Tyrosine-derived polymeric surfactant nanospheres insert cholesterol in cell membranes.

HYPOTHESIS: The design of biodegradable tyrosine-derived polymeric surfactants (TyPS) through the use of calculated thermodynamic parameters could lead to phospholipid membrane surface modifiers capable of controlling cellular properties such as viability. Delivery of cholesterol by TyPS nanospheres into membrane phospholipid domains could provide further controlled modulation of membrane physical and biological properties. EXPERIMENT: Calculated Hansen solubility parameters (∂T) and hydrophile:lipophile balances (HLB) were applied to design and synthesize a small family of diblock and triblock TyPS with different hydrophobic blocks and PEG hydrophilic blocks. Self-assembled TyPS/cholesterol nanospheres were prepared in aqueous media via co-precipitation. Cholesterol loading and Langmuir film balance surface pressures of phospholipid monolayers were obtained. TyPS and TyPS/cholesterol nanosphere effects on human dermal cell viability were evaluated by cell culture using poly(ethylene glycol) (PEG) and Poloxamer 188 as controls. FINDINGS: Stable TyPS nanospheres incorporated between 1% and 5% cholesterol. Triblock TyPS formed nanosphere with dimensions significantly smaller than diblock TyPS nanospheres. In accord calculated thermodynamic parameters, cholesterol binding increased with increasing TyPS hydrophobicity. TyPS inserted into phospholipid monolayer films in a manner consistent with their thermodynamic properties and TyPS/cholesterol nanospheres delivered cholesterol into the films. Triblock TyPS/cholesterol nanospheres increased human dermal cell viability, which was indicative of potentially beneficial TyPS effects on cell membrane surface properties.

How Predictable Are Human Stratum Corneum Lipid/Water Partition Coefficients? Assessment and Useful Correlations for Dermal Absorption.

Partition coefficients between human stratum corneum lipids and water (Ksclip/w) are collected or deduced from a variety of sources in a manner that approximately doubles the available data compared to the current state-of-the-art model (Hansen et al., Adv Drug Deliv Rev. 2013;65(2):251-264). An additional datum for water itself in porcine SC that considerably extends the molecular size and lipophilicity range of the data set is considered. The data are analyzed in terms of an extended linear free energy relationship involving octanol/water partition coefficients, Abraham solvation parameters, and a secondary, power law molecular weight dependence. The optimum fit to log Ksclip/w for the full data set reduces the standard error of prediction from 0.50 for a Hansen-like model to 0.39; corresponding multiplicative errors in Ksclip/w are reduced from a factor of 3.1 to one of 2.5. The difference in performance is driven by the water datum, which requires a more complex dependence on molecular size than that afforded by Abraham parameters. In the absence of the water value, the Hansen-like model, which does not include a dependence on molecular size, is essentially optimum. A comparison is presented to fluid-phase phospholipid-water systems, which have a demonstrably different structure-property relationship.

Rhodovulum algae sp. nov., isolated from an algal mat.

A reddish-brown-pigmented, phototrophic bacterium, designated strain JA877T, was isolated from a brown algae mat sample collected from Jalandhar beach, Gujarat, India. On the basis of the 16S rRNA gene sequence, strain JA877T belongs to the class Alphaproteobacteria and is closely related to the type strains Rhodovulum viride JA756T (99.0 %), Rhodovulum sulfidophilum Hansen W4T (98.9 %), Rhodovulumvisakhapatnamense JA181T (98.8 %),Rhodovulum kholense JA297T (97.5 %) and Rhodovulum salis JA746T (97.0). However, strain JA877T showed only 20-45 % relatedness with its phylogenetic neighbours and had a ∆Tm between 5.8 and 7.0 °C. The major respiratory quinone was ubiquinone-10 (Q10), and the polar lipid profile was composed of the major components phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, two unidentified sulfolipids and five unidentified lipids. The major fatty acids were C18 : 1ω5c, C18 : 1ω7c/C18 : 1ω6c, C16 : 0 and C18 : 0. The DNA G+C content was 64.5 mol%. On the basis of 16S rRNA gene sequence analysis, physiological data, and chemotaxonomic and molecular differences, strain JA877T is significantly different from other species of the genus Rhodovulum and represents a novel species, for which the name Rhodovulum algae sp. nov. is proposed. The type strain is JA877T (=LMG 29228T= KCTC 42963T).

Oxidative lipidomics coming of age: advances in analysis of oxidized phospholipids in physiology and pathology.

SIGNIFICANCE: Oxidized phospholipids are now well recognized as markers of biological oxidative stress and bioactive molecules with both pro-inflammatory and anti-inflammatory effects. While analytical methods continue to be developed for studies of generic lipid oxidation, mass spectrometry (MS) has underpinned the advances in knowledge of specific oxidized phospholipids by allowing their identification and characterization, and it is responsible for the expansion of oxidative lipidomics. RECENT ADVANCES: Studies of oxidized phospholipids in biological samples, from both animal models and clinical samples, have been facilitated by the recent improvements in MS, especially targeted routines that depend on the fragmentation pattern of the parent molecular ion and improved resolution and mass accuracy. MS can be used to identify selectively individual compounds or groups of compounds with common features, which greatly improves the sensitivity and specificity of detection. Application of these methods has enabled important advances in understanding the mechanisms of inflammatory diseases such as atherosclerosis, steatohepatitis, leprosy, and cystic fibrosis, and it offers potential for developing biomarkers of molecular aspects of the diseases. CRITICAL ISSUES AND FUTURE DIRECTIONS: The future in this field will depend on development of improved MS technologies, such as ion mobility, novel enrichment methods and databases, and software for data analysis, owing to the very large amount of data generated in these experiments. Imaging of oxidized phospholipids in tissue MS is an additional exciting direction emerging that can be expected to advance understanding of physiology and disease.

Rhodovulum salis sp. nov. and Rhodovulum viride sp. nov., phototrophic Alphaproteobacteria isolated from marine habitats.

Two strains (JA746(T) and JA756(T)) having yellowish brown-to-green pigment were isolated from a solar saltern and a pink pond, respectively. While both strains were non-motile and shared the presence of bacteriochlorophyll-a, major cellular fatty acids (C18 : 1ω7c, C16 : 0, C18 : 0), quinone (Q-10), polar lipids and hopanoids, they differed from each other in their carotenoid composition. The G+C content of genomic DNA of strains JA746(T) and 756(T) was 62.4 and 63.3 mol%, respectively. The 16S rRNA gene-based EzTaxon-e blast search analysis of strains JA746(T) and 756(T) indicated highest sequence similarity with members of the genus Rhodovulum in the family Rhodobacteraceae of the class Alphaproteobacteria. Strain JA746(T) has high sequence similarities with Rhodovulum visakhapatnamense JA181(T) (97.3 %), Rhodovulum steppense A-20s(T) (97.3 %), Rhodovulum phaeolacus JA580(T) (97 %), Rhodovulum strictum MB-G2(T) (97 %) and other members of the genus Rhodovulum (<97 %). Strain JA756(T) has high sequence similarities with Rhodovulum visakhapatnamense JA181(T) (99.8 %), Rhodovulum sulfidophilum Hansen W4(T) (99.1 %), Rhodovulum kholense JA297(T) (97.9 %) and other members of the genus Rhodovulum (<97 %). The sequence similarity between strains JA746(T) and JA756(T) was 97.5 %. However, these strains are not closely related to each other or to their phylogenetic neighbours since the DNA-DNA reassociation values were less than 56 %. The genomic information was also supported by phenotypic and chemotaxonomic results, leading us to classify strains JA746(T) ( = NBRC 108898(T) = KCTC 15180(T)) and JA756(T) ( = NBRC 109122(T) = KCTC 15223(T)) as the type strains of two novel species of the genus Rhodovulum, for which the names Rhodovulum salis sp. nov. and Rhodovulum viride sp. nov. are proposed, respectively.

Host-derived oxidized phospholipids and HDL regulate innate immunity in human leprosy.

Intracellular pathogens survive by evading the host immune system and accessing host metabolic pathways to obtain nutrients for their growth. Mycobacterium leprae, the causative agent of leprosy, is thought to be the mycobacterium most dependent on host metabolic pathways, including host-derived lipids. Although fatty acids and phospholipids accumulate in the lesions of individuals with the lepromatous (also known as disseminated) form of human leprosy (L-lep), the origin and significance of these lipids remains unclear. Here we show that in human L-lep lesions, there was preferential expression of host lipid metabolism genes, including a group of phospholipases, and that these genes were virtually absent from the mycobacterial genome. Host-derived oxidized phospholipids were detected in macrophages within L-lep lesions, and 1 specific oxidized phospholipid, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine (PEIPC), accumulated in macrophages infected with live mycobacteria. Mycobacterial infection and host-derived oxidized phospholipids both inhibited innate immune responses, and this inhibition was reversed by the addition of normal HDL, a scavenger of oxidized phospholipids, but not by HDL from patients with L-lep. The accumulation of host-derived oxidized phospholipids in L-lep lesions is strikingly similar to observations in atherosclerosis, which suggests that the link between host lipid metabolism and innate immunity contributes to the pathogenesis of both microbial infection and metabolic disease.

Plasma membrane composition of Debaryomyces hansenii adapts to changes in pH and external salinity.

Debaryomyces hansenii is a marine yeast that has to cope with different stress situations. Since changes in membrane properties can play an important function in adaptation, we have examined the fluidity and lipid composition of purified plasma membranes of D. hansenii grown at different external pH values and salt concentrations. Growth at low pH caused an increase in the sterol-to-phospholipid ratio and a decrease in fatty acid unsaturation which was reflected in decreased fluidity of the plasma membrane. High levels of NaCl increased the sterol-to-phospholipid ratio and fatty acid unsaturation, but did not significantly affect fluidity. The sterol-to-phospholipid ratios obtained in D. hansenii grown under any of these conditions were similar to the ratios that have been reported for halophilic/halotolerant black yeasts, but much smaller than those observed in the model yeast Saccharomyces cerevisiae.

Resuscitation of dormant Mycobacterium tuberculosis by phospholipids or specific peptides.

The presence of dormant tubercle bacilli presents a major problem for tuberculosis treatment. The culture supernatant of Mycobacterium tuberculosis was previously shown to resuscitate dormant bacilli in vitro. Here we report identification of active components as phospholipids and a tuberculosis protein Rv1174c. Remarkably, dormant bacilli from a one year old culture which failed to form any colonies could be resuscitated with peptides derived from Rv1174c and formed 10(5-7) colonies/ml. This finding represents the first unambiguous demonstration of resuscitation of dormant tubercle bacilli in vitro and may have implication for the study of mycobacterial dormancy and the design of novel strategies for improved treatment of tuberculosis.

The Rubino test for leprosy is a beta2-glycoprotein 1-dependent antiphospholipid reaction.

We describe the isolation and identification of three components required for the Rubino reaction (RR), which is the rapid sedimentation of formalinized sheep red-blood cells (SRBC) initiated by serum from leprosy patients with defective Mycobacterium leprae-specific cell immunity. The Rubino reaction factor (RRF) required for this phenomenon, previously identified as an immunoglobulin M (IgM), was purified from leprosy patient serum by adsorption to formalinized SRBC. Purified RRF IgM, when added to formalinized SRBC, did not produce a positive RR. However, when the contact was carried out in the presence of normal human serum (NHS), cells rapidly sedimented. The purified cofactor from NHS contained two components of 70 000 and 50 000 molecular weight (MW), as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The latter was recognized by the RRF IgM on immunoblot and its N-terminal sequence indicated that it was beta2-glycoprotein 1 (beta2-GP1), an anionic phospholipid-binding protein. Methanol-treated formalinized SRBC did not support the RR. Thin-layer chromatography of an extract of membranes indicated that the SRBC ligand was a cell-surface phospholipid. Cardiolipin inhibited the RR. These data demonstrate that the RR involves a trimolecular interaction in which IgM, beta2-GP1 and an SRBC phospholipid participate. By analogy with the antiphospholipid antibodies (anti-PL) that occur in autoimmune processes, serum samples from 29 systemic lupus erythematosus patients with high levels of anticardiolipin antibodies were submitted to the RR. A positive RR was obtained for 45% (13 of 29 patients). These results modify the paradigm of the absolute specificity of the RR for leprosy and demonstrate that RRF IgM is a beta2-GP1-dependent anti-PL.

Alterations of rat liver phospholipid composition induced by oral thalidomide.

In an attempt to elucidate the effects of oral thalidomide on liver phospholipid composition, doses of 1 and 3 mg/kg/day of thalidomide were orally administered to two groups of female Wistar rats (7 animals each), respectively, over a period of 60 days. Control animals (n = 7) received corresponding quantities of the vehicle alone. Chromatographic analysis and quantitative determination of the isolated phospholipid classes revealed statistically significant alterations of phospholipid fractions in the liver of the animals treated with the higher thalidomide dose (3 mg/kg/day). These alterations may be associated with changes in the metabolic activity, ionic transport and cell-cell interactions of the hepatic cellular components.

Do antibodies to phospholipid antigens play any role in murine leprosy?

In order to know whether antibodies to phospholipids and other host lipids play a role in the pathology of murine leprosy, we looked for the presence of antibodies to cardiolipin, cerebroside sulfatide, and to lipids extracted from normal murine spleen, liver and brain in the sera of mice bearing a 6-month infection with Mycobacterium lepraemurium. We also looked for the presence of antibodies to lipids isolated from M. lepraemurium. We found that all of the 16 animals examined contained high levels of antibodies to the mycobacterial lipids of intermediate polarity (mostly glycolipids) but none of them had antibodies to the other lipids tested, including those isolated from mouse liver, spleen and brain, bovine cardiolipin and sulfatide, nor any significant levels of antibodies to mycobacterial lipids of high or low polarity. The infected animals also had high levels of antibodies to antigens sonically extracted from the microorganism. Antibodies to the socially extracted antigens (mostly proteins) were mainly IgG, while antibodies to the lipid antigens were predominantly IgM. Despite the low but significant percentage (1%-3%) of infected animals developing bilateral paralysis of the rear limbs, autoimmunity (due to antibodies to phospholipids and other host lipids) does not seem to be a feature of murine leprosy.

Serodiagnostic efficiency of phospholipid associated protein of Mycobacterium tuberculosis H37Rv.

The phospholipid-associated protein (55-67 kDa) fraction of Mycobacterium tuberculosis H37Rv was purified as the DE-V protein fraction. This DE-V fraction was used for diagnosis of tuberculosis by enzyme-linked immunosorbent assay (ELISA), detecting IgG antibody in sera collected from different categories of tuberculosis patients, i.e. with acid fast bacilli (AFB) culture-positive pulmonary tuberculosis, with AFB culture-negative, but radiologically suspected, pulmonary tuberculosis, extrapulmonary tuberculosis, and control groups of patients suffering from diseases other than tuberculosis (asthma and/or rhinitis, lepromatous leprosy) as well as from healthy volunteers. Encouraging operational ELISA validity could be achieved with 93% sensitivity, 100% specificity, 97% efficiency, 100% positive predictivity and 95% negative predictability even among the extrapulmonary and suspected pulmonary tuberculosis patients. The above assay was insensitive but with 100% specificity among control group of patients suffering from diseases other than tuberculosis. The DE-V protein fraction was associated with phosphatidyl inositol and phosphatidyl inositol mannosides. The dissociation of phospholipid-protein complex decreased ELISA specificity. ELISA reactivity of the DE-V fraction appeared to be thermostable; thus, it may have serodiagnostic utility in developing countries.

Correlation of serum lipids and lipoproteins in leprosy.

Serum lipids and lipoproteins were assessed in sixty leprosy and forty age and sex matched healthy controls. The study subjects included cases of LL with reactions, LL without reactions, BL with reactions, BL without reactions, BT and TT types of leprosy. The levels of serum phospholipids, triglycerides, total cholesterol, LDL and VLDL fractions were significantly decreased in leprosy patients compared to control subjects. The levels of serum HDL cholesterol and HDL fraction were significantly elevated in leprosy patients. Maximum elevation in serum HDL cholesterol level and HDL fraction and maximum reduction in the levels of serum phospholipids, triglycerides, total cholesterol and LDL and VLDL fractions were observed in lepromatous leprosy (LL) patients with reactions.

Phospholipase activity of Mycobacterium leprae harvested from experimentally infected armadillo tissue.

Three types of phospholipase activity--phospholipase A1, A2, and lysophospholipase--were detected in Mycobacterium leprae harvested from armadillo tissue at about 25% of the specific activity found in a slowly growing mycobacterium, Mycobacterium microti, which was grown in medium to optimize its phospholipase activity. The highest activity found was lysophospholipase, which released fatty acid from 2-lyso-phosphatidylcholine. Phospholipase activity was detected by using phosphatidylcholine and phosphatidylethanolamine. Differences in relative activities with these three types of substrate distinguished phospholipase activity in M. leprae extracts from armadillo liver extracts. Furthermore, retention of activity in M. leprae after NaOH treatment showed that the activity associated with M. leprae was not host derived. The specific activity of phospholipase was 20 times higher in extracts of M. leprae than in intact M. leprae organisms. Diazotization, a treatment which abolishes activities of surface enzymes exposed to the environment by the formation of covalent azide bonds with exposed amino groups, did not affect M. leprae's phospholipase activity, with one exception: release of arachidonic acid from phosphatidylcholine, which was partially inhibited. Phenolic glycolipid I, the major excreted amphipathic lipid of M. leprae, inhibited phospholipase activity, including release of arachidonic acid, for both M. leprae- and armadillo-derived activity.

Essential fatty acids in plasma of patients with leprosy.

We have investigated the fatty-acid composition of plasma phospholipids in 61 patients with leprosy of various clinical types with either a short or long duration of treatment. All patients had significantly decreased levels of linoleic acid and alpha-linoleic acid, the parent fatty acids of the n-6 and n-3 families, respectively. Patients with a treatment duration of more than 6 months had significantly low levels of arachidonic acid and eicosapentaenoic acid compared to controls or to patients with a treatment duration of less than 6 months. There were no differences in the fatty-acid composition between multibacillary patients and paucibacillary patients. We conclude that dietary supplementation with essential fatty acids may be indicated in patients with leprosy, particularly in those with a long treatment duration.

Isolation and structural characteristics of a monoclonal antibody-defined cross-reactive phospholipid antigen from Mycobacterium tuberculosis and Mycobacterium leprae.

A low molecular weight antigen of Mycobacterium leprae and other mycobacteria was previously defined in our laboratory by means of IgG2a monoclonal antibody termed L4. The antigen had an apparent molecular mass of 4.5-6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was assumed to be a glycoprotein on the basis of its staining with periodic acid Schiff and sensitivity to periodate treatment. In the present work, the cross-reactive and phospholipidic nature of the antigen, present in Mycobacterium tuberculosis as well as in M. leprae sonicates, was demonstrated and this enabled us to undertake its purification from crude M. tuberculosis phospholipidic extracts. The L4-reactive antigen from M. tuberculosis called L4-PIM, was purified by means of silicic acid high pressure liquid chromatography. Its characterization by gas chromatography and FAB-MS showed the antigen to be the common mycobacterial dimannosylated phosphatidylinositol (PIM2), the structure of which had been previously established by others (Lee, Y. C., and Ballou, C. E. (1964) J. Biol. Chem. 239, 1316-1327; Lee, Y. C., and Ballou, C. E. (1965) J. Biochem. (Tokyo) 4, 1395-1404). Delineation of the L4 epitope on M. tuberculosis L4-PIM revealed the involvement of the axial 2-hydroxyl of the alpha-D-mannosyl residues, without any detectable contribution from the myo-inositol. Consequently, L4 was shown to react with PIM5, the structure of which contains twice the number of epitopes as does PIM2. By using both immunostained thin layer chromatography and indirect enzyme-linked immunosorbent assay, similar L4-PIM epitopes were demonstrated in M. leprae sonicate, thereby explaining the cross-reactive nature of the L4-monoclonal antibody. Antibodies of IgG class directed against M. tuberculosis L4-PIM were detectable in sera from patients with leprosy, but no evidence of T cell reactivity to L4-PIM was obtained. The demonstration of a correlation of anti-L4-PIM IgG and anti-disacharide-conjugated bovine serum albumin IgM antibody titers in the sera of leprosy patients indicates that measurement of antibodies directed against L4-PIM may have the potential to be used as a complementary assay to the disaccharide-conjugated bovine serum albumin test for diagnosis and monitoring of patients undergoing leprosy therapy.