Altered Membrane Composition Research Articles

The fabM gene product of Streptococcus mutans is responsible for the synthesis of monounsaturated fatty acids and is necessary for survival at low pH.

Previously, it has been demonstrated that the membrane fatty acid composition of Streptococcus mutans is affected by growth pH (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004; R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189:89-92, 2000). Specifically, the proportion of monounsaturated fatty acids increases when the organism is grown in acidic environments; if the shift to increased monounsaturated fatty acids is blocked by the addition of a fatty acid biosynthesis inhibitor, the organism is rendered more acid sensitive (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004). Recently, work with Streptococcus pneumoniae has identified a novel enzyme, FabM, responsible for the production of monounsaturated fatty acids (H. Marrakchi, K. H. Choi, and C. O. Rock, J. Biol. Chem. 277:44809-44816, 2002). Using the published S. pneumoniae sequence, a putative FabM was identified in the S. mutans strain UA159. We generated a fabM strain that does not produce unsaturated fatty acids as determined by gas chromatography of fatty acid methyl esters. The mutant strain was extremely sensitive to low pH in comparison to the wild type; however, the acid-sensitive phenotype was relieved by growth in the presence of long-chain monounsaturated fatty acids or through genetic complementation. The strain exhibited reduced glycolytic capability and altered glucose-PTS activity. In addition, the altered membrane composition was more impermeable to protons and did not maintain a normal DeltapH. The results suggest that altered membrane composition can significantly affect the acid survival capabilities, as well as several enzymatic activities, of S. mutans.

Temperature- and surfactant-induced membrane modifications that alter Listeria monocytogenes nisin sensitivity by different mechanisms.

Nisin interacts with target membranes in four sequential steps: binding, insertion, aggregation, and pore formation. Alterations in membrane composition might influence any of these steps. We hypothesized that cold temperatures (10 degrees C) and surfactant (0.1% Tween 20) in the growth medium would influence Listeria monocytogenes membrane lipid composition, membrane fluidity, and, as a result, sensitivity to nisin. Compared to the membranes of cells grown at 30 degrees C, those of L. monocytogenes grown at 10 degrees C had increased amounts of shorter, branched-chain fatty acids, increased fluidity (as measured by fluorescence anisotropy), and increased nisin sensitivity. When 0.1% Tween 20 was included in the medium and the cells were cultured at 30 degrees C, there were complex changes in lipid composition. They did not influence membrane fluidity but nonetheless increased nisin sensitivity. Further investigation found that these cells had an increased ability to bind radioactively labeled nisin. This suggests that the modification of the surfactant-adapted cell membrane increased nisin sensitivity at the binding step and demonstrates that each of the four steps can contribute to nisin sensitivity.

Effects of phytanic acid on the vitamin E status, lipid composition and physical properties of retinal cell membranes: implications for adult Refsum disease

Adult Refsum disease is an inherited disorder in which phytanic acid accumulates in tissues and serum. Two hypotheses have been proposed to explain the pathogenesis of this condition. The molecular distortion hypothesis suggests that phytanic acid may alter membrane composition and structure, thereby affecting membrane function(s). The anti-metabolite hypothesis suggests that an accumulation of phytanic acid in membranes may interfere with vitamin E function. These two hypotheses were investigated by studying the effects of modulating phytanic acid and alpha-tocopherol concentrations on the fatty acid composition and certain physical parameters of cultured retinal cells. Results showed that (a) the phospholipid fraction of retinal cells readily incorporated phytanic acid, (b) the incorporation of phytanic acid increased membrane fluidity, (c) there was no competition for uptake between phytanic acid and alpha-tocopherol, and (d) the incorporation of phytanic acid did not increase the susceptibility of membranes to lipid peroxidation in vitro. These results obtained with cultured retinal cells suggest that the molecular distortion hypothesis, but not the anti-metabolite hypothesis, could explain the pathogenesis of adult Refsum disease. In vitro tissue culture models can, however, only approximate to the much more complex situation that occurs in vivo.

The role of ceramide in receptor- and stress-induced apoptosis studied in acidic ceramidase-deficient Farber disease cells.

The activation of sphingomyelinases leading to the generation of ceramide has been implicated in various apoptotic pathways. However, the role of ceramide as an essential death mediator remains highly controversial. In the present study, we investigated the functional relevance of ceramide in a genetic model by using primary cells from a Farber disease patient. These cells accumulate ceramide as the result of an inherited deficiency of acidic ceramidase. We demonstrate that Farber disease lymphocytes and fibroblasts underwent apoptosis induced by various stress stimuli, including staurosporine, anticancer drugs and gamma-irradiation, equally as normal control cells. In addition, caspase activation by these proapoptotic agents occurred rather similarly in Farber disease and control fibroblasts. Interestingly, Farber disease lymphoid cells underwent apoptosis induced by the CD95 death receptor more rapidly than control cells. Our data therefore suggest that ceramide does not play an essential role as a second messenger in stress-induced apoptosis. However, in accordance with a role in lipid-rich microdomains, ceramide by altering membrane composition may function as an amplifier in CD95-mediated apoptosis.

Characterization of an OprF-deficient mutant suggests that OprF is an essential protein for Pseudomonas fluorescens strain MF0

A stable OprF-deficient mutant for Pseudomonas fluorescens strain MF0 was constructed using reverse genetics. This mutant, called MF372, showed a rounded morphology and grew more slowly in minimal medium, but not in rich medium. Contrary to other Pseudomonas strains, the loss of OprF for strain MF0 was accompanied by an altered outer membrane composition. At least three outer membrane proteins were overexpressed, apparently as a consequence of adaptive mutations. The N-terminal sequence of two of them revealed strong similarities with porins of the OprD family from P. aeruginosa. The data presented here shows that OprF may be an essential protein for this P. fluorescens strain.

Alzheimer's disease: its origin at the membrane, evidence and questions.

Numerous results on membrane lipid composition from different regions of autopsied Alzheimer's disease brains in comparison with corresponding fractions isolated from control brains revealed significant differences in serine- and ethanolamine-containing glycerophospholipid as well as in glycosphingolipid content. Changes in membrane lipid composition are frequently accompanied by alterations in membrane fluidity, hydrophobic mismatch, lipid signaling pathways, transient formation and disappearance of lipid microdomains, changes in membrane permeability to cations and variations of other membrane properties. In this review we focus on possible implications of altered membrane composition on beta-amyloid precursor protein (APP) and on proteolysis of APP leading eventually to the formation of neurotoxic beta-amyloid (A beta) peptides, the major proteinaceous component of extracellular senile plaques, directly involved in Alzheimer's disease pathogenesis.

Membrane traffic in sphingolipid storage diseases.

In this review, we summarize our studies of membrane lipid transport in sphingolipid storage disease (SLSD) fibroblasts. We recently showed that several fluorescent SL analogs were internalized from the plasma membrane predominantly to the Golgi complex of normal cells, while in ten different SLSD cell types, these lipids accumulated in endosomes and lysosomes (The Lancet 1999;354: 901-905). Additional studies showed that cholesterol homeostasis is perturbed in multiple SLSDs secondary to SL accumulation and that mistargeting of SL analogs was regulated by cholesterol (Nature Cell Biol 1999;1: 386-388). Based on these findings, we hypothesize that endogenous sphingolipids, which accumulate in SLSD cells due to primary defects in lipid catabolism, result in an altered intracellular distribution of cholesterol, and that this alteration in membrane composition then results in defective sorting and transport of SLs. The importance of SL/cholesterol interactions and potential mechanisms underlying the regulation of lipid transport and targeting are also discussed. These studies suggest a new paradigm for regulation of membrane lipid traffic along the endocytic pathway and could have important implications for future studies of protein trafficking as well as lipid transport. This work may also lead to important future clinical developments (e.g. screening tests for SLSD, new methodology for screening drugs which abrogate lipid storage, and possible therapeutic approaches to SLSD).

Combinatorial regulation of phospholipid biosynthetic gene expression by the UME6, SIN3 and RPD3 genes.

The Ume6p-Sin3p-Rpd3p complex negatively regulates expression of genes containing a Ume6p binding site. However, these regulatory proteins also function independently to regulate gene expression both negatively and positively. The model system for this combinatorial regulation is the yeast phospholipid biosynthetic pathway. Sin3p negatively regulates the INO1, CHO1, CHO2 and OPI3 genes while Ume6p negatively regulates the INO1 gene and positively regulates the other genes. We have suggested that the positive regulation results from indirect effects on expression of the INO2 transcriptional activator gene. Here, we demonstrate that the effect of Ume6p on INO2 gene expression is also indirect. We also show that Rpd3p is a negative regulator of phospholipid biosynthetic gene expression. The ability of Ume6p, Sin3p and Rpd3p to differentially regulate expression of the phospholipid biosynthetic genes affects phospholipid composition. A sin3 mutant strain lacks detectable levels of phosphatidylethanolamine and elevated levels of phosphatidylcholine (PC) and a rpd3 mutant strain has reduced levels of PC. These alterations in membrane composition suggest that there may exist additional differences in regulation of phospholipid biosynthetic gene expression and that membrane compositions may be coordinated with other biological processes regulated by Ume6p, Sin3p and Rpd3p.

INTRAHEPATIC CHOLESTASIS OF PREGNANCY

Intrahepatic cholestasis of pregnancy is one of the primary disorders of the liver that adversely affects maternal well-being and fetal outcome. Early identification of this condition, careful interdisciplinary monitoring, and prompt delivery at fetal maturity can improve outcomes in the mother and child. Although the cause is unclear, IHCP probably arises from a genetic predisposition for increased sensitivity to estrogens and progestogens and altered membrane composition and expression of bile ducts, hepatocytes, and canalicular transport systems. As a result, the elevations in maternal levels of bile acids and their molar ratios seen in healthy pregnancy rise further in IHCP patients. Also, as the normal fetal-to-maternal transfer of bile acids across the trophoblast is impaired, the excess bile acids with abnormal profiles accumulate and are toxic to the fetus. The management of IHCP is dictated by the increased risks of fetal distress, spontaneous preterm delivery, and sudden death, as well as by alleviating pruritus in the mother. These risks to the fetus rise progressively to delivery, regardless of serum levels of bile acids and ALT. Close monitoring of these markers is essential but does not prevent sudden fetal distress and death. Provision should be made to induce labor as soon as fetal lung maturity has been established. Ursodeoxycholic acid is the only therapy that has proven effective, albeit in small studies, in alleviating pruritus and restoring towards normal the abnormal profiles of bile acids and sulfated steroids in serum and other body fluids. Ursodeoxycholic acid seems to have no obvious adverse effects on the fetus, but experience is insufficient to draw conclusions regarding teratogenicity and prevention of adverse outcomes.

Effects of dietary fatty acids on lipid metabolism in streptozotocin-induced diabetic rats

We measured the activity of liver Δ 9- and Δ 6-desaturases and examined plasma and liver microsome phospholipid fatty acid composition in control and diabetic rats fed a basal diet supplemented with 5% (by weight) olive oil (OO), sunflower oil (SO), or fish oil (FO), respectively. Plasma glucose, cholesterol, triacylglyceride, and phospholipid levels were also measured. An increase in plasma and liver microsome oleic acid and a decrease in arachidonic acid were found in diabetes. In the liver, docosahexaenoic acid levels were higher in diabetic versus control rats. Diabetes increased liver Δ 9-desaturase in OO-fed rats and did not modify Δ 6-desaturase activity in OO- or SO-fed rats. Both enzymatic activities were decreased in diabetic rats fed the FO diet. As a main conclusion, it appears that diet-induced alterations in membrane composition provide a mechanism for improving the diabetic condition in animals and overcoming the effect of insulin deficiency on desaturase activities. Plasma cholesterol was not modified either by diabetes or by diet. In diabetes, OO-fed rats showed the lowest levels of triglycerides. Plasma phospholipids were significantly higher in OO-fed versus FO-fed rats. These findings suggest that OO contributes to a better control of the hypertriglyceridemia accompanying diabetes as compared with the other two diets in this rat model.

Biochemical alterations in Bradyrhizobium sp USDA 3187 induced by the fungicide Mancozeb.

We have previously shown that fungicide Mancozeb causes a 50% decrease in Bradyhizobium sp USDA 3187 growth rate and affects the bacteria-root symbiotic interaction. In order to elucidate the fungicide toxicity mechanism we determined the effects of Mancozeb on cell chemical composition, glutathione (GSH) content (molecule involved in the detoxification process), glutathione S-transferase (GST) activity and on polyamine, exopolysaccharides, capsular polysaccharides and lipopolysaccharides. Mancozeb produced biochemical alterations in membrane composition, polysaccharides and polyamines. In spite of the increment of GSH content and GST activity, they are not enough to prevent the growth diminution.

A dual labelling method for measuring uptake of low molecular weight compounds into the pathogenic yeastCandida albicans

In contrast to other eukaryotic cells the pathogenic yeast Candida albicans is resistant to many structurally unrelated metabolic inhibitors. Reduced permeability due to the cell wall and/or altered plasma membrane composition is thought to be at least partly responsible for this phenomenon. To study the uptake of low molecular weight compounds into C. albicans we developed a dual labelling method. Intact cells, metabolically inactivated cells, spheroplasts or membrane fragments of C. albicans were incubated with various [14C]-labelled compound in the presence of [3H]-labelled water. After separation of cells and supernatant isotope ratios [3H]/[14C] were determined. Quotients of the isotope ratios from cells and supernatant, called enrichment coefficients, were calculated under all four conditions. The enrichment coefficients indicated whether a compound can enter C. albicans cells, is trapped within the cell wall, is enriched in the lipophilic membrane compartment, is actively accumulated or actively exported by multidrug resistance carriers. We used six structurally unrelated compounds to test our method. We found no evidence for a general impermeability of C. albicans.

Dietary Oxidized Linoleic Acid Modifies Lipid Composition of Rat Liver Microsomes and Increases Their Fluidity

The effect of dietary oxidized oil on the lipid composition, fluidity and function of rat liver microsomes was studied. Male growing rats were fed diets containing 10 g/100 g of a fresh (control) or oxidized (experimental) linoleic acid–rich preparation for 4 wk. High levels of fluorescent compounds and of thiobarbituric acid reactive substances indicated the occurrence of substantial lipid peroxidation in the microsomes of the experimental rats. The fluidity of the liver microsomes derived from rats fed the experimental diet was significantly higher than that of the membranes of the controls. This was due to profound differences in lipid composition of the liver microsomes, namely, a lower cholesterol to phospholipid molar ratio and a greater arachidonic acid content in the phospholipids of the rats fed the experimental diet. The fluidity differences were accompanied by greater activity of the microsomal enzymes, aldehyde dehydrogenase and NADPH cytocrome C reductase. The study demonstrated that ingestion of oxidized lipids caused profound alterations in membrane composition, fluidity and function. These alterations are likely to be associated with an enhanced cholesterol turnover, as indicated by the greater cholesterol excretion observed for the experimental rats.

The effect of membrane cholesterol content on ion transport processes in plasma membranes.

Cholesterol is a prominent component of mammalian plasma membranes and is one of the factors that determine membrane function. In this review the effects of cholesterol content on transport processes in biological membranes are summarized. Membrane cholesterol affects a variety of membrane proteins, including ion channels, transporters, and receptors. Present concepts concerning the mechanistic basis of lipid-induced modulation of transport protein function range between two extremes: modulation by bulk properties or by specific interactions. Interest in bulk properties has been focussed mainly on membrane fluidity. The fluidity of biomembranes is diminished particularly by enrichment with cholesterol. As a change in membrane composition alters the environment in which the proteins are dissolved, any process which depends on membrane protein function may be affected by alterations in membrane composition, such as a change in cholesterol content. This review emphasizes the inhibitory effect of cholesterol enrichment on all membrane ATPases studied, and the stimulating effect of cholesterol enrichment on most other membrane transport proteins. Together with the intriguing feature that the cholesterol content of plasma membranes is considerably higher than that of subcellular membranes, there is ample evidence for a significant role of plasma membrane cholesterol in transmembrane protein function.

The relationship between altered membrane composition, eicosanoids and TNF-induced IL1 and IL6 production in macrophages of rats fed fats of different unsaturated fatty acid composition.

The study investigated the changes in individual molecular species in PE and the effects of a variety of dietary fats with varying proportions of saturated and unsaturated fatty acids on membrane composition, eicosanoid production and cytokine production in thioglycollate-elicited rat macrophages. The data obtained indicates that the greatest degree of modulation by dietary fats on cytokine production was observed after 8 weeks feeding and at this time, the total diacyl species containing linoleic acid (18:2 n-6) and arachidonic acid (20:4 n-6) at the sn-2 position related in a curvilinear fashion to total 18:2 n-6 intake and that IL1 and IL6 production related in a curvilinear fashion to the total diacyl species with 20:4 and 18:2 at the sn-2 position. After 4 weeks of feeding, fish and olive oils enhanced production of IL6 and LTB4, however, while IL1 production, after 8 weeks of dietary treatment, was greatest from macrophages of animals fed corn and olive oils, PGE2 production was greatest in the former group and LTB4 production in the latter. Thus an eicosanoid effect may explain the modulatory influence of olive oil and IL1 production but, cannot explain the effect of corn oil on production of the cytokine. The data from the present study provides some insight into how dietary fats could provide therapy for conditions in which inflammatory cytokines are implicated.

Aging-related decrease in liver peroxisomal fatty acid oxidation in control and clofibrate-treated mice. A biochemical study and mechanistic approach

Membrane fatty acid composition affects membrane structure and function. Alterations in membrane composition have been reported in old animals and it is now hypothesized that these alterations may contribute to the onset of age-related diseases. Previously, we proposed that peroxisomes might also be involved in these aging-related membrane alterations. In order to extend our previous work, we have assayed acyl-CoA oxidase activity and cyanide-insensitive fatty acid oxidation activity for both arachidonic 20:4(n-6) and docosa-hexaenoic 22:6(n-3) acids, catalase and urate oxidase activities, microsomal cytochrome P450 content and cytochrome P4504A1 laurate hydroxylase activity in the liver of young and old mice fed either a control or a clofibrate-supplemented diet. Our results suggest a progressive general decrease in peroxisomal function during aging, including a decrease in the fatty acid oxidation pathway that takes place via a specific decrease in acyl-CoA oxidase activity. The aging-related decrease in peroxisomal function is linked to a concomitant decrease in cytochrome P4504A laurate hydroxylase activity in control animals but not in clofibrate-treated mice. This suggests aging impairs a mechanism in peroxisome proliferation that is subsequent to the cytochrome P4504A step. Implications of the aging-related peroxisomal fatty acid oxidation decrease on health through possible alterations in membrane composition and function and very long chain fatty acid accumulation are discussed.

Crystal-membrane interaction in kidney stone disease.

Urolithiasis is a multifaceted process that initiates with the formation of microcrystals in the urine and terminates with the formation of mature renal calculi. The attachment of crystals by the urothelium is a major event in the successful formation of the mature stone. The papillary tip is the primary site for crystal attachment and stone maturation, and the attachment process appears to be mediated by specific molecular interactions between molecular structures on the surfaces of stone crystals and molecular arrays on the surfaces of cell membranes. Animal models have demonstrated the interaction between cells and crystals, and they have suggested a correlation between cellular damage and crystal interaction, especially when crystals bind to and then break free from the tubular epithelium. Cell culture studies on inner medullary late collecting duct (IMCD) cells have demonstrated that calcium oxalate monohydrate, hydroxyapatite, and uric acid crystals bind to IMCD cells in primary culture. The attachment of these crystals to IMCD cells was crystal structure dependent, saturable, and competitively inhibitable if more than one crystal type was present at the same time. The crystals preferentially attach to cells that have lost partial or complete intercellular junctional integrity. These crystal-attaching cells appear to have altered membrane composition and/or structure. Recent studies on red blood cells and IMCD cells that have been enriched with cholesterol and selected phospholipids suggest that crystal-membrane phospholipid interactions play a major role in crystal attachment.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary fat alters membrane composition in rod outer segments in normal and diabetic rats: Impact on content of very-long-chain ( C ⩾ 24) polyenoic fatty acids

The effect of high n − 3 (5.8%, w/w) vs. a low n − 3 (1.2%, w/w) fatty acids in a diet with a low ratio of polyunsaturated/saturated fatty acids ( P/ S = 0.27) content was investigated to determine the effect of diet on the level of long-and very-long-chain fatty acids ( VLCFA C ⩾ 24) in phospholipids of rod outer segments (ROS) of normal and diabetic rats. After 6 weeks of feeding, diets high in n − 3 fatty acids increased the levels of 22:5( n − 3) and 22:6( n − 3), while decreasing the 22:5( n − 6) level in all major phospholipid classes, n − 6 and n − 3 VLCFA of C24 to C34 with 4, 5 and 6 double bonds were found only in phosphatidylcholine (PC) while other phospholipid classes contained only C24 fatty acids as minor components. The content of VLCFA in PC was approx. 6.7% (w/w) of total fatty acids in the ROS. Feeding a high n − 3 fatty acid diet significantly reduced n − 6 tetraenoic VLCFA in all phospholipids. In the diabetic state, the levels of n − 6 tetraenes and pentaenes in individual phospholipids were different from control animals. This study demonstrates that the VLCFA content of photoreceptor cells reflects the dietary level of n − 3 fatty acids fed. The unique polyenoic n − 6 and n − 3 VLCFA appear to be synthesized from shorter chain precursors which respond to altering the ratio of soln − 6 n − 3 fatty acids fed.

Characterization of the heat-induced stimulation of Photosystem-I-mediated electron transport

SUMMARY Uncoupled Photosystem I (PS I) activity driven by either reduced 2,6-dichlorophenolindophenol (DCPIPH2) or N,N,N',N'-tetramethyl-p-phenylene diamine (TMPDH2) showed a stimulation following pre-treatment at 40–50°C, followed by inhibition at higher temperatures. The stimulation was more marked in thylakoids isolated from warm-grown plants than in those isolated from cool-grown plants. Approximately maximal rates of PS I activity were achieved at physiological temperatures (25–35°C) using highly lipophilic-reduced diaminodurene (DADH2) as an electron donor with little stimulation occurring as a result of high temperature pre-treatment. Electron transport driven by all three electron donors was markedly inhibited by KCN/HgCl2 following all pre-treatment temperatures, but relatively insensitive to ethyldimethylaminopropyl-carbodiimide (EDAC). It is suggested that the stimulation of PS I activity involves a phase change in the thylakoid membrane leading to an increase in permeability which allows enhanced access of DCPIPH2 and TMPDH2 to a common electron acceptor site located in the region of cytochrome f. The greater stimulation of electron transport in thylakoids isolated from warm-grown plants may be due to a more pronounced phase change occurring as a consequence of altered membrane composition as modified by growth temperature.

Influence of oral treatment with ursodeoxycholic and tauroursodeoxycholic acids on estrogen‐induced cholestasis in rats: effects on bile formation and liver plasma membranes

In this study, we examined whether ursodeoxycholic acid (UDC) and its taurine conjugate, tauroursodeoxycholic acid (TUDC), given per os, can prevent the cholestasis induced in rats by 17-alpha-ethynyl estradiol (EE) and whether this protection is mediated by choleretic activity or altered plasma membrane composition. EE (5 mg/kg body weight/day for 5 days) markedly reduced bile flow and bile salt secretion without significantly affecting plasma membrane composition and function. Treatment with UDC or TUDC (100, 150 or 200 (TUDC only) mumol/100 g body weight/day for 5 days) did not significantly modify bile flow, but the bile salt secretion rate increased in a dose-dependent manner. UDC was the main biliary bile acid secreted in groups given higher doses of UDC or TUDC. At these dose levels, bile acid treatment did not affect plasma membrane fluidity as assessed by fluorescence anisotropy, the cholesterol/phospholipid molar ratio as well as Na+K(+)- and Mg(++)-ATPase activities. The highest dose of UDC and TUDC prevented the reduction of both bile flow and bile salt secretion induced by EE, re-establishing these parameters to the values of the corresponding control for the UDC group. In conclusion, UDC and TUDC, given per os, improve EE-induced cholestasis, an effect that cannot be attributed to choleretic activity or altered plasma membrane composition.