Analysis of vaccine responses after anti-CD20 maintenance in B-cell lymphoma in the Balearic Islands. A single reference center experience.

Introduction: The use of maintenance approaches with anti-CD20 monoclonal antibodies has improved the outcomes of B-cell indolent lymphomas but may lead to significant peripheral B-cell depletion. This depletion can potentially hinder the serological response to neoantigens. Methods: Our objective was to analyze the effect of anti-CD20 maintenance therapy in a reliable model of response to neoantigens: SARS-CoV-2 vaccine responses and the incidence/severity ofCOVID-19 in a reference hospital. Results: In our series (n=118), the rate of vaccination failures was 31%. Through ROC curve analysis, we determined a cutoff for SARS-CoV-2 vaccine serologic response at 24 months from the last anti-CD20 dose. The risk of severe COVID-19 was notably higher within the first 24months following the last anti-CD20 dose (52%) compared to after this period (just 18%) (p=0.007). In our survival analysis, neither vaccine response nor hypogammaglobulinemia significantly affected OS. While COVID-19 led to a modest mortality rate of 2.5%, this figure was comparable to the OS reported in the general immunocompetent population. However, most patients with hypogammaglobulinemia received intravenous immunoglobulin therapy and all were vaccinated. In conclusion, anti-CD20 maintenance therapy impairs serological responses to SARS-CoV-2 vaccines. Discussion: We report for the first time that patients during maintenance therapy and up to 24 months after the last anti-CD20 dose are at a higher risk of vaccine failure and more severe cases of COVID-19. Nevertheless, with close monitoring, intravenous immunoglobulin supplementation or proper vaccination, the impact on survival due to the lack of serological response in this high-risk population can be mitigated, allowing for the benefits of anti-CD20 maintenance therapy, even in the presence of hypogammaglobulinemia.

Screening for Prognostic microRNAs Associated with Treatment Failure in Diffuse Large B Cell Lymphoma.

Diffuse large B cell lymphoma (DLBCL) treatment with R-CHOP regimen produces 5-year progression-free survival and overall survival of around 60-70%. Our objective was to discover prognostic biomarkers allowing early detection of the remaining 30-40% with poor long-term outcome. For this purpose, we applied a novel strategy: from a cohort of DLBCL patients, treated with standard therapy, a discovery group of 12 patients with poor prognosis (advanced stage III-IV, R-IPI > 2) was formed, consisting of six chemoresistant (refractory/early relapse < 12 months) and six chemosensitive (complete remission > 3 years) subjects. By using microarray assays, the most differentially expressed miRNAs were defined as an initial set of prognostic miRNA candidates. Their expression was then analyzed in a validation cohort of 68 patients and the three miRNAs with the most significant impact on event-free and overall survival were selected. In the DLBCL cell line U-2932 the transfection with miR-1244 and miR-193b-5p, but not miR-1231, blocked the effect of CHOP on cell viability. A subsequent gene set enrichment analysis in patients revealed the implication of the first two miRNAs in cell cycle control and chemoresistance-related pathways, whereas the last one was involved in immunological processes. In conclusion, this novel strategy identified three promising prognostic markers for DLBCL patients at high risk of failure with standard therapy.

WNT/ß-Catenin Pathway in Soft Tissue Sarcomas: New Therapeutic Opportunities?

Soft tissue sarcomas (STS) are a very heterogeneous group of rare tumors, comprising more than 50 different histological subtypes that originate from mesenchymal tissue. Despite their heterogeneity, chemotherapy based on doxorubicin (DXR) has been in use for forty years now and remains the standard first-line treatment for locally advanced unresectable or metastatic STS, although overall survival could not be improved by combination with other chemotherapeutics. In this sense, the development of new therapeutic approaches continues to be a largely unmatched goal. The WNT/ß-catenin signaling pathway is involved in various fundamental processes for embryogenic development, including the proliferation and differentiation of mesenchymal stem cells. Although the role of this pathway has been widely researched in neoplasms of epithelial origin, little is known about its relevance for mesenchymal neoplasms. This review covers the most important molecular alterations of the WNT signaling pathway in STS. The detection of these alterations and the understanding of their functional consequences for those pathways controlling sarcomagenesis development and progression are crucial to broaden the current knowledge about STS as well as to identify novel drug targets. In this regard, the current therapeutic options and drug candidates to modulate WNT signaling, which are usually classified by their interaction site upstream or downstream of ß-catenin, and their presumable clinical impact on STS are also discussed.

Impact of Wnt/ß-Catenin Inhibition on Cell Proliferation through CDC25A Downregulation in Soft Tissue Sarcomas.

The Wnt signaling pathway is an important cellular mechanism for regulating differentiation processes as well as cell cycle events, and different inhibitors of this pathway, for example, PRI-724, are showing promising results in clinical trials for treatment of advanced pancreatic adenocarcinoma or ovarian cancer. Growing evidence suggests that Wnt signaling may also be crucial for tumorigenesis and progression of soft tissue sarcomas (STS), a malignant neoplasm with few therapeutic options at an advanced state. Our study with several STS cell lines and primary cultures shows that inhibition of Wnt/ß-catenin signaling with PRI-724 is able to suppress cell viability/proliferation and to increase cell death rates. TCF/ß-catenin-mediated transcriptional activity is decreased in treated cells, leading to downregulation of its target genes CCND1 and CDC25A. The latter was critical because its downregulation via siRNA was able to mimic the effect of PRI-724 on cell cycle arrest and cell death induction. An evaluation of NCBI/GenBank data confirmed that CDC25A mRNA is elevated in STS patients. Importantly, PRI-724 in combination with standard STS chemotherapeutics doxorubicin or trabectedin enhanced their antitumoral effect in a synergistic manner according to isobolographic analysis, suggesting that Wnt inhibition through PRI-724 could be a beneficial combination regime in patients with advanced STS.

Disruption of TCF/ß-Catenin Binding Impairs Wnt Signaling and Induces Apoptosis in Soft Tissue Sarcoma Cells.

Soft tissue sarcomas (STS) are malignant tumors of mesenchymal origin and represent around 1% of adult cancers, being a very heterogeneous group of tumors with more than 50 different subtypes. The Wnt signaling pathway is involved in the development and in the regulation, self-renewal, and differentiation of mesenchymal stem cells, and plays a role in sarcomagenesis. In this study, we have tested pharmacologic inhibition of Wnt signaling mediated by disruption of TCF/ß-catenin binding and AXIN stabilization, being the first strategy more efficient in reducing cell viability and downstream effects. We have shown that disruption of TCF/ß-catenin binding with PKF118-310 produces in vitro antitumor activity in a panel of prevalent representative STS cell lines and primary cultures. At the molecular level, PKF118-310 treatment reduced ß-catenin nuclear localization, reporter activity, and target genes, resulting in an increase in apoptosis. Importantly, combination of PKF118-310 with doxorubicin resulted in enhanced reduction of cell viability, suggesting that Wnt inhibition could be a new combination regime in these patients. Our findings support the usefulness of Wnt inhibitors as new therapeutic strategies for the prevalent STS. Mol Cancer Ther; 16(6); 1166-76. ©2017 AACR.

Down-Regulation of AKT Signalling by Ursolic Acid Induces Intrinsic Apoptosis and Sensitization to Doxorubicin in Soft Tissue Sarcoma.

Several important biological activities have been attributed to the pentacyclic triterpene ursolic acid (UA), being its antitumoral effect extensively studied in human adenocarcinomas. In this work, we focused on the efficacy and molecular mechanisms involved in the antitumoral effects of UA, as single agent or combined with doxorubicin (DXR), in human soft tissue sarcoma cells. UA (5-50 µM) strongly inhibited (up to 80%) the viability of STS cells at 24 h and its proliferation in soft agar, with higher concentrations increasing apoptotic death up to 30%. UA treatment (6-9 h) strongly blocked the survival AKT/GSK3ß/ß-catenin signalling pathway, which led to a concomitant reduction of the anti-apoptotic proteins c-Myc and p21, altogether resulting in the activation of intrinsic apoptosis. Interestingly, UA at low concentrations (10-15 µM) enhanced the antitumoral effects of DXR by up to 2-fold, while in parallel inhibiting DXR-induced AKT activation and p21 expression, two proteins implicated in antitumoral drug resistance and cell survival. In conclusion, UA is able to induce intrinsic apoptosis in human STS cells and also to sensitize these cells to DXR by blocking the AKT signalling pathway. Therefore, UA may have beneficial effects, if used as nutraceutical adjuvant during standard chemotherapy treatment of STS.

Fluorescence resonance energy transfer-based real-time polymerase chain reaction method without DNA extraction for the genotyping of F5, F2, F12, MTHFR, and HFE.

Blood samples are extensively used for the molecular diagnosis of many hematological diseases. The daily practice in a clinical laboratory of molecular diagnosis in hematology involves using a variety of techniques, based on the amplification of nucleic acids. Current methods for polymerase chain reaction (PCR) use purified genomic DNA, mostly isolated from total peripheral blood cells or white blood cells (WBC). In this paper we describe a real-time fluorescence resonance energy transfer-based method for genotyping directly from blood cells. Our strategy is based on an initial isolation of the WBCs, allowing the removal of PCR inhibitors, such as the heme group, present in the erythrocytes. Once the erythrocytes have been lysed, in the LightCycler(®) 2.0 Instrument, we perform a real-time PCR followed by a melting curve analysis for different genes (Factors 2, 5, 12, MTHFR, and HFE). After testing 34 samples comparing the real-time crossing point (CP) values between WBC (5×10(6) WBC/mL) and purified DNA (20 ng/µL), the results for F5 Leiden were as follows: CP mean value for WBC was 29.26±0.566 versus purified DNA 24.79±0.56. Thus, when PCR was performed from WBC (5×10(6) WBC/mL) instead of DNA (20 ng/µL), we observed a delay of about 4 cycles. These small differences in CP values were similar for all genes tested and did not significantly affect the subsequent analysis by melting curves. In both cases the fluorescence values were high enough, allowing a robust genotyping of all these genes without a previous DNA purification/extraction.

Oleanolic and maslinic acid sensitize soft tissue sarcoma cells to doxorubicin by inhibiting the multidrug resistance protein MRP-1, but not P-glycoprotein.

The pentacyclic triterpenes oleanolic acid (OLA) and maslinic acid (MLA) are natural compounds present in many plants and dietary products consumed in the Mediterranean diet (e.g., pomace and virgin olive oils). Several nutraceutical activities have been attributed to OLA and MLA, whose antitumoral effects have been extensively evaluated in human adenocarcinomas, but little is known regarding their effectiveness in soft tissue sarcomas (STS). We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of OLA and MLA as single agents or in combination with doxorubicin (DXR) in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound, MLA (10-100 µM) was more potent than OLA, inhibiting the growth of SW982 and SK-UT-1 cells by 70.3 ± 1.11% and 68.8 ± 1.52% at 80 µM, respectively. Importantly, OLA (80 µM) or MLA (30 µM) enhanced the antitumoral effect of DXR (0.5-10 µM) by up to 2.3-fold. On the molecular level, efflux activity of the multidrug resistance protein MRP-1, but not of the P-glycoprotein, was inhibited. Most probably as a consequence, DXR accumulated in these cells. Kinetic studies showed that OLA behaved as a competitive inhibitor of substrate-mediated MRP-1 transport, whereas MLA acted as a non-competitive one. Moreover, none of both triterpenes induced a compensatory increase in MRP-1 expression. In summary, OLA or MLA sensitized cellular models of STS to DXR and selectively inhibited MRP-1 activity, but not its expression, leading to a higher antitumoral effect possibly relevant for clinical treatment.

Effect of a 2-hydroxylated fatty acid on cholesterol-rich membrane domains.

2-Hydroxyoleic acid (2OHOA) is a synthetic fatty acid with antihypertensive properties that is able to alter structural membranes properties. The main purpose of this study was to analyze the effect of 2OHOA on the membrane architecture in cholesterol (Cho)-rich domains. For this purpose, model membranes mimicking the composition of lipid rafts and PC- or PE-Cho-rich domains were examined in the absence and presence of 2OHOA by synchrotron X-ray diffraction, atomic force microscopy (AFM) and microcalorimetry (DSC) techniques. Our results demonstrate that 2OHOA phase separates from lipid raft domains and affects the lateral organization of lipids in the membrane. In model raft membranes, 2OHOA interacted with the sphingomyelin (SM) gel phase increasing the thickness of the water layer, which should lead to increased bilayer fluidity. The hydrogen binding competition between 2OHOA and Cho could favour the enrichment of 2OHOA in SM domains separated from the SM-Cho domains, resulting in an enhanced phase separation into SM-2OHOA-rich liquid-disordered (non-raft) and SM-Cho-rich liquid-ordered (raft) domains. The segregation into 2OHOA-rich/Cho-poor and 2OHOA-poor/Cho-rich domains was also observed in PC bilayers.

Nilotinib counteracts P-glycoprotein-mediated multidrug resistance and synergizes the antitumoral effect of doxorubicin in soft tissue sarcomas.

The therapeutic effect of doxorubicin (DXR) in the treatment of soft tissue sarcomas (STS) is limited by its toxicity and the development of multidrug resistance (MDR), the latter mainly induced by high expression of efflux pumps (e.g., P-glycoprotein [P-gp]). Therefore, the search for alternative therapies, which sensitize these tumors to chemotherapy while maintaining a low toxicity profile, is a rational approach. We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of the tyrosine kinase inhibitors, nilotinib and imatinib, as single agents or in combination with DXR, in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound nilotinib (1-10 µM) was more potent than imatinib inhibiting the growth of SK-UT-1 and SW982 cells by 33.5-59.6%, respectively. Importantly, only nilotinib synergized the antitumoral effect of DXR (0.05-0.5 µM) by at least 2-fold, which clearly surpassed the mere sum of effects according to isobolographic analysis. Moreover, nilotinib in combination with DXR had a sustained effect on cell number (-70.3±5.8%) even 12 days after withdrawal of drugs compared to DXR alone. On the molecular level, only nilotinib fully blocked FBS-induced ERK1 and p38 MAPK activation, hence, reducing basal and DXR-induced up-regulation of P-gp levels. Moreover, efflux activity of the MDR-related proteins P-gp and MRP-1 was inhibited, altogether resulting in intracellular DXR retention. In high-risk STS tumors 53.8% and 15.4% were positive for P-gp and MRP-1 expression, respectively, with high incidence of P-gp in synovial sarcoma (72.7%). In summary, nilotinib exhibits antiproliferative effects on cellular models of STS and sensitizes them to DXR by reverting DXR-induced P-gp-mediated MDR and inhibiting MRP-1 activity, leading to a synergistic effect with potential for clinical treatment.

Yondelis® (ET-743, Trabectedin) sensitizes cancer cell lines to CD95-mediated cell death: new molecular insight into the mechanism of action.

Trabectedin, a naturally occurring substance isolated from the Caribbean marine invertebrate Ecteinascidia turbinata, is the active compound of the antitumor drug Yondelis®. The mechanism of action of Trabectedin has been attributed to interactions with the minor groove of the DNA double helix, thereby affecting transcription of different genes involved in DNA repair and thus facilitating lethal DNA strand breaks. Nevertheless, the existence of other clinically important molecular mechanisms has not yet been fully explored. In this paper we demonstrate how Yondelis®, apart from activating the caspase-8-dependent cascade of apoptosis, sensitizes cancer cells to Fas-mediated cell death at achievable concentrations similar to those found in the plasma of patients. In addition we show that the facilitated apoptosis activated through the Fas death receptor, is associated with a significant increase of membrane Fas/FasL, as well as the modulation of accessory proteins regulating this route, such as FLIP (L) or Akt. Thus, our results propose that the sensitization of the death receptor pathway is an essential mechanism amplifying the cytotoxic properties of Yondelis® that could explain the hepatotoxicity observed in patients treated with this drug. Finally, we also show how the use of dexamethasone as a prophylactic agent that protects against hepatotoxicity induced by Yondelis® may also inhibit some of the cytotoxic properties described in this work. The study of this important mechanism of action should set up the basis for reassessing clinical therapy with Yondelis® in order to improve antitumor treatment outcome.

Plant pentacyclic triterpenic acids as modulators of lipid membrane physical properties.

Free triterpenic acids (TTPs) present in plants are bioactive compounds exhibiting multiple nutriceutical activities. The underlying molecular mechanisms have only been examined in part and mainly focused on anti-inflammatory properties, cancer and cardiovascular diseases, in all of which TTPs frequently affect membrane-related proteins. Based on the structural characteristics of TTPs, we assume that their effect on biophysical properties of cell membranes could play a role for their biological activity. In this context, our study is focused on the compounds, oleanolic (3ß-hydroxy-12-oleanen-28-oic acid, OLA), maslinic (2α,3ß-dihydroxy-12-oleanen-28-oic acid, MSL) and ursolic ((3ß)-3-hydroxyurs-12-en-28-oic acid, URL) as the most important TTPs present in orujo olive oil. X-ray diffraction, differential scanning calorimetry, (31)P nuclear magnetic resonance and Laurdan fluorescence data provide experimental evidence that OLA, MSL and URL altered the structural properties of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and DPPC-Cholesterol (Cho) rich membranes, being located into the polar-hydrophobic interphase. Specifically, in DPPC membranes, TTPs altered the structural order of the L(ß'), phase without destabilizing the lipid bilayer. The existence of a nonbilayer isotropic phase in coexistence with the liquid crystalline L(α) phase, as observed in DPPC:URL samples, indicated the presence of lipid structures with high curvature (probably inverted micelles). In DPPC:Cho membranes, TTPs affected the membrane phase properties increasing the Laurdan GP values above 40°C. MSL and URL induced segregation of Cho within the bilayer, in contrast to OLA, that reduced the structural organization of the membrane. These results strengthen the relevance of TTP interactions with cell membranes as a molecular mechanism underlying their broad spectrum of biological effects.

Interactions of fatty acids with phosphatidylethanolamine membranes: X-ray diffraction and molecular dynamics studies.

An experimental and theoretical study on 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes containing fatty acids (FAs) was performed by means of X-ray diffraction analysis and molecular dynamics (MD) simulations. The study was aimed at understanding the interactions of several structurally related FAs with biomembranes, which is necessary for further rational lipid drug design in membrane-lipid therapy. The main effect of FAs was to promote the formation of a H(II) phase, despite a stabilization of the coexisting L(alpha) + H(II) phases. Derivatives of OA exhibited a specific density profile in the direction perpendicular to the bilayer that reflects differences in the relative localization of the carboxylate group within the polar region of the membrane as well as in the degree of membrane penetration of the FA acyl chain. Hydroxyl and methyl substituents at carbon-2 in the FA acyl chain were identified as effective modulators of the position of carboxylate group in the lipid bilayer. Our data highlight the specific potential of each FA in modulating the membrane structure properties.

Olive oils modulate fatty acid content and signaling protein expression in apolipoprotein E knockout mice brain.

Atherosclerosis contributes to disruption of neuronal signaling pathways by producing lipid-dependent modifications of brain plasma membranes, neuroinflammation and oxidative stress. We investigated whether long-term (11 weeks) consumption of refined- (ROO) and pomace- (POO) olive oil modulated the fatty acid composition and the levels of membrane signaling proteins in the brain of apolipoprotein E (apoE) knockout (KO) mice, an animal model of atherosclerosis. Both of these oils are rich in bioactive molecules with anti-inflammatory and antioxidant effects. ROO and POO long-term consumption increased the proportion of monounsaturated fatty acids (MUFAs), particularly of oleic acid, while reducing the level of the saturated fatty acids (SFAs) palmitic and stearic acid. As a result, the MUFA:SFA ratio was higher in apoE KO mice brain fed with ROO and POO. Furthermore, both oils reduced the level of arachidonic and eicosapentaenoic acid, suggesting a decrease in the generation of pro- and anti-inflammatory eicosanoids. Finally, ROO and POO induced an increase in the density of membrane proteins implicated in both the Galphas/PKA and Galphaq/PLCbeta1/PKCalpha signaling pathways. The combined effects of long-term ROO and POO consumption on fatty acid composition and the level of signaling proteins involved in PKA and PKC activation, suggest positive effects on neuroinflammation and brain function in apoE KO mice brain, and convert these oils into promising functional foods in diseases involving apoE deficiency.

Membrane interactions of G proteins and other related proteins.

Guanine nucleotide-binding proteins, G proteins, propagate incoming messages from receptors to effector proteins. They switch from an inactive to active state by exchanging a GDP molecule for GTP, and they return to the inactive form by hydrolyzing GTP to GDP. Small monomeric G proteins, such as Ras, are involved in controlling cell proliferation, differentiation and apoptosis, and they interact with membranes through isoprenyl moieties, fatty acyl moieties, and electrostatic interactions. This protein-lipid binding facilitates productive encounters of Ras and Raf proteins in defined membrane regions, so that signals can subsequently proceed through MEK and ERK kinases, which constitute the canonical MAP kinase signaling cassette. On the other hand, heterotrimeric G proteins undergo co/post-translational modifications in the alpha (myristic and/or palmitic acid) and the gamma (farnesol or geranylgeraniol) subunits. These modifications not only assist the G protein to localize to the membrane but they also help distribute the heterotrimer (Galphabetagamma) and the subunits generated upon activation (Galpha and Gbetagamma) to appropriate membrane microdomains. These proteins transduce messages from ubiquitous serpentine receptors, which control important functions such as taste, vision, blood pressure, body weight, cell proliferation, mood, etc. Moreover, the exchange of GDP by GTP is triggered by nucleotide exchange factors. Membrane receptors that activate G proteins can be considered as such, but other cytosolic, membranal or amphitropic proteins can accelerate the rate of G protein exchange or even activate this process in the absence of receptor-mediated activation. These and other protein-protein interactions of G proteins with other signaling proteins are regulated by their lipid preferences. Thus, G protein-lipid interactions control the features of messages and cell physiology.

Inhibition of c-Myc down-regulation by sustained extracellular signal-regulated kinase activation prevents the antimetabolite methotrexate- and gemcitabine-induced differentiation in non-small-cell lung cancer cells.

Non-small-cell lung cancer (NSCLC) is characterized by severe resistance to chemotherapy. Here, we demonstrate that A549 adenocarcinoma cells permanently differentiate with the antimetabolites methotrexate (MTX) and gemcitabine (GE) when blocking the resistance mechanism that normally counteracts this process. MTX (1-10 microM) and GE (1 microM) induced growth arrest accompanied by sustained extracellular signal-regulated kinase (ERK1/2) phosphorylation and moderate reduction of c-Myc levels after 96 h, whereas only a low percentage of the cells differentiated. Combination with the mitogen-activated protein kinase kinase (MEK) inhibitor 1,4-diamino-2,3-dicyano-1,4-bis-(methylthio)butadiene (U0126) reduced MTX- or GE-induced ERK1/2 over-phosphorylation, nearly abolished c-Myc expression, and provoked radical morphological changes in all cells. Besides the appearance of multilamellar bodies and intracellular cytokeratin reorganization, modulation of molecular markers occurred in a manner consistent with differentiation (gelsolin, +300%; surfactant protein A and C, -70%). Similar to U0126, c-Myc inactivation with specific small interfering RNA initiated differentiation only in the presence of MTX, demonstrating that inhibition of the mitogen-activated protein kinase/ERK pathway alone or down-regulation of c-Myc is not sufficient to induce this process. It is noteworthy that withdrawal of antitumoral drugs and U0126 neither reversed differentiation nor reactivated proliferation. Our results reveal that maintenance of a certain threshold of c-Myc expression through sustained ERK1/2 activation represents a molecular mechanism that confers resistance to antimetabolite-induced differentiation in A549 cells, and provide a novel molecular basis for therapeutic strategies based on irreversible differentiation of cancer cells using conventional chemotherapeutic antimetabolites in combination with inhibitors of the MEK/ERK pathway or c-Myc.

Effects of 2-hydroxyoleic acid on the structural properties of biological and model plasma membranes.

Genetic hypertension is associated with alterations in lipid metabolism, membrane lipid composition and membrane-protein function. 2-Hydroxyoleic acid (2OHOA) is a new antihypertensive molecule that regulates the structure of model membranes and their interaction with certain peripheral signalling proteins in vitro. While the effect of 2OHOA on elevated blood pressure is thought to arise through its influence on signalling proteins, its effects on membrane lipid composition remain to be assessed. 2OHOA administration altered the lipid membrane composition of hypertensive and normotensive rat plasma membranes, and increased the fluidity of reconstituted liver membranes from hypertensive rats. In spontaneously hypertensive rats (SHR), treatment with 2OHOA increased the cholesterol and sphingomyelin content while decreasing that of phosphatidylserine-phosphatidylinositol lipids. In addition, monounsaturated fatty acid levels increased as well as the propensity of reconstituted membranes to form HII-phases. These data suggest that 2OHOA regulates lipid metabolism that is altered in hypertensive animals, and that it affects the structural properties of liver plasma membranes in SHR. These changes in the structural properties of the plasma membrane may modulate the activity of signalling proteins that associate with the cell membrane such as the Galphaq/11 protein and hence, signal transduction.

Interaction of the C-terminal region of the Ggamma protein with model membranes.

Heterotrimeric G-proteins interact with membranes. They accumulate around membrane receptors and propagate messages to effectors localized in different cellular compartments. G-protein-lipid interactions regulate G-protein cellular localization and activity. Although we recently found that the Gbetagamma dimer drives the interaction of G-proteins with nonlamellar-prone membranes, little is known about the molecular basis of this interaction. Here, we investigated the interaction of the C-terminus of the Ggamma(2) protein (P(gamma)-FN) with model membranes and those of its peptide (P(gamma)) and farnesyl (FN) moieties alone. X-ray diffraction and differential scanning calorimetry demonstrated that P(gamma)-FN, segregated into P(gamma)-FN-poor and -rich domains in phosphatidylethanolamine (PE) and phosphatidylserine (PS) membranes. In PE membranes, FN increased the nonlamellar phase propensity. Fourier transform infrared spectroscopy experiments showed that P(gamma) and P(gamma)-FN interact with the polar and interfacial regions of PE and PS bilayers. The binding of P(gamma)-FN to model membranes is due to the FN group and positively charged amino acids near this lipid. On the other hand, membrane lipids partially altered P(gamma)-FN structure, in turn increasing the fluidity of PS membranes. These data highlight the relevance of the interaction of the C-terminal region of the Ggamma protein with the cell membrane and its effect on membrane structure.

Consumption of virgin olive oil influences membrane lipid composition and regulates intracellular signaling in elderly adults with type 2 diabetes mellitus.

We aimed to define changes in membrane fatty acids and signaling proteins induced by virgin olive oil (VOO) consumption in elderly persons with type 2 diabetes (n = 16) compared to a control group (n = 28). The fatty acid composition was determined by gas chromatography and G-protein subunits and protein kinase C alpha (PKCalpha) by immunoblotting. VOO consumption increased the monounsaturated fatty acid content in phospholipids and cholesterol esters in both groups. In contrast, saturated fatty acids were decreased only in phospholipids. The levels of Galphao, Gbeta, and PKCalpha were significantly lower in diabetics than in controls. However, whereas VOO consumption reduced Galphas, Gbeta, and PKCalpha in both groups, reduction in Galphai was observed only in diabetics. These results indicate that long-term VOO consumption modifies the fatty acid composition of plasma membrane, which influences the association of G proteins and PKCalpha with the lipid bilayer. These combined effects probably account for the positive effects of VOO on glycemic homeostasis.