Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Deficiency of the surfactant leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. The generation of DPPtdCho in cells occurs via two integral routes: the de novo and remodeling pathways. The interplay between these pathways has not been investigated. Overexpression of the remodeling enzyme, acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT1), in epithelia decreases de novo PtdCho synthesis without significantly altering cellular phospholipid mass; this occurs through increased degradation of cholinephosphotransferase (CPT1), the terminal enzyme of the de novo pathway. CPT1 is degraded by multi-ubuiquitination and trafficking via the lysosomal pathway. When expressed in lung epithelia, CPT1 mutants harboring arginine substitutions at multiple carboxyl-terminal lysine residues exhibited proteolytic resistance to effects of LPCAT1 overexpression. Cellular expression of these CPT1 mutants also restores de novo PtdCho synthesis to levels normally observed in lung epithelia. Further studies demonstrate that the SCF (Skip-Cullen-F-box) ubiquitin E3 ligase component, β-TrCP, was sufficient to degrade CPT1. Similar to CPT1, LPCAT1 levels are also regulated at the level of protein stability. However, LPCAT1 is a polyubiquitinated enzyme processed within the proteasome. Similar to CPT1, β-TrCP is the putative E3 ubiquitin ligase subunit responsible for LPCAT1 ubiquitination. β-TrCP appears to dock and ubiquitinate LPCAT1 within its amino-terminus. Collectively, these observations indicate the presence of cross-talk between the phospholipid remodeling and de novo pathways; this involves tight regulation by site-specific ubiquitination of indispensable regulatory enzymes catalyzed by SCF ubiquitin E3 ligase members that mechanistically provide homeostatic control of cellular phospholipid content.

Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Surfactant deficiency leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. These are features of the acute lung injury syndrome, characterized by a strong pro-inflammatory component where cytokines or bacteria infections greatly impair surfactant DPPtdCho biosynthesis. The key enzyme needed to produce surfactant DPPtdCho is a rate-limiting enzyme CTP: phosphocholine cytidylyltransferase (CCTalpha). Calmodulin (CaM), rather than disruption of an NH2-terminal PEST sequence, stabilizes CCTalpha from actions of the proteinase, calpain. Mapping and site-directed mutagenesis of CCTalpha uncovered a motif (LQERVDKVK) harboring a vital recognition site, Q243, whereby CaM directly binds to the enzyme. Mutagenesis of CCTalpha Q243 not only resulted in loss of CaM binding, but also led to complete calpain resistance in vitro and in vivo. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTalpha that stabilizes the enzyme under pro-inflammatory stress. We further show that CCTalpha does not undergo polyubiquitination and proteasomal degradation. Rather, the enzyme is monoubiquitinated at a molecular site (K57) juxtaposed near its NLS resulting in disruption of its interaction with importin, nuclear exclusion, and subsequent degradation within the lysosome. Importantly, by using CCTalpha-ubiquitin hybrid constructs that vary in the intermolecular distance between ubiquitin and the NLS, we show that CCTalpha monoubiquitination masks its NLS resulting in cytoplasmic retention. These results unravel a unique molecular mechanism whereby monoubiquitination governs the trafficking of a critical regulatory enzyme in vivo. Last, we identify FBXL2 as a novel F-box E3 ubiquitin ligase that targets CCTalpha for degradation. Interestingly, FBXL2 also interacts with CaM, and CaM directly disrupts CCTalpha and FBXL2 interaction. This study demonstrates in the first time that adenoviral gene transfer of CaM attenuates the deleterious effects of P. aeruginosa infection by improving several parameters of pulmonary mechanics in animal models of sepsis-induced acute pulmonary injury. Collectively, these studies reveal a novel regulatory mechanism for phosphatidylcholine synthesis that may provide important clues to understanding the pathobiology of acute lung injury.

Otitis Media is one of the most common childhood diseases. Recurrent acute otitis media RAOM is characterized by repeated episodes of inflammation of the middle ear in conjunction with middle ear fluid, and often with an inflamed or bulging eardrum. Defective clearance by the Eustachian tube results in mucus build-up and is characteristic of otitis media with effusion (OME). Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, respiratory syncytial virus, and rhinovirus are the most common contributors to otitis media pathogenesis. In New Zealand, OME has been implicated with conductive hearing loss in childhood and has been shown to significantly impact on speech and language development. New Zealand Māori and Polynesian children have displayed significantly higher hearing test failure rates than European-Caucasian children. The collectins, Surfactant Protein (SP)-A and -D are encoded by three genes (SP-A1, SP-A2, and SP-D) and are host defense proteins present in the middle ear and Eustachian tube. Single nucleotide polymorphisms (SNPs) in SP-A1 and SP-A2 have been associated with increased or decreased susceptibility to otitis media, meningococcal disease, and range of respiratory diseases. Using allele-specific primers and real-time PCR with SYBR Green I melting curve analysis, four groups of individuals were genotyped for eleven SP-A1, SP-A2, and SP-D SNPs: European-Caucasian individuals with RAOM/OME; New Zealand Māori/Polynesian individuals with RAOM/OME; individuals with meningococcal disease; and a control group. The computer program, Haploview, was employed to perform χ2 analyses and identify statistically significant associations of alleles/haplotypes with RAOM/OME or meningococcal disease. In the European-Caucasian population, two SP-A1 alleles, one SP-A2 allele, and four haplotypes (CGAGC, 1A3, 1A9, and 1A10) were found to be associated with increased risk of RAOM/OME (P lt; 0.05). Conversely, haplotypes 6A2 and 1A2 were found to be protective against susceptibility to RAOM/OME (P lt; 0.05). In New Zealand Māori and Polynesian individuals, two SP-A1 alleles, three SP-A2 alleles, one SP-D allele, and four haplotypes (6A8, 6A10, 1A3, and 1A10) were found to be associated with increased risk of RAOM/OME (P lt; 0.05). An additional four haplotypes (6A2, 1A0, 1A2, and TA) were determined to be protective against susceptibility to RAOM/OME (P lt; 0.05). However, protective SPA1/SPA2/SPD haplotype 6A2-1A0-TA was significantly under-represented in the New Zealand Māori and Polynesian population (P lt; 0.05). A single allele and haplotype were associated with increased risk of meningococcal disease (P lt; 0.05). The findings of this study confirm that specific genetic variants of SP-A and SP-D are associated with either increased or decreased risk of developing RAOM and/or OME. Furthermore, it was demonstrated that New Zealand Māori and Polynesian individuals appear to exhibit more haplotypes susceptible to RAOM/OME. This may provide a partial explanation for the higher RAOM/OME-related failure rates of hearing tests in New Zealand Māori and Polynesian children. However, there are numerous socio-economic and environmental factors that also contribute to otitis media pathogenesis which were not considered in this study. The effects of the SP-A1, SP-A2, and SP-D alleles and haplotypes on the bacterial/viral binding efficiencies of SP-A and SP-D need to be investigated by further research, using a large population, to confirm the association with susceptibility or resistance with RAOM/OME.

Pulmonary surfactant is a complex mixture of lipids and proteins that lowers surface tension, increases lung compliance, and prevents the adhesion of respiratory surfaces and pulmonary oedema. Pressure can have an enormous impact on respiratory function, by mechanically compressing tissues, increasing gas tension resulting in increased gas absorption and by increasing dissolved gas tensions during diving, resulting in the formation of bubbles in the blood and tissues. The lungs of diving mammals have a huge range of morphological adaptations to enable them to endure the extremely high pressures associated with deep diving. Here, I hypothesise that surfactant will also be modified, to complement the morphological changes and enable more efficient lung function during diving. Molecular adaptations to diving were examined in surfactant protein C (SP-C) using phylogenetic analyses. The composition and function of pulmonary surfactant from several species of diving mammals was examined using biochemical assays, mass spectrometry and captive bubble surfactometry. The development of surfactant in one species of diving mammal (California sea lion), and the control of surfactant secretion using chemical and mechanical stimuli were also determined. Diving mammals showed modifications to SP-C, which are likely to lead to stronger binding to the monolayer, thereby increasing its fluidity. Phospholipid molecular species concentrations were altered to increase the concentration of more fluid species. There was also an increase in the percentage of alkyl molecular species, which may increase the stability of the monolayer during compression and facilitate rapid respreading. Levels of SP-B were much lower in the diving species, and cholesterol was inversely proportional to the maximum dive depth of the three species. Surface activity of surfactant from diving mammals was very poor compared to surfactant from terrestrial mammals. The newborn California sea lion surfactant was similar to terrestrial mammal surfactant, suggesting that these animals develop the diving-type of surfactant after they first enter the water. The isolated cells of California sea lions also showed a similar response to neuro-hormonal stimulation as terrestrial mammals, but were insensitive to pressure. These findings showed diving mammal surfactant to have a primarily anti-adhesive function that develops after the first entry into the water, with a surfactant monolayer, which would be better suited to repeated collapse and respreading. / Thesis (Ph.D.)--School of Earth and Environmental Sciences, 2005.

<p>The aim of the project was to modify polydimethylsiloxane (PDMS) surfaces in order to minimize adsorption of proteins. PDMS is used in micro-fluidic devices that control the delivery of samples to a sensor chip in Biacore instrumentation. These instruments are used to characterize interactions between biomolecules with a detection principle based on surface plasmon resonance (SPR). To minimize adsorption of proteins poly-ethylene-oxide (PEO) based surfactants, were added to the buffer. The added PEO surfactants were P20, Pluronic F-127 and Brij 35. Interaction of these surfactants with the sensor chip in Biacore instruments was also examined. Creating a more hydrophilic surface layer on PDMS by oxidation was also examined.</p><p>When surfactants were continuously added to protein samples, as in dynamically coating of PDMS surfaces, Brij 35 resulted in the strongest reduction in protein adsorption. Brij 35 was also the surfactant that was easiest to remove from both PDMS and the sensor surfaces. Pluronic bound strongest to surfaces, and is most suitable when only adding surfactant to the buffer in a pre-coating step. All surfactants did reduce protein adsorption considerably (99% or more) and addition is necessary when working with protein solutions and hydrophobic surfaces as PDMS. Another alternative is oxidation of PDMS surface, which is an easy procedure that decreased the protein adsorption to about 10% compared to adsorption to untreated surface.</p>

Recently, surface tension has been shown to be important in emerging high technologies, such as in pumping and control of flow in microfluidic devices, in microchemical analysis of complex fluids, and in rapid DNA screening, etc. Advances in these new technologies will depend strongly on the availability of flexible methods for controlling surface tension. Photo-control using a photoresponsive surfactant is a potentially attractive route to accomplishing many of the tasks required in these processes. Photoresponsive surfactants typically incorporate an azobenzene group as the functional unit which experiences reversible trans-to-cis photoisomerization under different irradiation conditions. The photoisomerization usually causes a change in surface tension. Obviously, a large change in surface tension under different illumination conditions will be highly desirable in practical applications. However, the largest change in surface tension as reported in the literature is only 3 mN/m which is too small to generate any significant effect. In this presentation, we report a new class of photoresponsive surfactants which exhibit excellent performance in surface tension control. Under different illumination conditions, the change in surface tension can be as large as 11.0 mN/m. Experimental results are presented for two new photoresponsive surfactants. A discussion of experimental results follows. / Singapore-MIT Alliance (SMA)

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 117-129.

Drag reduction is a well-observed phenomenon, it was first observed by the British chemist Toms in 1946, yet its mechanism is still unknown to this day. Polymer Drag reduction has found application in reducing pumping costs for oil pipelines (its use in the Trans Alaska Pipeline has resulted in an increase from 1.44 million bbl./day to 2.1356 million bbl./day), increasing the flow rate in firefighting equipment , and in supporting irrigation and drainage systems. Surfactant drag reducers are used industrially in district heating and cooling systems. Though the fields of Surfactant Drag Reduction and Polymer Drag Reduction are each independently well-developed the effect of their interaction on drag reduction is a less explored phenomenon. Through a well chosen pairing of surfactant and polymer, drag reduction can be maximized while minimizing surfactant and polymer concentrations cutting down on cost and environmental impact. The focus of this work was to determine if there was any positive interaction between the polymers Polyethylene Oxide (PEO) and Anionic PolyAcrylAmide (PAM) and the surfactant Amphosol CG (Cocamidopropyl Betaine) as well as any interaction between the polymers themselves. Both polymers are popular drag reducers while Amphosol is a practically nontoxic (LD50=5g/kg) zwitterionic surfactant and is readily biodegradable. In order to determine if any interaction was present and at what concentration was this most notable 4 techniques were used: Surface tension, Conductivity, Relative Viscosity and Shear Viscosity measurement. From this analysis the polymer Saturation point (PSP), Critical aggregation concentration (CAC) and Critical micelle concentration (CMC) were found as well as the concentrations that optimized the viscosity for the pilot plant runs. The bench scale results were used to pick the optimum concentrations for the polymer surfactant solutions. Pressure readings and flowrate measurements were used to plot the Fanning Friction Factor against the Generalized Reynolds Number for the surfactant polymer mixtures and compared to their pure polymer and surfactant counterparts. The Blasius line was found to hold for water measurements taken and is the base to determine percentage drag reduction. The effect of the presence of amphosol on degradation and overall drag reduction were noted. Other factors considered were pipe diameter and the effect of ionic impurities in the solvent.

Drag reduction in turbulent pipe flow using polymeric and surfactant additives is well known. Although extensive research work has been carried out on the drag reduction behavior of polymers and surfactants in isolation, little progress has been made on the synergistic effects of combined polymers and surfactants. In this work the interactions between drag-reducing polymers and surfactants were studied. The drag-reducing polymers studied were nonionic polyethylene oxide (referred to as PEO) and anionic copolymer of acrylamide and sodium acrylate (referred to as CPAM). The drag-reducing surfactants studied were nonionic ethoxylated alcohol - Alfonic 1412-7 (referred to as EA), cationic surfactant - Octadecyltrimethylammonium chloride in pure powder form (referred to as OTAC-p) and commercial grade cationic surfactant - Octadecyltrimethylammonium chloride in isopropanol solvent - Arquad 18-50 (referred to as OTAC-s). The interactions between polymers and surfactant were reflected in the measurements of the physical properties such as electrical conductivity, surface tension, viscosity and turbidity. The critical micelle concentration (cmc) of the mixed polymer / surfactant system was found to be different from that of the surfactant alone. The viscosity of a polymer solution was significantly affected by the addition of surfactant. Weak interactions were observed for the mixed systems of nonionic polymer - nonionic surfactant and anionic polymer - nonionic surfactant. Due to the wrapping of polymer chains around the developing micelles, a minimum in the viscosity is observed in these two cases. In the case of nonionic polymer / cationic surfactant system, the change in the viscosity was found to depend on the polymer concentration (C) and the critical entanglement concentration (C*). When the polymer concentration (C) was less than C* (C < C*), the plot of the viscosity versus surfactant concentration exhibited a minimum. When C > C*, a maximum in the viscosity versus surfactant concentration plot was observed. The interactions between nonionic polymer and cationic surfactant were observed to increase with the increase in temperature. A large drop in the viscosity occurred in the case of anionic-polymer / cationic-surfactant system when surfactant was added to the polymer solution. The observed changes in the viscosity are explained in terms of the changes in the extension of polymeric chains resulting from polymer-surfactant interactions. The anionic CPAM chains collapsed upon the addition of cationic OTAC-p, due to charge neutralization. The presence of counterion sodium salicylate (NaSal) stabilized the cationic surfactant monomers in the solution, resulting in micelle formation at a surfactant concentration well below the concentration where complete charge neutralization of anionic polymer occurred. Preliminary results are reported on the pipeline drag reduction behavior of mixed polymer-surfactant system. The results obtained using combinations of CPAM / OTAC-p in pipeline flow are found to be in harmony with the interaction study. Due to the shrinkage of CPAM chains upon the addition of OTAC-p, the drag reducing ability of CPAM is compromised.

Drag reduction in turbulent pipe flow using polymeric and surfactant additives is well known. Although extensive research work has been carried out on the drag reduction behavior of polymers and surfactants in isolation, little progress has been made on the synergistic effects of combined polymers and surfactants. In this work the interactions between drag-reducing polymers and surfactants were studied. The drag-reducing polymers studied were nonionic polyethylene oxide (referred to as PEO) and anionic copolymer of acrylamide and sodium acrylate (referred to as CPAM). The drag-reducing surfactants studied were nonionic ethoxylated alcohol - Alfonic 1412-7 (referred to as EA), cationic surfactant - Octadecyltrimethylammonium chloride in pure powder form (referred to as OTAC-p) and commercial grade cationic surfactant - Octadecyltrimethylammonium chloride in isopropanol solvent - Arquad 18-50 (referred to as OTAC-s). The interactions between polymers and surfactant were reflected in the measurements of the physical properties such as electrical conductivity, surface tension, viscosity and turbidity. The critical micelle concentration (cmc) of the mixed polymer / surfactant system was found to be different from that of the surfactant alone. The viscosity of a polymer solution was significantly affected by the addition of surfactant. Weak interactions were observed for the mixed systems of nonionic polymer - nonionic surfactant and anionic polymer - nonionic surfactant. Due to the wrapping of polymer chains around the developing micelles, a minimum in the viscosity is observed in these two cases. In the case of nonionic polymer / cationic surfactant system, the change in the viscosity was found to depend on the polymer concentration (C) and the critical entanglement concentration (C*). When the polymer concentration (C) was less than C* (C < C*), the plot of the viscosity versus surfactant concentration exhibited a minimum. When C > C*, a maximum in the viscosity versus surfactant concentration plot was observed. The interactions between nonionic polymer and cationic surfactant were observed to increase with the increase in temperature. A large drop in the viscosity occurred in the case of anionic-polymer / cationic-surfactant system when surfactant was added to the polymer solution. The observed changes in the viscosity are explained in terms of the changes in the extension of polymeric chains resulting from polymer-surfactant interactions. The anionic CPAM chains collapsed upon the addition of cationic OTAC-p, due to charge neutralization. The presence of counterion sodium salicylate (NaSal) stabilized the cationic surfactant monomers in the solution, resulting in micelle formation at a surfactant concentration well below the concentration where complete charge neutralization of anionic polymer occurred. Preliminary results are reported on the pipeline drag reduction behavior of mixed polymer-surfactant system. The results obtained using combinations of CPAM / OTAC-p in pipeline flow are found to be in harmony with the interaction study. Due to the shrinkage of CPAM chains upon the addition of OTAC-p, the drag reducing ability of CPAM is compromised.