Jiao, Xiujun

Keating, Eleonora

Petersen, Nils O.

Possmayer, Fred

Veldhuizen, Ruud A.

Zhao, Lin

Zuo, Yi Y.

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Comparative Studies On Bovine And Rat Pulmonary Surfactants Using AFM 

352a Monday, March 2, 2009both lateral tension and bending rigidity of the nanotube membrane can be ex-
tracted. The obtained results are in good agreement with the data reported by
different techniques for similar lipid compositions. Hence the electric field
can be utilized for measurement of mechanical parameters of tubular mem-
brane, specifically, short and/or narrow tubules which are not readily accessible
by conventional techniques.
1807-Pos Board B651
Surface Behaviour of Peptoid Mimics of Pulmonary Surfactant Protein
SP-C: Captive Bubble Surfactometry
Olga L. Ospina1, David Schu¨rch1, Nate Brown2, Annelise Barron2,
Jesu´s Pe´rez-Gil1.
1Universidad Complutense, Madrid, Spain, 2Bioengineering Dept., Stanford
University, Palo Alto, CA, CA, USA.
Pulmonary surfactant lipopeptide SP-C modulates the surface properties of in-
terfacial films required to stabilize the respiratory interface along breathing dy-
namics. Several attempts have been made to produce entirely synthetic analogs
of SP-C suitable to develop potentially useful therapeutic preparations.
In this study we have tested the potential of five different poly-N-substituted
glycines, or peptoids, designed to mimic (roughly) the primary and secondary
structure and hydrophobicity of SP-C, to produce acceptable surfactant-like be-
haviour once incorporated into lipid/peptoid suspensions and assessed in a cap-
tive bubble surfactometer (CBS). The surface activities of different peptoids
were compared in two model lipid mixtures: DPPC/POPG/Palmitic acid (68/
22/9), which resembles the lipid composition of several clinical surfactants cur-
rently in use, and DPPC/POPC/POPG/Chol (50/25/15/10), which mimics the
balance of saturated/unsaturated and zwitterionic/anionic phospholipids and
the cholesterol content of natural surfactant as purified from bronchoalveolar
lavage. We have assessed the ability of the different lipid/peptoid suspensions
to i) rapidly adsorb at the bubble air-liquid interface, ii) stably produce very low
surface tensions upon relatively slow repetitive quasi-static compressions and
iii) maintain the lowest surface tensions with minimal compression and hyster-
esis under rapid physiological-like compression-expansion dynamics. Signifi-
cant differences were found between different peptoids differing in their back-
bone structure and hydrophobicity, with some of the peptoids mimicking
efficiently the effect of native SP-C, usually at larger proportions of peptoids
than required for the natural protein.
1808-Pos Board B652
Looking at Lipid Domains in StratumCorneum LipidModels using Vibra-
tional Microspectroscopy
Michel Lafleur, Sungjong Kwak, Aı¨cha Ouakrim, Amine Touggant.
Universite´ de Montre´al, Montre´al, QC, Canada.
The impermeability of the skin is intimately related to the structure of the stra-
tum corneum (SC), the top layer of the epidermis. The large fraction of SC
lipids existing in a solid/crystalline form is believed to be a key factor in the
low permeability of the skin barrier. We have characterized, using Raman
and infrared microspectroscopies, the mixing properties of model mixtures
that included ceramide, free fatty acids, and sterol, the 3 main lipid components
of SC. We show that, in ternary mixtures with palmitic acid and cholesterol, the
transformation of sphingomyeline, a precursor of ceramide, into ceramide leads
to an increase of the heterogeneity of the spatial lipid distribution, in parallel
with an increase of the chain order. Therefore the enzymatic conversion of
sphingomyeline in ceramide leads to the transformation of a homogeneous
and relatively disordered matrix into a heterogeneous matrix containing crystal-
line domains. This heterogeneity in lipid composition was observed from the
microscopic local variations of the relative areas of the C-H stretching and
the C-D stretching bands, the fatty acids being deuterated in our model mix-
tures. The thermal evolution of the mixing properties of the ceramide/palmitic
acid/cholesterol mixtures indicated that an increase of temperature (above
50 C) leads to the disordering of the fatty acid and, to a lesser extent, of ceram-
ide. In parallel to this melting, a mixing of the lipid species is observed as the
areas enriched in palmitic acid were also enriched in cholesterol. These results
suggest the formation of a liquid ordered phase mainly composed of palmitic
acid and cholesterol; this phase may ensure the cohesion between the solid
domains. The recording of spectra from several microscopic voxels provides
a unique description of the phase composition of these model mixtures.
1809-Pos Board B653
Comparative Studies On Bovine And Rat Pulmonary Surfactants Using
AFM
Fred Possmayer1, Xiujun Jiao1, Lin Zhao1, Eleonora Keating1,
Ruud A. Veldhuizen1, Nils O. Petersen2, Yi Y. Zuo3.
1University of Western Ontario, London, ON, Canada, 2National Institute of
Nanotechnology, Edmonton, AB, Canada, 3University of Hawaii, Honolulu,
HI, USA.Recent studies employing a variety of microscopic techniques have re-
vealed that phospholipid (PL) phase separations and transitions may play
important roles in determining the biophysical properties of pulmonary sur-
factant. Most of these microscopic studies used model systems which were
composed of simple mixtures of PL with or without hydrophobic surfactant
proteins and cholesterol. The present work compared modified natural (lipid
extract) bovine and rat surfactants using atomic force microscopy (AFM).
AFM revealed PL phase separation upon compression of both surfactant
monolayers, and a monolayer-to-multilayer transition at surface pressure
40-50 mN/m. Similar to bovine surfactant, the tilted-condensed (TC) phase
in rat surfactant consisted of domains both on micrometer and nanometer
scales. Upon film compression, the microdomains were dissociated into
nanodomains, thus forming a more homogeneous two-phase mixture. Dif-
ferences between rat and bovine surfactants were: (1) more TC domains
were formed at lower surface pressures in rat than in bovine surfactant; and
(2) an interesting domain-in-domain structure was exclusively observed in rat
surfactant. These structural differences were attributed to the higher cholesterol
content of rat surfactant (~ 10 vs ~2.5 wt%). To further investigate the effects of
cholesterol on the structure of surfactant films, we have studied cholesterol-de-
pleted bovine surfactant (~ 0%) prepared by repetitive acetone extraction. Re-
moval of cholesterol from bovine surfactant induced significant variations in
film structure. More importantly, the film structure can be effectively restored
by recombining cholesterol with the cholesterol-depleted bovine surfactant. Re-
combinant bovine surfactant with 10% cholesterol showed domain-in-domain
structures similar to those found with rat surfactant. These interspecies studies
of the micro- and nano- structures of natural pulmonary surfactants add insight
into the biophysical interpretation of phospholipid phase transition and separa-
tion, in particular the role of cholesterol.
1810-Pos Board B654
Differences in Lateral Membrane Organization in Fibroblasts Expressing
Low and High Levels of the Influenza Viral Protein Hemagglutinin
Manasa V. Gudheti, Travis J. Gould, Samuel T. Hess.
University of Maine, Orono, ME, USA.
Lateral organization in cell membranes is crucial for biological processes
such as endocytosis, signaling, protein transport, membrane trafficking and
viral infection. Hemagglutinin (HA) is an influenza viral envelope transmem-
brane protein which has been shown to be associated with the liquid ordered
(lo) phase. Fibroblasts that constitutively express HA, referred to as HAb2
cells, were used to characterize the lateral organization of HA. Since
HAb2 cells have cell-to-cell variability in the membrane density of HA, cells
with low and high expression levels of HA containing more consistent den-
sities of HA were also used. Fluorescence correlation spectroscopy (FCS),
confocal microscopy and fluorescence photoactivation localization micros-
copy (FPALM) were used to characterize membrane organization after label-
ing cells with fluorescent probes and/or transfecting with either EGFP-HA or
Dendra2-HA. Preliminary FCS results show that the diffusion of the liquid-
disordered (ld) phase probe Lissamine Rhodamine DOPE is similar in cells
with high and low HA expression levels i.e. the amount of HA present
does not influence the diffusion time. Confocal microscopy was used to
study the effect of HA expression level on the extent of phase separation
observed after blebbing was induced by DMSO treatment. FPALM was
used to obtain details about membrane organization at the nanometer length
scale.
1811-Pos Board B655
Determination Of The Lipid Membrane Composition Of J774 Macro-
phages Cells Surexpressing Mrp Protein (resistant To Ciprofloxacin)
Hayet Bensikaddour.
Catholic University of Louvain, Brussels, Belgium.
Ciprofloxacin (CIP) is a fluoroquinolone antibiotic with an activity towards
both extracellular and intracellular bacteria (Seral et al., 2005). Diffusion
and efflux processes modulate accumulation of this drug within eukaryotic
cells. When J774 macrophages were grown in presence of ciprofloxacin, the an-
tibiotic is subject to constitutive efflux through the activity of an MRP-related
transporter (Michot et al., 2004).
In view of the critical role of lipids for both drug uptake and activity of MRP
proteins (Hinrichs et al, 2004), together with the ability of fluoroquinolones to
interact with lipids (Bensikaddour et al., 2008 (a,b)), we investigated the com-
position of lipids in resistant and sensitive J 774 macrophages to ciprofloxacin.
Firstly, we characterized by thin layer chromatography the phospholipids com-
position of J774 macrophages cells sensitive (WT) and resistant to ciprofloxa-
cin (CIP). Results showed that sphingomyelin (SM) decreased 2 times whereas
phosphatidylinositol increased 1.5 fold in resistant cells. Phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine and cholesterol didn’t show
any significant change. Secondly, we studied membrane fluidity of liposomes


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Comparative Studies On Bovine And Rat Pulmonary Surfactants Using AFM

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