis was quantified by the Drabkin method. Oxidative stress on purified hemoglobin solutions induced an early formation of Hb +. In intact erythrocytes, no modified form of haemoglobin was found. Only the hemoglobin released by hemolysis in the extracellular medium was notified in the same way as purified haemoglobin. Thus, the cell membrane appears to protect intraerythrocytic hemoglobin toward an extracellular oxidative stress. Oxidative stress-induced by hemolysis does not seem to be due to changes in intraerythrocytic hemoglobin forms.

Lactococcal 936-Species Phage Attachment to Surface of Lactococcus lactis

The interactions of the 936-species phages sk1, jj50, and 64 with the cell surface of Lactococcus lactis LM0230 were analyzed. Cell envelopes (walls + plasma membrane), cell wall, or plasma membrane from L. lactis ssp. lactis LM0230 each inactivated the phages in vitro. However, other 936-species phages kh and P008, which do not infect strain LM0230, were not inactivated by any of the subcellular fractions. Treating cell walls or plasma membrane with the cell wall hydrolase mutanolysin eliminated inactivation of phage sk1. This suggested that intact cell wall fragments were required for inactivation. A role for plasma membrane in phage sk1 inactivation was further investigated. Boiling, washing in 2 M KCl, 8 M urea, or 0.1 M Na2CO3/pH 11, or treating the plasma membrane with proteases did not reduce adsorption or inactivation of phage. Adding lipoteichoic acid or antibodies to lipoteichoic acid did not reduce inactivation of phage in a mixture with membrane, suggesting that lipoteichoic acid was not involved. Inactivation by envelopes or cell wall correlated with ejection of DNA from the phage sk1 capsid. Although calcium is required for plaque formation, it was not required for adsorption, inactivation, or ejection of phage DNA by envelopes or cell wall. The results suggest that at least for phages sk1, jj50, and 64, adsorption and phage DNA injection into the host does not require a host membrane protein or lipoteichoic acid, and that cell wall components are sufficient for these initial steps of phage infection.

Chlamydia pneumoniae infected macrophages exhibit enhanced plasma membrane fluidity and show increased adherence to endothelial cells

Chlamydia pneumoniae, an intracellular prokaryote, is known to have requirement for some lipids which it is incapable of synthesizing, and these lipids have important fluidizing roles in plasma membrane. We decided to examine if the trafficking of these lipids to C. pneumoniae alters the physicochemical properties of macrophage plasma membrane, affects the expression of genes and proteins of enzymes associated with metabolism of some of these lipids and assess if Ca2+ signaling usually induced in macrophages infected with C. pneumoniae modulates the genes of these selected enzymes. Chlamydia pneumoniae induced the depletion of macrophage membrane cholesterol, phosphatidylinositol and cardiolipin but caused an increase in phosphotidylcholine resulting in a relative increase in total phospholipids. There was increased membrane fluidity, enhanced macrophage fragility and heightened adherence of macrophages to endothelial cells despite the application of inhibitor of adhesion molecules. Also, there was impairment of macrophage 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase gene and protein expression independent of Ca2+ signaling, while phospholipase C gene and protein were up-regulated in a manner minimally dependent on Ca2+ signaling. The implications of these findings are that macrophages infected with C. pneumoniae have altered membrane physicochemical characteristics which may render them atherogenic.

Effect of Soluble Calcium and IBA Treatments on Fruit Characteristics and Browning of Blossom End Part in `Fuyu' Sweet Persimmon

Sweet persimmon, `Fuyu', is the major cultivar for MA storage, but browning of blossom end part (BBEP) and darkening are occurring during storage and decrease fruit qualities in fresh fruit markets in Korea. The symptom of BBEP of fruit started to occur on the blossom end part and spread to the middle and stem end part. These fruits lose their marketable values. Calcium has very important role in cell membrane in physical and physiological. This study was conducted to investigate the effect of soluble Ca treatments of fertigation (FG) and foliar application (FA) on soil properties, tree growth and fruit quality and on the occurrence of BBEP. Ca content was high in Ca FA and Ca + IBA FG compared to other treatments. This increase might affect the soil pH, and so pH followed the same pattern of Ca content in soil. In leaves and fruits, Ca content was much higher in Ca Ca FA and Ca + IBA, Ca FG, respectively. Ca content in fruit parts showed stem end part “middle part” blossom end part. In fruit quality, fruit firmness, soluble solid content and fruit weight did not show any tendency in treatments. The occurrence rate of BBEP was very low 14% in Ca FA and 20% in Ca FG than 50% in control. These results showed that Ca content in soil, leaves and fruits was increased by soil FG and FA and the increased Ca content in blossom end part have a negative relationship with BBEP in sweet persimmon.

Real-Time Analysis of the Effects of Cholesterol on Lipid Raft Behavior Using Atomic Force Microscopy

Cholesterol plays a crucial role in cell membranes, and has been implicated in the assembly and maintenance of sphingolipid-rich rafts. We have examined the cholesterol-dependence of model rafts (sphingomyelin-rich domains) in supported lipid monolayers and bilayers using atomic force microscopy. Sphingomyelin-rich domains were observed in lipid monolayers in the absence and presence of cholesterol, except at high cholesterol concentrations, when separate domains were suppressed. The effect of manipulating cholesterol levels on the behavior of these sphingomyelin-rich domains in bilayers was observed in real time. Depletion of cholesterol resulted in dissolution of the model lipid rafts, whereas cholesterol addition resulted in an increased size of the sphingomyelin-rich domains and eventually the formation of a single raftlike lipid phase. Cholesterol colocalization with sphingomyelin-rich domains was confirmed using the sterol binding agent filipin.

Polar lipid and fatty acid distribution in carotenolipoprotein complexes extracted from sea buckthorn fruits.

Sea buckthorn (Hippophae rhamnoides L., fam. Elaeagnaceae) fruits are rich in pigments and lipoproteins located in membranes and the fleshy mesocarp. In spite of many reports concerning the neutral lipids in the mesocarp, no data about the polar lipids and their fatty acid composition are available even though they play important structural and physiological roles in cell membranes and may offer interesting applications as emulsifiers and nutrients in cosmetic preparations. Carotenolipoprotein complexes are located particularly in fruit membranes where polar lipids may function as bridge compounds between the polar (protein) and non-polar (carotenoid) moieties. The fatty acid compositions of total and individual polar lipids separated from carotenolipoprotein complexes were determined by HPTLC and GC. The polar lipids included 61% phospholipids and 39% galactolipids, which contained mainly 16:0, 16:1 (9c), 18:1 (9c), 18:1 (11c) and 18:2 (9c, 12c) fatty acids. Almost all polar lipids showed high ratios of 16:0/16:1 (11c) and 18:1 (9c)/18:1 (11c), and higher quantities of 18 carbon unsaturated fatty acids than of the saturated analogue. Galactolipids proved to be richest in 18:1 (9c) and 18:3 (9c, 12c, 15c) fatty acids, while phospholipids contained higher concentrations of 16:0 and 18:1 (9c).

Possible role of plasma membrane H +-ATPase in the elicitation of phytoalexin and related isoflavone root secretion in chickpea ( Cicer arietinum L.) seedlings

Etiolated 3-day old chickpea seedlings constitute a very simple, organized intact-plant system, whose response to elicitors acting at root level is characterized by the excretion rather than the tissue accumulation of phytoalexins (medicarpin and maackiain) and constitutive isoflavones (formononetin and biochanin A). By using this system, it was concluded that seedling incubation in buffered media with high pH values (higher than 6.5) causes a marked induction of isoflavonoid excretion after 24 h. The response was higher for phosphate buffers than for Mes and Pipes buffers, and, with the phosphate buffers, an optimal response was obtained at pH 7.5 (instead of 6.0, 6.5, 7.0 or 8.0), at a concentration of 80 or 120 mM (instead of 40 mM) and potassium salt (instead of sodium salt). Based on the above facts, we tested the hypothesis that the decrease in proton efflux and/or membrane depolarisation caused by plasma membrane H +-ATPase inhibition elicits the phytoalexin response in chickpea. The ATPase inhibitors orthovanadate and N, N′-dicyclohexylcarbodiimide and the ionophores carbonyl cyanide 3-chlorophenylhydrazone and nigericin were all effective elicitors whereas the ionophore valynomycin, which specifically promotes K + transport and thus should affect the charge but not the pH component of the transmembrane electrochemical gradient, showed little eliciting activity. These data indicate that the inhibition of the plasma membrane H +-ATPase, and more specifically the proton efflux, may be a key step in the signalling pathway leading to the activation of phytoalexin and isoflavone excretion in chickpea seedlings.

The Role of Plasma Membrane in Bioreduction of Two Tetrazolium Salts, MTT, and CTC

Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. We investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. We provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. We postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc.

Membrane, intracellular, and plasma magnesium and calcium concentrations in preeclampsia

Changes in intracellular calcium and magnesium concentrations seem to be involved in the pathogenesis of preeclampsia, whereas the role of cell membranes has not been studied in detail yet. To investigate the changes in calcium and magnesium metabolism in normal pregnancy and preeclampsia, plasma, intracellular, and membrane calcium and magnesium concentrations were determined in a clinical study. Twenty-five control, 18 untreated healthy pregnant, and 16 nulliparas preeclamptic women were investigated. Plasma, cellular, and membrane (erythrocytes) calcium and magnesium contents were measured by atomic absorption spectroscopy. Plasma and intracellular magnesium concentrations were significantly lower in the healthy pregnant group and the preeclamptic group as compared to controls ( P < .01). In erythrocyte membranes magnesium content was found significantly decreased in the preeclamptic women as compared to healthy subjects ( P < .001). There was a significant decrease in the plasma calcium concentration in the preeclamptic group compared to controls or healthy pregnant women ( P < .05). Membranous calcium content was significantly increased in the preeclamptic group versus controls or healthy pregnant women ( P < .001) and an inverse correlation with membranous magnesium content was found (r = −0.79, P < .01). Lowered plasma, intracellular, and membrane magnesium concentrations in preeclampsia may contribute to the development in hypertension in pregnancy. In addition, a disturbed calcium homeostasis is observed in preeclampsia.

Molecular approaches to study plasma membrane H+-ATPases in arbuscular mycorrhizas

The activity of H+-ATPases of plant and fungi generates an electrochemical gradient of H+ across the cell plasma membrane that drives a number of secondary transport systems, including those responsible for the translocation of cations, anions, amino acids and sugars. During the last years, several studies have been aimed at elucidating the role of plasma membrane H+-ATPases in the nutrient exchange processes taking place between the plant and the fungus in arbuscular mycorrhizal (AM) symbiosis. This paper reviews present knowledge about plasma membrane H+-ATPases and experimental evidence supporting the involvement of H+-ATPases of both organisms in the bidirectional transport of nutrients between partners. Molecular strategies that will provide further information on the function and regulation of plasma membrane H+-ATPases in AM symbiosis are presented and discussed.

Plasma and Membrane Ca2+ and Mg2+ Concentrations in Normal Pregnancy and in Preeclampsia

Objective: Changes in intracellular Ca²⁺ and Mg²⁺ concentrations seem to be involved in the pathogenesis of preeclampsia, whereas the role of cell membranes has not been studied in detail yet. To investigate the changes in Ca²⁺ and Mg²⁺ metabolism in normal pregnancy and preeclampsia, plasma and membrane Ca²⁺ and Mg²⁺ concentrations were determined in a clinical study as compared to healthy subjects. Study Design: 25 healthy female subjects, 22 untreated healthy pregnant and 20 preeclamptic women were investigated. In each patient, plasma and membrane Ca²⁺ and Mg²⁺ content were measured. Ca²⁺ and Mg²⁺ concentrations were measured by atomic absorption spectroscopy. Erythrocyte membranes were chosen for membranous Ca²⁺ and Mg²⁺ determination. Results: Plasma Mg²⁺ concentrations were significantly lowered in the healthy pregnant group and the preeclamptic group as compared to contols (p < 0.0001). In erythrocyte membranes, Mg²⁺ content was found significantly decreased in the preeclamptic women as compared to healthy subjects (p < 0.001). In plasma Ca²⁺ concentrations there was a significant decrease in the preeclamptic group as compared to controls or healthy pregnant women (p < 0.05). Membranous Ca²⁺ content was significantly increased in the preeclamptic group versus controls or healthy pregnant women (p < 0.001). Conclusion: Lowered plasma and membrane Mg²⁺ concentrations in preeclampsia may contribute to the development of hypertension in pregnancy. Additionally, a disturbed Ca²⁺ homeostasis is observed in preeclampsia.

JC-1, but not DiOC 6(3) or rhodamine 123, is a reliable fluorescent probe to assess Δ Ψ changes in intact cells: implications for studies on mitochondrial functionality during apoptosis

The sensitivity and specificity of three fluorescent probes used for cytofluorimetric analysis of mitochondrial membrane potential (Δ Ψ) were studied in the U937 human cell line. First, the role of plasmamembrane in influencing the binding of the probes to mitochondria has been investigated. The depolarization of plasmamembrane with high doses of extracellular KCl had no immediate effects on the loading of JC-1, DiOC 6(3) and rhodamine 123 (R123). However, after a few hours of culture in the presence of KCl, significant changes were observed only in cells stained with DiOC 6(3). Second, a comparative study was performed concerning the effects of agents capable of collapsing Δ Ψ. While adding FCCP to cell cultures resulted in consistent changes in the fluorescence emission of both JC-1 and DiOC 6(3) – but not of R123 – only cells stained with JC-1 responded to valinomycin. On the whole, our data indicate that JC-1 is a reliable probe for analyzing Δ Ψ changes with flow cytometry, while the others show a lower sensitivity (R123), or a non-coherent behaviour, due to a high sensitivity to changes in plasmamembrane potential [DiOC 6(3)]. These data cast some doubts on those studies that, using fluorescent probes that have a low sensitivity to Δ Ψ, hypothesized that the fall in Δ Ψ is one of the early events, if not one of the main causes, of apoptosis.