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Here we report the characterization and optimization of a peptide/non-cationic lipid gene delivery system that successfully produces high levels of gene expression when delivered by microinjection into chicken embryos in vivo. In addition to plasmid DNA, the delivery complex consisted of four components: 1) a "condensing" peptide with both hydrophobic and cationic amino acid segments; 2) a "fusogenic" peptide with both membrane insertion and amphipathic helical segments; 3) a relatively short-chain phosphatidylcholine (14:1 cis-9); and 4) polyethyleneglycol conjugated to dioleoylphosphatidylethanolamine through a disulfide linkage. Optimum amounts of each component were determined by measuring expression of a luciferase reporter gene following a 24-hour incubation with chick embryo fibroblast (CEF) cells in culture. When relatively low amounts of condensing peptide, fusogenic peptide, or lipid were assembled into the complexes, relatively large concentrations of complex were required to reach maximum gene expression. When the amounts of peptide or lipid were increased, less complex was required to achieve maximum expression, but expression fell substantially with higher amounts of added complex. The polyethyleneglycol component significantly increased gene expression. With some preparations, luciferase activities in the CEF cells reached 1x10(10) relative light units per second per mg protein within 24 hours. Following the optimization experiments with the CEF cells, formulations containing low levels, intermediate levels, and high levels of the delivery system components were assembled with green fluorescent protein plasmid DNA, then microinjected into somite regions of chicken embryos in vivo. It was found that intermediate levels of the components gave the most reliable formulations for inducing localized gene expression in the somitic cells.

A small library of monovalent and bivalent Smac mimics was synthesized based on 2 types of monomers, with general structure NMeAla-Xaa-Pro-BHA (Xaa=Cys or Lys). Position 2 of the compounds was utilized to dimerize both types of monomers employing various bis-reactive linkers, as well as to modify selected compounds with lipids. The resulting library was screened in vitro against metastatic human breast cancer cell line MDA-MB-231, and the two most active compounds selected for in vivo studies. The most active lipid-conjugated analogue M11, showed in vivo activity while administered both subcutaneously and orally. Collectively, our findings suggest that lipidation may be a viable approach in the development of new Smac-based therapeutic leads.

Background: Optimal functionality of synthetic lung surfactant for treatment of respiratory distress syndrome in preterm infants largely depends on the quality and quantity of the surfactant protein B (SP-B) peptide mimic and the lipid mixture. B-YL peptide is a 41-residue sulfur-free SP-B mimic with its cysteine and methionine residues replaced by tyrosine and leucine, respectively, to enhance its oxidation resistance.Aim: Testing the structural and functional stability of the B-YL peptide in synthetic surfactant lipids after long-term storage.Methods: The structural and functional properties of B-YL peptide in surfactant lipids were studied using three production runs of B-YL peptides in synthetic surfactant lipids. Each run was held at 5 °C ambient temperature for three years and analyzed with structural and computational techniques, i.e., MALDI-TOF mass spectrometry, ATR-Fourier Transform Infrared Spectroscopy (ATR-FTIR), secondary homology modeling of a preliminary B-YL structure, and tertiary Molecular Dynamic simulations of B-YL in surfactant lipids, and with functional methods, i.e., captive bubble surfactometry (CBS) and retesting in vivo surface activity in surfactant-deficient young adult rabbits.Results: MALDI-TOF mass spectrometry showed no degradation of the B-YL peptide as a function of stored time. ATR-FTIR studies demonstrated that the B-YL peptide still assumed stable alpha-helical conformations in synthetic surfactant lipids. These structural findings correlated with excellent in vitro surface activity during both quasi-static and dynamic cycling on CBS after three years of cold storage and in vivo surface activity of the aged formulations with improvements in oxygenation and dynamic lung compliance approaching those of the positive control surfactant Curosurf®.Conclusions: The structure of the B-YL peptide and the in vitro and in vivo functions of the B-YL surfactant were each maintained after three years of refrigeration storage. [ABSTRACT FROM AUTHOR]

Breathing is enabled by lung surfactant, a mixture of proteins and lipids that forms a surface-active layer and reduces surface tension at the air-water interface in lungs. Surfactant protein B (SP-B) is an essential component of lung surfactant. In this study we probe the mechanism underlying the important functional contributions made by the N-terminal 7 residues of SP-B, a region sometimes called the “insertion sequence”. These studies employed a construct of SP-B, SP-B (1–25,63–78), also called Super Mini-B, which is a 41-residue peptide with internal disulfide bonds comprising the N-terminal 7-residue insertion sequence and the N- and C-terminal helices of SP-B. Circular dichroism, solution NMR, and solid state 2H NMR were used to study the structure of SP-B (1–25,63–78) and its interactions with phospholipid bilayers. Comparison of results for SP-B (8–25,63–78) and SP-B (1–25,63–78) demonstrates that the presence of the 7-residue insertion sequence induces substantial disorder near the centre of the lipid bilayer, but without a major disruption of the overall mechanical orientation of the bilayers. This observation suggests the insertion sequence is unlikely to penetrate deeply into the bilayer. The 7-residue insertion sequence substantially increases the solution NMR linewidths, most likely due to an increase in global dynamics. [ABSTRACT FROM AUTHOR]

The structural basis for the Gram selectivity of two disulfide-bonded β-hairpin antimicrobial peptides (AMPs) is investigated using solid-state nuclear magnetic resonance (NMR) spectroscopy. The hexa-arginine PG-i exhibits potent activities against both Gram-positive and Gram-negative bacteria, while a mutant of PG-1 with only three cationic residues maintains Gram-positive activity but is 30-fold less active against Gram-negative bacteria. We determined the topological structure and lipid interactions of these two peptides in a lipopolysaccharide (LPS)-rich membrane that mimics the outer membrane of Gram-negative bacteria and in the POPE/POPG membrane, which mimics the membrane of Gram-positive bacteria. 31P NMR line shapes indicate that both peptides cause less orientational disorder in the LPS-rich membrane than in the POPE/POPG membrane. 13C chemical shifts and 13C-¹H dipolar couplings show that both peptides maintain their/I-hairpin conformation in these membranes and are largely immobilized, but the mutant exhibits noticeable intermediate-time scale motion in the LPS membrane at physiological temperature, suggesting shallow insertion. tndeed, ¹H spin diffusion from lipid chains to the peptides shows that PG-i fully inserts into the LPS-rich membrane whereas the mutant does not. The 13C-31P distances between the most hydrophobically embedded Mg of PG-1 and the lipid 31P are significantly longer in the LPS membrane than in the POPE! POPG membrane, indicating that PG-1 does not cause toroidal pore defects in the LPS membrane, in contrast to its behavior in the POPE/POPG membrane. Taken together, these data indicate that PG-1 causes transmembrane pores of the barrel-stave type in the LPS membrane, thus allowing further translocation of the peptide into the inner membrane of Gram-negative bacteria to kill the cells. In comparison, the less cationic mutant cannot fully cross the LPS membrane because of weaker electrostatic attractions, thus causing weaker antimicrobial activities. Therefore, strong electrostatic attraction between the peptide and the membrane surface, ensured by having a sufficient number of Arg residues, is essential for potent antimicrobial activities against Gram-negative bacteria. The data provide a rational basis for controlling Gram selectivity of AMPs by adjusting the charge densities. [ABSTRACT FROM AUTHOR]

Lung surfactant protein B (SP-B) is required for proper surface activity of pulmonary surfactant. In model lung surfactant lipid systems composed of saturated and unsaturated lipids, the unsaturated lipids are removed from the film at high compression. It is thought that SP-B helps anchor these lipids closely to the monolayer in three-dimensional cylindrical structures termed "nanosilos" seen by atomic force microscopy imaging of deposited monolayers at high surface pressures. Here we explore the role of the SP-B NH2 terminus in the formation and stability of these cylindrical structures, specifically the distribution of lipid stack height, width, and density with four SP-B truncation peptides: SP-B 1-25, SP-B 9-25, SP-B 11-25, and SP-B 1-25Nflex (prolines 2 and 4 substituted with alanine). The first nine amino acids, termed the insertion sequence and the interface seeking tryptophan residue 9, are shown to stabilize the formation of nanosilos while an increase in the insertion sequence flexibility (SP-B 1-25Nflex) may improve peptide functionality. This provides a functional understanding of the insertion sequence beyond anchoring the protein to the two-dimensional membrane lining the lung, as it also stabilizes formation of nanosilos, creating reversible repositories for fluid lipids at high compression. In lavaged, surfactant-deficient rats, instillation of a mixture of SP-B 1-25 (as a monomer or dimer) and synthetic lung lavage lipids quickly improved oxygenation and dynamic compliance, whereas SP-B 11-25 surfactants showed oxygenation and dynamic compliance values similar to that of lipids alone, demonstrating a positive correlation between formation of stable, but reversible, nanosilos and in vivo efficacy. [ABSTRACT FROM AUTHOR]

Background: Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., ∼residues 8-25 and 63-78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1-7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity. Methodology/Results: FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary α-helix and secondary β-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a ''saposin-like'' fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B. Conclusion: Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B. [ABSTRACT FROM AUTHOR]

Surfactant protein B (SP-B) is an essential component of pulmonary surfactant. Synthetic dimeric SP-B 1–25 (SP-B 1–25 ), a peptide based on the N-terminal domain of human SP-B, efficiently mimics the functional properties of SP-B. The authors investigated the optimum lipid composition for SP-B 1–25 by comparing the effects of natural lung lavage lipids (NLL), a synthetic equivalent of NLL (synthetic lavage lipids SLL), and a standard lipid mixture (TL) on the activities of SP-B 1–25 . Surfactant preparations were formulated by mixing 2 mol% SP-B 1–25 in NNL, SLL, and TL. Calfactant, a calf lung lavage extract with SP-B and SP-C, was a positive control and lipids without peptide were negative controls. Minimum surface tension measured on a captive bubble surfactometer was similar for the three SP-B 1–25 surfactant preparations and calfactant. The effects on lung function were compared in ventilated, lavaged, surfactant-deficient rats. Oxygenation and lung volumes were consistently higher in rats treated with calfactant and SP-B 1–25 in NLL or SLL than in rats treated with SP-B 1–25 in TL. Fourier transform infrared spectra observed abnormal secondary conformations for SP-B 1–25 in TL as a possible cause for the reduced lung function. Lipid composition plays a crucial role in the in vitro and in vivo functions of SP-B 1–25 in surfactant preparations. [ABSTRACT FROM AUTHOR]

Presents information on a study which examined the multiple states of beta-sheet peptide protegrin in lipid bilayers. Methodology of the study; Results and discussion.

Abstract: The ability to selectively target the harmful microbial membrane over that of the host cell is one of the most important characteristics of the antimicrobial peptides (AMPs). This selectivity strongly depends on the chemical and structural properties of the lipids that make up the cell membrane. A systematic study of the initial membrane selectivity of protegrin-1 (PG-1), a β-sheet AMP, was performed using Langmuir monolayers. Constant pressure insertion assay was used to quantify the amount of PG-1 insertion and fluorescence microscopy was employed to observe the effect of PG-1 on lipid ordering. Charge and packing properties of the monolayer were altered by using lipids with different head groups, substituting saturated with unsaturated lipid tail group(s) and incorporating spacer molecules. PG-1 inserted most readily into anionic films composed of phosphatidylglycerol (PG) and lipid A, consistent with its high selectivity for microbial membranes. It also discriminated between zwitteranionic phospholipids, inserting more readily into phosphatidylcholine (PC) monolayers than those composed of phosphatidylethanolamine, potentially explaining why PG-1 is hemolytic for PC-rich human erythrocytes and not for the PE-rich erythrocytes of ruminants. Increased packing density of the monolayer by increased surface pressure, increased tail group saturation or incorporation of dihydrocholesterol diminishes the insertion of PG-1. Fluorescence microscopy shows that lipid packing is disordered upon PG-1 insertion. However, the presence of PG-1 can still affect lipid morphology even with no observed PG-1 insertion. These results show the important role that lipid composition of the cell membrane plays in the activity of AMPs. [Copyright &y& Elsevier]

The formation of lipid bilayers, lifted from the solid substrate by layer-by-layer polyion cushions, on self-assembled monolayers (SAMs) on gold was investigated by surface plasmon resonance (SPR) and fluorescence recovery after photobleaching (FRAP). The polyions poly(diallyldimethylammonium chloride) (PDDA) and polystyrene sulfonate (PSS) sodium salt were used for the layer-by-layer polyion macromolecular assembly. The cushion was formed by electrostatic interaction of PDDA/PSS/PDDA layers with a negatively charged surface of an SAM of 11-mercaptoundecanoic acid (MUA) on gold. The lipid bilayer membranes were deposited by vesicle fusion with different compositions of SOPS (an anionic lipid, 1-stearoyl-2-oleoyl-phosphatidylserine) and POPC (a zwitterionic lipid, 1-palmitoyl-2-oleoylphosphatidylcholine). In the case of pure SOPS and for lipid mixtures with a POPC composition up to 25%, single bilayers were deposited. FRAP experiments showed that single bilayers supported on PDDA/PSS/PDDA/MUA were mobile at room temperature, with lateral coefficients of approximately (1.2–2.1)×10−9 cm2/s. The kinetics of the addition of the ion-channel-forming peptide protegrin-1 to the supported bilayers was detected by SPR. A two-step interaction was observed, similar to the association behavior of protegrin-1 with bilayers supported on PDDA/MUA. The results are similar to that of supported lipid bilayers without a layer-by-layer cushion. The model membrane system in this work is a potential biosensor for mimicking the natural activities of biomolecules and is a possible tool to investigate the fundamental properties of biomembranes. [Copyright &y& Elsevier]

Focuses on plicatamide, an antimicrobial octapeptide from Styla plicata hemocytes. Formation of cation-selective channels in model lipid bilayers composed of bacterial lipids; Finding that plicatamide was potently hemolytic for human red blood cells but did not lyse ovine erythrocytes.

Examines the two states of cyclic antimicrobial peptide RTD-1 in lipid layers. Difference of the placement and connectivity of cysteine residues; Versatility of peptide and membrane interactions; Formation of amide linking the amino and carboxy-termini.

Antimicrobial peptides (AMPs) are a promising class of innate host defense molecules for next-generation antibiotics, as they uniquely target and permeabilize membranes of pathogens. This selectivity has been explained by the electrostatic attraction between these predominantly cationic peptides and the bacterial membrane, which is heavily populated with anionic lipids. However, AMP-resistant bacteria have non-electrostatic countermeasures that modulate membrane rigidity and thickness. We explore how variations in physical properties affect the membrane affinity and disruption process of protegrin-1 (PG-1) in phosphatidylcholine (PC) membranes with altered lipid packing densities and thicknesses. From isothermal titration calorimetry and atomic force microscopy, our results showed that PG-1 could no longer insert into membranes of increasing cholesterol amounts nor into monounsaturated PC membranes of increasing thicknesses with similar fluidities. Prevention of PG-1's incorporation consequently made the membranes more resistant to peptide-induced structural transformations like pore formation. Our study provides evidence that AMP affinity and activity are strongly correlated with the fluidity and thickness of the membrane. A basic understanding of how physical mechanisms can regulate cell selectivity and resistance towards AMPs will aid in the development of new antimicrobial agents. • PG-1 has detergent-like behavior, inducing membrane structural transformations. • Membrane rigidification by cholesterol lowers the affinity and activity of PG-1. • Thicker membranes, even when fluid, deter PG-1 insertion and membrane poration. • AMP affinity/activity are correlated with the fluidity and thickness of the membrane. • AMP selectivity is more complicated than simple electrostatics suggests. Unlabelled Image [ABSTRACT FROM AUTHOR]