Biomedical subjects

Alan Mackie

Publications and source records attributed to Alan Mackie.

8 recordsLinked to original sources

PubMed2006

Structure modification in hen egg yolk low density lipoproteins layers between 30 and 45 mN/m observed by AFM.

We have studied the structure of films made by low density lipoproteins (LDL) from hen egg yolk, which are composed of apoproteins, neutral lipids and phospholipids. These LDL have been deposited on air-water interface to form a monolayer which has been compressed to measure an isotherm using Langmuir balance. This isotherm presented three transitions (neutral lipid (surface pressure, pi=19 mN/m), apoprotein-lipid (pi=41 mN/m) and phospholipid (pi=51 mN/m) transitions). We have studied only the apoprotein-lipid transition. In order to observe the LDL film structure before (pi=30 mN/m) and after (pi=45 mN/m) the apoprotein-lipid transition, the formed films were transferred and visualised by atomic force microscopy (AFM). Our results have shown that the structures observed in the LDL film were different depending on the surface pressure. The apoproteins and neutral lipids appeared to be miscible up to the apoprotein-lipid transition, when demixing occurred. The structures observed after the apoprotein-lipid transition should be due to the demixing between apoproteins and neutral lipids. On the other hand, apoproteins and phospholipids seemed miscible whatever the surface pressure. Hence, the first transition (pi=19 mN/m) should be attributed to the free neutral lipid collapse; the second transition (pi=41 mN/m) should be attributed to the demixing of apoprotein-neutral lipid complexes; and the last transition (pi=51 mN/m) should be attributed to phospholipid collapse or to demixing of apoprotein-phospholipid complexes.

Animals↗

PubMed2005

The role of interactions in defining the structure of mixed protein-surfactant interfaces.

The behaviour of dispersed systems is often controlled by the composition of the interface between the different phases. Because of the inherent differences between proteins and surfactants, when both are present at the interface complex pseudo two-dimensional structures can be formed. This review brings together work that allows these structures to be explained in terms of the interactions involved. The significance of both the interactions and the structures is discussed in terms of the effect on bulk properties in colloidal systems.

Adsorption↗

PubMed2005

Binding of lipopeptide to CD14 induces physical proximity of CD14, TLR2 and TLR1.

Lipoproteins or lipopeptides (LP) are bacterial cell wall components detected by the innate immune system. For LP, it has been shown that TLR2 is the essential receptor in cellular activation. However, molecular mechanisms of LP recognition are not yet clear. We used a FLAG-labeled derivative of the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine (Pam(3)CSK(4)) to study the roles of CD14, TLR2 and TLR1 in binding and signaling of LP and their molecular interactions in human cells. The activity of Pam(3)CSK(4)-FLAG was TLR2 dependent, whereas the binding was enabled by CD14, as evaluated by flow cytometry and confocal microscopy. Using FRET and FRAP imaging techniques to study molecular associations, we could show that after Pam(3)CSK(4)-FLAG binding, CD14 and Pam(3)CSK(4)-FLAG associate with TLR2 and TLR1, and TLR2 is targeted to a low-mobility complex. Thus, LP binding to CD14 is the first step in the LP recognition, inducing physical proximity of CD14 and LP with TLR2/TLR1 and formation of the TLR2 signaling complex.

Animals↗

PubMed2004

Lateral diffusion of Toll-like receptors reveals that they are transiently confined within lipid rafts on the plasma membrane.

The innate immune system utilises pattern recognition receptors in order to recognise microbial conserved molecular patterns. The family of Toll-like receptors (TLRs) has been shown to act as the main pattern recognition receptors for the innate immune system. Using biochemical as well as fluorescence imaging techniques, TLR2 and TLR4 were found to be recruited within microdomains upon stimulation by bacterial products. Furthermore their lateral diffusion in the cell membrane as determined by fluorescence recovery after photobleaching revealed that upon stimulation by bacterial products TLRs encounter barriers to their lateral movement, thus supporting the notion that specialised domains on the plasma membrane facilitate the innate recognition.

Animals↗

PubMed2004

Combinational clustering of receptors following stimulation by bacterial products determines lipopolysaccharide responses.

The innate immune system has the capacity to recognize a wide range of pathogens based on conserved PAMPs (pathogen-associated molecular patterns). In the case of bacterial LPS (lipopolysaccharide) recognition, the best studied PAMP, it has been shown that the innate immune system employs at least three cell-surface receptors: CD14, TLR4 (Toll-like receptor 4) and MD-2 protein. CD14 binds LPS from Enterobacteriaceae and then transfers it to MD-2, leading to TLR4 aggregation and signal transduction. LPS analogues such as lipid IVa seem to act as LPS antagonists in human cells, but exhibit LPS mimetic activity in mouse cells. Although TLR4 has been shown to be involved in this species-specific discrimination, the mechanism by which this is achieved has not been elucidated. The questions that remain are how the innate immune system can discriminate between LPS from different bacteria as well as different LPS analogues, and whether or not the structure of LPS affects its interaction with the CD14-TLR4-MD-2 cluster. Is it possible that the 'shape' of LPS induces the formation of different receptor clusters, and thus a different immune response? In the present study, we demonstrate using biochemical as well as fluorescence-imaging techniques that different LPS analogues trigger the recruitment of different receptors within microdomains. The composition of each receptor cluster as well as the number of TLR4 molecules that are recruited within the cluster seem to determine whether an immune response will be induced or inhibited.

Animals↗

PubMed2004

Lipoteichoic acid and toll-like receptor 2 internalization and targeting to the Golgi are lipid raft-dependent.

Lipoteichoic acid (LTA), a key cell wall component of Gram-positive bacteria, seems to function as an immune activator with characteristics very similar to lipopolysaccharide from Gram-negative bacteria. It has been shown that LTA binds CD14 and triggers activation via Toll-like receptor 2, but whether the activation occurs at the cell surface or internalization is required to trigger signaling has yet to be demonstrated. In this work we have investigated LTA binding and internalization and found that LTA and its receptor molecules accumulate in lipid rafts and are subsequently targeted rapidly to the Golgi apparatus. This internalization seems to be lipid raft-dependent because raft-disrupting drugs inhibited LTA/Toll-like receptor 2 colocalization in the Golgi. Similarly to lipopolysaccharide, LTA activation occurs at the cell surface, and the observed trafficking is independent of signaling.

Animals↗

PubMed2004

Proteins and emulsifiers at liquid interfaces.

The interfacial properties of proteins and emulsifiers have been studied extensively in the field of food colloid research. Emulsions form the basis of a huge range of food products and are generally stabilised by either protein and/or emulsifiers. Proteins have been shown to stabilise emulsions by forming a viscoelastic, adsorbed layer on the oil droplets, which form a physical barrier to coalescence. Emulsifiers can be oil or water soluble, forming a fluid, close-packed layer at the interface with a low interfacial tension. This results in an emulsion with a small droplet size distribution, stabilised by the fluid Gibbs-Marangoni mechanism or weak electrostatic repulsion. In real food emulsions, there is usually a mixture of proteins and emulsifiers competing for the interfacial area. This can produce a finer emulsion, however, the emulsifiers break down the viscoelastic protein-adsorbed layer, resulting in an emulsion with reduced stability. We present a review recent work that aims to characterise the composition, structure and physical properties of mixed protein-emulsifier interfaces, in an effort to understand the mechanisms behind the stability behaviour of food emulsion systems.

Colloids↗

PubMed2004

Molecular diffusion in sperm plasma membranes during epididymal maturation.

Fluorescence recovery after photobleaching (FRAP) analysis has been used to measure lipid diffusion in different regions of the sperm plasma membrane. Our goal has been to understand how some membrane components are confined to specific surface domains, whilst others are freely diffusing and in some cases are able to migrate against large concentration gradients. Results with a variety of fluorescent lipid reporter probes (ODAF, NBD-PC, NBD-cholesterol) show that diffusion coefficients (D) are generally three to four times higher on the sperm acrosome than on the principal piece of the tail and increase significantly during epididymal maturation (ram, mouse, goat, dog and monkey sperm). Cholesterol diffusion is approximately 10 times faster on the sperm head than the tail and has a heterogenous distribution when detected with filipin. Lipid diffusion is very temperature sensitive but remarkably insensitive to changes in external pH and osmotic pressure. There was no evidence that the posterior ring or annulus functioned as diffusion barriers to lipids. On this basis it was possible to construct models of increasing complexity to describe the behaviour of a lipid molecule on the sperm surface, beginning with simple linear diffusion progressing to random diffusion and eventually to constrained diffusion.

Animals↗