Transbilayer Phospholipid Asymmetry in Plasmodium knowlesi- Infected Host Cell Membrane

The membranes from normal and Plasmodium knowlesi-infected rhemsus monkey erythrocytes (90 to 95 percent infected with early ring stage) were analyzed for transbilayer distribution of phosphatidylcholine (PC). hosphatidylethanolamine (PE). and hosphatidylserine (PS). by means of chemical and enzymatic probes. The external monolayer of the normal red cell membrane contained at least 68 to 72 percent of the total phosphatidylcholine and 15 to 20 percent of the total phosphati dylethanolamine. In the infected cell, the transmembrane phosphatidylcholine distribution appeared to be reversed, with only 20 to 30 percent of it being externally localized, whereas roughly equal amounts of phosphatidylethanolamine were present in the outer and'inner surfaces. However, total pho.~phatid)'lserine in both the infected and normal red cells was exc/usi~'ely internal. Unlike that in the normal intact cell, external phosphatidylethanolamine in the parasitized cell was readily accessible to phospholipase A2. These results indicate that significant changes in molecular architecture of the host cell membrane are the result of varasitization.

ECHOGENIC LIPOSOMES FOR NITRIC OXIDE DELIVERY AND BREAST CANCER TREATMENT

Liposomes, also known as nontoxic, biodegradable, and non-immunogenic therapeutic delivery vehicles, have been proposed as a carrier for drugs and antitumor agents in cancer chemotherapy. Echogenic liposomes (ELIP) have the potential to entrap air or bioactive gas to enhance acoustic reflectivity in ultrasound and are used as a contrast agent. The innovative part of this study is based on a novel concept to encapsulate nitric oxide (NO) gas into ELIP, deliver it to breast cancer cells, and control its release via direct ultrasound exposure. Studies on the effect of NO in tumor biology have shown that a high levels of NO (> 300 nM) leads to cytostasis or apoptosis by decreasing the translation of several cell cycle proteins and stimulating cancer cell death by activating the p53 pathway. The central hypothesis is that NO gas can be packaged and delivered through a delivery methodology to breast cancer cells to facilitate tumor regression with minimal systemic toxicity. The primary goal of this thesis is to develop an echogenic liposomal solution that has the ability to encapsulate NO, to release NO locally upon ultrasound exposure, and to induce breast cancer cell death. NO-containing echogenic liposomes (NO-ELIP) were prepared by the freezing-under-pressure method previously developed in our laboratory. It was necessary to evaluate stability of NO-ELIP and release of NO from NO-ELIP by measuring echogenicity using intravascular ultrasound images. Breast cancer cell lines, MDA-MB-231 and MDA-MB-468, were selected to investigate the cytotoxic effects of NO liberated from NO-ELIP and their response to NO concentration. Ultrasound-triggered NO release from NO-ELIP using ultrasound activation was studied. It was demonstrated that NO-ELIP remained stable for 5 hours in bovine serum albumin. Delivery of NO using NO-ELIP induced cytotoxicity and programmed cell death of MDA-MB-231 and MDA-MB-468 after 5 hours of incubation. Enhancement of the NO-ELIP effect for therapeutic application was observed with ultrasound activation. This work demonstrates that NO-ELIP can incorporate and deliver NO to breast cancer cells providing increased NO stability and ultrasound-controlled NO release. Improved therapeutic effect with the use of NO-ELIP is expected to be found for breast cancer treatment.

Calcification of a collagen-liposome complex

The purpose of the study was to evaluate in vitro calcification potential among liposomes composed of phospholipids with variations in fatty acid chains and polar head groups. The liposome was also modified by utilizing mixed phospholipids, incorporation of different types of protein to the liposome, or complexing with various collagen preparations. The samples were then incubated in a metastable calcium phosphate solution for the proposed time period. Calcium and phosphate uptake were measured. Resulting precipitates were processed for x-ray diffraction and electron microscopy. Acidic phospholipid, Dioleoylphosphatidic acid and mixed phospholipids, Dioleoylphosphatidic acid/Dipalmitoylphosphatidylethanolamine liposomes calcified at a faster rate and to a greater degree than other phospholipids tested. The incorporation of polylysine, fibronectin, bone protein, or the complexing with collagen decreased the rate and amount of calcification. Electron microscopy demonstrated the similarity of the calcified collagen-liposome complex to the natural calcification matrix. These preparations may be used as a model to study the role of membrane lipids and collagen-phospholipid during the process of calcification.^ The in vivo study was designed to determine whether the potential existed for the promotion of bone healing by the synthetic liposome-collagen complex. The implant materials were modified to provide decreased antigenicity, biocompatability while maintaining their bone conduction properties. The samples were placed subcutaneously and/or subperiosteally and/or in 8 mm calvarium defects of adult rats. Histological and immunological studies demonstrated that the implant itself retained minimal antigenicity and did not inhibit bone formation. However, modification of the implant may contain the bone induction property and be utilized to stimulate bony healing. ^

The inhibition of lipid transfer protein by negatively charged liposomes modifies the therapeutic index of amphotericin B

We have shown that liposomal amphotericin B (L-AmpB) decreased renal toxicity and maintains the antifungal activity of amphotericin B (AmpB). We have also observed that L-AmpB is predominantly associated with high density lipoproteins (HDL) as compared to Fungizone (AmpB + deoxycholate). The present experiments were designed to assess the biological relevance of transferring AmpB to HDL. We observed that AmpB was less toxic to kidney cells when associated with HDL, however AmpB toxicity was maintained when associated with LDL. To further understand how HDL-associated AmpB reduces renal cell toxicity the presence of HDL and LDL receptors in this cell line was determined. We observed that these cells expressed high and low affinity LDL receptors, but only low affinity HDL receptors. The reduced renal cell toxicity of HDL-associated AmpB may be due to its lack of interaction with renal cells because of the absence of HDL receptors. Since AmpB interacts with cholesteryl esters whose transfer among lipoproteins is regulated by Lipid transfer Protein (LTP), the role of LTP on the distribution of AmpB to HDL and LDL was next examined. We found that negatively charged liposomes significantly reduced LTP-mediated transfer of CE between HDL and LDL, independent of the presence of AmpB, while Fungizone only significantly inhibited CE transfer at one concentration tested (20$\mu$g/ml). Therefore, we believe that the decreased renal toxicity of L-AmpB is related to its predominant distribution to HDL which is regulated by the inhibition of LTP activity. ^

Regulation of phospholipid biosynthesis in {\it Saccharomyces cerevisiae\/} by CDP-diacylglycerol synthase

Amine-containing phospholipid synthesis in Saccharomyces cerevisiae starts with the conversion of CDP-diacylglycerol (CDP-DAG) and serine to phosphatidylserine (PS) while phosphatidylinositol (PI) is formed from CDP-DAG and inositol (derived from inositol-1-phosphate). In this study a gene (CDS1) encoding CDP-DAG synthase in S. cerevisiae was isolated and identified. The CDS1 gene encodes the majority, if not all, of the synthase activity, and is essential for cell growth. Overexpression of the CDS1 gene resulted in an elevation in the apparent initial rate of synthesis and also steady-state level of PI relative to PS in both wild type yeast and the cds1 mutant. Down-regulation of CDS1 expression resulted in an inositol excretion phenotype and an opposite effect on the above phospholipid synthesis in the cds1 mutant. This regulation of phospholipid biosynthesis is mediated by changes of the phospholipid biosynthetic enzymes via a mechanism independent of the expression of the INO2-OPI1 regulatory genes. Reduction in the level of CDP-DAG synthase activity resulted in an increase in PS synthase activity which followed a similar change in the CHO1/PSS (encodes PS synthase) mRNA level. INO1 (encodes inositol-1-phosphate synthase) mRNA also increased but only after CDP-DAG synthase activity fell below the wild type level. PI synthase activity followed the decrease of the CDP-DAG synthase activity, but there was no parallel change in the level of PIS1 mRNA. A G$\sp{305}$/A$\sp{305}$ point mutation within the CDS1 gene which causes the cdg1 phenotype was identified. A human cDNA clone encoding CDP-DAG synthase activity was characterized by complementation of the yeast cds1 null mutant. ^

Identification and characterization of genes encoding phospholipid biosynthetic enzymes of {\it Saccharomyces cerevisiae\/}

Phospholipids are the major component of cellular membranes. In addition to its structural role, phospholipids play an active and diverse role in cellular processes. The goal of this study is to identify the genes involved in phospholipid biosynthesis in a model eukaryotic system, Saccharomyces cerevisiae. We have focused on the biosynthetic steps localized in the inner mitochondrial membrane; hence, the identification of the genes encoding phosphatidylserine decarboxylase (PSD1), cardiolipin synthase (CLS1), and phosphatidylglycerophosphate synthase (PGS1).^ The PSD1 gene encoding a phosphatidylserine decarboxylase was cloned by complementation of a conditional lethal mutation in the homologous gene in Escherichia coli strain EH150. Overexpression of the PSD1 gene in wild type yeast resulted in 20-fold amplification of phosphatidylserine decarboxylase activity. Disruption of the PSD1 gene resulted in 20-fold reduction of decarboxylase activity, but the PSD1 null mutant exhibited essentially normal phenotype. These results suggest that yeast has a second phosphatidylserine decarboxylation activity.^ Cardiolipin is the major anionic phospholipid of the inner mitochondrial membrane. It is thought to be an essential component of many biochemical functions. In eukaryotic cells, cardiolipin synthase catalyzes the final step in the synthesis of cardiolipin from phosphatidylglycerol and CDP-diacylglycerol. We have cloned the gene CLS1. Overexpression of the CLS1 gene product resulted in significantly elevated cardiolipin synthase activity, and disruption of the CLS1 gene, confirmed by PCR and Southern blot analysis, resulted in a null mutant that was viable and showed no petite phenotype. However, phospholipid analysis showed undetectable cardiolipin level and an accumulation of phosphatidylglycerol. These results support the conclusion that CLS1 encodes the cardiolipin synthase of yeast and that normal levels of cardiolipin are not absolutely essential for survival of the cell.^ Phosphatidylglycerophosphate (PGP) synthase catalyzes the synthesis of PGP from CDP-diacylglycerol and glycerol-3-phosphate and functions as the committal and rate limiting step in the biosynthesis of cardiolipin. We have identified the PGS1 gene as encoding the PGP synthase. Overexpression of the PGS1 gene product resulted in over 15-fold increase in in vitro PGP synthase activity. Disruption of the PGS1 gene in a haploid strain of yeast, confirmed by Southern blot analysis, resulted in a null mutant strain that was viable but had significantly altered phenotypes, i.e. inability to grow on glycerol and at $37\sp\circ$C. These cells showed over a 10-fold decrease in PGP synthase activity and a decrease in both phosphatidylglycerol and cardiolipin levels. These results support the conclusion that PGS1 encodes the PGP synthase of yeast and that neither phosphatidylglycerol nor cardiolipin are absolutely essential for survival of the cell. ^

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24-33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance

Variation of the detergent-binding capacity and phospholipid content of membrane proteins when purified in different detergents.

Purified membrane proteins are ternary complexes consisting of protein, lipid, and detergent. Information about the amounts of detergent and endogenous phospholipid molecules bound to purified membrane proteins is largely lacking. In this systematic study, three model membrane proteins of different oligomeric states were purified in nine different detergents at commonly used concentrations and characterized biochemically and biophysically. Detergent-binding capacities and phospholipid contents of the model proteins were determined and compared. The insights on ternary complexes obtained from the experimental results, when put into a general context, are summarized as follows. 1), The amount of detergent and 2) the amount of endogenous phospholipids bound to purified membrane proteins are dependent on the size of the hydrophobic lipid-accessible protein surface areas and the physicochemical properties of the detergents used. 3), The size of the detergent and lipid belt surrounding the hydrophobic lipid-accessible surface of purified membrane proteins can be tuned by the appropriate choice of detergent. 4), The detergents n-nonyl-β-D-glucopyranoside and Cymal-5 have exceptional delipidating effects on ternary complexes. 5), The types of endogenous phospholipids bound to membrane proteins can vary depending on the detergent used for solubilization and purification. 6), Furthermore, we demonstrate that size-exclusion chromatography can be a suitable method for estimating the molecular mass of ternary complexes. The findings presented suggest a strategy to control and tune the numbers of detergent and endogenous phospholipid molecules bound to membrane proteins. These two parameters are potentially important for the successul crystallization of membrane proteins for structure determination by crystallographic approaches.

Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2.

Exposure of biological membranes to reactive oxygen species creates a complex mixture of distinct oxidized phospholipid (OxPL) species, which contribute to the development of chronic inflammatory diseases and metabolic disorders. While the ability of OxPL to modulate biological processes is increasingly recognized, the nature of the biologically active OxPL species and the molecular mechanisms underlying their signaling remain largely unknown. We have employed a combination of mass spectrometry, synthetic chemistry, and immunobiology approaches to characterize the OxPL generated from the abundant phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and investigated their bioactivities and signaling pathways in vitro and in vivo. Our study defines epoxycyclopentenones as potent anti-inflammatory lipid mediators that mimic the signaling of endogenous, pro-resolving prostanoids by activating the transcription factor nuclear factor E2-related factor 2 (Nrf2). Using a library of OxPL variants, we identified a synthetic OxPL derivative, which alleviated endotoxin-induced lung injury and inhibited development of pro-inflammatory T helper (Th) 1 cells. These findings provide a molecular basis for the negative regulation of inflammation by lipid peroxidation products and propose a novel class of highly bioactive compounds for the treatment of inflammatory diseases.