Surfactant Phospholipids Research Articles

Double-Tailed Cystine Derivatives as Novel Substitutes of Phospholipids with Special Reference to Liposomes.

Cystine-based gemini surfactants with dodecyl, tetradecyl, hexadecyl, and octadecyl hydrocarbon chains were synthesized, and their interactions with unsaturated (soy phosphatidylcholine, SPC)/saturated (hydrogenated SPC, HSPC) soy phosphatidylcholines in the forms of a monolayer and a model liposome were estimated for different combinations of the components in the mixed systems. Studies of Langmuir monolayers at the air-aqueous buffer interface revealed condensation of the monomolecular films with the addition of surfactants. The effect of surfactants decreased according to the following order: octadecyl > hexadecyl > tetradecyl > dodecyl homologs. The nonideal mixing between the components was estimated using the deviation of the experimental molecular area from the ideal area per molecule. The excess molecular area increased with the increase in the surfactant chain length and phospholipid saturation. The 50 mol % mixture of cystine derivatives and phospholipids formed thermodynamically stable monolayers. The surfactants increased the rigidity of SPC monolayers and decreased that of HSPC monolayers, as observed by the studies of surface dialational rheology. The film structure at the air-water interface could differentiate the SPC- and HSPC-comprising systems through the formation of organized regions, especially at a higher surface pressure. The constriction of surfactant/phospholipid hybrid vesicles was observed with an increase in the length of surfactant hydrocarbon chains. The negative zeta potential of vesicles took the highest values and did not change with time for 20 and 50 mol % surfactant. The spherical shape of the vesicles was confirmed by transmission electron microscopy. Differential scanning calorimetry revealed an increase in fluidity of HSPC bilayers and rigidity of SPS bilayers under the influence of surfactants. These effects were confirmed by fluorescence spectroscopy. All of the vesicle formulations were found to be nontoxic from the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assay, suggesting their potential as a novel membranous system for the delivery of drugs, genetic materials, vaccines, and other therapeutic agents.

L-α-Phosphatidylglycerol Chlorohydrins as Potential Biomarkers for Chlorine Gas Exposure.

Chlorine is a widely available toxic chemical that has been repeatedly used in armed conflict globally. The Organization for the Prohibition of Chemical Weapons (OPCW) have on numerous occasions found "compelling confirmation" that chlorine gas has been used against civilians in northern Syria. However, currently, there are no analytical methods available to unambiguously prove chlorine gas exposure. In this study, we describe the screening for chlorinated biomolecules by the use of mass isotope ratio filters followed by the identification of two biomarkers present in bronchoalveolar lavage fluid (BALF) from chlorine gas exposed mice. The relevance of these markers for human exposure was verified by their presence in in vitro chlorinated human BALF. The biomarkers were detectable for 72 h after exposure and were absent in nonexposed control animals. Furthermore, the biomarkers were not detected in humans diagnosed with chronic respiratory diseases. The potential chlorine specific markers were all chlorohydrins of unsaturated pulmonary surfactant phospholipids; phosphatidylglycerols, and phosphatidylcholines. Mass spectrometry fragmentation characteristics were favorable for the phosphatidylglycerol chlorohydrins, and they were therefore proposed as the best biomarker candidates.

Stability of an amphipathic helix-hairpin surfactant peptide in liposomes

Surfactant protein B (SP-B; 79 residues) is a member of the saposin superfamily and plays a pivotal role in lung function. The N- and C-terminal regions of SP-B, cross-linked by two disulfides, were theoretically predicted to fold as charged amphipathic helices, suggesting participation in surfactant activities. Previous studies with oxidized Super Mini-B (SMB), a construct based on the N- and C-regions of SP-B (i.e., residues 1–25 and 63–78) joined with a designer turn (-PKGG-) and two disulfides, indicated that freshly prepared SMB in lipids folded as a surface active, α-helix-hairpin. Because other peptides modeled on α-helical SP domains lost helicity and surfactant activity on storage, experiments were here performed on oxidized SMB in surfactant liposomes stored at ~2–8°C for ≤5.5years. Captive bubble surfactometry confirmed low minimum surface tensions for fresh and stored SMB preparations. FTIR spectroscopy of fresh and stored SMB formulations showed secondary structures compatible with the peptide folding as α-helix-hairpin. A homology (I-TASSER) model of oxidized SMB demonstrated a globular protein, exhibiting a core of hydrophobic residues and a surface of polar residues. Since mass spectroscopy indicated that the disulfides were maintained on storage, the stability of SMB may be partly due to the disulfides bringing the N- and C-α-helices closer. Mass spectroscopy of stored SMB preparations showed some methionine oxidation, and also partial deacylation of surfactant phospholipids to form lyso-derivatives. However, the stable conformation and activity of stored SMB surfactant suggest that the active helix-hairpin resists these chemical changes which otherwise may lead to surfactant inhibition.

Inhaled Vitamin D: A Novel Strategy to Enhance Neonatal Lung Maturation

The physiologic vitamin D (VD), 1α,25(OH)2D3 (1,25D) is a local paracrine/autocrine effecter of fetal lung maturation. By stimulating alveolar type II cell and lipofibroblast proliferation and differentiation, parenterally administered 1,25D has been shown to enhance neonatal lung maturation; but due to the potential systemic side effects of the parenteral route, the translational value of these findings might be limited. To minimize the possibility of systemic toxicity, we examined the effects of VD on neonatal lung maturation, when delivered directly to lungs via nebulization. One-day-old rat pups were administered three different doses of 1,25D and its physiologic precursor 25(OH)D (25D), or the diluent, via nebulization daily for 14days. Pups were sacrificed for lung, kidneys, and blood collection to determine markers of lung maturation, and serum 25D and calcium levels. Compared to controls, nebulized 25D and 1,25D enhanced lung maturation as evidenced by the increased expression of markers of alveolar epithelial (SP-B, leptin receptor), mesenchymal (PPARγ, C/EBPα), and endothelial (VEGF, FLK-1) differentiation, surfactant phospholipid synthesis, and lung morphology without any significant increases in serum 25D and calcium levels. Inhaled VD is a potentially safe and effective novel strategy to enhance neonatal lung maturation.

What is the identity of fibroblast-pneumocyte factor?

Glucocorticoid induction of pulmonary surfactant involves a mesenchyme-derived protein first characterized in 1978 by Smith and termed fibroblast-pneumocyte factor (FPF). Despite a number of agents having been postulated as being FPF, its identity has remained obscure. In the past decade three strong candidates for FPF have arisen. This review examines the evidence that keratinocyte growth factor (KGF), leptin or neuregulin-1β (NRG-1β) act as FPF or components of it. As with FPF production, glucocorticoids enhance the concentration of each of these agents in fibroblast-conditioned media. Moreover, each stimulates the synthesis of surfactant-associated phospholipids and proteins in type II pneumocytes. Further, some have unique activities, for example, KGF also minimizes lung injury through enhanced epithelial cell proliferation and NRG-1β enhances surfactant phospholipid secretion and β-adrenergic receptor activity in type II cells. However, even though these agents have attributes in common with FPF, it is inappropriate to specify any one of these agents as FPF. Rather, it appears that each contributes to separate mesenchymal-epithelial signaling mechanisms involved in different aspects of lung development. Given that the production of pulmonary surfactant is essential for postnatal survival, it is reasonable to suggest that several mechanisms independently regulate surfactant synthesis.

Optical Regulation of Carbon Nanodots by Chemical Functionalization

The chemical functionalization of carbon nanodots (CNDs) with a bis-amino-modified silicone reagent enhanced their fluorescence properties. Further treatment with a poly(ethylene glycol)–phospholipid surfactant increased the water dispersibility and promoted the nanoassembly of the silicone-modified CNDs. The synthesized nanocomplexes were found to be biologically compatible and useful for cellular fluorescence imaging.

Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit toll-like receptor 2 and 4 signaling

The pulmonary surfactant phospholipid, 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), potently inhibits toll-like receptor (TLR)2 and TLR4 signaling from the cell surface of macrophages. Analogs of POPG that vary in polar head group length, hydroxylation, and alkyl branching were synthesized using a phospholipase D-catalyzed transphosphatidylation reaction and a 1-palmitoyl-2-oleoyl phosphatidylcholine substrate. Lipid analogs with C3 and C4 alkyl head group length (POP-propanol and POP-butanol) are less effective than POPG as TLR2 and TLR4 antagonists. However, adding a hydroxyl group at the alkyl chain 3- or 4-position (POP-propanediols or POP-butanediols) greatly increased their inhibitory effects against TLR2 and TLR4. POP-2',2'-dimethylpropanediol is a weak inhibitor of TLR2 and TLR4 activation that results in arachidonic acid release, but an effective inhibitor of TLR4 activation that results in TNF-α production. Addition of an amino group at the alkyl-2 position (POP-2'-aminopropanediol) completely abolished the antagonism of TLRs 2 and 4. Multiple analogs strongly bind to the TLR4 coreceptors, cluster of differentiation 14 (CD14) and myeloid differentiation 2, but competition for di[3-deoxy-D-manno-octulosonyl]-lipid A binding to CD14 is the best predictor of biological activity at the cellular level. Collectively, these findings identify new compounds for antagonizing TLR2 and TLR4 activation and define structural properties of POPG analogs for discriminating between two TLR systems.

35. Establishment of the Dose-Response Relationship Needed for Human Translation of Pulmonary Macrophage Transplantation (PMT) Therapy of Hereditary Pulmonary Alveolar Proteinosis (hPAP)

Hereditary pulmonary alveolar proteinosis (hPAP) is a severe pediatric lung disease caused by mutations in CSF2RA/B (encoding GM-CSF receptor α/β, respectively) without pharmacologic therapy. Disease patho-genesis is mediated by loss of GM-CSF-dependent clearance by alveolar macrophages (AMs) resulting in progressive pulmonary alveolar surfactant accumulation and hypoxemic respiratory failure. We recently reported a conceptually and technically novel, exceptionally efficient gene and cell therapy approach -PMT- as a promising alternative for the existing highly invasive, inefficient whole lung lavage procedure for hPAP. Our preclinical studies revealed that macrophage cell doses from 0.5 to 4 x106/mouse were similarly highly efficacious as therapy of hPAP in Csf2rb-/- mice, an authentic model of human hPAP (Nature, 2014, 514: 450-5). The present study was undertaken to identify a dose-response relationship and to address the hypothesis of an expected ‘trade-off’ between the minimum effective dose and time to treatment effect. We isolated Lineage-, Sca1+, cKit+ hematopoietic stem/progenitors from the bone marrow of wild-type (WT) CD45.1+ mice, differentiated them in vitro into mature macrophages and sorted for highly homogenous CD11cHi, F4/80Hi macrophage population by flow cytometry. We administered cell doses (2.5 x103, 2.5 x104, 2.5 x105, 1 x106 cells/mouse; n=3-4/dose) by PMT to CD45.2+ Csf2rb-/- recipients. At 8 weeks after PMT, therapeutic efficacy was evaluated by measuring the optical density (OD) of bronchoalveolar lavage (BAL turbidity) - an excellent measure of overall PAP disease severity. The CD45.1+ donor macrophages recovered from the BAL were of CD11cHi, F4/80Hi phenotype. BAL turbidity decreased smoothly with increasing PMT cell dose over the entire range but reached significance only at the two highest doses compared to age-matched, untreated Csf2rb-/- controls (OD λ=600 nm = 0.88±0.07 and 0.5±0.08 vs. 2.32±0.18; P<0.001, P<0.0001; respectively). BAL turbidity correlated inversely with the percentage of CD45.1+ donor macrophage engraftment in CD45.2+ Csf2rb-/- recipients (Spearman rank correlation, R2=0.74). Since we have identified an increased ratio of cholesterol (relative to phospholipid) in surfactant as the primary lipid abnormality in hPAP, we also evaluated the relationship of BAL cholesterol level and PMT cell dose. Like turbidity, BAL cholesterol levels decreased smoothly with PMT cell dose over the entire range but reached significance only at the two highest cell doses compared to age-matched, untreated Csf2rb-/- controls (ng/ml BAL = 25±3 and 10±0.3 versus 54±0.3; P<0.01, P<0.001; respectively, R2=0.67). Our pre-clinical results establish a direct relationship between the number of macrophages transplanted and efficacy of PMT therapy in Csf2rb-/- mice and facilitate the design of a clinical trial to test PMT therapy in children with hPAP. Based on these results and the previously demonstrated strong survival advantage of transplanted over endogenous macrophages, we anticipate a ‘trade-off’ between the minimum effective dose and the time to equivalent treatment effect.

Mutation of Serine 32 to Threonine in Peroxiredoxin 6 Preserves Its Structure and Enzymatic Function but Abolishes Its Trafficking to Lamellar Bodies

Peroxiredoxin 6 (Prdx6), a bifunctional protein with phospholipase A2 (aiPLA2) and GSH peroxidase activities, protects lungs from oxidative stress and participates in lung surfactant phospholipid turnover. Prdx6 has been localized to both cytosol and lamellar bodies (LB) in lung epithelium, and its organellar targeting sequence has been identified. We propose that Prdx6 LB targeting facilitates its role in the metabolism of lung surfactant phosphatidylcholine (PC). Ser-32 has been identified as the active site in Prdx6 for aiPLA2 activity, and this activity was abolished by the mutation of serine 32 to alanine (S32A). However, aiPLA2 activity was unaffected by mutation of serine 32 in Prdx6 to threonine (S32T). Prdx6 protein expression and aiPLA2 activity were normal in the whole lung of a "knock-in" mouse model carrying an S32T mutation in the Prdx6 gene but were absent from isolated LB. Analyses by proximity ligation assay in lung sections demonstrated the inability of S32T Prdx6 to bind to the chaperone protein, 14-3-3ϵ, that is required for LB targeting. The content of total phospholipid, PC, and disaturated PC in lung tissue homogenate, bronchoalveolar lavage fluid, and lung LB was increased significantly in Prdx6-S32T mutant lungs, whereas degradation of internalized [(3)H]dipalmitoyl-PC was significantly decreased. Thus, Thr can substitute for Ser for the enzymatic activities of Prdx6 but not for its targeting to LB. These results confirm an important role for LB Prdx6 in the degradation and remodeling of lung surfactant phosphatidylcholine.

Neonatal Respiratory Failure with Retarded Perinatal Lung Maturation in Mice Caused by Reticulocalbin 3 Disruption.

Reticulocalbin 3 (Rcn3) is an endoplasmic reticulum lumen protein localized to the secretory pathway. As a Ca2t-binding protein of 45 kDa (Cab45)/Rcn/ER Ca2t-binding protein of 55 kDa (ERC45)/calumenin (CREC) family member, Rcn3 is reported to function as a chaperone protein involved in protein synthesis and secretion; however, the biological role of Rcn3 is largely unknown. The results presented here, for the first time, depict an indispensable physiological role of Rcn3 in perinatal lung maturation by using an Rcn3 gene knockout mouse model. These mutant mice die immediately at birth owing to atelectasis-induced neonatal respiratory distress, although these embryos are produced with grossly normal development. This respiratory distress results from a failure of functional maturation of alveolar epithelial type II cells during alveogenesis. This immaturity of type II cells is associated with a dramatic reduction in surfactant protein A and D, a disruption in surfactant phospholipid homeostasis, and a disorder in lamellar body. In vitro studies further show that Rcn3 deficiency blunts the secretion of surfactant proteins and phospholipids from lung epithelial cells, suggesting a decrease in availability of surfactants for their surface activity. Collectively, these observations indicate an essential role of Rcn3 in perinatal lung maturation and neonatal respiratory adaptation as well as shed additional light on the mechanism of neonatal respiratory distress syndrome development.

Meconium-induced inflammation and surfactant inactivation: specifics of molecular mechanisms.

This review summarizes neonatal meconium aspiration syndrome in light of meconium-induced inflammation and inflammatory surfactant inactivation, related to both endogenous and therapeutic exogenous surfactant. The wide effect of meconium on surfactant properties is divided into three points. Direct effect of meconium on surfactant properties refers mainly to fragmentation of dipalmitoylphosphatidylcholine and other surfactant phospholipids together with cleavage of surfactant proteins. Initiation of inflammatory response due to activation of receptors by yet unspecified compounds involves complement and Toll-like receptor activation. A possible role of lung collectins, surfactant proteins A and D, which can exert both pro- and anti-inflammatory reactions, is discussed. Initiation of inflammatory response by specified compounds in meconium reflects inflammatory functioning of cytokines, bile acids, and phospholipases contained in meconium. Unifying sketch of many interconnections in all these actions aims at providing integrated picture of inflammatory surfactant inactivation.

The miR-200 Family and Its Targets Regulate Type II Cell Differentiation in Human Fetal Lung

Type II cell differentiation and expression of the major surfactant protein, SP-A, in mid-gestation human fetal lung (HFL) are induced by cAMP and inhibited by TGF-β. cAMP induction of SP-A promoter activity is mediated by increased phosphorylation and DNA binding of thyroid transcription factor-1 (TTF-1/Nkx2.1), a master regulator of lung development. To further define mechanisms for developmental induction of surfactant synthesis in HFL, herein, we investigated the potential roles of microRNAs (miRNAs, miRs). To identify and characterize differentially regulated miRNAs in mid-gestation HFL explants during type II pneumocyte differentiation in culture, we performed miRNA microarray of RNA from epithelial cells isolated from mid-gestation HFL explants before and after culture with or without Bt2cAMP. Interestingly, the miR-200 family was significantly up-regulated during type II cell differentiation; miR-200 induction was inversely correlated with expression of known targets, transcription factors ZEB1/2 and TGF-β2. miR-200 antagonists inhibited TTF-1 and surfactant proteins and up-regulated TGF-β2 and ZEB1 expression in type II cells. Overexpression of ZEB1 in type II cells decreased DNA binding of endogenous TTF-1, blocked cAMP stimulation of surfactant proteins, and inhibited miR-200 expression, whereas cAMP markedly inhibited ZEB1/2 and TGF-β. Importantly, overexpression of ZEB1 or miR-200 antagonists in HFL type II cells also inhibited LPCAT1 and ABCA3, enzymes involved in surfactant phospholipid synthesis and trafficking, and blocked lamellar body biogenesis. Our findings suggest that the miR-200 family and ZEB1, which exist in a double-negative feedback loop regulated by TGF-β, serve important roles in the developmental regulation of type II cell differentiation and function in HFL.

Lysophosphatidylcholine Acyltransferase 1 (LPCAT1) Specifically Interacts with Phospholipid Transfer Protein StarD10 to Facilitate Surfactant Phospholipid Trafficking in Alveolar Type II Cells

Pulmonary surfactant, a mixture of proteins and phospholipids, plays an important role in facilitating gas exchange by maintaining alveolar stability. Saturated phosphatidylcholine (SatPC), the major component of surfactant, is synthesized both de novo and by the remodeling of unsaturated phosphatidylcholine (PC) by lyso-PC acyltransferase 1 (LPCAT1). After synthesis in the endoplasmic reticulum, SatPC is routed to lamellar bodies (LBs) for storage prior to secretion. The mechanism by which SatPC is transported to LB is not understood. The specificity of LPCAT1 for lyso-PC as an acyl acceptor suggests that formation of SatPC via LPCAT1 reacylation is a final step in SatPC synthesis prior to transport. We hypothesized that LPCAT1 forms a transient complex with SatPC and specific phospholipid transport protein(s) to initiate trafficking of SatPC from the endoplasmic reticulum to the LB. Herein we have assessed the ability of different StarD proteins to interact with LPCAT1. We found that LPCAT1 interacts with StarD10, that this interaction is direct, and that amino acids 79-271 of LPCAT1 and the steroidogenic acute regulatory protein-related lipid transfer (START) domain of START domain-containing protein 10 (StarD10) are sufficient for this interaction. The role of StarD10 in trafficking of phospholipid to LB was confirmed by the observation that knockdown of StarD10 significantly reduced transport of phospholipid to LB. LPCAT1 also interacted with one isoform of StarD7 but showed no interaction with StarD2/PC transfer protein.

Amniotic fluid LPCAT1 mRNA correlates with the lamellar body count.

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is required in the biosynthesis of pulmonary surfactant. This short communication describes our assessment of LPCAT1 mRNA levels in human amniotic fluid. We found a direct correlation between LPCAT1 mRNA copies and the amniotic fluid lamellar body count (LBC). This finding corroborates an association between LPCAT1 and surfactant phospholipid biosynthesis in humans. It may provide a model for future research in perinatal medicine.

Pitfalls in flow cytometric analyses of surfactant-exposed human leukocytes

BackgroundSurfactant replacement treatment is the standard of care for the prevention and treatment of neonatal respiratory distress syndrome in preterm infants and may also improve oxygenation in acute respiratory distress syndrome in children, adolescents and adults. Beside surface tension- and mechanical shear-reducing functions, natural surfactants have been ascribed immunomodulatory capacities. Current in vitro studies on immunomodulatory effects of pulmonary surfactant preparations on human leukocytes rely on ELISA, Western blot and polymerase chain reaction. Data obtained by flow cytometry are missing, so far, most likely due to confounding phospholipid residues. Intracellular cytokine flow cytometry in surfactant-exposed immune cells would provide information on pro- and anti-inflammatory immunomodulation at the single-cell level and would allow for integrating detailed immunophenotyping, functional assays and assessment of viability. AimWe implemented a flow cytometry protocol for reliable quantitative assessment of in vitro intracellular cytokine production in surfactant-exposed human lymphocytes (CD4+) and monocytes (CD14+). MethodsTwo different permeabilization techniques were tested for their ability to provide intracellular cytokine staining in surfactant-exposed CD14+ monocytes and CD4+ lymphocytes. Both a commercially available solution containing saponin and ice-cold methanol were used as permeabilization reagents. ResultsFor both cell types, flow cytometry following saponin-based permeabilization revealed pronounced unspecific fluorescence signals in surfactant-exposed samples overlapping with the fluorescence spectra of the majority of conjugates. Autofluorescence of surfactant phospholipid particles interfered significantly with reliable quantification of fluorochrome-specific signals and conclusive analysis. Implementation of a methanol-based permeabilization protocol resulted in the elimination of confounding non-cell particle signals allowing for an accurate quantification of intracellular cytokine production. ConclusionReliable detection of intracellular cytokines by flow cytometry may be challenging in surfactant-exposed cell samples due to significant autofluorescence of aggregated phospholipid particles. This issue has been addressed for the first time and may be of high relevance for all types of surfactant research. We demonstrate that a methanol-based permeabilization approach completely removes interfering fluorescent surfactant micelles and allows for correct evaluation of data. The successful removal of confounding surfactant phospholipids opens up a wide variety of multiparameter flow cytometry; a method that has not been applied in the field of surfactant research, yet.

Analysis of pulmonary surfactant in rat lungs after inhalation of nanomaterials: Fullerenes, nickel oxide and multi-walled carbon nanotubes

The health risks of inhalation exposure to engineered nanomaterials in the workplace are a major concern in recent years, and hazard assessments of these materials are being conducted. The pulmonary surfactant of lung alveoli is the first biological entity to have contact with airborne nanomaterials in inhaled air. In this study, we retrospectively evaluated the pulmonary surfactant components of rat lungs after a 4-week inhalation exposure to three different nanomaterials: fullerenes, nickel oxide (NiO) nanoparticles and multi-walled carbon nanotubes (MWCNT), with similar levels of average aerosol concentration (0.13–0.37 mg/m3). Bronchoalveolar lavage fluid (BALF) of the rat lungs stored after previous inhalation studies was analyzed, focusing on total protein and the surfactant components, such as phospholipids and surfactant-specific SP-D (surfactant protein D) and the BALF surface tension, which is affected by SP-B and SP-C. Compared with a control group, significant changes in the BALF surface tension and the concentrations of phospholipids, total protein and SP-D were observed in rats exposed to NiO nanoparticles, but not in those exposed to fullerenes. Surface tension and the levels of surfactant phospholipids and proteins were also significantly different in rats exposed to MWCNTs. The concentrations of phospholipids, total protein and SP-D and BALF surface tension were correlated significantly with the polymorphonuclear neutrophil counts in the BALF. These results suggest that pulmonary surfactant components can be used as measures of lung inflammation.

(Invited) Developing Short-Wave Infrared Fluorescent Immunoprobes Based on Single-Walled Carbon Nanotubes

Progress will be reported on a project to develop a new type of fluorescent immunoprobe containing antibodies conjugated to structurally sorted single-walled carbon nanotubes (SWCNTs). The (n,m)-sorted SWCNTs display sharp and distinct fluorescence at wavelengths in the short-wave infrared (SWIR) region, allowing effective spectral multiplexing and minimal interference from autofluorescence backgrounds. To avoid fluorescence quenching from covalent functionalization of nanotube sidewalls, the antibodies are linked to PEG side chains of a phospholipid surfactant that noncovalently coats individualized SWCNTs. We are preparing SWCNT-based immunoprobes containing the OC125 antibody, which binds specifically to the CA125 ovarian cancer biomarker. SWIR fluorescence microscopy is being used to assess specificity of binding to cultured ovarian cancer cells. We will also describe new developments that aid in the testing of SWCNT-based probes. One is a custom optical apparatus that scans specimens in two dimensions to sensitively measure SWIR fluorescence spectra as a function of position. The device is used to read dot blots of selected antigens spotted onto a cellulose membrane and probed by SWCNT-conjugated antibodies. Using this approach, one can quickly and quantitatively assess efficiencies of SWCNT conjugation and antigen binding.

(692) - Surfactant Phospholipids as a Marker of Chronic Lung Allograft Dysfunction: A Targeted Lipidomics Approach

Lung transplantation is and established treatment option for end stage lung disease, although long-term clinical outcomes are halted by the onset of bronchiolitis obliterans syndrome and chronic lung allograft dysfunction (CLAD). To date diagnosis of CLAD relies on pulmonary function tests. No biologic marker has yet been clearly established to corroborate or anticipate the diagnosis. We sought to investigate role of broncho-alveolar surfactant phospholipids as a marker of CLAD by using a targeted lipidomic methodology.

Serine 32 to threonine mutation preserves peroxiredoxin 6 structure and enzymatic function but abolishes its trafficking to lamellar bodies in vivo

Peroxiredoxin 6 (Prdx6), a bifunctional protein with lysosomal‐type phospholipase A2 (aiPLA2) and GSH peroxidase activities, protects lungs from oxidative stress and participates in lung surfactant phospholipid (PL) turnover. Prdx6 has been localized to both cytosol and acidic organelles (lamellar bodies [LB] and lysosomes) in lung epithelium and its organellar targeting sequence has been identified (31DSWGILFSHP40, Am.J.Physiol.297 : L871, 2009). Interestingly, an S32T mutation fully preserved secondary structure of recombinant protein and had no effect on either its peroxidase or aiPLA2 activity. In a “knock‐in” mouse model carrying the S32T mutation in the Prdx6 gene, Prdx6 protein and aiPLA2 activity were present in lung epithelial cells. However,the protein was absent from epithelial cell LB. Lung surfactant dipalmitoylphosphatidylcholine (DPPC) is endocytosed by alveolar epithelial cells and degraded by aiPLA2. The content of total phospholipid, PC, and disaturated phosphatidylcholine (DSPC) in bronchoalveolar lavage fluid, lung lamellar bodies, and lung homogenate was increased in S32T mutant animals and degradation of internalized [3H]DPPC in isolated mouse lungs was significantly decreased. Thus, Thr can substitute for Ser for the enzymatic activities of Prdx6 but not for its targeting to LB. These results confirm an important role for lamellar body Prdx6 in the degradation and remodeling of lung surfactant phosphatidylcholine. [HL‐019737, HL102016 and HL105509]

Infection Tunes Stability of Key Molecules in Lung Diseases

efficiently fulfill a vast range of life processes. In the processes, proteins are dynamic in responding to the environmental changes. If a given protein is needed, de novo synthesis from mRNA will be augmented. Of note, a less emphasized but probably more efficacious way is to enhance the protein stability through preventing from degradation. If redundant, proteins will be promptly dumped out by degradation. Regulation of protein stability via degradation plays an important role in a vast range of life processes. Most proteins destined for turnover are via ubiquitin proteasomal/lysosomal degradation machinery. Protein turnover is not a randomly happened event but highly regulated and is a substrate-specific signal transduction cascade. Firstly, targeted proteins are posttranslational modified for recognition by the ubiquitin proteasomal/lysosomal degradation machinery. A well-studied prototype is phosphorylation that may directly affects ubiquitin proteasome system recognition of the substrate. Other posttranslational modifications, such as acetylation, methylation, or palmitoylation are important modifications involved in ubiquitination process as well. Secondly, the destined protein is ubiquitinated. Ubiquitination covalently adds a ubiquitin or polyubiquitin moieties to a lysine residue within the protein substrate. The process of ubiquitination is an enzymatic cascade involving E1 ubiquitin activation enzyme, E2 ubiquitin conjugation enzyme, and E3 ubiquitin ligase. Among the ubiquitin enzymes, E3 ubiquitin ligase recognizes the substrate that is substrate specific. Strikingly, the ubiquitin can be removed from the ubiquitinated protein by a family of enzymes called de ubiquitin enzymes. Finally, the ubiquitinated protein is then escorted into proteasome or lysosome for degradation. Bacterial infection fine-turns proteins involving in cell surface sensors, signal mediator proteins, and effect or proteins. For example, endotoxin lipopolysaccharide treatment or bacterial infection regulates IL22R, IL33, kinases, and TRAF proteins in lung epithelial cells. Epigenetic enzymes that control essential life process such as DNA replication and gene transcription are also deregulated in these models. Enzymes involved in active lung surfactant or other phospholipids synthesis, metabolism, and energy production are also regulated in this system. Understanding the molecular mechanism(s) of protein turnover in infectious lung disease has shed light on the development of new pharmaceutical therapeutic approaches. Successful results have been achieved to screen small molecules against novel molecular targets including E3 ubiquitin ligases in infectious lung diseases.