Altered Membrane Composition Research Articles

Modulation of platelet membrane function via exogenous lipid moiety exposure alters platelet responsiveness to shear.

Shear-induced platelet activation may cause life-threatening thrombosis, particularly in patients with mechanical support devices or coronary atherosclerosis. The majority of present anti-platelet agents target or interfere with biochemical, rather than physical mechanisms of platelet activation. Less data and understanding exists with regard to pharmacologic modulation of shear-mediated platelet activation. In this work, we hypothesized that modulating cell membrane properties, via alteration of membrane composition through addition of exogenous lipid moieties, would alter platelet responsiveness to shear. Here we tested fatty acids, lecithin and cholesterol as additive lipid compounds. We demonstrated that incorporation of fatty acids (DHA/EPA) or lecithin into the platelet membrane triggered enhanced sensitivity of platelets to shear-mediated activation. On the other hand, cholesterol incorporation provides significant protection, limiting the effect of shear on platelet activation. These findings provide valuable insight for the development of therapeutic strategies that can modulate shear-mediated platelet activation.

Constitutive production of c-di-GMP is associated with mutations in a variant of Pseudomonas aeruginosa with altered membrane composition.

Most bacteria can form multicellular communities called biofilms on biotic and abiotic surfaces. This multicellular response to surface contact correlates with an increased resistance to various adverse environmental conditions, including those encountered during infections of the human host and exposure to antimicrobial compounds. Biofilm formation occurs when freely swimming (planktonic) cells encounter a surface, which stimulates the chemosensory-like, surface-sensing system Wsp and leads to generation of the intracellular second messenger 3',5'-cyclic-di-guanosine monophosphate (c-di-GMP). We identified adaptive mutations in a clinical small colony variant (SCV) of Pseudomonas aeruginosa and correlated their presence with self-aggregating growth behavior and an enhanced capacity to form biofilms. We present evidence that a point mutation in the 5' untranslated region of the accBC gene cluster, which encodes components of an enzyme responsible for fatty acid biosynthesis, was responsible for a stabilized mRNA structure that resulted in reduced translational efficiency and an increase in the proportion of short-chain fatty acids in the plasma membrane. We propose a model in which these changes in P. aeruginosa serve as a signal for the Wsp system to constitutively produce increased amounts of c-di-GMP and thus play a role in the regulation of adhesion-stimulated bacterial responses.

Dietary fats shift mechanisms underlying vasodilation to acetylcholine

Dietary fatty acids may be an effective target for reversing vascular abnormalities associated with diet‐induced obesity. We tested the hypothesis that dietary fats modify mechanisms underlying vascular responses by altering membrane composition and the contribution from nitric oxide synthase (NOS) and cyclooxygenase (Cox) in vascular responses. We fed male mice (C57BL/6J) normal (13% kcal fat) or a high saturated fat diet from lard (HF, 60% kcal fat) for 12 weeks before substituting half of the saturated fats with n‐3 polyunsaturated fatty acid enriched menhaden oil (MO) while still maintaining the same total kcal from fat. After an additional 6‐9 weeks, weights of mice on HF and MO‐enriched diets were similar and more than normal diet mice. MO‐enriched diet increased percentage of n‐3 long chain fatty acids DHA and EPA over three fold in aorta and the unsaturation index (1.07±0.03 vs 1.63±0.14, p<0.05). Relaxation to acetylcholine (ACh) was similar in aorta from all groups but was reduced by inhibition of NOS only in normal and high fat and not MO fed mice. Inhibition of Cox had no effect on aortic responses in any group. ACh responses were not different in gracilis arteries from HF but enhanced in MO compared to normal diet mice. Inhibition of NOS reduced ACh relaxation only in normal and MO fed mice, but Cox contributed to dilation to ACh in gracilis arteries in MO mice. Relaxation to nitroprusside was not affected by diet. Despite shifts in the contribution from NOS versus Cox in responses, diet did not affect total expression of endothelial NOS or Cox1 in arteries from either group. We conclude that a diet high in unsaturated n‐3 fatty acids improved small artery vascular responses but not large artery, in part, through an increase in the Cox contribution.

Evolution for exogenous octanoic acid tolerance improves carboxylic acid production and membrane integrity

Carboxylic acids are an attractive biorenewable chemical, but as with many biorenewables, their toxicity to microbial biocatalysts limits their fermentative production. While it is generally accepted that membrane damage is the main mechanism of fatty acid toxicity, previous metabolic engineering efforts that increased membrane integrity did not enable increased carboxylic acid production. Here we used an evolutionary approach to improve tolerance to exogenous octanoic acid, with the goal of learning design strategies from this evolved strain. This evolution of an Escherichia coli MG1655 derivative at neutral pH in minimal media produced a strain with increased tolerance not only to octanoic acid, but also to hexanoic acid, decanoic acid, n-butanol and isobutanol. This evolved strain also produced carboxylic acids at a 5-fold higher titer than its parent strain when expressing the Anaerococcus tetradius thioesterase. While it has been previously suggested that intracellular acidification may contribute to carboxylic acid toxicity, we saw no evidence that the evolved strain has increased resistance to this acidification. Characterization of the evolved strain membrane showed that it had significantly altered membrane polarization (fluidity), integrity (leakage) and composition relative to its parent. The changes in membrane composition included a significant increase in average lipid length in a variety of growth conditions, including 30°C, 42°C, carboxylic acid challenge and ethanol challenge. The evolved strain has a more dynamic membrane composition, showing both a larger number of significant changes and larger fold changes in the relative abundance of membrane lipids. These results highlight the importance of the cell membrane in increasing microbial tolerance and production of biorenewable fuels and chemicals.

Contribution of resistance-nodulation-cell division efflux systems to antibiotic resistance and biofilm formation in Acinetobacter baumannii.

ABSTRACTAcinetobacter baumannii is a nosocomial pathogen of increasing importance due to its multiple resistance to antibiotics and ability to survive in the hospital environment linked to its capacity to form biofilms. To fully characterize the contribution of AdeABC, AdeFGH, and AdeIJK resistance-nodulation-cell division (RND)-type efflux systems to acquired and intrinsic resistance, we constructed, from an entirely sequenced susceptible A. baumannii strain, a set of isogenic mutants overexpressing each system following introduction of a point mutation in their cognate regulator or a deletion for the pump by allelic replacement. Pairwise comparison of every derivative with the parental strain indicated that AdeABC and AdeFGH are tightly regulated and contribute to acquisition of antibiotic resistance when overproduced. AdeABC had a broad substrate range, including β-lactams, fluoroquinolones, tetracyclines-tigecycline, macrolides-lincosamides, and chloramphenicol, and conferred clinical resistance to aminoglycosides. Importantly, when combined with enzymatic resistance to carbapenems and aminoglycosides, this pump contributed in a synergistic fashion to the level of resistance of the host. In contrast, AdeIJK was expressed constitutively and was responsible for intrinsic resistance to the same major drug classes as AdeABC as well as antifolates and fusidic acid. Surprisingly, overproduction of AdeABC and AdeIJK altered bacterial membrane composition, resulting in decreased biofilm formation but not motility. Natural transformation and plasmid transfer were diminished in recipients overproducing AdeABC. It thus appears that alteration in the expression of efflux systems leads to multiple changes in the relationship between the host and its environment, in addition to antibiotic resistance.

Mechanical and molecular basis for the symmetrical division of the fission yeast nuclear envelope.

In fission yeast Schizosaccharomyces pombe, the nuclear envelope remains intact throughout mitosis and undergoes a series of symmetrical morphological changes when the spindle pole bodies (SPBs), embedded in the nuclear envelope, are pushed apart by elongating spindle microtubules. These symmetrical membrane shape transformations do not correspond to the shape behavior of an analogous system based on lipid vesicles. Here we report that the symmetry of the dividing fission yeast nucleus is ensured by SPB-chromosome attachments, as loss of kinetochore clustering in the vicinity of SPBs results in the formation of abnormal asymmetric shapes with long membrane tethers. We integrated these findings in a biophysical model, which explains the symmetry of the nuclear shapes on the basis of forces exerted by chromosomes clustered at SPBs on the extending nuclear envelope. Based on this analysis we conclude that the fission yeast nuclear envelope exhibits the same mechanical properties as simple lipid vesicles, but interactions with other cellular components, such as chromosomes, influence the nuclear shape during mitosis, allowing the formation of otherwise energetically unfavorable symmetrical dumbbell structures upon spindle elongation. The model allows us to explain the appearance of abnormal asymmetric shapes in fission yeast mutants with mis-segregated chromosomes as well as with altered nuclear membrane composition.

P6 - Impaired metabolism of senescent muscle satellite cells is associated with oxidative modifications of glycolytic enzymes

Accumulation of damaged macromolecules, including irreversibly oxidized proteins, is a hallmark of cellular and organismal ageing. Failure of protein homesotasis is a major contributor to the age-related accumulation of damaged proteins. In skeletal muscle, tissue maintenance and regeneration is assured by resident adult stem cells known as satellite cells. During senescence their replication and differentiation is compromised contributing to sarcopenia. In this study we have addressed the impact of oxidatively modified proteins in the impaired metabolism of senescent human satellite cells. By using a targeted proteomics analysis we have found that proteins involved in protein quality control and glycolytic enzymes are the main targets of oxidation (carbonylation) and modification with advanced glycation/lipid peroxidation end products during replicative senescence of satellite cells. Inactivation of the proteasome in aged cells appeared as a key contributor to the accumulation of such damaged proteins. Untargeted metabolomic profiling and functional analyses indicated glucose metabolism impairment in senescent cells, although mitochondrial respiration remained unaffected. A metabolic shift leading to increased mobilization of non-carbohydrate substrates as branched chain amino acids or long chain fatty acids was observed in senescent cells. In addition, phospho-and glycerolipids metabolism was altered. Increased levels of acyl-carnitines indicated augmented turnover of storage and membrane lipids for energy production. Such changes reflect alterations in membrane composition and dysregulation of sphingolipids signaling during senescence. This study establishes a new concept connecting oxidative protein modifications with the altered cellular metabolism associated with the senescent phenotype. In addition, these findings highlight the molecular mechanisms implicated in satellite cells dysfunction during ageing, paving the road for future therapeutic interventions aimed at preventing oxidative modifications of proteins and/or stimulating their elimination.

Binding profiles and cytokine-inducing effects of fish rhamnose-binding lectins on Burkitt's lymphoma Raji cells.

Rhamnose-binding lectin (RBL) is one of the animal lectin categories which take part in the innate immune responses of fish. Osmerus lanceolatus lectin (OLL) from shishamo smelt eggs is an RBL composed of two tandem-repeated domains, both of which are considered to be a carbohydrate-recognition domain. SAL, catfish (Silurus asotus) egg RBL composed of three domains, binds to Burkitt's lymphoma Raji cells through globotriaosylceramide (Gb3) carbohydrate chain and to reduce cell size and growth by altering membrane composition without causing cell death. In this experiment, we tried to compare the binding effects of these two RBLs on Raji cells. Flow cytometric and fluorescence microscopic analyses revealed that OLL also directly bound to and shrunk Raji cells with ten times less reactivity than SAL but reduced cell growth with decreasing cell viability. Anti-Gb3 antibody completely blocked the binding of SAL to Raji cells but not that of OLL. In addition, the direct bindings of OLL and SAL to Raji cells were comparably inhibited by melibiose, but lactose was more effective inhibitor for the binding of OLL than that of SAL. These results suggest that OLL has slightly different cell-binding property compared with SAL and binds not only to Gb3 but also to the other carbohydrate receptor-bearing β-galactoside chains. The quantitative RT-PCR analysis revealed that SAL induced the expression of TNF-α but not of IFN-γ, IL-1β, and IL-10. Thus, SAL-induced cytostatic effect on Raji cells might be partially caused by TNF-α-mediated signaling pathway.

Synthetic Multivalent Antifungal Peptides Effective against Fungi

Taking advantage of the cluster effect observed in multivalent peptides, this work describes antifungal activity and possible mechanism of action of tetravalent peptide (B4010) which carries 4 copies of the sequence RGRKVVRR through a branched lysine core. B4010 displayed better antifungal properties than natamycin and amphotericin B. The peptide retained significant activity in the presence of monovalent/divalent cations, trypsin and serum and tear fluid. Moreover, B4010 is non-haemolytic and non-toxic to mice by intraperitoneal (200 mg/kg) or intravenous (100 mg/kg) routes. S. cerevisiae mutant strains with altered membrane sterol structures and composition showed hyper senstivity to B4010. The peptide had no affinity for cell wall polysaccharides and caused rapid dissipation of membrane potential and release of vital ions and ATP when treated with C. albicans. We demonstrate that additives which alter the membrane potential or membrane rigidity protect C. albicans from B4010-induced lethality. Calcein release assay and molecular dynamics simulations showed that the peptide preferentially binds to mixed bilayer containing ergosterol over phophotidylcholine-cholesterol bilayers. The studies further suggested that the first arginine is important for mediating peptide-bilayer interactions. Replacing the first arginine led to a 2–4 fold decrease in antifungal activities and reduced membrane disruption properties. The combined in silico and in vitro approach should facilitate rational design of new tetravalent antifungal peptides.

Erythrocyte osmotic resistance recovery after lung volume reduction surgery

Alteration of erythrocyte osmotic resistance, with increment of reticulocytes, is common in emphysema. This fragility is probably due to an altered fatty acid membrane composition from lipid peroxidation, a reaction triggered by the disease-related increment of reactive oxidative species. We analysed the effects of lung volume reduction surgery (LVRS) on this anomaly compared with respiratory rehabilitation (RR) therapy. We retrospectively compared 58 male patients with moderate-to-severe emphysema who underwent LVRS with 56 similar patients who underwent standardized RR. Respiratory function parameters, erythrocyte osmotic resistance and antioxidant enzymes levels were evaluated before and 6 months after treatments. Significant improvements in respiratory function, exercise capacity, unsaturated fatty acid content (+10.0%, P = 0.035), erythrocyte osmotic resistance (hyperosmolar resistance -21.0%, P = 0.001; hyposmolar resistance -18.0%, P = 0.007) and erythrocyte antioxidant enzymes [superoxide dismutase (SOD) +60.0%, P < 0.001; glutathione peroxidase +39.0%, P = 0.004 and glutathione reductase +24.5%, P = 0.008] were observed after surgery. In the RR group, we did not find any significant improvements in osmotic resistance, although respiratory and functional parameters were significantly improved. Correlation analysis in the surgical group showed that the reduction in residual volume (RV) significantly correlated the normalization of hyperosmotic (P = 0.019) and hyposmotic resistances (P = 0.006), the decrease in the absolute number of reticulocytes (P = 0.037) and increase in SOD (P < 0.001). LVRS improved unsaturated fatty acid content, erythrocyte osmotic resistance and levels of erythrocyte antioxidant enzymes compared with RR. Correlations between erythrocyte osmotic resistance and antioxidant intracellular enzymes with RV suggest that reduction in lung hyperinflation with the elimination of inflammatory emphysematous tissue may explain such improvements after surgery.

Rapeseed oil-rich diet alters in vitro menadione and nimesulide hepatic mitochondrial toxicity

Diet-induced changes in the lipid composition of mitochondrial membranes have been shown to influence physiological processes. However, the modulation effect of diet on mitochondrially-active drugs has not yet received the deserved attention. Our hypothesis is that modulation of membrane dynamics by diet impacts drug-effects on liver mitochondrial functioning. In a previous work, we have shown that a diet rich in rapeseed oil altered mitochondrial membrane composition and bioenergetics in Wistar rats. In the present work, we investigated the influence of the modified diet on hepatic mitochondrial activity of two drugs, menadione and nimesulide, and FCCP, a classic protonophore, was used for comparison. The results showed that the effects of menadione and nimesulide were less severe on liver mitochondria for rats fed the modified diet than on rats fed the control diet. A specific effect on complex I seemed to be involved in drug-induced mitochondria dysfunction. Liver mitochondria from the modified diet group were more susceptible to nimesulide effects on MPT induction. The present work demonstrates that diet manipulation aimed at modifying mitochondrial membrane properties alters the toxicity of mitochondria active agents. This work highlights that diet may potentiate mitochondrial pharmacologic effects or increase drug-induced liabilities.

Antifungal activity of azole compounds CPA18 and CPA109 against azole-susceptible and -resistant strains of Candida albicans

In this study we investigated the in vitro fungistatic and fungicidal activities of CPA18 and CPA109, two azole compounds with original structural features, alone and in combination with fluconazole against fluconazole-susceptible and -resistant Candida albicans strains. Antifungal activities were measured by MIC evaluation and time-kill studies. Azole binding analysis was performed by UV-Vis spectroscopy. Hyphal growth inhibition and filipin and propidium iodide staining assays were used for morphological analysis. An analysis of membrane lipids was also performed to gauge alterations in membrane composition and integrity. Synergism was calculated using fractional inhibitory concentration indices (FICIs). Evaluation of cytotoxicity towards murine macrophages was performed to verify selective antifungal activity. Even though their binding affinity to C. albicans Erg11p is comparable to that of fluconazole, CPA compounds are active against resistant strains of C. albicans with a mutation in ERG11 sequences and/or overexpressing the ABC transporter genes CDR1 and CDR2, which encode ATP-dependent efflux pumps. Moreover, CPA18 is fungistatic, even against the two resistant strains, and was found to be synergistic with fluconazole. Differently from fluconazole and other related azoles, CPA compounds induced marked changes in membrane permeability and dramatic alterations in membrane lipid composition. Our outcomes suggest that CPA compounds are able to overcome major mechanisms of resistance in C. albicans. Also, they are promising candidates for combination treatment that could reduce the toxicity caused by high fluconazole doses, particularly in immunocompromised patients.

Obese mice incur greater myofiber membrane disruption in response to mechanical load compared with lean mice

Obesity is associated with modified transmembrane signaling events in skeletal muscle, such as insulin signaling and glucose transport. The underlying cause of these obesity-related effects on transmembrane signaling is still unknown. In general, the function of membrane proteins responsible for transmembrane signaling is modulated by the biochemical makeup of the membrane, such as lipid composition, in which they are embedded. Any obesity-related alterations in membrane composition would also be predicted to modify membrane biomechanical properties and membrane susceptibility to mechanical load-induced damage. The primary objective of this study was to investigate whether obesity influences myofiber membrane susceptibility to mechanical damage in skeletal muscle. Myofiber membrane damage was compared between 12-week-old obese, hypercholesterolemic (B6.V Lep(ob) /J) and isogenic, normocholesterolemic control (C57BL6/J) male mice following either normal cage activity or strenuous eccentric exercise (downhill running). Myofiber membrane damage was quantified in perfusion-fixed frozen sections of the gastrocnemius muscle via sarcoplasmic concentration of either albumin (cage activity experiment) or a fluorescent marker that had been injected immediately before activity (eccentric exercise experiment). Obese mice exhibited evidence of increased myofiber membrane damage compared with lean mice after both normal cage activity and eccentric exercise indicating that myofiber membranes of obese mice are more susceptible to mechanical damage in general and that eccentric exercise exacerbates this effect. These observations are consistent with the notion that obesity influences the biochemical and biomechanical properties of myofiber membranes.

Abstract P6-04-22: Regulation of Notch localization by endocrine therapy in Estrogen Receptor positive breast cancer cells: Clinical implications for endocrine resistance

Abstract Background: Estrogen receptors occur in about two-thirds of breast tumors and endocrine therapy is probably the most important systemic therapy for hormone receptor positive breast cancer. However, after an initial response to hormonal therapy, most tumors develop resistance leading to disease progression. Central mechanisms thought to be involved in this process include: 1) alterations in tumor cell physiology causing insensitivity to drug-induced apoptosis or induction of drug-detoxifying mechanisms; 2) expression of energy-dependent transporters that eject anti-cancer drugs from cells and 3) the emergence of cancer stem-like cell clones that are estrogen-independent and/or antiestrogen resistant. We and others have shown that Notch signaling mediates survival signals in ER+ breast cancer cells; induces expression of drug transporter ABCG2 (BCRP), activates ERα in the absence of estrogen and mediates survival of breast cancer stem-like cells. Previously, we demonstrated a crosstalk between ERα and Notch signaling whereby estrogen inhibits Notch activation and estrogen withdrawal or tamoxifen re-activate Notch, a possible mechanism of resistance. The mechanism of these effects has remained elusive. Methods: we used sub-cellular fractionation and immunofluorescence staining to investigate the total expression and cellular distribution of Notch signaling components in MCF-7 cells treated with 17-β-estradiol, or estrogen-free medium. Results: Our data shows that in cells treated with estrogen-free medium, Notch1 is located in the Lipid Rafts (LR) in the plasma membrane, along with Notch signaling components such as presenilin 1 (the catalytic subunit of γ-secretase), ADAM10 and Notch negative regulator NUMB. There is published evidence that γ-secretase is most active in lipid rafts.17-β-Estradiol modifies the membrane lipid composition and change the localization of Notch and Notch regulators (such as ADAM10, presenilin-1 and NUMB) at the plasma membrane, thereby altering the activation of Notch. These observations are consistent with a model in which the lipid composition of the plasma membrane is a critical factor in regulating the rate of Notch activation. FDA-approved cholesterol lowering drugs affect Notch signaling in ways consistent with this model. Conclusions: Our findings indicate that endocrine therapy affects Notch signaling at least in part through alterations in membrane composition and lipid raft localization of Notch signaling components. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-22.

Biochemical Characterization of Cardiolipin Synthase Mutations Associated with Daptomycin Resistance in Enterococci

Daptomycin (DAP) resistance in enterococci has been linked to mutations in genes that alter the cell envelope stress response (CESR) (liaFSR) and changes in enzymes that directly affect phospholipid homeostasis, and these changes may alter membrane composition, such as that of cardiolipin synthase (Cls). While Cls substitutions are observed in response to DAP therapy, the effect of these mutations on Cls activity remains obscure. We have expressed, purified, and characterized Cls enzymes from both Enterococcus faecium S447 (residues 52 to 482; Cls447a) and Enterococcus faecalis S613 (residues 53 to 483; Cls613a) as well as Cls variants harboring a single-amino-acid change derived from DAP-resistant isolates of E. faecium. E. faecium Cls447a and E. faecalis Cls613a are tightly associated with the membrane and copurify with their substrate, phosphatidylglycerol (PG), and product, cardiolipin (CL). The amount of PG that copurifies with Cls is in molar excess to protein, suggesting that the enzyme localizes to PG-rich membrane regions. Both Cls447a(H215R) and Cls447a(R218Q) showed an increase in V(max) (μM CL/min/μM protein) from 0.16 ± 0.01 to 0.26 ± 0.02 and 0.26 ± 0.04, respectively, indicating that mutations associated with adaptation to DAP increase Cls activity. Modeling of Cls447a to Streptomyces sp. phospholipase D indicates that the adaptive mutations Cls447a(H215R) and Cls447a(R218Q) are proximal to the phospholipase domain 1 (PLD1) active site and near the putative nucleophile H217. As mutations to Cls are part of a larger genomic adaptation process, increased Cls activity is likely to be highly epistatic with other changes to facilitate DAP resistance.

Abstract 5165: Fatty acid diversification in prostate cancer progression

Abstract Two clinically significant indices of prostate cancer progression are the dedifferentiation of the gland (as measured by histopathology and graded by Gleason scoring, GS) and extension beyond the prostatic capsule (extracapsular extension, ECE). In order to better understand the underlying biology of prostate cancer progression, we undertook the metabolomic characterization of prostatectomy tissues and correlated our metabolite findings with these post-surgical endpoints. Briefly, frozen tissues from 298 prostatectomy cases were characterized by frozen sections and then disaggregated and the biochemical constituents were extracted into cold methanol as previously reported. These extracts were analyzed in three parallel workflows: gas chromatography-mass spectrometry and ultrahigh performance liquid chromatography and tandem mass spectrometry. The resulting data sets were compared against an in-house chemical reference library of over 2500 authentic standards. Three hundred and fifty-six metabolites were identified across all biochemical classes (amino acids, carbohydrates, lipids, nucleotides, cofactors and xenobiotics (primarily dietary and pharmaceutical compounds)). The steady state abundance profiles of numerous long chain fatty acids increased with Gleason pattern progression. The products of α-oxidation of long chain fatty acids (long chain α-hydroxy fatty acids (2-hydroxypalmitate and 2-hydroxystearate) and odd chain length fatty acids (such as C19:0) increased in tissue samples in conjunction with the amount of Gleason pattern 4 tissue that was present in the tumor. Branched chain fatty acids such as isopalmitate and polyunsaturated C22 species (22:2n6 and 22:3n3) increased in abundance from Gleason 7 to 8. In addition to these changes with Gleason pattern progression, extracapsular extension was found to have biochemical correlates in fatty acid metabolism. Specifically, the monounsaturated fatty acids (14:1n5, 16:1n7, 17:1n7, 18:1n9 and 19:1n9) were all found to increase in abundance in prostate tissue samples where capsular integrity was compromised compared to those tissues where the tumor was confined to the gland. These data demonstrate that during the progression of human prostate cancer, there is tremendous fatty acid diversification. Taken together with other data demonstrating phospholipid turnover and membrane remodeling in prostate cancer, these data suggest that prostate cancer cells can dramatically alter membrane composition. The reliance of many prostate cancers on these fatty acid diversification pathways can be appreciated by the demonstration of chemotherapeutic activities of inhibitors of both fatty acid synthase and stearoyl-coA dehydrogenase. In addition to target validation, metabolomic profiling may also provide insight into therapeutic selection for and monitoring of the prostate cancer patient. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5165. doi:1538-7445.AM2012-5165

The Influence of dietary lipid composition on skeletal muscle mitochondria from mice following eight months of calorie restriction

Calorie restriction (CR) has been shown to decrease reactive oxygen species (ROS) production, attenuate oxidative damage and retard aging in a variety of species. CR has also been observed to alter membrane composition towards saturation. This has led to a theory that changes in membrane saturation are central to the actions of CR. As a first step towards testing this theory, C57BL/6 mice were assigned to 4 dietary groups (control and 3 CR groups) and fed AIN‐93G diets at 95% (control) or 60% (CR) of ad libitum. To manipulate membrane composition, the primary dietary fats for the CR groups were soybean oil (also used in the control diet), fish oil or lard. The diets were fed for 8 months and then mitochondria from hindlimb skeletal muscle were isolated for measures of mitochondrial lipid composition, proton leak, and H2O2 production. Changes in mitochondrial phospholipid fatty acids in the CR mice reflected the dietary lipid sources. The capacity of mitochondrial complex III to produce ROS was decreased in the CR lard compared to either the CR soybean or fish oil groups. However, diet lipid composition did not alter proton leak kinetics between the CR groups. The results of this study indicate that mitochondrial lipid composition can influence ROS production in the CR regimens. It remains to be determined if lard or other mixtures of dietary oils can maximize the CR‐induced decrease in ROS production. (Supported by NIH RO1 AG028125)

Effects of Long-Term Space Flight on Erythrocytes and Oxidative Stress of Rodents

Erythrocyte and hemoglobin losses have been frequently observed in humans during space missions; these observations have been designated as “space anemia”. Erythrocytes exposed to microgravity have a modified rheology and undergo hemolysis to a greater extent. Cell membrane composition plays an important role in determining erythrocyte resistance to mechanical stress and it is well known that membrane composition might be influenced by external events, such as hypothermia, hypoxia or gravitational strength variations. Moreover, an altered cell membrane composition, in particular in fatty acids, can cause a greater sensitivity to peroxidative stress, with increase in membrane fragility. Solar radiation or low wavelength electromagnetic radiations (such as gamma rays) from the Earth or the space environment can split water to generate the hydroxyl radical, very reactive at the site of its formation, which can initiate chain reactions leading to lipid peroxidation. These reactive free radicals can react with the non-radical molecules, leading to oxidative damage of lipids, proteins and DNA, etiologically associated with various diseases and morbidities such as cancer, cell degeneration, and inflammation. Indeed, radiation constitutes on of the most important hazard for humans during long-term space flights. With this background, we participated to the MDS tissue-sharing program performing analyses on mice erythrocytes flown on the ISS from August to November 2009. Our results indicate that space flight induced modifications in cell membrane composition and increase of lipid peroxidation products, in mouse erythrocytes. Moreover, antioxidant defenses in the flight erythrocytes were induced, with a significant increase of glutathione content as compared to both vivarium and ground control erythrocytes. Nonetheless, this induction was not sufficient to prevent damages caused by oxidative stress. Future experiments should provide information helpful to reduce the effects of oxidative stress exposure and space anemia, possibly by integrating appropriate dietary elements and natural compounds that could act as antioxidants.

Neurodegeneration Induced by Clustering of Sialylated Glycosylphosphatidylinositols of Prion Proteins

The transmissible spongiform encephalopathies, more commonly known as the prion diseases, are associated with the production and aggregation of disease-related isoforms of the prion protein (PrP(Sc)). The mechanisms by which PrP(Sc) accumulation causes neurodegeneration in these diseases are poorly understood. In cultured neurons, the addition of PrP(Sc) alters cell membranes, increasing cholesterol, activating cytoplasmic phospholipase A(2) (cPLA(2)), and triggering synapse damage. These effects of PrP(Sc) are dependent upon its glycosylphosphatidylinositol (GPI) anchor, suggesting that it is the increased density of GPIs that occurs following the aggregation of PrP(Sc) molecules that triggers neurodegeneration. This hypothesis was supported by observations that cross-linkage of the normal cellular prion protein (PrP(C)) also increased membrane cholesterol, activated cPLA(2), and triggered synapse damage. These effects were not seen after cross-linkage of Thy-1, another GPI-anchored protein, and were dependent on the GPI anchor attached to PrP(C) containing two acyl chains and sialic acid. We propose that the aggregation of PrP(Sc), or the cross-linkage of PrP(C), causes the clustering of sialic acid-containing GPI anchors at high densities, resulting in altered membrane composition, the pathological activation of cPLA(2), and synapse damage.

Methylation of glycosylated sphingolipid modulates membrane lipid topography and pathogenicity of Cryptococcus neoformans

In previous studies we showed that the replication of Cryptococcus neoformans in the lung environment is controlled by the glucosylceramide (GlcCer) synthase gene (GCS1), which synthesizes the membrane sphingolipid GlcCer from the C9-methyl ceramide. Here, we studied the effect of the mutation of the sphingolipid C9 methyltransferase gene (SMT1), which adds a methyl group to position 9 of the sphingosine backbone of ceramide. The C. neoformans Δsmt1 mutant does not make C9-methyl ceramide and, thus, any methylated GlcCer. However, it accumulates demethylated ceramide and demethylated GlcCer. The Δsmt1 mutant loses more than 80% of its virulence compared with the wild type and the reconstituted strain. Interestingly, growth of C. neoformans Δsmt1 in the lung was decreased and C. neoformans cells were contained in lung granulomas, which significantly reduced the rate of their dissemination to the brain reducing the onset of meningoencephalitis. Thus, using fluorescent spectroscopy and atomic force microscopy we compared the wild type and Δsmt1 mutant and found that the altered membrane composition and GlcCer structure affects fungal membrane rigidity, suggesting that specific sphingolipid structures are required for proper fungal membrane organization and integrity. Therefore, we propose that the physical structure of the plasma membrane imparted by specific classes of sphingolipids represents a critical factor for the ability of the fungus to establish virulence.