The Central PXXP Motif Is Crucial for PMAP-23 Translocation across the Lipid Bilayer.

PMAP-23, a cathelicidin-derived host defense peptide, does not cause severe membrane permeabilization, but exerts strong and broad-spectrum bactericidal activity. We have previously shown that it forms an amphipathic α-helical structure with a central hinge induced by the PXXP motif, which is implicated in the interaction of PMAP-23 with negatively charged bacterial membranes. Here, we studied the potential roles of the PXXP motif in PMAP-23 translocation across the lipid bilayer by replacing Pro residues with either α-helix former Ala (PMAP-PA) or α-helix breaker Gly (PMAP-PG).

Release pattern of dexamethasone after administration through an implant-mediated drug delivery device with an active plunger of super absorbent polymer.

The implant-mediated drug delivery system (IMDDS) is a novel, innovative device that allows drug delivery through bone marrow. The purpose of this study was to investigate the effect of an active plunger component made of super absorbent polymer (SAP) on the plasma concentration of dexamethasone released from the IMDDS. The IMDDSs were installed in a total of 18 rabbits.

Asymmetric Phosphatidylethanolamine Distribution Controls Fusion Pore Lifetime and Probability.

Little attention has been given to how the asymmetric lipid distribution of the plasma membrane might facilitate fusion pore formation during exocytosis. Phosphatidylethanolamine (PE), a cone-shaped phospholipid, is predominantly located in the inner leaflet of the plasma membrane and has been proposed to promote membrane deformation and stabilize fusion pores during exocytotic events. To explore this possibility, we modeled exocytosis using plasma membrane SNARE-containing planar-supported bilayers and purified neuroendocrine dense core vesicles (DCVs) as fusion partners, and we examined how different PE distributions between the two leaflets of the supported bilayers affected SNARE-mediated fusion.

HIV virions sense plasma membrane heterogeneity for cell entry.

It has been proposed that cholesterol in host cell membranes plays a pivotal role for cell entry of HIV. However, it remains largely unknown why virions prefer cholesterol-rich heterogeneous membranes to uniformly fluid membranes for membrane fusion. Using giant plasma membrane vesicles containing cholesterol-rich ordered and cholesterol-poor fluid lipid domains, we demonstrate that the HIV receptor CD4 is substantially sequestered into ordered domains, whereas the co-receptor CCR5 localizes preferentially at ordered/disordered domain boundaries.

Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein.

Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein's folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons.

The role of cholesterol in membrane fusion.

Cholesterol modulates the bilayer structure of biological membranes in multiple ways. It changes the fluidity, thickness, compressibility, water penetration and intrinsic curvature of lipid bilayers. In multi-component lipid mixtures, cholesterol induces phase separations, partitions selectively between different coexisting lipid phases, and causes integral membrane proteins to respond by changing conformation or redistribution in the membrane.

Line tension at lipid phase boundaries as driving force for HIV fusion peptide-mediated fusion.

Lipids and proteins are organized in cellular membranes in clusters, often called 'lipid rafts'. Although raft-constituent ordered lipid domains are thought to be energetically unfavourable for membrane fusion, rafts have long been implicated in many biological fusion processes. For the case of HIV gp41-mediated membrane fusion, this apparent contradiction can be resolved by recognizing that the interfaces between ordered and disordered lipid domains are the predominant sites of fusion.

Supported lipid bilayers as models for studying membrane domains.

Supported lipid bilayers have been in use for over 30 years. They have been employed to study the structure, composition, and dynamics of lipid bilayer phases, the binding and distribution of soluble, integral, and lipidated proteins in membranes, membrane fusion, and interactions of membranes with elements of the cytoskeleton. This review focuses on the unique ability of supported lipid bilayers to study liquid-ordered and liquid-disordered domains in membranes.

HIV gp41-mediated membrane fusion occurs at edges of cholesterol-rich lipid domains.

Lipid rafts in plasma membranes have emerged as possible platforms for the entry of HIV and other viruses into cells. However, little is known about how lipid phase heterogeneity contributes to viral entry because of the fine-grained and still poorly understood complexity of biological membranes. We used model systems mimicking HIV envelopes and T cell membranes and found that raft-like liquid-ordered (Lo-phase) lipid domains were necessary and sufficient for efficient membrane targeting and fusion.

A three dimensional observation of palatal vault growth in children using mixed effect analysis: a 9 year longitudinal study.

The understanding of palatine vault growth in normal subjects is important to orthodontists. The aim of this study was to evaluate three dimensional (3D) longitudinal changes in the palatal vault from 6 to 14 years of age. Complete dental stone casts were biennially prepared for 50 subjects (25 girls and 25 boys) followed up from 6 to 14 years of age.

Mechanistic study of broadly neutralizing human monoclonal antibodies against dengue virus that target the fusion loop.

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection.

Cell-penetrating peptide induces leaky fusion of liposomes containing late endosome-specific anionic lipid.

Cationic cell-penetrating peptides (CPPs) are a promising vehicle for the delivery of macromolecular drugs. Although many studies have indicated that CPPs enter cells by endocytosis, the mechanisms by which they cross endosomal membranes remain elusive. On the basis of experiments with liposomes, we propose that CPP escape into the cytosol is based on leaky fusion (i.

Dengue virus ensures its fusion in late endosomes using compartment-specific lipids.

Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN), the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycero)phosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery.

Analysis of the solution structure of the human antibiotic peptide dermcidin and its interaction with phospholipid vesicles.

Dermcidin is a human antibiotic peptide that is secreted by the sweat glands and has no homology to other known antimicrobial peptides. As an initial step toward understanding dermcidin's mode of action at bacterial membranes, we used homonuclear and heteronuclear NMR to determine the conformation of the peptide in 50% trifluoroethanol solution. We found that dermcidin adopts a flexible amphipathic alpha-helical structure with a helix-hinge-helix motif, which is a common molecular fold among antimicrobial peptides.

Cell selectivity and anti-inflammatory activity of a Leu/Lys-rich alpha-helical model antimicrobial peptide and its diastereomeric peptides.

To investigate the effect of the number and distribution of d-amino acids introduced into non-cell-selective alpha-helical antimicrobial peptides on the cell selectivity, protease stability and anti-inflammatory activity, we synthesized an 18-meric Leu/Lys-rich alpha-helical model peptide (K(9)L(8)W) and d-amino acid-containing diastereomeric peptides. Increasing in cell selectivity of the peptides was increased in parallel with increasing in the number of d-amino acids introduced. Despite having the same number of d-amino acids, D(9)-K(9)L(8)W-1 had better cell selectivity than D(9)-K(9)L(8)W-2, indicating that a dispersed distribution of d-amino acids in diastereomeric peptides is more effective for cell selectivity than their segregated distribution.

Different modes of antibiotic action of homodimeric and monomeric bactenecin, a cathelicidin-derived antibacterial peptide.

The bactenecin is an antibacterial peptide with an intramolecular disulfide bond. We recently found that homodimeric bactenecin exhibits more potent antibacterial activity than the monomeric form and retains its activity at physiological conditions. Here we assess the difference in the modes of antibiotic action of homodimeric and monomeric bactenecins.

Molecular interaction between kisspeptin decapeptide analogs and a lipid membrane.

Kisspeptin-10 is the C-terminal decapeptide amide of kisspeptin, an endogenous ligand for GPR54, and exhibits the same binding and agonist activity as the parent molecule. Although GPR54 is a membrane-embedded protein, details of the molecular interaction between kisspeptin-10 and lipid membranes remain unclear. Here, we performed a series of structural analyses using alanine-scanning analogs of kisspeptin-10 in membrane-mimetic medium.

Effect of dimerization of a beta-turn antimicrobial peptide, PST13-RK, on antimicrobial activity and mammalian cell toxicity.

PST13-RK (KKKFPWWWPFKKK-NH(2)) is an improved derivative of tritrpticin adopting a beta-turn structure. In order to investigate the effect of dimerization of PST13-RK on antimicrobial activity and mammalian cell toxicity, we designed and synthesized its Cys- and Lys-linked dimers. The dimerization of PST13-RK resulted in a 2-4 fold decreased antimicrobial activity against Gram-positive and Gram-negative bacteria.

Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies.

Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)(4) (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)(4)-PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy.

Salt-resistant homodimeric bactenecin, a cathelicidin-derived antimicrobial peptide.

The cathelicidin antimicrobial peptide bactenecin is a beta-hairpin molecule with a single disulfide bond and broad antimicrobial activity. The proform of bactenecin exists as a dimer, however, and it has been proposed that bactenecin is released as a dimer in vivo, although there has been little study of the dimeric form of bactenecin. To investigate the effect of bactenecin dimerization on its biological activity, we characterized the dimer's effect on phospholipid membranes, the kinetics of its bactericidal activity, and its salt sensitivity.

Structural analysis of immunotherapeutic peptides for autoimmune Myasthenia gravis.

Myasthenia gravis (MG) and its animal model, experimental MG (EAMG), are autoimmune disorders in which major pathogenic antibodies are directed against the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (nAChR). In an earlier attempt to develop peptide mimotopes capable of preventing the anti-MIR-mediated pathogenicity, the peptide Pep.1 was initially identified from phage display, and subsequently, Cyclic extended Pep.

Development of potent inhibitors of the coxsackievirus 3C protease.

Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2' pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP.

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action.

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action.

A rationally designed macrocyclic cavitand that kills bacteria with high efficacy and good selectivity.

An amphiphilic macrocyclic cavitand that shows good antibacterial activity, comparable to that of peptide-based antibiotics was developed by rational design and its antibacterial selectivity over mammalian cells was examined.