Detection of surfactant protein A (SP-A) and surfactant protein D (SP-D) in equine synovial fluid with immunoblotting

Once considered unique to the lung, surfactant proteins have been clearly identified in the intestine and peritoneum and are suggested to exist in several other organs. In the lung, surfactant proteins assist in the formation of a monolayer of surface-active phospholipid at the liquid-air interface of the alveolar lining, reducing the surface tension at this surface. In contrast, surface-active phospholipid adsorbed to articular surfaces has been identified as the load-bearing boundary lubricant of the joint. This raises the question of whether surfactant proteins in synovial fluid (SF) are required for the formation of the adsorbed layer in normal joints. Proteins from small volumes of equine SF were resolved by 1- and 2-dimensional polyacrylamide gel electrophoresis and detected by Western blotting to investigate the presence of surfactant proteins. The study showed that surfactant proteins A and D (SP-A and SP-D) are present in the SF of normal horses. We suggest that, like surface-active phospholipid, SP-A and SP-D play a significant role in the functioning of joints. Next will be clarification of the roles of surfactant proteins as disease markers in a variety of joint diseases, such as degenerative joint disease and inflammatory problems.

Role of LBPA and Alix in multivesicular liposome formation and endosome organization

What are the components that control the assembly of subcellular organelles in eukaryotic cells? Although membranes can clearly be distorted by cytosolic factors, very little is known about the intrinsic mechanisms that control the biogenesis, shape, and organization of organellar membranes. Here, we found that the unconventional phospholipid lysobisphosphatidic acid (LBPA) could induce the formation of multivesicular liposomes that resembled the multivesicular endosomes that exist where this lipid is found in vivo. This process depended on the same pH gradient that exists across endosome membranes in vivo and was selectively controlled by Alix. In turn, Alix regulated the organization of LBPA-containing endosomes in vivo.

The origin and evolution of the surfactant system in fish: Insights into the evolution of lungs and swim bladders

Several times throughout their radiation fish have evolved either lungs or swim bladders as gas-holding structures. Lungs and swim bladders have different ontogenetic origins and can be used either for buoyancy or as an accessory respiratory organ. Therefore, the presence of air-filled bladders or lungs in different groups of fishes is an example of convergent evolution. We propose that air breathing could not occur without the presence of a surfactant system and suggest that this system may have originated in epithelial cells lining the pharynx. Here we present new data on the surfactant system in swim bladders of three teleost fish ( the air-breathing pirarucu Arapaima gigas and tarpon Megalops cyprinoides and the non-air-breathing New Zealand snapper Pagrus auratus). We determined the presence of surfactant using biochemical, biophysical, and morphological analyses and determined homology using immunohistochemical analysis of the surfactant proteins (SPs). We relate the presence and structure of the surfactant system to those previously described in the swim bladders of another teleost, the goldfish, and those of the air-breathing organs of the other members of the Osteichthyes, the more primitive air-breathing Actinopterygii and the Sarcopterygii. Snapper and tarpon swim bladders are lined with squamous and cuboidal epithelial cells, respectively, containing membrane-bound lamellar bodies. Phosphatidylcholine dominates the phospholipid (PL) profile of lavage material from all fish analyzed to date. The presence of the characteristic surfactant lipids in pirarucu and tarpon, lamellar bodies in tarpon and snapper, SP-B in tarpon and pirarucu lavage, and SPs ( A, B, and D) in swim bladder tissue of the tarpon provide strong evidence that the surfactant system of teleosts is homologous with that of other fish and of tetrapods. This study is the first demonstration of the presence of SP-D in the air-breathing organs of nonmammalian species and SP-B in actinopterygian fishes. The extremely high cholesterol/disaturated PL and cholesterol/PL ratios of surfactant extracted from tarpon and pirarucu bladders and the poor surface activity of tarpon surfactant are characteristics of the surfactant system in other fishes. Despite the paraphyletic phylogeny of the Osteichthyes, their surfactant is uniform in composition and may represent the vertebrate protosurfactant.

Encapsulation of lipopeptides within liposomes: Effect of number of lipid chains, chain length and method of liposome preparation

The purpose of this study was to systematically investigate the effect of lipid chain length and number of lipid chains present on lipopeptides on their ability to be incorporated within liposomes. The peptide KAVYNFATM was synthesized and conjugated to lipoamino acids having acyl chain lengths of C-8, C-12 and C-16. The C-12 construct was also prepared in the monomeric, dimeric and trimeric form. Liposomes were prepared by two techniques: hydration of dried lipid films (Bangham method) and hydration of freeze-dried monophase systems. Encapsulation of lipopeptide within liposomes prepared by hydration of dried lipid films was incomplete in all cases ranging from an entrapment efficiency of 70% for monomeric lipoamino acids at a 5% (w/w) loading to less than 20% for di- and trimeric forms at loadings of 20% (w/w). The incomplete entrapment of lipopeptides within liposomes appeared to be a result of the different solubilities of the lipopeptide and the phospholipids in the solvent used for the preparation of the lipid film. In contrast, encapsulation of lipopeptide within liposomes prepared by hydration of freeze-dried monophase systems was high, even up to a loading of 20% (w/w) and was much less affected by the acyl chain length and number than when liposomes were prepared by hydration of dried lipid films. Freeze drying of monophase systems is better at maintaining a molecular dispersion of the lipopeptide within the solid phospholipid matrix compared to preparation of lipid film by evaporation, particularly if the solubility of the lipopeptide in solvents is markedly different from that of the polar lipids used for liposome preparation. Consequently, upon hydration, the lipopeptide is more efficiently intercalated within the phospholipid bilayers. (C) 2005 Elsevier B.V. All rights reserved.

Individual Palmitoyl Residues Serve Distinct Roles in H-ras Trafficking, Microlocalization, and Signaling

H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopallmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cbolesterol-dependent and cholesterol-independent microdomains. In contrast, monopallmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-pahnitate is weaker. Thus, membrane affinity of a pallmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopahnitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

Farnesoid X receptor agonism - a new approach to the treatment of cholesterol gallstone disease

Gallstone disease is very common among native Americans and Hispanics, and similar to 20 million patients are treated for this disease annually in the US. The nuclear farnesoid X receptor (FXR) is the receptor for bile acids, and GW4064 is a synthetic agonist at the FXR. FXR-/- mice fed a lithogenic diet (high fat, cholesterol and cholic acid) are more susceptible to gallstone disease than wild-type mice with the same mixed background, thus establishing that the ablation of FXR is associated with this disease. The C57L mouse is susceptible to gallstone formation. When C57L mice are fed a lithogenic diet for a week, the bile contains large aggregates of cholesterol precipitates, and two of five C57L mice had macroscopic cholesterol crystals. in contrast, when C57L mice were fed the lithogenic diet and administered GW4064 100 mg/kg/day by oral gavage, there was no precipitation of cholesterol. Treatment with this agent also increased bile salt and phospholipid concentration, and prevented gallbladder epithelium damage. As FXR agonism with GW4064 has been shown to be useful in a mouse model of cholesterol gallstone disease, it should undergo further development for the treatment of this condition.

Quil A-lipid powder formulations releasing ISCOMs and related colloidal stuctures upon hydration

The aim of the present study was to prepare solid Quil A-cholesterol-phospholid formulations (as powder mixtures or compressed to pellets) by physical mixing or by freeze-drying of aqueous dispersions of these components in ratios that allow spontaneous formation of ISCOMs and other colloidal stuctures upon hydration. The effect of addition of excess cholesterol to the lipid mixtures on the release of a model antigen (PE-FITC-OVA) from the pellets was also investigated. Physical properties were evaluated by X-ray powder diffractometry (XPRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and polarized light microscopy (PLM). Characterization of aqueous colloidal dispersions was performed by negative staining transmission electron microscopy (TEM). Physically mixed powders (with or without PE-FITC-OVA) and pellets prepared from the same powders did not spontaneously form ISCOM matrices and related colloidal structures such as worm-like micelles, ring-like micelles, lipidic/layered structures and lamellae (hexagonal array of ring-like micelles) upon hydration as expected from the pseudo-temary diagram for aqueous mixtures of Quil A, cholesterol and phospholipid. In contrast, spontaneous formation of the expected colloids was demonstrated for the freeze-dried lipid mixtures. Pellets prepared by compression of freeze-dried powders released PE-FITC-OVA slower than those prepared from physically mixed powders. TEM investigations revealed that the antigen was released in the form of colloidal particles (ISCOMs) from pellets prepared by compression of freeze-dried powders. The addition of excess cholesterol slowed down the release of antigen. The findings obtained in this study are important for the formulation of solid Quil A-containing lipid articles as controlled particulate adjuvant containing antigen delivery systems. (c) 2004 Elsevier B.V. All rights reserved.

Studies on the membrane interactions of the cyclotides kalata B1 and kalata B6 on model membrane systems by surface plasmon resonance

In this study we have demonstrated the interactions of kalata B1 and its naturally occurring analogue kalata B6 with five model lipid membranes and have analyzed the binding kinetics using surface plasmon resonance. Two kalata peptides showed a higher affinity for the phosphatidylethanolamine-containing membranes, indicating that the peptides would bind selectively to bacterial membranes. Also we have optimized the procedure for the immobilization of five liposome mixtures and have shown that the procedure provides reproducible levels of immobilized liposomes and could be used to screen the selective binding of putative antimicrobial peptides to model mammalian or microbial phospholipid membranes. (C) 2004 Elsevier Inc. All rights reserved.

Versatile peptide dendrimers for nucleic acid delivery

Dendrimers are nonviral vectors that have attracted interest on account of a number of features. They are structurally versatile because their size, shape, and surface charge can be selectively altered. Here we examine the functions of a new family of composite dendrimers that were synthesized with lipidic amino acid cores. These dendrimers are bifunctional because they are characterized by positively charged (lysine) modules for interaction with nucleic acids and neutral lipidic moieties for membrane lipid-bilayer transit. We assessed their structure-function correlations by a combination of molecular and biophysical techniques. Our assessment revealed an unexpected pleitropy of functions subserved by these vectors that included plasmid and oligonucleotide delivery. We also generated a firefly luciferase cell line in which we could modulate luciferase activity by RNA interference. We found that these vectors could also mediate RNA suppression of luciferase expression by delivering double-stranded luciferase transcripts generated in vitro. The structural uniqueness of these lipidic peptide dendrimers coupled with their ease and specificity of assembly and the versatility in their choice of cargo, puts them in a new category of macromolecule carriers. These vectors, therefore, have potential applications as epigenetic modifiers of gene function. (C) 2004 Wiley-Liss, Inc. and the American Pharmacists Association.

The structure of ultrathin Langmuir-Blodgett films of cadmium behenate

Grazing incidence x-ray-diffraction investigations of the structures of Langmuir-Blodgett films of cadmium behenate with 1, 2, 3, 5, and 21 monolayers are reported. The single monolayer film, deposited on a hydrophilic substrate, showed a hexagonal structure, whereas the bilayer film, deposited on a hydrophobic substrate, had a rectangular structure with herringbone orientation of the acyl chains. With multilayer films formed on a hydrophilic substrate, it was possible to detect that the hexagonal structure of the first layer was retained when additional layers were deposited and that the additional layers had the same rectangular structure as the bilayer. (c) 2005 American Institute of Physics.

Regulated localization of rab18 to lipid droplets - Effects of lipolytic stimulation and inhibition of lipid droplet catabolism

Rab GTPases are crucial regulators of membrane traffic. Here we have examined a possible association of Rab proteins with lipid droplets (LDs), neutral lipid-containing organelles surrounded by a phospholipid monolayer, also known as lipid bodies, which have been traditionally considered relatively inert storage organelles. Although we found close apposition between LDs and endosomal compartments labeled by expressed Rab5, Rab7, or Rab11 constructs, there was no detectable labeling of the LD surface itself by these Rab proteins. In contrast, GFP-Rab18 localized to LDs and immunoelectron microscopy showed direct association with the monolayer surface. Green fluorescent protein (GFP)-Rab18-labeled LDs underwent oscillatory movements in a localized area as well as sporadic, rapid, saltatory movements both in the periphery of the cell and toward the perinuclear region. In both adipocytes and non-adipocyte cell lines Rab18 localized to a subset of LDs. To gain insights into this specific localization, Rab18 was co-expressed with Cav3(DGV), a truncation mutant of caveolin-3 shown to inhibit the catabolism and motility of lipid droplets. GFP-Rab18 and mRFP-Cav3(DGV) labeled mutually exclusive subpopulations of LDs. Moreover, in 3T3-L1 adipocytes, stimulation of lipolysis increased the localization of Rab18 to LDs, an effect reversed by beta-adrenergic antagonists. These results show that a Rab protein localizes directly to the monolayer surface of LDs. In addition, association with the LD surface was increased following stimulation of lipolysis and inhibited by a caveolin mutant suggesting that recruitment of Rab18 is regulated by the metabolic state of individual LDs.

Differences in surfactant lipids collected from pleural and pulmonary lining fluids

Purpose. The type and relative importance of saturated and unsaturated phospholipid components of surfactant within the epithelial lining fluid (ELF) of the inner and outer surfaces of the lung is not known. Methods. Seven healthy dogs were anesthetized and a bronchoalveolar lavage (BAL) was performed, immediately followed by a pleural lavage (PL). Lipid was extracted from lavage fluid and then analyzed for saturated, primarily dipalmitoylphosphatidylcholine (DPPC), and unsaturated phosphatidylcholine (PC) species using high-performance liquid chromatography (HPLC) with combined fluorescence and ultraviolet detection. Dilution of ELF in lavage fluids was corrected for using the urea method. Results. DPPC (494.7 +/- 213.9 mu g/mL) was the predominant PC present in ELF collected from the alveolar surface. In contrast, significantly higher (p = 0.028) proportions of unsaturated PC species were measured in PL fluid (similar to 105 mu g/mL), particularly stearoyl-linoleoyl-phosphatidylcholine (SLPC), which could not be measured in fluid collected from the alveoli, compared to DPPC (2.6 +/- 2.0 mu g/mL). Conclusions. This study indicates that unsaturated PC species seem to be more important than saturated species, particularly DPPC, in the pleural cavity, which has implications for surfactant replenishment following pleural disease or thoracic surgery.

Plasma delipidation process induces rapid regression of atherosclerosis and mobilisation of adipose tissue

Cholesterol is a major component of atherosclerotic plaques. Cholesterol accumulation within the arterial intima and atherosclerotic plaques is determined by the difference of cellular cholesterol synthesis and/or influx from apo B-containing lipoproteins and cholesterol efflux. In humans, apo A-I Milano infusion has led to rapid regression of atherosclerosis in coronary arteries. We hypothesised that a multifunctional plasma delipidation process (PDP) would lead to rapid regression of experimental atherosclerosis and probably impact on adipose tissue lipids. In hyperlipidemic animals, the plasma concentrations of cholesterol, triglyceride and phospholipid were, respectively, 6-, 157-, and 18-fold higher than control animals, which consequently resulted in atherosclerosis. PDP consisted of delipidation of plasma with a mixture of butanol-diisopropyl ether (DIPE). PDP removed considerably more lipid from the hyperlipidemic animals than in normolipidemic animals. PDP treatment of hyperlipidemic animals markedly reduced intensity of lipid staining materials in the arterial wall and led to dramatic reduction of lipid in the adipose tissue. Five PDP treatments increased apolipoprotein A1 concentrations in all animals. Biochemical and hematological parameters were unaffected during PDP treatment. These results show that five PDP treatments led to marked reduction in avian atherosclerosis and removal of lipid from adipose tissue. PDP is a highly effective method for rapid regression of atherosclerosis.

Comparison of surfactant lipids between pleural and pulmonary lining fluids

Saturated phospholipids (PCs), particularly dipalmitoylphosphatidylcholine (DPPC), predominate in surfactant lining the alveoli, although little is known about the relationship between saturated and unsaturated PCs on the outer surface of the lung, the pleura. Seven healthy cats were anesthetized and a bronchoalveolar lavage (BAL) was performed, immediately followed by a pleural lavage (PL). Lipid was extracted from lavage fluid and then analyzed for saturated, primarily dipalmitoylphosphatidylcholine (DPPC), and unsaturated PC species using high-performance liquid chromatography (HPLC) with combined fluorescence and ultraviolet detection. Dilution of epithelial lining fluid (ELF) in lavage fluids was corrected for using the urea method. The concentration of DPPC in BAL fluid (85.3 +/- 15.7 mu g/mL) was significantly higher (P=0.021) than unsaturated PCs (similar to 40 mu g/mL). However, unsaturated PCs (similar to 34 mu g/mL), particularly stearoyl-linoleoyl-phosphatidylcholine (SLPC; 17.4 +/- 6.8), were significantly higher (P = 0.021) than DPPC (4.3 +/- 1.8 mu g/mL) in PL fluid. These results show that unsaturated PCs appear functionally more important in the pleural cavity, which may have implications for surfactant replenishment following pleural disease or thoracic surgery. (c) 2005 Published by Elsevier Ltd.

Solubilisation of drugs within liposomal bilayers:alternatives to cholesterol as a membrane stabilising agent

Objectives The aim of this work was to investigate the effect of cholesterol on the bilayer loading of drugs and their subsequent release and to investigate fatty alcohols as an alternative bilayer stabiliser to cholesterol. Methods The loading and release rates of four low solubility drugs (diazepam, ibuprofen, midazolam and propofol) incorporated within the bilayer of multilamellar liposomes which contained a range of cholesterol (0–33 mol/mol%) or a fatty alcohol (tetradecanol, hexadecanol and octadecanol) were investigated. The molecular packing of these various systems was also investigated in Langmuir monolayer studies. Key findings Loading and release of drugs within the liposome bilayer was shown to be influenced by their cholesterol content: increasing cholesterol content was shown to reduce drug incorporation and inclusion of cholesterol in the bilayer changed the release profile of propofol from zero-order, for phosphatidyl choline only liposomes, to a first-order model when 11 to 33 total molar % of cholesterol was present in the formulation. At higher bilayer concentrations substitution of cholesterol with tetradecanol was shown to have less of a detrimental impact on bilayer drug loading. However, the presence of cholesterol within the liposome bilayer was shown to reduce drug release compared with fatty alcohols. Monolayer studies undertaken showed that effective mean area per molecule for a 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) : cholesterol mixture deviated by 9% from the predicted area compared with 5% with a similar DSPC : tetradecanol mixture. This evidence, combined with cholesterol being a much more bulky structure, indicated that the condensing influence of tetradecanol was less compared with cholesterol, thus supporting the reduced impact of tetradecanol on drug loading and drug retention. Conclusions Liposomes can be effectively formulated using fatty alcohols as an alternative bilayer stabiliser to cholesterol. The general similarities in the characteristics of liposomes containing fatty alcohols or cholesterol suggest a common behavioural influence for both compounds within the bilayer.