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Sample records for human surfactant protein

  1. The detection of surfactant proteins A, B, C and D in the human brain and their regulation in cerebral infarction, autoimmune conditions and infections of the CNS.

    PubMed

    Schob, Stefan; Schicht, Martin; Sel, Saadettin; Stiller, Dankwart; Kekulé, Alexander S; Kekulé, Alexander; Paulsen, Friedrich; Maronde, Erik; Bräuer, Lars

    2013-01-01

    Surfactant proteins (SP) have been studied intensively in the respiratory system. Surfactant protein A and surfactant protein D are proteins belonging to the family of collectins each playing a major role in the innate immune system. The ability of surfactant protein A and surfactant protein D to bind various pathogens and facilitate their elimination has been described in a vast number of studies. Surfactant proteins are very important in modulating the host's inflammatory response and participate in the clearance of apoptotic cells. Surfactant protein B and surfactant protein C are proteins responsible for lowering the surface tension in the lungs. The aim of this study was an investigation of expression of surfactant proteins in the central nervous system to assess their specific distribution patterns. The second aim was to quantify surfactant proteins in cerebrospinal fluid of healthy subjects compared to patients suffering from different neuropathologies. The expression of mRNA for the surfactant proteins was analyzed with RT-PCR done with samples from different parts of the human brain. The production of the surfactant proteins in the brain was verified using immunohistochemistry and Western blot. The concentrations of the surfactant proteins in cerebrospinal fluid from healthy subjects and patients suffering from neuropathologic conditions were quantified using ELISA. Our results revealed that surfactant proteins are present in the central nervous system and that the concentrations of one or more surfactant proteins in healthy subjects differed significantly from those of patients affected by central autoimmune processes, CNS infections or cerebral infarction. Based on the localization of the surfactant proteins in the brain, their different levels in normal versus pathologic samples of cerebrospinal fluid and their well-known functions in the lungs, it appears that the surfactant proteins may play roles in host defense of the brain, facilitation of

  2. Assignment of the human pulmonary surfactant protein D gene (SFTP4) to 10q22-q23 close to the surfactant protein A gene cluster

    SciTech Connect

    Koelble, K.; Kaluz, S.; Reid, K.B.M. ); Mole, S.E. )

    1993-08-01

    Pulmonary surfactant consists of a complex mixture of phospholipids and several proteins essential to normal respiratory function. Two of the surfactant proteins, SP-A and SP-D, appear to have lectin-like activity relevant to the local phagocytic defense. Using polymerase chain reaction (PCR)-based somatic cell hybrid mapping, the human SP-D gene (SFTP4) was assigned to chromosome 10. A regional mapping panel was assembled and characterized using sequence tagged sites for five loci previously mapped to 10q. SFTP4, the SP-A gene (SFTP1), and the microsatellite D10S109 were placed in the interval 10q22-q23. Low-stringency PCR using the SFTP1 primer pair suggested the presence of at least two additional SP-A-related genes in the same region. With the locus for mannose-binding lectin (MBL) at 10q21, this may be indicative of this region's central role in the evolutionary history of carbohydrate-binding proteins containing collagen-like regions. 41 refs., 3 figs., 1 tab.

  3. Pulmonary surfactant protein A and surfactant lipids upregulate IRAK-M, a negative regulator of TLR-mediated inflammation in human macrophages

    PubMed Central

    Nguyen, Huy A.; Rajaram, Murugesan V. S.; Meyer, Douglas A.

    2012-01-01

    Alveolar macrophages (AMs) are exposed to frequent challenges from inhaled particulates and microbes and function as a first line of defense with a highly regulated immune response because of their unique biology as prototypic alternatively activated macrophages. Lung collectins, particularly surfactant protein A (SP-A), contribute to this activation state by fine-tuning the macrophage inflammatory response. During short-term (10 min–2 h) exposure, SP-A's regulation of human macrophage responses occurs through decreased activity of kinases required for proinflammatory cytokine production. However, AMs are continuously exposed to surfactant, and the biochemical pathways underlying long-term reduction of proinflammatory cytokine activity are not known. We investigated the molecular mechanism(s) underlying SP-A- and surfactant lipid-mediated suppression of proinflammatory cytokine production in response to Toll-like receptor (TLR) 4 (TLR4) activation over longer time periods. We found that exposure of human macrophages to SP-A for 6–24 h upregulates expression of IL-1 receptor-associated kinase M (IRAK-M), a negative regulator of TLR-mediated NF-κB activation. Exposure to Survanta, a natural bovine lung extract lacking SP-A, also enhances IRAK-M expression, but at lower magnitude and for a shorter duration than SP-A. Surfactant-mediated upregulation of IRAK-M in macrophages suppresses TLR4-mediated TNF-α and IL-6 production in response to LPS, and IRAK-M knockdown by small interfering RNA reverses this suppression. In contrast to TNF-α and IL-6, the surfactant components upregulate LPS-mediated immunoregulatory IL-10 production, an effect reversed by IRAK-M knockdown. In conclusion, these data identify an important signaling regulator in human macrophages that is used by surfactant to control the long-term alveolar inflammatory response, i.e., enhanced IRAK-M activity. PMID:22886503

  4. An alternatively spliced surfactant protein B mRNA in normal human lung: disease implication.

    PubMed Central

    Lin, Z; Wang, G; Demello, D E; Floros, J

    1999-01-01

    We identified an alternatively-spliced surfactant protein B (SP-B) mRNA from normal human lung with a 12 nt deletion at the beginning of exon 8. This deletion causes a loss of four amino acids in the SP-B precursor protein. Sequence comparison of the 3' splice sites reveals only one difference in the frequency of U/C in the 11 predominantly-pyrimidine nucleotide tract, 73% for the normal and 45% for the alternatively-spliced SP-B mRNA (77-99% for the consensus sequence). Analysis of SP-B mRNA in lung indicates that the abundance of the alternatively-spliced form is very low and varies among individuals. Although the relative abundance of the deletion form of SP-B mRNA remains constant among normal lungs, it is found with relatively higher abundance in the lungs of some individuals with diseases such as congenital alveolar proteinosis, respiratory distress syndrome, bronchopulmonary dysplasia, alveolar capillary dysplasia and hypophosphatasia. This observation points to the possibility that the alternative splicing is a potential regulatory mechanism of SP-B and may play a role in the pathogenesis of disease under certain circumstances. PMID:10493923

  5. Structural Studies of Protein-Surfactant Complexes

    SciTech Connect

    Chodankar, S. N.; Aswal, V. K.; Wagh, A. G.

    2008-03-17

    The structure of protein-surfactant complexes of two proteins bovine serum albumin (BSA) and lysozyme in presence of anionic surfactant sodium dodecyl sulfate (SDS) has been studied using small-angle neutron scattering (SANS). It is observed that these two proteins form different complex structures with the surfactant. While BSA protein undergoes unfolding on addition of surfactant, lysozyme does not show any unfolding even up to very high surfactant concentrations. The unfolding of BSA protein is caused by micelle-like aggregation of surfactant molecules in the complex. On the other hand, for lysozyme protein there is only binding of individual surfactant molecules to protein. Lysozyme in presence of higher surfactant concentrations has protein-surfactant complex structure coexisting with pure surfactant micelles.

  6. Biofoams and natural protein surfactants

    PubMed Central

    Cooper, Alan; Kennedy, Malcolm W.

    2010-01-01

    Naturally occurring foam constituent and surfactant proteins with intriguing structures and functions are now being identified from a variety of biological sources. The ranaspumins from tropical frog foam nests comprise a range of proteins with a mixture of surfactant, carbohydrate binding and antimicrobial activities that together provide a stable, biocompatible, protective foam environment for developing eggs and embryos. Ranasmurfin, a blue protein from a different species of frog, displays a novel structure with a unique chromophoric crosslink. Latherin, primarily from horse sweat, but with similarities to salivary, oral and upper respiratory tract proteins, illustrates several potential roles for surfactant proteins in mammalian systems. These proteins, together with the previously discovered hydrophobins of fungi, throw new light on biomolecular processes at air–water and other interfaces. This review provides a perspective on these recent findings, focussing on structure and biophysical properties. PMID:20615601

  7. Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice

    SciTech Connect

    Korfhagen, T.R.; Glasser, S.W.; Wert, S.E.; Bruno, M.D.; Daugherty, C.C.; McNeish, J.D.; Stock, J.L.; Potter, S.S.; Whitsett, J.A. )

    1990-08-01

    Pulmonary surfactant is produced in late gestation by developing type II epithelial cells lining the alveolar epithelium of the lung. Lack of surfactant at birth is associated with respiratory distress syndrome in premature infants. Surfactant protein C (SP-C) is a highly hydrophobic peptide isolated from pulmonary tissue that enhances the biophysical activity of surfactant phospholipids. Like surfactant phospholipid, SP-C is produced by epithelial cells in the distal respiratory epithelium, and its expression increases during the latter part of gestation. A chimeric gene containing 3.6 kilobases of the promoter and 5{prime}-flanking sequences of the human SP-C gene was used to express diphtheria toxin A. The SP-C-diphtheria toxin A fusion gene was injected into fertilized mouse eggs to produce transgenic mice. Affected mice developed respiratory failure in the immediate postnatal period. Morphologic analysis of lungs from affected pups showed variable but severe cellular injury confined to pulmonary tissues. Ultrastructural changes consistent with cell death and injury were prominent in the distal respiratory epithelium. Proximal components of the tracheobronchial tree were not severely affected. Transgenic animals were of normal size at birth, and structural abnormalities were not detected in nonpulmonary tissues. Lung-specific diphtheria toxin A expression controlled by the human SP-C gene injured type II epithelial cells and caused extensive necrosis of the distal respiratory epithelium. The absence of type I epithelial cells in the most severely affected transgenic animals supports the concept that developing type II cells serve as precursors to type I epithelial cells.

  8. The Interactions between SP-B Protein and Anionic Lipids Found in Human Lung Surfactant

    NASA Astrophysics Data System (ADS)

    Lee, Ka Yee C.; Lipp, Michael M.; Zasadzinski, Joseph A.; Waring, Alan J.

    1997-03-01

    Several lung pathologies, including neonatal respiratory distress syndrome, are characterized by a failure of the lung surfactant (LS) system to function properly. Utilizing fluorescence and Brewster angle microscopy, we have investigated the phase behavior of monolayers of binary mixtures of anionic lipids found in LS (palmitic acid, and both saturated and unsaturated phosphatidylglycerol) with both the full length SP-B protein and a shorter, 25-amino acid sequence based on its amino terminus. We found that both protein candidates interact specifically yet differently with each of the lipid components, altering their phase behavior to resemble more closely to that of an ideal LS monolayer. The SP-B protein incorporates itself in the lipid monolayer in all cases, and partitions preferentially into the fluid-type phases during phase transitions; its presence drastically changes the collapse mechanism of the monolayer.

  9. Biomimicry of surfactant protein C.

    PubMed

    Brown, Nathan J; Johansson, Jan; Barron, Annelise E

    2008-10-01

    Since the widespread use of exogenous lung surfactant to treat neonatal respiratory distress syndrome, premature infant survival and respiratory morbidity have dramatically improved. Despite the effectiveness of the animal-derived surfactant preparations, there still remain some concerns and difficulties associated with their use. This has prompted investigation into the creation of synthetic surfactant preparations. However, to date, no clinically used synthetic formulation is as effective as the natural material. This is largely because the previous synthetic formulations lacked analogues of the hydrophobic proteins of the lung surfactant system, SP-B and SP-C, which are critical functional constituents. As a result, recent investigation has turned toward the development of a new generation of synthetic, biomimetic surfactants that contain synthetic phospholipids along with a mimic of the hydrophobic protein portion of lung surfactant. In this Account, we detail our efforts in creating accurate mimics of SP-C for use in a synthetic surfactant replacement therapy. Despite SP-C's seemingly simple structure, the predominantly helical protein is extraordinarily challenging to work with given its extreme hydrophobicity and structural instability, which greatly complicates the creation of an effective SP-C analogue. Drawing inspiration from Nature, two promising biomimetic approaches have led to the creation of rationally designed biopolymers that recapitulate many of SP-C's molecular features. The first approach utilizes detailed SP-C structure-activity relationships and amino acid folding propensities to create a peptide-based analogue, SP-C33. In SP-C33, the problematic and metastable polyvaline helix is replaced with a structurally stable polyleucine helix and includes a well-placed positive charge to prevent aggregation. SP-C33 is structurally stable and eliminates the association propensity of the native protein. The second approach follows the same design

  10. Surfactant protein A expression in human normal and neoplastic breast epithelium.

    PubMed

    Braidotti, P; Cigala, C; Graziani, D; Del Curto, B; Dessy, E; Coggi, G; Bosari, S; Pietra, G G

    2001-11-01

    We studied the presence of surfactant protein A (Sp-A) immunoreactivity and messenger RNA in 62 normal and abnormal breast samples. Sections were immunostained with polyclonal anti-Sp-A antibody. The association between Sp-A immunoreactivity and histologic grade of 32 invasive ductal carcinomas was assessed by 3 pathologists who scored the intensity of Sp-A immunoreactivity times the percentage of tumor immunostained; individual scores were averaged, and the final scores were correlated with tumor grade, proliferative index, and expression of estrogen and progesterone receptors. Strong Sp-A immunoreactivity was present at the luminal surface of ductal epithelial cells in normal breast samples and in benign lesions; carcinomas displayed variable immunoreactivity, inversely proportional to the degree of differentiation. Sp-A messenger RNA was detected by reverse transcriptase-polymerase chain reaction in 3 of 3 normal breast samples and 9 of 9 carcinomas. The significance of Sp-A expression in breast epithelium requires further study; possibly it has a role in native host defense or epithelial differentiation.

  11. Differential susceptibility of transgenic mice expressing human surfactant protein B genetic variants to Pseudomonas aeruginosa induced pneumonia

    PubMed Central

    Ge, Lin; Liu, Xinyu; Chen, Rimei; Xu, Yongan; Zuo, Yi Y.; Cooney, Robert N; Wang, Guirong

    2015-01-01

    Surfactant protein B (SP-B) is essential for lung function. Previous studies have indicated that a SP-B 1580C/T polymorphism (SNP rs1130866) was associated with lung diseases including pneumonia. The SNP causes an altered N-linked glycosylation modification at Asn129 of proSP-B, e.g. the C allele with this glycosylation site but not in the T allele. This study aimed to generate humanized SP-B transgenic mice carrying either SP-B C or T allele without a mouse SP-B background and then examine functional susceptibility to bacterial pneumonia in vivo. A total of 18 transgenic mouse founders were generated by the DNA microinjection method. These founders were back-crossed with SP-B KO mice to eliminate mouse SP-B background. Four founder lines expressing similar SP-B levels to human lung were chosen for further investigation. After intratracheal infection with 50μl of P. aeruginosa solution (1×107 CFU/mouse) or saline in SP-B-C, SP-B-T mice the mice were sacrificed 24 hours post-infection and tissues were harvested. Analysis of surfactant activity revealed differential susceptibility between SP-B-C and SP-B-T mice to bacterial infection, e.g. higher minimum surface tension in infected SP-B-C versus infected SP-B-T mice. These results demonstrate for the first time that human SP-B C allele is more susceptible to bacterial pneumonia than SP-B T allele in vivo. PMID:26620227

  12. Genetic Complexity of the Human Innate Host Defense Molecules, Surfactant Protein A1 (SP-A1) and SP-A2—Impact on Function

    PubMed Central

    Floros, Joanna; Wang, Guirong; Mikerov, Anatoly N.

    2010-01-01

    Innate immunity mechanisms play a critical role in the primary response to invading pathogenic microorganisms and other insulting agents. The innate lung immune system includes lung surfactant, a lipoprotein complex that carries out a function essential for life, that is, reduction of the surface tension at the air–liquid interphase of the alveolar space. By means of this function, pulmonary surfactant prevents lung collapse, therefore ensuring normal lung function and lung health. Pulmonary surfactant contains a number of host-defense molecules that are involved in the elimination of pathogens, viruses, particles, allergens, and other insults, as well as in the control of inflammation. This review is concerned with one of the surfactant proteins, the human (h) surfactant protein A (hSP-A), which, in addition to its role in surfactant-related functions, plays an important role in the modulation of lung host defense. The hSP-A locus has been identified with extensive complexity that may have an impact on its function, structure, and regulation. In humans, two genes—SP-A1 (SFTPA1) and SP-A2 (SFTPA2)—encode SP-A, with SP-A2 gene products being more biologically active than SP-A1 in most of the in vitro assays investigated. Although the two hSP-A genes share a high level of sequence similarity, differences in the structure and function between SP-A1 and SP-A2 have been observed in recent studies. In this review, we discuss the human SP-A complexity and how this may affect SP-A function. PMID:19392648

  13. Contributions of Phenylalanine 335 to Ligand Recognition by Human Surfactant Protein D: Ring Interactions with SP-D Ligands

    SciTech Connect

    Crouch,E.; McDonald, B.; Smith, K.; Cararella, T.; Seaton, B.; Head, J.

    2006-01-01

    Surfactant Protein D (SP-D) is an innate immune effector that contributes to antimicrobial host defense and immune regulation. Interactions of SP-D with microorganisms and organic antigens involve binding of glycoconjugates to the C-type lectin carbohydrate recognition domain (CRD). A trimeric fusion protein encoding the human neck+CRD (hNCRD) bound to the aromatic glycoside, p-nitrophenyl-alpha-D-maltoside, with nearly a log-fold higher affinity than maltose, the prototypical competitor. Maltotriose, which has the same linkage pattern as the maltoside, bound with intermediate affinity. Site-directed substitution of leucine for phenylalanine 335 (Phe335) decreased affinities for the maltoside and maltotriose without significantly altering the affinity for maltose or glucose, and substitution of tyrosine or tryptophan for leucine restored preferential binding to maltotriose and the maltoside. A mutant with alanine at this position failed to bind to mannan or maltose-substituted solid supports. Crystallographic analysis of the hNCRD complexed with maltotriose or p-nitrophenyl-maltoside showed stacking of the terminal glucose or nitrophenyl ring with the aromatic ring of Phe335. Our studies indicate that Phe335, which is evolutionarily conserved in all known SP-Ds, plays important - if not critical roles - in SP-D function.

  14. Lipid-restricted recognition of mycobacterial lipoglycans by human pulmonary surfactant protein A: a surface-plasmon-resonance study.

    PubMed Central

    Sidobre, Stéphane; Puzo, Germain; Rivière, Michel

    2002-01-01

    The human pulmonary surfactant protein A (hSP-A), a member of the mammalian collectin family, is thought to play a key defensive role against airborne invading pulmonary pathogens, among which is Mycobacterium tuberculosis, the aetiologic agent of tuberculosis. hSP-A has been shown to promote the uptake and the phagocytosis of pathogenic bacilli through the recognition and the binding of carbohydrate motifs on the invading pathogen surface. Recently we identified lipomannan and mannosylated lipoarabinomannan (ManLAM), two major mycobacterial cell-wall lipoglycans, as potential ligands for binding of hSP-A. We demonstrated that both the terminal mannose residues and the fatty acids are critical for binding, whereas the inner arabinosyl and mannosyl domains do not participate. In the present study we developed a surface-plasmon-resonance assay to analyse the molecular basis for the recognition of ManLAM by hSP-A and to try to define further the role of the lipidic aglycone moiety. Binding of ManLAM to immobilized hSP-A was consistent with the simplest one-to-one interaction model involving a single class of carbohydrate-binding site. This observation strongly suggests that the lipid moiety of ManLAM does not directly interact with hSP-A, but is rather responsible for the macromolecular organization of the lipoglycan, which may be necessary for efficient recognition of the terminal mannosyl epitopes. The indirect, structural role of the lipoglycan lipidic component is further supported by the complete lack of interaction with hSP-A in the presence of a low concentration of mild detergent. PMID:12071842

  15. Surfactant-associated proteins: structure, function and clinical implications.

    PubMed

    Ketko, Anastasia K; Donn, Steven M

    2014-01-01

    Surfactant replacement therapy is now the standard of care for infants with respiratory distress syndrome. As the understanding of surfactant structure and function has evolved, surfactant-associated proteins are now understood to be essential components of pulmonary surfactant. Their structural and functional diversity detail the complexity of their contributions to normal pulmonary physiology, and deficiency states result in significant pathology. Engineering synthetic surfactant protein constructs has been a major research focus for replacement therapies. This review highlights what is known about surfactant proteins and how this knowledge is pivotal for future advancements in treating respiratory distress syndrome as well as other pulmonary diseases characterized by surfactant deficiency or inactivation.

  16. Interfacial reactions of ozone with surfactant protein B in a model lung surfactant system.

    PubMed

    Kim, Hugh I; Kim, Hyungjun; Shin, Young Shik; Beegle, Luther W; Jang, Seung Soon; Neidholdt, Evan L; Goddard, William A; Heath, James R; Kanik, Isik; Beauchamp, J L

    2010-02-24

    Oxidative stresses from irritants such as hydrogen peroxide and ozone (O(3)) can cause dysfunction of the pulmonary surfactant (PS) layer in the human lung, resulting in chronic diseases of the respiratory tract. For identification of structural changes of pulmonary surfactant protein B (SP-B) due to the heterogeneous reaction with O(3), field-induced droplet ionization (FIDI) mass spectrometry has been utilized. FIDI is a soft ionization method in which ions are extracted from the surface of microliter-volume droplets. We report structurally specific oxidative changes of SP-B(1-25) (a shortened version of human SP-B) at the air-liquid interface. We also present studies of the interfacial oxidation of SP-B(1-25) in a nonionizable 1-palmitoyl-2-oleoyl-sn-glycerol (POG) surfactant layer as a model PS system, where competitive oxidation of the two components is observed. Our results indicate that the heterogeneous reaction of SP-B(1-25) at the interface is quite different from that in the solution phase. In comparison with the nearly complete homogeneous oxidation of SP-B(1-25), only a subset of the amino acids known to react with ozone are oxidized by direct ozonolysis in the hydrophobic interfacial environment, both with and without the lipid surfactant layer. Combining these experimental observations with the results of molecular dynamics simulations provides an improved understanding of the interfacial structure and chemistry of a model lung surfactant system subjected to oxidative stress.

  17. Experimental Study on How Human Lung Surfactant Protein SP-B1-25 is Oxidized by Ozone in the Presence of Fe(II) and Ascorbic Acid

    NASA Astrophysics Data System (ADS)

    Colussi, A. J.; Enami, S.; Hoffmann, M. R.

    2014-12-01

    We will report the results of experiments on the chemical fate of the human lung surfactant protein SP-B1-25 upon exposure to gaseous ozone in realistic aqueous media simulating the conditions prevalent in epithelial lining fluids in polluted ambient air. Our experiments consist of exposing aqueous microjets containing SP-B1-25, the natural antioxidant ascorbic acid, and the Fe2+ carried by most atmospheric fine particulates, under mild acidic conditions, such as those created by the innate lung host defense response. Reactants and the products of such interactions are detected via online electrospray ionization mass spectrometry. We will show that ascorbic acid largely inhibits the ozonation of SP-B1-25 in the absence of Fe2+, leading to the formation of an ascorbic acid ozonide (Enami et al., PNAS 2008). In the presence of Fe2+, however, the ozonide decomposes into reactive intermediates that result in the partial oxidation of SP-B1-25, presumable affecting its function as surfactant. We infer that these experimental results establish a plausible causal link for the observed synergic adverse health effects of ambient ozone and fine particulates

  18. Impact of C-reactive protein (CRP) on surfactant function

    SciTech Connect

    Li, J.J.; Sanders, R.L.; McAdam, K.P.; Hales, C.A.; Thompson, B.T.; Gelfand, J.A.; Burke, J.F. )

    1989-12-01

    Plasma levels of the acute-phase reactant, C-reactive protein (CRP), increase up to one thousand-fold as a result of trauma or inflammation. CRP binds to phosphorylcholine (PC) in a calcium-ion dependent manner. The structural homology between PC and the major phospholipid component of surfactant, dipalmitoyl phosphatidylcholine (DPPC), led to the present study in which we examined if CRP levels might be increased in patients with adult respiratory distress syndrome (ARDS), and subsequently interfere with surfactant function. Our results showed that CRP levels in the bronchoalveolar fluid (BALF) was increased in patients with ARDS (97.8 +/- 84.2 micrograms/mg total protein vs. 4.04 +/- 2.2 micrograms/mg total protein in normals). Our results show that CRP binds to liposomes containing DPPC and phosphatidylglycerol (PG). As a result of this interaction, CRP inhibits the surface activity of a PG-DPPC mixture when tested with a Wilhelmy surfactometer or with the Enhorning pulsating bubble apparatus. Furthermore, the surface activity of a clinically used surfactant replacement, Surfactant TA (2 mg/ml), was also severely impaired by CRP in a dose-dependent manner (doses used ranging from 24.5 to 1,175 micrograms/ml). In contrast, human serum albumin (HSA) at 500 and 900 micrograms/ml had no inhibitory effect on Surfactant TA surface activity. These results suggest that CRP, although not an initiating insult in ARDS, may contribute to the subsequent abnormalities of surfactant function and thus the pathogenesis of the pulmonary dysfunction seen in ARDS.

  19. Molecular simulation of surfactant-assisted protein refolding

    NASA Astrophysics Data System (ADS)

    Lu, Diannan; Liu, Zheng; Liu, Zhixia; Zhang, Minlian; Ouyang, Pingkai

    2005-04-01

    Protein refolding to its native state in vitro is a challenging problem in biotechnology, i.e., in the biomedical, pharmaceutical, and food industry. Protein aggregation and misfolding usually inhibit the recovery of proteins with their native states. These problems can be partially solved by adding a surfactant into a suitable solution environment. However, the process of this surfactant-assisted protein refolding is not well understood. In this paper, we wish to report on the first-ever simulations of surfactant-assisted protein refolding. For these studies, we defined a simple model for the protein and the surfactant and investigated how a surfactant affected the folding behavior of a two-dimensional lattice protein molecule. The model protein and model surfactant were chosen such that we could capture the important features of the folding process and the interaction between the protein and the surfactant, namely, the hydrophobic interaction. It was shown that, in the absence of surfactants, a protein in an "energy trap" conformation, i.e., a local energy minima, could not fold into the native form, which was characterized by a global energy minimum. The addition of surfactants created folding pathways via the formation of protein-surfactant complexes and thus enabled the conformations that fell into energy trap states to escape from these traps and to form the native proteins. The simulation results also showed that it was necessary to match the hydrophobicity of surfactant to the concentration of denaturant, which was added to control the folding or unfolding of a protein. The surfactants with different hydrophobicity had their own concentration range on assisting protein refolding. All of these simulations agreed well with experimental results reported elsewhere, indicating both the validity of the simulations presented here and the potential application of the simulations for the design of a surfactant on assisting protein refolding.

  20. Structural study of surfactant-dependent interaction with protein

    SciTech Connect

    Mehan, Sumit; Aswal, Vinod K.; Kohlbrecher, Joachim

    2015-06-24

    Small-angle neutron scattering (SANS) has been used to study the complex structure of anionic BSA protein with three different (cationic DTAB, anionic SDS and non-ionic C12E10) surfactants. These systems form very different surfactant-dependent complexes. We show that the structure of protein-surfactant complex is initiated by the site-specific electrostatic interaction between the components, followed by the hydrophobic interaction at high surfactant concentrations. It is also found that hydrophobic interaction is preferred over the electrostatic interaction in deciding the resultant structure of protein-surfactant complexes.

  1. Nintedanib modulates surfactant protein-D expression in A549 human lung epithelial cells via the c-Jun N-terminal kinase-activator protein-1 pathway.

    PubMed

    Kamio, Koichiro; Usuki, Jiro; Azuma, Arata; Matsuda, Kuniko; Ishii, Takeo; Inomata, Minoru; Hayashi, Hiroki; Kokuho, Nariaki; Fujita, Kazue; Saito, Yoshinobu; Miya, Toshimichi; Gemma, Akihiko

    2015-06-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a high mortality rate. Signalling pathways activated by several tyrosine kinase receptors are known to be involved in lung fibrosis, and this knowledge has led to the development of the triple tyrosine kinase inhibitor nintedanib, an inhibitor of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), for the treatment of IPF. Pulmonary surfactant protein D (SP-D), an important biomarker of IPF, reportedly attenuates bleomycin-induced pulmonary fibrosis in mice. In this study, we investigated whether nintedanib modulates SP-D expression in human lung epithelial (A549) cells using quantitative real-time reverse transcriptase polymerase chain reaction and western blotting. To investigate the mechanisms underlying the effects of nintedanib, we evaluated the phosphorylation of c-Jun N-terminal kinase (JNK) and its downstream target c-Jun. The effect of the JNK inhibitor SP600125 on c-Jun phosphorylation was also tested. Activation of activator protein-1 (AP-1) was examined using an enzyme-linked immunosorbent assay-based test, and cell proliferation assays were performed to estimate the effect of nintedanib on cell proliferation. Furthermore, we treated mice with nintedanib to examine its in vivo effect on SP-D levels in lungs. These experiments showed that nintedanib up-regulated SP-D messenger RNA expression in a dose-dependent manner at concentrations up to 5 μM, with significant SP-D induction observed at concentrations of 3 μM and 5 μM, in comparison with that observed in vehicle controls. Nintedanib stimulated a rapid increase in phosphorylated JNK in A549 cells within 30 min of treatment and stimulated c-Jun phosphorylation, which was inhibited by the JNK inhibitor SP600125. Additionally, nintedanib was found to activate AP-1. A549 cell proliferation was not affected by nintedanib at any of the tested

  2. Lung Surfactant Levels are Regulated by Ig-Hepta/GPR116 by Monitoring Surfactant Protein D

    PubMed Central

    Fukuzawa, Taku; Ishida, Junji; Kato, Akira; Ichinose, Taro; Ariestanti, Donna Maretta; Takahashi, Tomoya; Ito, Kunitoshi; Abe, Jumpei; Suzuki, Tomohiro; Wakana, Shigeharu; Fukamizu, Akiyoshi; Nakamura, Nobuhiro; Hirose, Shigehisa

    2013-01-01

    Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta+/+ and Ig-Hepta−/− mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i) balanced synthesis of surfactant lipids and proteins and (ii) surfactant secretion, and (iii) a stimulating effect on recycling (uptake) in response to elevated levels of Sp-D in alveolar space. PMID:23922714

  3. “SP-G”, a Putative New Surfactant Protein – Tissue Localization and 3D Structure

    PubMed Central

    Paulsen, Friedrich; Ngueya, Ivan; Bräuer, Lars; Brandt, Wolfgang

    2012-01-01

    Surfactant proteins (SP) are well known from human lung. These proteins assist the formation of a monolayer of surface-active phospholipids at the liquid-air interface of the alveolar lining, play a major role in lowering the surface tension of interfaces, and have functions in innate and adaptive immune defense. During recent years it became obvious that SPs are also part of other tissues and fluids such as tear fluid, gingiva, saliva, the nasolacrimal system, and kidney. Recently, a putative new surfactant protein (SFTA2 or SP-G) was identified, which has no sequence or structural identity to the already know surfactant proteins. In this work, computational chemistry and molecular-biological methods were combined to localize and characterize SP-G. With the help of a protein structure model, specific antibodies were obtained which allowed the detection of SP-G not only on mRNA but also on protein level. The localization of this protein in different human tissues, sequence based prediction tools for posttranslational modifications and molecular dynamic simulations reveal that SP-G has physicochemical properties similar to the already known surfactant proteins B and C. This includes also the possibility of interactions with lipid systems and with that, a potential surface-regulatory feature of SP-G. In conclusion, the results indicate SP-G as a new surfactant protein which represents an until now unknown surfactant protein class. PMID:23094088

  4. "SP-G", a putative new surfactant protein--tissue localization and 3D structure.

    PubMed

    Rausch, Felix; Schicht, Martin; Paulsen, Friedrich; Ngueya, Ivan; Bräuer, Lars; Brandt, Wolfgang

    2012-01-01

    Surfactant proteins (SP) are well known from human lung. These proteins assist the formation of a monolayer of surface-active phospholipids at the liquid-air interface of the alveolar lining, play a major role in lowering the surface tension of interfaces, and have functions in innate and adaptive immune defense. During recent years it became obvious that SPs are also part of other tissues and fluids such as tear fluid, gingiva, saliva, the nasolacrimal system, and kidney. Recently, a putative new surfactant protein (SFTA2 or SP-G) was identified, which has no sequence or structural identity to the already know surfactant proteins. In this work, computational chemistry and molecular-biological methods were combined to localize and characterize SP-G. With the help of a protein structure model, specific antibodies were obtained which allowed the detection of SP-G not only on mRNA but also on protein level. The localization of this protein in different human tissues, sequence based prediction tools for posttranslational modifications and molecular dynamic simulations reveal that SP-G has physicochemical properties similar to the already known surfactant proteins B and C. This includes also the possibility of interactions with lipid systems and with that, a potential surface-regulatory feature of SP-G. In conclusion, the results indicate SP-G as a new surfactant protein which represents an until now unknown surfactant protein class.

  5. Pseudomonas aeruginosa protease IV degrades surfactant proteins and inhibits surfactant host defense and biophysical functions.

    PubMed

    Malloy, Jaret L; Veldhuizen, Ruud A W; Thibodeaux, Brett A; O'Callaghan, Richard J; Wright, Jo Rae

    2005-02-01

    Pulmonary surfactant has two distinct functions within the lung: reduction of surface tension at the air-liquid interface and participation in innate host defense. Both functions are dependent on surfactant-associated proteins. Pseudomonas aeruginosa is primarily responsible for respiratory dysfunction and death in cystic fibrosis patients and is also a leading pathogen in nosocomial pneumonia. P. aeruginosa secretes a number of proteases that contribute to its virulence. We hypothesized that P. aeruginosa protease IV degrades surfactant proteins and results in a reduction in pulmonary surfactant host defense and biophysical functions. Protease IV was isolated from cultured supernatant of P. aeruginosa by gel chromatography. Incubation of cell-free bronchoalveolar lavage fluid with protease IV resulted in degradation of surfactant proteins (SP)-A, -D, and -B. SPs were degraded in a time- and dose-dependent fashion by protease IV, and degradation was inhibited by the trypsin-like serine protease inhibitor Nalpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK). Degradation by protease IV inhibited SP-A- and SP-D-mediated bacterial aggregation and uptake by macrophages. Surfactant treated with protease IV was unable to reduce surface tension as effectively as untreated surfactant, and this effect was inhibited by TLCK. We speculate that protease IV may be an important contributing factor to the development and propagation of acute lung injury associated with P. aeruginosa via loss of surfactant function within the lung.

  6. Recognition of Mannosylated Ligands and Influenza A Virus by Human Surfactant Protein D: Contributions of an Extended Site and Residue 343

    SciTech Connect

    Crouch, E.; Hartshorn, K; Horlacher, T; McDonald, B; Smith, K; Cafarella, T; Seaton, B; Seeberger, P; Head, J

    2009-01-01

    Surfactant protein D (SP-D) plays important roles in antiviral host defense. Although SP-D shows a preference for glucose/maltose, the protein also recognizes d-mannose and a variety of mannose-rich microbial ligands. This latter preference prompted an examination of the mechanisms of mannose recognition, particularly as they relate to high-mannose viral glycans. Trimeric neck plus carbohydrate recognition domains from human SP-D (hNCRD) preferred ?1-2-linked dimannose (DM) over the branched trimannose (TM) core, ?1-3 or ?1-6 DM, or d-mannose. Previous studies have shown residues flanking the carbohydrate binding site can fine-tune ligand recognition. A mutant with valine at 343 (R343V) showed enhanced binding to mannan relative to wild type and R343A. No alteration in affinity was observed for d-mannose or for ?1-3- or ?1-6-linked DM; however, substantially increased affinity was observed for ?1-2 DM. Both proteins showed efficient recognition of linear and branched subdomains of high-mannose glycans on carbohydrate microarrays, and R343V showed increased binding to a subset of the oligosaccharides. Crystallographic analysis of an R343V complex with 1,2-DM showed a novel mode of binding. The disaccharide is bound to calcium by the reducing sugar ring, and a stabilizing H-bond is formed between the 2-OH of the nonreducing sugar ring and Arg349. Although hNCRDs show negligible binding to influenza A virus (IAV), R343V showed markedly enhanced viral neutralizing activity. Hydrophobic substitutions for Arg343 selectively blocked binding of a monoclonal antibody (Hyb 246-05) that inhibits IAV binding activity. Our findings demonstrate an extended ligand binding site for mannosylated ligands and the significant contribution of the 343 side chain to specific recognition of multivalent microbial ligands, including high-mannose viral glycans.

  7. Relating Surfactant Properties to Activity and Solubilization of the Human Adenosine A3 Receptor

    PubMed Central

    Berger, Bryan W.; García, Roxana Y.; Lenhoff, Abraham M.; Kaler, Eric W.; Robinson, Clifford R.

    2005-01-01

    The effects of various surfactants on the activity and stability of the human adenosine A3 receptor (A3) were investigated. The receptor was expressed using stably transfected HEK293 cells at a concentration of 44 pmol functional receptor per milligram membrane protein and purified using over 50 different nonionic surfactants. A strong correlation was observed between a surfactant's ability to remove A3 from the membrane and the ability of the surfactant to remove A3 selectively relative to other membrane proteins. The activity of A3 once purified also correlates well with the selectivity of the surfactant used. The effects of varying the surfactant were much stronger than those achieved by including A3 ligands in the purification scheme. Notably, all surfactants that gave high efficiency, selectivity and activity fall within a narrow range of hydrophile-lipophile balance values. This effect may reflect the ability of the surfactant to pack effectively at the hydrophobic transmembrane interface. These findings emphasize the importance of identifying appropriate surfactants for a particular membrane protein, and offer promise for the development of rapid, efficient, and systematic methods to facilitate membrane protein purification. PMID:15849244

  8. Molecular dynamics for surfactant-assisted protein refolding

    NASA Astrophysics Data System (ADS)

    Lu, Diannan; Liu, Zheng; Wu, Jianzhong

    2007-02-01

    Surfactants are widely used to refold recombinant proteins that are produced as inclusion bodies in E. Coli. However, the microscopic details of the surfactant-assisted protein refolding processes are yet to be uncovered. In the present work, the authors aim to provide insights into the effect of hydrophobic interactions of a denatured protein with surfactant molecules on the refolding kinetics and equilibrium by using the Langevin dynamics for coarse-grained models. The authors have investigated the folding behavior of a β-barrel protein in the presence of surfactants of different hydrophobicities and concentrations. It is shown that the protein folding process follows a "collapse-rearrangement" mechanism, i.e., the denatured protein first falls into a collapsed state before acquiring the native conformation. In comparison with the protein folding without surfactants, the protein-surfactant hydrophobic interactions promote the collapse of a denatured protein and, consequently, the formation of a hydrophobic core. However, the surfactants must be released from the hydrophobic core during the rearrangement step, in which the native conformation is formed. The simulation results can be qualitatively reproduced by experiments.

  9. Key interactions of surfactants in therapeutic protein formulations: A review.

    PubMed

    Khan, Tarik A; Mahler, Hanns-Christian; Kishore, Ravuri S K

    2015-11-01

    Proteins as amphiphilic, surface-active macromolecules, demonstrate substantial interfacial activity, which causes considerable impact on their multifarious applications. A commonly adapted measure to prevent interfacial damage to proteins is the use of nonionic surfactants. Particularly in biotherapeutic formulations, the use of nonionic surfactants is ubiquitous in order to prevent the impact of interfacial stress on drug product stability. The scope of this review is to convey the current understanding of interactions of nonionic surfactants with proteins both at the interface and in solution, with specific focus to their effects on biotherapeutic formulations.

  10. Molecular basis for premature senescence induced by surfactants in normal human cells.

    PubMed

    Yamakami, Yoshimi; Miki, Kensuke; Yonekura, Ryuzo; Kudo, Ikuru; Fujii, Michihiko; Ayusawa, Dai

    2014-01-01

    Sublethal doses of surfactants as exemplified by NP-40 clearly induce premature senescence in normal human cells. To understand molecular basis for this phenomenon, we tried to suppress it with use of various inhibitors. An inhibitor of p38 of the MAPK family almost completely suppressed growth arrest and morphological changes induced by surfactants; however, other inhibitors tested had no effect. Oleic acid, a weak inducer of premature senescence, was found to suppress the effect of NP-40. Fluorescein-labeled oleic acid rapidly bound to the cell surface, and this binding was clearly blocked by pre-treatment with surfactants, suggesting that surfactants and oleic acid compete for binding to the cell surface. Moderate concentrations of cycloheximide, an inhibitor of protein synthesis, also suppressed the senescent features induced by NP-40. These results suggest that surfactants activate p38 signaling pathway by binding to the cell surface, and induce cellular senescence.

  11. Cell-specific modulation of surfactant proteins by ambroxol treatment

    SciTech Connect

    Seifart, Carola . E-mail: zwiebel@mailer.uni-marburg.de; Clostermann, Ursula; Seifart, Ulf

    2005-02-15

    Ambroxol [trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexanole hydrochloride], a mucolytic agent, was postulated to provide surfactant stimulatory properties and was previously used to prevent surfactant deficiency. Currently, the underlying mechanisms are not exactly clear. Because surfactant homeostasis is regulated by surfactant-specific proteins (SP), we analyzed protein amount and mRNA expression in whole lung tissue, isolated type II pneumocytes and bronchoalveolar lavage of Sprague-Dawley rats treated with ambroxol i.p. (75 mg/kg body weight, twice a day [every 12 h]). The methods used included competitive polymerase chain reaction (RT-PCR), Northern blotting, Western immunoblotting, and immunohistochemistry. In isolated type II pneumocytes of ambroxol-treated animals, SP-C protein and mRNA content were increased, whereas SP-A, -B and -D protein, mRNA, and immunoreactivity remained unaffected. However, ambroxol treatment resulted in a significant increase of SP-B and in a decrease of SP-D in whole lung tissue with enhanced immunostaining for SP-B in Clara Cells. SP-A and SP-D were significantly decreased in BAL fluid of ambroxol-treated animals. The data suggest that surfactant protein expression is modulated in a cell-specific manner by ambroxol, as type II pneumocytes exhibited an increase in SP-C, whereas Clara cells exhibited an increase in the immunoreactivity for SP-B accounting for the increased SP-B content of whole lung tissue. The results indicate that ambroxol may exert its positive effects, observed in the treatment of diseases related to surfactant deficiency, via modulation of surfactant protein expression.

  12. Cell-specific modulation of surfactant proteins by ambroxol treatment.

    PubMed

    Seifart, Carola; Clostermann, Ursula; Seifart, Ulf; Müller, Bernd; Vogelmeier, Claus; von Wichert, Peter; Fehrenbach, Heinz

    2005-02-15

    Ambroxol [trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexanole hydrochloride], a mucolytic agent, was postulated to provide surfactant stimulatory properties and was previously used to prevent surfactant deficiency. Currently, the underlying mechanisms are not exactly clear. Because surfactant homeostasis is regulated by surfactant-specific proteins (SP), we analyzed protein amount and mRNA expression in whole lung tissue, isolated type II pneumocytes and bronchoalveolar lavage of Sprague-Dawley rats treated with ambroxol i.p. (75 mg/kg body weight, twice a day [every 12 h]). The methods used included competitive polymerase chain reaction (RT-PCR), Northern blotting, Western immunoblotting, and immunohistochemistry. In isolated type II pneumocytes of ambroxol-treated animals, SP-C protein and mRNA content were increased, whereas SP-A, -B and -D protein, mRNA, and immunoreactivity remained unaffected. However, ambroxol treatment resulted in a significant increase of SP-B and in a decrease of SP-D in whole lung tissue with enhanced immunostaining for SP-B in Clara Cells. SP-A and SP-D were significantly decreased in BAL fluid of ambroxol-treated animals. The data suggest that surfactant protein expression is modulated in a cell-specific manner by ambroxol, as type II pneumocytes exhibited an increase in SP-C, whereas Clara cells exhibited an increase in the immunoreactivity for SP-B accounting for the increased SP-B content of whole lung tissue. The results indicate that ambroxol may exert its positive effects, observed in the treatment of diseases related to surfactant deficiency, via modulation of surfactant protein expression.

  13. Deduced amino acid sequence of human pulmonary surfactant proteolipid: SPL(pVal)

    SciTech Connect

    Whitsett, J.A.; Glasser, S.W.; Korfhagen, T.R.; Weaver, T.E.; Clark, J.; Pilot-Matias, T.; Meuth, J.; Fox, J.L.

    1987-05-01

    Hydrophobic, proteolipid-like protein of Mr 6500 was isolated from ether/ethanol extracts of human, canine and bovine pulmonary surfactant. Amino acid composition of the protein demonstrated a remarkable abundance of hydrophobic residues, particularly valine and leucine. The N-terminal amino acid sequence of the human protein was determined: N-Leu-Ile-Pro-Cys-Cys-Pro-Val-Asn-Leu-Lys-Arg-Leu-Leu-Ile-Val4... An oligonucleotide probe was used to screen an adult human lung cDNA library and resulted in detection of cDNA clones with predicted amino acid sequence with close identity to the N-terminal amino acid sequence of the human peptide. SPL(pVal) was found within the reading frame of a larger peptide. SPL(pVal) results from proteolytic processing of a larger preprotein. Northern blot analysis detected in a single 1.0 kilobase SPL(pVal) RNA which was less abundant in fetal than in adult lung. Mixtures of purified canine and bovine SPL(pVal) and synthetic phospholipids display properties of rapid adsorption and surface tension lowering activity characteristic of surfactant. Human SPL(pVal) is a pulmonary surfactant proteolipid which may therefore be useful in combination with phospholipids and/or other surfactant proteins for the treatment of surfactant deficiency such as hyaline membrane disease in newborn infants.

  14. Whey protein coating efficiency on surfactant-modified hydrophobic surfaces.

    PubMed

    Lin, Shih-Yu D; Krochta, John M

    2005-06-15

    Whey protein oxygen-barrier coatings on peanuts are not effective, due to incomplete peanut-surface coverage, as well as some cracking and flaking of the coating. Addition of sorbitan laurate (Span 20) in the whey protein coating solution up to the critical micelle concentration (cmc) of 0.05% (w/w) significantly improved coating coverage to 88% of the peanut surface. Increasing the Span 20 concentration in the coating solution to 3 times the cmc (0.15% w/w) produced a substantial increase in peanut surface energy (>70 dyn/cm), indicating adsorption of the surfactant to the peanut surface. With this level of Span 20, the whey protein coating coverage on peanuts increased to 95%. These results suggest that a concentration of surfactant above the cmc in the coating solution is required for formation of self-assembled structures of surfactant molecules on peanut surfaces, which significantly increases the hydrophilicity, and thus coatability, of peanut surfaces.

  15. A Function of Lung Surfactant Protein SP-B

    NASA Astrophysics Data System (ADS)

    Longo, M. L.; Bisagno, A. M.; Zasadzinski, J. A. N.; Bruni, R.; Waring, A. J.

    1993-07-01

    The primary function of lung surfactant is to form monolayers at the alveolar interface capable of lowering the normal surface tension to near zero. To accomplish this process, the surfactant must be capable of maintaining a coherent, tightly packed monolayer that avoids collapse during expiration. The positively charged amino-terminal peptide SP-B1-25 of lung surfactant-specific protein SP-B increases the collapse pressure of an important component of lung surfactant, palmitic acid (PA), to nearly 70 millinewtons per meter. This alteration of the PA isotherms removes the driving force for "squeeze-out" of the fatty acids from the primarily dipalmitoylphosphatidylcholine monolayers of lung surfactant. An uncharged mutant of SP-B1-25 induced little change in the isotherms, suggesting that a specific charge interaction between the cationic peptide and the anionic lipid is responsible for the stabilization. The effect of SP-B1-25 on fatty acid isotherms is remarkably similar to that of simple poly-cations, suggesting that such polymers might be useful as components of replacement surfactants for the treatment of respiratory distress syndrome.

  16. The importance of surfactant proteins-New aspects on macrophage phagocytosis.

    PubMed

    Tschernig, Thomas; Veith, Nils T; Diler, Ebru; Bischoff, Markus; Meier, Carola; Schicht, Martin

    2016-11-01

    Surfactant and its components have multiple functions. The so called collectins are surfactant proteins which opsonize bacteria and improve pulmonary host defense via the phagocytosis and clearance of microorganisms and particles. In this special issue of the Annals of Anatomy a new surfactant protein, Surfactant Associated 3, is highlighted. As outlined in this mini review Surfactant Associated 3 is regarded as an enhancer of phagocytosis. In addition, the role played by SP-A is updated and open research questions raised.

  17. cDNA and deduced amino acid sequence of human pulmonary surfactant-associated proteolipid SPL(Phe)

    SciTech Connect

    Glasser, S.W.; Korfhagen, T.R.; Weaver, T.; Pilot-Matias, T.; Fox, J.L.; Whitsett, J.A.

    1987-06-01

    Hydrophobic surfactant-associated protein of M/sub r/ 6000-14,000 was isolated from either/ethanol or chloroform/methanol extracts of mammalian pulmonary surfactant. Automated Edman degradation in a gas-phase sequencer showed the major N-terminus of the human low molecular weight protein to be Phe-Pro-Ile-Pro-Leu-Pro-Try-Cys-Trp-Leu-Cys-Arg-Ala-Leu-. Because of the N-terminal phenylalanine, the surfactant protein was designated SPL(Phe). Antiserum generated against hydrophobic surfactant protein(s) from bovine pulmonary surfactant recognized protein of M/sub r/ 6000-14,000 in immunoblot analysis and was used to screen a lambdagt11 expression library constructed from adult human lung poly(A)/sup +/ RNA. This resulted in identification of a 1.4-kilobase cDNA clone that was shown to encode the N-terminus of the surfactant polypeptide SPL(Phe) (Phe-Pro-Ile-Pro-Leu-Pro-) within an open reading frame for a larger protein. Expression of a fused ..beta..-galactosidase-SPL (Phe) gene in Escherichia coli yielded an immunoreactive M/sub r/ 34,000 fusion peptide. Hybrid-arrested translation with the cDNA and immunoprecipitation of (/sup 35/S)methionine-labeled in vitro translation products of human poly(A)/sup +/ RNA with a surfactant polyclonal antibody resulted in identification of a M/sub r/ 40,000 precursor protein. Blot hybridization analysis of electrophoretically fractionated RNA from human lung detected a 2.0-kilobase RNA that was more abundant in adult lung than in fetal lung. These proteins, and specifically SPL(Phe), may therefore be useful for synthesis of replacement surfactants for treatment of hyaline membrane disease in newborn infants or of other surfactant-deficient states.

  18. Environmental and human safety of major surfactants. Volume 1. Anionic surfactants. Part 1. Linear alkylbenzene sulfonates. Final report

    SciTech Connect

    Not Available

    1991-02-01

    The report discusses critical reviews of published literature and unpublished company data on major surfactants. Part 1 of Vol. 1 discusses the chemistry, biodegradation, environmental effects and safety and human safety of linear alkylbenzene sulfonates. The information presented updates and supplements similar data included in two predecessor studies, Human Safety and Environmental Aspects of Major Surfactants (1977) NTIS Accession Number PB-301193 and Human and Environmental Aspects of Major Surfactants (Supplement) (1981) NTIS Accession Number PB-81-182453.

  19. Environmental and human safety of major surfactants. Volume 1. Anionic surfactants. Part 2. Alcohol ethoxy sulfates. Final report

    SciTech Connect

    Not Available

    1991-02-01

    The report discusses critical reviews of published literature and unpublished company data on major surfactants. Part 2 of Vol. 1 discusses the chemistry, biodegradation, environmental effects and safety and human safety of alcohol ethoxy sulfates. The information presented updates and supplements similar data included in two predecessor studies, Human Safety and Environmental Aspects of Major Surfactants (1977) NTIS Accession Number PB301193 and Human and Environmental Aspects of Major Surfactants (Supplement) (1981) NTIS Accessiion Number PB81-182453.

  20. Environmental and human safety of major surfactants. Volume 1. Anionic surfactants. Part 3. Alkyl sulfates. Final report

    SciTech Connect

    Not Available

    1991-02-01

    The report discusses critical reviews of published literature and unpublished company data on major surfactants. Part 3 of Vol. 1 discusses the chemistry, biodegradation, environmental effects and safety and human safety of alkyl sulfates. The information presented updates and supplements similar data included in two predecessor studies, Human Safety and Environmental Aspects of Major Surfactants (1977) NTIS Accession Number PB301193 and Human and Environmental Aspects of Major Surfactants (Supplement) (1981) NTIS Accession Number PB81-182453.

  1. Surfactant enhanced disinfection of the human norovirus surrogate, tulane virus with organic acids and surfactant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Human infection with foodborne viruses can occur following consumption of contaminated food, person-to-person body contact, or release of aerosols. Combinatorial treatments of surfactants and organic acids may have synergistic or additive mechanisms to inactivate foodborne viruses and prevent outbr...

  2. The interplay of lung surfactant proteins and lipids assimilates the macrophage clearance of nanoparticles.

    PubMed

    Ruge, Christian A; Schaefer, Ulrich F; Herrmann, Jennifer; Kirch, Julian; Cañadas, Olga; Echaide, Mercedes; Pérez-Gil, Jesús; Casals, Cristina; Müller, Rolf; Lehr, Claus-Michael

    2012-01-01

    The peripheral lungs are a potential entrance portal for nanoparticles into the human body due to their large surface area. The fact that nanoparticles can be deposited in the alveolar region of the lungs is of interest for pulmonary drug delivery strategies and is of equal importance for toxicological considerations. Therefore, a detailed understanding of nanoparticle interaction with the structures of this largest and most sensitive part of the lungs is important for both nanomedicine and nanotoxicology. Astonishingly, there is still little known about the bio-nano interactions that occur after nanoparticle deposition in the alveoli. In this study, we compared the effects of surfactant-associated protein A (SP-A) and D (SP-D) on the clearance of magnetite nanoparticles (mNP) with either more hydrophilic (starch) or hydrophobic (phosphatidylcholine) surface modification by an alveolar macrophage (AM) cell line (MH-S) using flow cytometry and confocal microscopy. Both proteins enhanced the AM uptake of mNP compared with pristine nanoparticles; for the hydrophilic ST-mNP, this effect was strongest with SP-D, whereas for the hydrophobic PL-mNP it was most pronounced with SP-A. Using gel electrophoretic and dynamic light scattering methods, we were able to demonstrate that the observed cellular effects were related to protein adsorption and to protein-mediated interference with the colloidal stability. Next, we investigated the influence of various surfactant lipids on nanoparticle uptake by AM because lipids are the major surfactant component. Synthetic surfactant lipid and isolated native surfactant preparations significantly modulated the effects exerted by SP-A and SP-D, respectively, resulting in comparable levels of macrophage interaction for both hydrophilic and hydrophobic nanoparticles. Our findings suggest that because of the interplay of both surfactant lipids and proteins, the AM clearance of nanoparticles is essentially the same, regardless of different

  3. Fluorescently labeled pulmonary surfactant protein C in spread phospholipid monolayers.

    PubMed Central

    Nag, K; Perez-Gil, J; Cruz, A; Keough, K M

    1996-01-01

    Pulmonary surfactant, a lipid-protein complex, secreted into the fluid lining of lungs prevents alveolar collapse at low lung volumes. Pulmonary surfactant protein C (SP-C), an acylated, hydrophobic, alpha-helical peptide, enhances the surface activity of pulmonary surfactant lipids. Fluorescein-labeled SP-C (F-SP-C) (3, 6, 12 wt%) in dipalmitoylphosphatidylcholine (DPPC), and DPPC:dipalmitoylphosphatidylglycerol (DPPG) [DPPC:DPPG 7:3 mol/mol] in spread monolayers was studied by epifluorescence microscopy. Mass spectometry of F-SP-C indicated that the protein is partially deacylated and labeled with 1 mol fluorescein/1 mol protein. The protein partitioned into the fluid, or liquid expanded, phase. Increasing amounts of F-SP-C in DPPC or DPPC:DPPG monolayers decreased the size and total amounts of the condensed phase at all surface pressures. Calcium (1.6 mM) increased the amount of the condensed phase in monolayers of DPPC:DPPG but not of DPPC alone, and such monolayers were also perturbed by F-SP-C. The study indicates that SP-C perturbs the packing of neutral and anionic phospholipid monolayers even when the latter systems are condensed by calcium, indicating that interactions between SP-C and the lipids are predominantly hydrophobic in nature. Images FIGURE 2 FIGURE 4 FIGURE 7 PMID:8804608

  4. Impact of a surfactant on the electroactivity of proteins at an aqueous-organogel microinterface array.

    PubMed

    O'Sullivan, Shane; Arrigan, Damien W M

    2013-02-05

    The impact of surfactant addition to the organic phase on the electroactivity of proteins at the aqueous-organogel interface was examined by voltammetry. The presence of bis(2-ethylhexyl)sulfosuccinate (AOT) in the organogel phase, as the sodium salt, caused marked changes in the peak currents for myoglobin detection. The protein desorption voltammetric peak exhibited a 6-fold increase in the current compared to the corresponding experiment without surfactant. Interfacial coverage showed a 17-fold increase in the adsorbed protein at the interface, from 50 pmol cm(-2), in the absence of surfactant, to 850 pmol cm(-2), in the presence of 10 mM surfactant. Additionally, the presence of the surfactant resulted in a second pair of adsorption/desorption peaks at lower potentials and in a change in the capacitance of the system. The formation of surfactant-protein and surfactant-protein-organic anion deposits is proposed on the basis of these features, leading to increased voltammetric signals for myoglobin, hemoglobin, and cytochrome c. The mechanism of protein-surfactant interaction was probed by using the surfactant as the anion in the organic phase electrolyte salt. Repetitive cyclic voltammetry of cytochrome c showed that in the presence of surfactant there was an enhancement of the signal, caused by a buildup of the protein-surfactant-electrolyte anion assembly at the interface. These findings provide the basis for surfactant-modified interfaces to enhance the electroanalytical performance for protein detection.

  5. Multi-block poloxamer surfactants suppress aggregation of denatured proteins.

    PubMed

    Mustafi, Devkumar; Smith, Catherine M; Makinen, Marvin W; Lee, Raphael C

    2008-01-01

    On the basis of elastic light scattering, we have compared the capacity of the multi-block, surfactant copolymers Poloxamer 108 (P108), Poloxamer 188 (P188), and Tetronic 1107 (T1107), of average molecular weight 4700, 8400, and 15,000, respectively, with that of polyethylene glycol (PEG, molecular weight 8000) to suppress aggregation of heat-denatured hen egg white lysozyme (HEWL) and bovine serum albumin (BSA). We also compared the capacity of P188 to that of PEG to suppress aggregation of carboxypeptidase A denatured in the presence of trifluoroethanol and to facilitate recovery of catalytic activity. In contrast to the multi-block copolymers, PEG had no effect in inhibiting aggregation of HEWL or of carboxypeptidase A with the recovery of catalytic activity. At very high polymer:protein ratios (>or=10:1), PEG increased aggregation of heat-denatured HEWL and BSA, consistent with its known properties to promote macromolecular crowding and crystallization of proteins. At a polymer:protein ratio of 2:1, the tetra-block copolymer T1107 was the most effective of the three surfactant copolymers, completely suppressing aggregation of heat-denatured HEWL. At a T1107:BSA ratio of 10:1, the poloxamer suppressed aggregation of heat-denatured BSA by 50% compared to that observed in the absence of the polymer. We showed that the extent of suppression of aggregation of heat-denatured proteins by multi-block surfactant copolymers is dependent on the size of the protein and the copolymer:protein molar ratio. We also concluded that at least one of the tertiary nitrogens in the ethylene-1,2-diamine structural core of the T1107 copolymer is protonated, and that this electrostatic factor underlies its capacity to suppress aggregation of denatured proteins more effectively than nonionic, multi-block poloxamers. These results indicate that amphiphilic, surfactant, multi-block copolymers are efficient as additives to suppress aggregation and to facilitate refolding of denatured

  6. Structural requirements for palmitoylation of surfactant protein C precursor.

    PubMed Central

    ten Brinke, Anja; Vaandrager, Arie B; Haagsman, Henk P; Ridder, Anja N J A; van Golde, Lambert M G; Batenburg, Joseph J

    2002-01-01

    Pulmonary surfactant protein C (SP-C) propeptide (proSP-C) is a type II transmembrane protein that is palmitoylated on two cysteines adjacent to its transmembrane domain. To study the structural requirements for palmitoylation of proSP-C, His-tagged human proSP-C and mutant forms were expressed in Chinese hamster ovary cells and analysed by metabolic labelling with [3H]palmitate. Mutations were made in the amino acid sequence representing mature SP-C, as deletion of the N- and C-terminal propeptide parts showed that this sequence by itself could already be palmitoylated. Substitution of the transmembrane domain by an artificial transmembrane domain had no effect on palmitoylation. However, an inverse correlation was found between palmitoylation of proSP-C and the number of amino acids present between the cysteines and the transmembrane domain. Moreover, substitution by alanines of amino acids localized on the N-terminal side of the cysteines had drastic effects on palmitoylation, probably as a result of the removal of hydrophobic amino acids. These data, together with the observation that substitution by alanines of the amino acids localized between the cysteines and the transmembrane domain had no effect on palmitoylation, suggest that the palmitoylation of proSP-C depends not on specific sequence motifs, but more on the probability that the cysteine is in the vicinity of the membrane surface. This is probably determined not only by the number of amino acids between the cysteines and the transmembrane domain, but also by the hydrophobic interaction of the N-terminus with the membrane. This may also be the case for the palmitoylation of other transmembrane proteins. PMID:11802797

  7. PLUNC Is a Novel Airway Surfactant Protein with Anti-Biofilm Activity

    PubMed Central

    Penterman, Jon; Mizrachi, Dario; Singh, Pradeep K.; Mallampalli, Rama K.; Ramaswamy, S.; McCray, Paul B.

    2010-01-01

    Background The PLUNC (“Palate, lung, nasal epithelium clone”) protein is an abundant secretory product of epithelia present throughout the conducting airways of humans and other mammals, which is evolutionarily related to the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family. Two members of this family - the bactericidal/permeability increasing protein (BPI) and the lipopolysaccharide binding protein (LBP) - are innate immune molecules with recognized roles in sensing and responding to Gram negative bacteria, leading many to propose that PLUNC may play a host defense role in the human airways. Methodology/Principal Findings Based on its marked hydrophobicity, we hypothesized that PLUNC may be an airway surfactant. We found that purified recombinant human PLUNC greatly enhanced the ability of aqueous solutions to spread on a hydrophobic surface. Furthermore, we discovered that PLUNC significantly reduced surface tension at the air-liquid interface in aqueous solutions, indicating novel and biologically relevant surfactant properties. Of note, surface tensions achieved by adding PLUNC to solutions are very similar to measurements of the surface tension in tracheobronchial secretions from humans and animal models. Because surfactants of microbial origin can disperse matrix-encased bacterial clusters known as biofilms [1], we hypothesized that PLUNC may also have anti-biofilm activity. We found that, at a physiologically relevant concentration, PLUNC inhibited biofilm formation by the airway pathogen Pseudomonas aeruginosa in an in vitro model. Conclusions/Significance Our data suggest that the PLUNC protein contributes to the surfactant properties of airway secretions, and that this activity may interfere with biofilm formation by an airway pathogen. PMID:20161732

  8. Effect of surfactants on preformed fibrils of human serum albumin.

    PubMed

    Pandey, Nitin Kumar; Ghosh, Sudeshna; Dasgupta, Swagata

    2013-08-01

    The central reason behind pathogenesis of various neurological disorders is usually attributed to the accumulation of aggregated proteins particularly in fibrillar morphology in vivo. One of the plausible remedial treatments for such disorders may be to identify molecules which are capable of either preventing formation of fibrils or disintegrating formed fibrils. The effect of cationic surfactants cetyl trimethylammonium bromide (CTAB), dodecyl trimethylammonium bromide (DTAB) and the anionic surfactant sodium dodecyl sulfate (SDS) in vitro toward mature HSA fibrils has been investigated. The process has been monitored using ThT fluorescence, FTIR, circular dichroism, fluorescence microscopy and HRTEM. It was observed that the micelles of cationic surfactants were able to effectively disrupt the HSA fibrils, among which CTAB was found to be the most potent.

  9. SANS and DLS Studies of Protein Unfolding in Presence of Urea and Surfactant

    SciTech Connect

    Aswal, V. K.; Chodankar, S. N.; Wagh, A. G.; Kohlbrecher, J.; Vavrin, R.

    2008-03-17

    Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) have been used to study conformational changes in protein bovine serum albumin (BSA) during its unfolding in presence of protein denaturating agents urea and surfactant. On addition of urea, the BSA protein unfolds for urea concentrations greater than 4 M and acquires a random coil configuration with its radius of gyration increasing with urea concentration. The addition of surfactant unfolds the protein by the formation of micelle-like aggregates of surfactants along the unfolded polypeptide chains of the protein. The fractal dimension of such a protein-surfactant complex decreases and the overall size of the complex increases on increasing the surfactant concentration. The conformation of the unfolded protein in the complex has been determined directly using contrast variation SANS measurements by contrast matching the surfactant to the medium. Results of DLS measurements are found to be in good agreement with those obtained using SANS.

  10. Pulmonary surfactant protein A (SP-A) specifically binds dipalmitoylphosphatidylcholine

    SciTech Connect

    Kuroki, Y.; Akino, T. )

    1991-02-15

    Phospholipids are the major components of pulmonary surfactant. Dipalmitoylphosphatidylcholine is believed to be especially essential for the surfactant function of reducing the surface tension at the air-liquid interface. Surfactant protein A (SP-A) with a reduced denatured molecular mass of 26-38 kDa, characterized by a collagen-like structure and N-linked glycosylation, interacts strongly with a mixture of surfactant-like phospholipids. In the present study the direct binding of SP-A to phospholipids on a thin layer chromatogram was visualized using 125I-SP-A as a probe, so that the phospholipid specificities of SP-A binding and the structural requirements of SP-A and phospholipids for the binding could be examined. Although 125I-SP-A bound phosphatidylcholine and sphingomyeline, it was especially strong in binding dipalmitoylphosphatidylcholine, but failed to bind phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. Labeled SP-A also exhibited strong binding to distearoylphosphatidylcholine, but weak binding to dimyristoyl-, 1-palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine. Unlabeled SP-A readily competed with labeled SP-A for phospholipid binding. SP-A strongly bound dipalmitoylglycerol produced by phospholipase C treatment of dipalmitoylphosphatidylcholine, but not palmitic acid. This protein also failed to bind lysophosphatidylcholine produced by phospholipase A2 treatment of dipalmitoylphosphatidylcholine. 125I-SP-A shows almost no binding to dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylethanolamine. The addition of 10 mM EGTA into the binding buffer reduced much of the 125I-SP-A binding to phospholipids. Excess deglycosylated SP-A competed with labeled SP-A for binding to dipalmitoylphosphatidylcholine, but the excess collagenase-resistant fragment of SP-A failed.

  11. Weak interactive forces govern the interaction between a non-ionic surfactant with human serum albumin

    NASA Astrophysics Data System (ADS)

    Ghosh, Narayani; Mondal, Ramakanta; Deshmukh, Arundhati; Dutta, Sanjay; Mukherjee, Saptarshi

    2015-08-01

    The effect of the non-ionic surfactant Tween 40 (TW40) on Human Serum Albumin (HSA) has been studied by spectroscopic and isothermal titration calorimetric (ITC) methods. Our steady-state and time-resolved spectroscopic results reveal the perturbation of the native protein conformation upon interaction with TW40. The interaction of TW40 with HSA does not occur in a sequential manner unlike another anionic surfactant, sodium dodecyl sulfate (SDS). Our major conclusion is that the HSA-TW40 interaction is mainly driven by weak forces like van der Waal/hydrogen bonding interactions. This is also generalized from the results of interaction of HSA with another non-ionic surfactant TW80.

  12. Lung remodeling in aging surfactant protein D deficient mice.

    PubMed

    Schneider, Jan Philipp; Arkenau, Martina; Knudsen, Lars; Wedekind, Dirk; Ochs, Matthias

    2017-02-07

    Pulmonary surfactant, a mixture of lipids and proteins at the air-liquid interface of alveoli, prevents the lungs from collapsing due to surface tension. One constituent is surfactant-associated protein-D (SP-D), a protein involved in surfactant homeostasis and innate immunity. Mice deficient in SP-D (SP-D (-/-)) has been described as developing a characteristic phenotype which affects the surfactant system (including changes in the intra-cellular and intra-alveolar surfactant pool, alveolar epithelial type II cells and alveolar macrophages), lung architecture and its inflammatory state (development of an emphysema-like pathology, inflammatory cell infiltration). Furthermore, it has been described that these mice develop sub-pleural fibrosis and a thickening of alveolar septal walls. The aim of the present study was to systematically investigate the long term progression of this phenotype with special focus on parenchymal remodeling, whether there are progressive emphysematous changes and whether there is progressive septal wall thickening which might indicate the development of pulmonary fibrosis. By means of design-based stereology and light microscopy, lungs of wild type (wt) and SP-D (-/-) mice of four age groups (3, 6, 12 and ∼18 months) were investigated. The data do not suggest a relevant spontaneous pro-fibrotic remodeling or a destructive process in the aging SP-D (-/-) mice. We demonstrated neither a significant destructive emphysema nor significant thickening of alveolar septal walls, but the data suggest an increase in the number weighted mean alveolar volume in aging SP-D (-/-) mice without loss of alveoli or alveolar epithelial surface area per lung. This increase may reflect over-distension due to altered mechanical properties of alveoli. In the light of our findings and data from the literature, the question arises as to whether a lack of SP-D promotes structural changes in the lung which have been described as being associated with aging lungs

  13. Mature Surfactant Protein-B Expression by Immunohistochemistry as a Marker for Surfactant System Development in the Fetal Sheep Lung.

    PubMed

    Lock, Mitchell C; McGillick, Erin V; Orgeig, Sandra; Zhang, Song; McMillen, I Caroline; Morrison, Janna L

    2015-11-01

    Evaluation of the number of type II alveolar epithelial cells (AECs) is an important measure of the lung's ability to produce surfactant. Immunohistochemical staining of these cells in lung tissue commonly uses antibodies directed against mature surfactant protein (SP)-C, which is regarded as a reliable SP marker of type II AECs in rodents. There has been no study demonstrating reliable markers for surfactant system maturation by immunohistochemistry in the fetal sheep lung despite being widely used as a model to study lung development. Here we examine staining of a panel of surfactant pro-proteins (pro-SP-B and pro-SP-C) and mature proteins (SP-B and SP-C) in the fetal sheep lung during late gestation in the saccular/alveolar phase of development (120, 130, and 140 days), with term being 150 ± 3 days, to identify the most reliable marker of surfactant producing cells in this species. Results from this study indicate that during late gestation, use of anti-SP-B antibodies in the sheep lung yields significantly higher cell counts in the alveolar epithelium than SP-C antibodies. Furthermore, this study highlights that mature SP-B antibodies are more reliable markers than SP-C antibodies to evaluate surfactant maturation in the fetal sheep lung by immunohistochemistry.

  14. Modifications in structure and interaction of nanoparticle-protein-surfactant complexes in electrolyte solution

    NASA Astrophysics Data System (ADS)

    Mehan, Sumit; Kumar, S.; Aswal, V. K.; Schweins, R.

    2016-05-01

    SANS experiments of three-component system of anionic silica nanoparticles, anionic BSA protein and anionic SDS surfactants have been carried out without and with electrolyte in aqueous solution. In both the cases, the interaction of surfactant with protein results in formation of bead-necklace structure of protein-surfactant complexes in solution. These protein-surfactant complexes interact very differently with nanoparticles in absence and presence of electrolyte. In absence of electrolyte, nanoparticles remain in dispersed phase in solution, whereas with the addition of electrolyte the nanoparticles fractal aggregates are formed. SANS describes the phase behavior to be governed by competition of electrostatic and depletion interactions among the components solution.

  15. Surfactant proteins A and D in the genital tract of mares.

    PubMed

    Kankavi, Orhan; Ata, Ayhan; Gungor, Orsan

    2007-04-01

    The presence of surface-active material in the lung alveolus has been known for several decades as being essential for normal lung function. Surfactant is essential for reducing the surface tension at the alveolar air-liquid interface. Pulmonary surfactant is composed of 90% lipids and 10% proteins. There are four non-serum proteins surfactant protein-A (SP-A), surfactant protein-B (SP-B), surfactant protein-C (SP-C) and surfactant protein-D (SP-D) named in chronologic order of discovery. Lung SP-A and SP-D belong to a family of collagen-containing C-type lectin family called collectins. The host defence and controlling inflammatory processes of the lung are the major functions of SP-A and SP-D. SP-A and SP-D were originally demonstrated in alveolar type II cells, but recent studies have shown extrapulmonary expression of SP-A and SP-D indicating systemic roles of these proteins. Present study describes the presence of SP-A and SP-D in the mare genital tract, vulva, vagina, ovarium, uterus and tuba uterina using immunohistochemistry and Western blotting. The aim of this study was to characterize surfactant proteins in terms of: (i) whether surfactant proteins were present in the various structures of the mare genital system, (ii) if so, identifying and locating the surfactant proteins and finally (iii) determining the differences from those previously characterized for the lung. Although beyond the scope of this report, it is recognized that there are also some potential implications for better defining the reproductive defence mechanisms in mare. Therefore, genital system organs and tissues from mares were examined. We were able to show that proteins reactive with surfactant-specific antibodies were present in the mare genital tract. Thus, surfactant proteins are present not in just lamellar bodies associated with lung, but also genital system of mare.

  16. Observation of two different fractal structures in nanoparticle, protein and surfactant complexes

    SciTech Connect

    Mehan, Sumit Kumar, Sugam Aswal, V. K.

    2014-04-24

    Small angle neutron scattering has been carried out from a complex of nanoparticle, protein and surfactant. Although all the components are similarly (anionic) charged, we have observed strong interactions in their complex formation. It is characterized by the coexistence of two different mass fractal structures. The first fractal structure is originated from the protein and surfactant interaction and second from the depletion effect of first fractal structure leading the nanoparticle aggregation. The fractal structure of protein-surfactant complex represents to bead necklace structure of micelle-like clusters of surfactant formed along the unfolded protein chain. Its fractal dimension depends on the surfactant to protein ratio (r) and decreases with the increase in r. However, fractal dimension of nanoparticle aggregates in nanoparticle-protein complex is found to be independent of protein concentration and governed by the diffusion limited aggregation like morphology.

  17. Interaction of two imidazolium gemini surfactants with two model proteins BSA and HEWL.

    PubMed

    Gospodarczyk, W; Kozak, M

    Gemini surfactants and their interactions with proteins have gained considerable scientific interest, especially when amyloidogenic proteins are taken into account. In this work, the influence of two selected dicationic (gemini) surfactants (3,3'-[1,8-(2,7-dioxaoctane)]bis(1-dodecylimidazolium) chloride and 3,3'-[1,12-(2,11-dioxadodecane)]bis(1-dodecylimidazolium) chloride) on two model proteins, bovine serum albumin (BSA) and hen egg white lysozyme (HEWL), have been investigated. A pronounced and sophisticated influence on BSA structure has been revealed, including a considerable change of protein radius of gyration as well as substantial alteration of its secondary structure. Radius of gyration has been found to rise significantly with addition of surfactants and to fall down for high surfactants concentration. Similarly, a remarkable fall of secondary structure (α-helix content) has been observed, followed by its partial retrieval for high surfactants concentration. A strong aggregation of BSA has been observed for a confined range of surfactants concentrations as well. In case of HEWL-gemini system, on the other hand, the protein-surfactant interaction was found to be weak. Molecular mechanisms explaining such behaviour of protein-surfactant systems have been proposed. The differences of properties of both studied surfactants have also been discussed.

  18. Binding of Alkyl Polyglucoside Surfactants to Bacteriorhodopsin and its Relation to Protein Stability

    PubMed Central

    Santonicola, M. Gabriella; Lenhoff, Abraham M.; Kaler, Eric W.

    2008-01-01

    The binding of alkyl polyglucoside surfactants to the integral membrane protein bacteriorhodopsin (BR) and the formation of protein-surfactant complexes are investigated by sedimentation equilibrium via analytical ultracentrifugation and by small-angle neutron scattering (SANS). Contrast variation techniques in SANS enable measurement of the composition of the protein-surfactant complexes and determination of the thickness of the surfactant shell bound to the protein. The results indicate that alkyl polyglucosides can bind to BR as single surfactant layers or as a thicker shell. The thickness of the surfactant shell increases with increasing surfactant tail length, and it is generally unrelated to the aggregation number of the micelles even for a small and predominantly hydrophobic membrane protein such as BR. The aggregation numbers determined by sedimentation equilibrium methods match those measured by SANS, which also allows reconstruction of the shape of the protein-detergent complex. When the surfactant is present as a single layer, the BR loses activity, as measured by absorption spectroscopy, more quickly than it does when the surfactant forms a thicker shell. PMID:18234822

  19. Binding of alkyl polyglucoside surfactants to bacteriorhodopsin and its relation to protein stability.

    PubMed

    Santonicola, M Gabriella; Lenhoff, Abraham M; Kaler, Eric W

    2008-05-01

    The binding of alkyl polyglucoside surfactants to the integral membrane protein bacteriorhodopsin (BR) and the formation of protein-surfactant complexes are investigated by sedimentation equilibrium via analytical ultracentrifugation and by small-angle neutron scattering (SANS). Contrast variation techniques in SANS enable measurement of the composition of the protein-surfactant complexes and determination of the thickness of the surfactant shell bound to the protein. The results indicate that alkyl polyglucosides can bind to BR as single surfactant layers or as a thicker shell. The thickness of the surfactant shell increases with increasing surfactant tail length, and it is generally unrelated to the aggregation number of the micelles even for a small and predominantly hydrophobic membrane protein such as BR. The aggregation numbers determined by sedimentation equilibrium methods match those measured by SANS, which also allows reconstruction of the shape of the protein-detergent complex. When the surfactant is present as a single layer, the BR loses activity, as measured by absorption spectroscopy, more quickly than it does when the surfactant forms a thicker shell.

  20. DECREASED PRODUCTION OF SURFACTANT PROTEINS AFTER DIESEL EXHAUST EXPOSURE INCREASES SUSCEPTIBILITY TO INFLUENZA INFECTION

    EPA Science Inventory

    Pulmonary surfactant proteins A and D (SP-A and SP-D), termed collectins, enhance the opsonization of foreign particles and pathogens by phagocytic cells. Inhaled pollutants such as diesel exhaust (DE) have a possible role in suppressing the production of surfactant proteins whic...

  1. Surfactant protein A, exposure to endotoxin, and asthma in garbage collectors and in wastewater workers.

    PubMed

    Widmeier, Susanne; Bernard, Alfred; Tschopp, Alois; Jeggli, Stefan; Dumont, Xavier; Hilfiker, Silvia; Oppliger, Anne; Hotz, Philippe

    2007-04-01

    Endotoxin causes an inflammation at the bronchial and alveolar level. The inflammation-induced increase in permeability of the bronchoalveolar epithelial barrier is supposed to cause a leakage of pneumoproteins. Therefore, their concentrations are expected to increase in the bloodstream. This study aimed at examining the association between occupational exposure to endotoxin and a serum pneumoprotein, surfactant protein A, to look for nonoccupational factors capable of confounding this association, and examine the relation between surfactant protein A and spirometry. There were 369 control subjects, 325 wastewater workers, and 84 garbage collectors in the study. Exposure to endotoxin was assessed through personal sampling and the Limulus amebocytes lysate assay. Surfactant protein A was determined by an in house sandwich enzyme-linked immunosorbent assay (ELISA) in 697 subjects. Clinical and smoking history were ascertained and spirometry carried out according to American Thoracic Society criteria. Multiple linear regression was used for statistical analysis. Exposure was fairly high during some tasks in wastewater workers but did not influence surfactant protein A. Surfactant protein A was lower in asthmatics. Interindividual variability was large. No correlation with spirometry was found. Endotoxin has no effect on surfactant protein A at these endotoxin levels and serum surfactant protein A does not correlate with spirometry. The decreased surfactant protein A secretion in asthmatics requires further study.

  2. Tween surfactants: Adsorption, self-organization, and protein resistance

    NASA Astrophysics Data System (ADS)

    Shen, Lei; Guo, Athena; Zhu, Xiaoyang

    2011-03-01

    Tween surfactants, each containing hydrophilic ethylene glycol head groups and a hydrophobic alkyl tail, are being actively explored as protein-resistant surface coatings, but little is known about how they adsorb on surfaces. We carry out a comparative study of the adsorption of two Tween molecules (same hydrophilic head group, but a shorter dodecyl tail for Tween 20 and a longer octadecyl tail for Tween 40) on Au and polystyrene surfaces. Despite the similarity between these two molecules, there is a drastic difference in their protein resistance: a monolayer of Tween 20 on a hydrophobic surface is repulsive against protein adsorption but that of Tween 40 is not. The difference in protein resistance can be attributed to two distinctly different adsorption mechanisms. While the adsorption of Tween 40 is described by a simple first-order mechanism, that of Tween 20 consists of a fast adsorption step and a slower reorganization process at a high surface coverage. The latter leads to the formation of a high-density and self-organized monolayer, which is responsible for the enhanced stability and resistance against non-specific protein adsorption.

  3. Molecular biological characterization of equine surfactant protein A.

    PubMed

    Hospes, R; Hospes, B I L; Reiss, I; Bostedt, H; Gortner, L

    2002-12-01

    In the following, we describe the isolation and sequencing of the equine surfactant protein A (Sp-A) as found in both the cDNA and the genomic DNA. We found a length of the cDNA sequence of 747 bp (base pairs), in translation into amino acids of 248. Compared with the known molecular biological facts about Sp-A in other species, the cDNA sequence obtained showed highest homology with that of sheep (85.01%). The genomic DNA of equine Sp-A, as in other species, includes three introns. There were no hints for the existence of two different Sp-A genes. These results should form the basis for a better understanding of respiratory failure in foals and adult horses, and also lead to further studies on this item.

  4. Protein binding onto surfactant-based synthetic vesicles.

    PubMed

    Letizia, Caterina; Andreozzi, Patrizia; Scipioni, Anita; La Mesa, Camillo; Bonincontro, Adalberto; Spigone, Elisabetta

    2007-02-01

    Synthetic vesicles were prepared by mixing anionic and cationic surfactants, aqueous sodium dodecylsulfate with didodecyltrimethylammonium or cetyltrimethylammonium bromide. The overall surfactant content and the (anionic/cationic) mole ratios allow one to obtain negatively charged vesicles. In the phase diagram, the vesicular region is located between a solution phase, a lamellar liquid crystalline dispersion, and a precipitate area. Characterization of the vesicles was performed by electrophoretic mobility, NMR, TEM, and DLS and we determined their uni-lamellar character, size, stability, and charge density. Negatively charged vesicular dispersions, made of sodium dodecylsulfate/didodecyltrimethylammonium bromide or sodium dodecylsulfate/cetyltrimethylammonium bromide, were mixed with lysozyme, to form lipoplexes. Depending on the protein/vesicle charge ratio, binding, surface saturation, and lipoplexes flocculation, or precipitation, occurs. The free protein in excess remains in solution, after binding saturation. The systems were investigated by thermodynamic (surface tension and solution calorimetry), DLS, CD, TEM, 1H NMR, transport properties, electrophoretic mobility, and dielectric relaxation. The latter two methods give information on the vesicle charge neutralization by adsorbed protein. Binding is concomitant to modifications in the double layer thickness of vesicles and in the surface charge density of the resulting lipoplexes. This is also confirmed by developing the electrophoretic mobility results in terms of a Langmuir-like adsorption isotherm. Charges in excess with respect to the amount required to neutralize the vesicle surface promote lipoplexes clustering and/or flocculation. Protein-vesicle interactions were observed by DLS, indicating changes in particle size (and in their distribution functions) upon addition of LYSO. According to CD, the bound protein retains its native conformation, at least in the SDS/CTAB vesicular system. In fact

  5. Impact of the New Generation Reconstituted Surfactant CHF5633 on Human CD4+ Lymphocytes

    PubMed Central

    Fehrholz, Markus; Glaser, Kirsten; Seidenspinner, Silvia; Ottensmeier, Barbara; Curstedt, Tore; Speer, Christian P.; Kunzmann, Steffen

    2016-01-01

    Background Natural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP-) B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown. Aim The aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4+ lymphocytes. Methods Purified human CD4+ T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf®). Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry. Results Neither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4+ lymphocytes under the given conditions. IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFNγ, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4+ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf®, expression levels of anti-inflammatory IL-4 and IL-10 m

  6. Effects of ozone and acid aerosol exposures on surfactant-associated protein A in the lung

    SciTech Connect

    Su, W.Y.

    1993-01-01

    This study examined the effect of ozone and/or acid aerosol exposure on the level of surfactant associated protein A (SP-A), its gene expression and functionality in the lung. Guinea pigs were exposed to (1) a single exposure to 0.2 to 0.8 ppm ozone for 6 hr and sacrificed at 0 to 120 hr postexposure, (2) 0.8 ppm ozone, 6 hr/day for 3 to 5 days and sacrificed immediately postexposure, or (3) 0.8 ppm ozone, 600 [mu]g/m[sup 3] sulfuric acid, or ozone plus acid for 6 hr and sacrificed at 72 hr postexposure. The concentration of SP-A was determined by ELISA in lavage fluid, lavage cell pellets, and lung tissue compartments. SP-A gene expression was examined in lung tissue by Northern and slot blot analysis. Effect of ozone exposure on functionality of surfactant was tested by its ability to modulate phagocytic cell respiratory burst in a luminol-amplified chemiluminescence (CL) assay of phagocytic cells simulated by PMA or opsonized-zymosan. There were isolated, but significant, changes in SP-A concentrations in the lavage cell and the lavage fluid compartments at 24 and 48 hr after single exposure to 0.8 ppm ozone, respectively. Exposure to ozone and ozone plus acid also slightly increased total SP-A level in the lung. No change in SP-A gene expression was detected under the exposure conditions examined. However, surfactant from ozone exposed animals significantly enhanced CL response of phagocytic cells stimulated by either PMA or opsonized-zymosan. Blocking of the enhancement of CL by a rabbit anti-human SP-A antibody strongly suggested that SP-A may contribute in the altered respiratory burst of phagocytic cells induced by surfactant from ozone exposed animals.

  7. Surfactant protein A (SP-A) and SP-A-derived peptide attenuate chemotaxis of mast cells induced by human β-defensin 3.

    PubMed

    Uehara, Yasuaki; Takahashi, Motoko; Murata, Masaki; Saito, Atsushi; Takamiya, Rina; Hasegawa, Yoshihiro; Kuronuma, Koji; Chiba, Hirofumi; Hashimoto, Jiro; Sawada, Norimasa; Takahashi, Hiroki; Kuroki, Yoshio; Ariki, Shigeru

    2017-02-08

    Human β-defensin 3 (hBD3) is known to be involved in mast cell activation. However, molecular mechanisms underlying the regulation of hBD3-induced mast cell activation have been poorly understood. We previously reported that SP-A and SP-A-derived peptide 01 (SAP01) regulate the function of hBD3. In this study, we focused on the effects of SP-A and SAP01 on the activation of mast cells induced by hBD3. SAP01 directly bound to hBD3. Mast cell-mediated vascular permeability and edema in hBD3 administered rat ears were decreased when injected with SP-A or SAP01. Compatible with the results in rat ear model, both SP-A and SAP01 inhibited hBD3-induced chemotaxis of mast cells in vitro. Direct interaction between SP-A or SAP01 and hBD3 seemed to be responsible for the inhibitory effects on chemotaxis. Furthermore, SAP01 attenuated hBD3-induced accumulation of mast cells and eosinophils in tracheas of the OVA-sensitized inflammatory model. SP-A might contribute to the regulation of inflammatory responses mediated by mast cells during infection.

  8. Surfactant protein A is a principal and oxidation-sensitive microbial permeabilizing factor in the alveolar lining fluid.

    PubMed

    Kuzmenko, Alexander I; Wu, Huixing; Wan, Sijue; McCormack, Francis X

    2005-07-08

    We have reported that surfactant protein A kills some Gram-negative organisms by increasing membrane permeability. In this study, we investigated the physiologic importance of this activity and the effect of oxidative stress on the antimicrobial functions of SP-A in vitro and in vivo. Concentrated bronchoalveolar lavage fluids from SP-A+/+ mice increased the permeability of the Escherichia coli K12 cell membrane to a greater extent than lavage from SP-A-/- animals. Similarly, calcium-dependent surfactant-binding proteins of SP-A+/+ mice increased membrane permeability more than those from SP-A-/- mice and produced greater zonal killing of agar-embedded bacteria in a radial diffusion assay. Exposure of human SP-A to copper-initiated surfactant phospholipid peroxidation or to free radicals generated by human neutrophils in vitro increased the level of SP-A-associated carbonyl moieties and blocked the permeabilizing function of the protein. We also found that exposure of mice to 90% O2 for 4 days, sufficient to lead to consumption of glutathione, oxidation of protein thiols, and accumulation of airspace protein-associated carbonyl moieties, blocked the permeabilizing activity of lavage fluid from SP-A+/+ mice. We conclude that SP-A is a major microbial permeablizing factor in lavage fluid and that oxidative stress inhibits the antibacterial activity of SP-A by a mechanism that includes oxidative modification and functional inactivation of the protein.

  9. Protective Effect of Surfactant Protein D in Pulmonary Vaccinia Virus Infection: Implication of A27 Viral Protein

    PubMed Central

    Julien, Perino; Thielens, Nicole M.; Crouch, Erika; Spehner, Danièle; Crance, Jean-Marc; Favier, Anne-Laure

    2013-01-01

    Vaccinia virus (VACV) was used as a surrogate of variola virus (VARV) (genus Orthopoxvirus), the causative agent of smallpox, to study Orthopoxvirus infection. VARV is principally transmitted between humans by aerosol droplets. Once inhaled, VARV first infects the respiratory tract where it could encounter surfactant components, such as soluble pattern recognition receptors. Surfactant protein D (SP-D), constitutively present in the lining fluids of the respiratory tract, plays important roles in innate host defense against virus infection. We investigated the role of SP-D in VACV infection and studied the A27 viral protein involvement in the interaction with SP-D. Interaction between SP-D and VACV caused viral inhibition in a lung cell model. Interaction of SP-D with VACV was mediated by the A27 viral protein. Binding required Ca2+ and interactions were blocked in the presence of excess of SP-D saccharide ligands. A27, which lacks glycosylation, directly interacted with SP-D. The interaction between SP-D and the viral particle was also observed using electron microscopy. Infection of mice lacking SP-D (SP-D-/-) resulted in increased mortality compared to SP-D+/+ mice. Altogether, our data show that SP-D participates in host defense against the vaccinia virus infection and that the interaction occurs with the viral surface protein A27. PMID:23518578

  10. Thermodynamics, interfacial pressure isotherms and dilational rheology of mixed protein-surfactant adsorption layers.

    PubMed

    Fainerman, V B; Aksenenko, E V; Krägel, J; Miller, R

    2016-07-01

    Proteins and their mixtures with surfactants are widely used in many applications. The knowledge of their solution bulk behavior and its impact on the properties of interfacial layers made great progress in the recent years. Different mechanisms apply to the formation process of protein/surfactant complexes for ionic and non-ionic surfactants, which are governed mainly by electrostatic and hydrophobic interactions. The surface activity of these complexes is often remarkably different from that of the individual protein and has to be considered in respective theoretical models. At very low protein concentration, small amounts of added surfactants can change the surface activity of proteins remarkably, even though no strongly interfacial active complexes are observed. Also small added amounts of non-ionic surfactants change the surface activity of proteins in the range of small bulk concentrations or surface coverages. The modeling of the equilibrium adsorption behavior of proteins and their mixtures with surfactants has reached a rather high level. These models are suitable also to describe the high frequency limits of the dilational viscoelasticity of the interfacial layers. Depending on the nature of the protein/surfactant interactions and the changes in the interfacial layer composition rather complex dilational viscoelasticities can be observed and described by the available models. The differences in the interfacial behavior, often observed in literature for studies using different experimental methods, are at least partially explained by a depletion of proteins, surfactants and their complexes in the range of low concentrations. A correction of these depletion effects typically provides good agreement between the data obtained with different methods, such as drop and bubble profile tensiometry.

  11. Surfactant protein A2 mutations associated with pulmonary fibrosis lead to protein instability and endoplasmic reticulum stress.

    PubMed

    Maitra, Meenakshi; Wang, Yongyu; Gerard, Robert D; Mendelson, Carole R; Garcia, Christine Kim

    2010-07-16

    Rare heterozygous mutations in the gene encoding surfactant protein A2 (SP-A2, SFTPA2) are associated with adult-onset pulmonary fibrosis and adenocarcinoma of the lung. We have previously shown that two recombinant SP-A2 mutant proteins (G231V and F198S) remain within the endoplasmic reticulum (ER) of A549 cells and are not secreted into the culture medium. The pathogenic mechanism of the mutant proteins is unknown. Here we analyze all common and rare variants of the surfactant protein A2, SP-A2, in both A549 cells and in primary type II alveolar epithelial cells. We show that, in contrast with all other SP-A2 variants, the mutant proteins are not secreted into the medium with wild-type SP-A isoforms, form fewer intracellular dimer and trimer oligomers, are partially insoluble in 0.5% Nonidet P-40 lysates of transfected A549 cells, and demonstrate greater protein instability in chymotrypsin proteolytic digestions. Both the G231V and F198S mutant SP-A2 proteins are destroyed via the ER-association degradation pathway. Expression of the mutant proteins increases the transcription of a BiP-reporter construct, expression of BiP protein, and production of an ER stress-induced XBP-1 spliced product. Human bronchoalveolar wash samples from individuals who are heterozygous for the G231V mutation have similar levels of total SP-A as normal family members, which suggests that the mechanism of disease does not involve an overt lack of secreted SP-A but instead involves an increase in ER stress of resident type II alveolar epithelial cells.

  12. Comparison of positional surfactant isomers for displacement of rubisco protein from the air-water interface.

    PubMed

    He, Lizhong; Onaizi, Sagheer A; Dimitrijev-Dwyer, Mirjana; Malcolm, Andrew S; Shen, Hsin-Hui; Dong, Chuchuan; Holt, Stephen A; Thomas, Robert K; Middelberg, Anton P J

    2011-08-15

    Protein-surfactant interaction, which is a function of the protein and surfactant characteristics, is a common phenomenon in a wide range of industrial applications. In this work, we used rubisco, the most abundant protein in nature, as a model protein and sodium dodecylbenzenesulfonate (SDOBS), one of the most widely used commercial surfactants, with two positional isomers (SDOBS-2 and SDOBS-6), as a model surfactant. We first examined the surface tension and the mechanical properties of interfacial mixed rubisco-SDOBS films adsorbed at the air-water interface. The concentration of rubisco in solution was fixed at 0.1 mg mL(-1) while the SDOBS concentration varied from 0 to 150 μM. Both the surface tension and the mechanical strength of the interfacial film decreased with increasing SDOBS concentration. Overall, the surface tension of a rubisco-SDOBS-6 mixture is lower than that of rubisco-SDOBS-2, while the mechanical strength of both systems is similar. Neutron reflection data suggest that rubisco protein is likely denatured at the interface. The populations of rubisco and SDOBS of the mixed systems at the interface were determined by combining non-deuterated and deuterated SDOBS to provide contrast variation. At a low surfactant concentration, SDOBS-6 has a stronger ability to displace rubisco from the air-water interface than SDOBS-2. However, when surfactant concentration reaches 50 μM, SDOBS-2 has a higher population than SDOBS-6, with more rubisco displaced from the interface. The results presented in this work suggest that the extent of protein displacement from the air-water interface, and hence the nature of the protein-surfactant interactions at the interface, are strongly affected by the position of surfactant isomerisation, which might allow the design of formulations for efficient removal of protein stains.

  13. Interactions between Surfactants in Solution and Electrospun Protein Fibers: Effects on Release Behavior and Fiber Properties.

    PubMed

    Stephansen, Karen; García-Díaz, María; Jessen, Flemming; Chronakis, Ioannis S; Nielsen, Hanne M

    2016-03-07

    Intermolecular interaction phenomena occurring between endogenous compounds, such as proteins and bile salts, and electrospun compounds are so far unreported, despite the exposure of fibers to such biorelevant compounds when applied for biomedical purposes, e.g., tissue engineering, wound healing, and drug delivery. In the present study, we present a systematic investigation of how surfactants and proteins, as physiologically relevant components, interact with insulin-loaded fish sarcoplasmic protein (FSP) electrospun fibers (FSP-Ins fibers) in solution and thereby affect fiber properties such as accessible surface hydrophilicity, physical stability, and release characteristics of an encapsulated drug. Interactions between insulin-loaded protein fibers and five anionic surfactants (sodium taurocholate, sodium taurodeoxycholate, sodium glycocholate, sodium glycodeoxycholate, and sodium dodecyl sulfate), a cationic surfactant (benzalkonium chloride), and a neutral surfactant (Triton X-100) were studied. The anionic surfactants increased the insulin release in a concentration-dependent manner, whereas the neutral surfactant had no significant effect on the release. Interestingly, only minute amounts of insulin were released from the fibers when benzalkonium chloride was present. The FSP-Ins fibers appeared dense after incubation with this cationic surfactant, whereas high fiber porosity was observed after incubation with anionic or neutral surfactants. Contact angle measurements and staining with the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid indicated that the FSP-Ins fibers were hydrophobic, and showed that the fiber surface properties were affected differently by the surfactants. Bovine serum albumin also affected insulin release in vitro, indicating that also proteins may affect the fiber performance in an in vivo setting.

  14. Pulmonary surfactant: hydrophobic nature of the mucosal surface of the human amnion.

    PubMed

    Cotton, D B; Hills, B A

    1984-04-01

    The contact angle has been measured for a drop of saline placed upon the rinsed mucosal surface of the amnion in eleven human placental membranes obtained from normal births at full term. The contact angle averaged 70 degrees, indicating a hydrophobic surface comparable with graphite (86 degrees), polyethylene (94 degrees) or oxyntic tissue (85 degrees) which is also exposed to endogenous surface-active phospholipids in vivo. By comparison, four pre-term placentas with an average gestation period of 29.5 weeks gave a mean contact angle of 32 degrees, indicating that hydrophobicity of the placenta increases with maturity (41 weeks) and might well be imparted by adsorbed surfactants present in amniotic fluid and known to render other surfaces hydrophobic. Since the mucosal epithelium of the amnion is exposed to the same surfactants in the same physical state as the fetal alveolar wall, the above results imply that this surface may also be hydrophobic, as indicated in the adult lung by other studies. The concept of surfactant directly adsorbed to the pulmonary tissue surfaces is discussed in connexion with its possible functional advantages in 'de-watering' the lung at birth, maintaining homeostasis by water repellency , releasing airway surfaces and lymph ducts glued by protein and lubricating tissue respiratory movement.

  15. Interactions of gemini surfactants with two model proteins: NMR, CD, and fluorescence spectroscopies.

    PubMed

    Amiri, Razieh; Bordbar, Abdol-Khalegh; García-Mayoral, Ma Flor; Khosropour, Ahmad Reza; Mohammadpoor-Baltork, Iraj; Menéndez, Margarita; Laurents, Douglas V

    2012-03-01

    Gemini surfactants have two polar head groups and two hydrocarbon tails. Compared with conventional surfactants, geminis have much lower (μM vs. mM) critical micelle concentrations and possess slower (ms vs. μs) monomer <-- / --> micelle kinetics. The structure of the gemini surfactants studied is [HOCH(2)CH(2)-, CH(3)-, CH(3)(CH(2))(15)-N(+)-(CH(2))(s)-N(+)-(CH(2))(15)CH(3),-CH(3),-CH(2)CH(2)OH]·2Br(-) where s=4, 5, or 6. Our objective is to reveal the effect of these cationic gemini surfactants on the structure and stability of two model proteins: Ribonuclease A (RNase A) and Hen Egg White Lysozyme (HEWL). 2D (1)H NMR and Circular Dichroism (CD) spectroscopies show that the conformation of RNase A and HEWL is unaffected at low to neutral pH where these proteins are positively charged, although hydrogen exchange shows that RNase A's conformational stability is slightly lowered. At alkaline pH, where these proteins lose their net positive charge, fluorescence and CD spectroscopies and ITC experiments show that they do interact with gemini surfactants, and multiple protein•gemini complexes are observed. Based on the results, we conclude that these cationic gemini surfactants neither interact strongly with nor severely destabilize these well folded proteins in physiological conditions, and we advance that they can serve as useful membrane mimetics for studying the interactions between membrane components and positively charged proteins.

  16. Exposure of surfactant protein A to ozone in vitro and in vivo impairs its interactions with alveolar cells

    SciTech Connect

    Oosting, R.S.; Van Iwaarden, J.F.; Van Bree, L.; Verhoef, J.; Van Golde, L.M.; Haagsman, H.P. )

    1992-01-01

    This study focused on the question of whether exposure of surfactant protein A (SP-A) to ozone affected properties of this protein that may be involved in regulating alveolar type II cell and alveolar macrophage functions. In vitro exposure of human or canine SP-A to ozone reduced the ability of this protein to inhibit phorbol-ester induced secretion of (3H)phosphatidylcholine by alveolar type II cells in culture. Ozone-exposed human SP-A showed a decreased ability to enhance phagocytosis of herpes simplex virus and to stimulate superoxide anion production by alveolar macrophages. Experiments with elastase showed that ozone-exposed canine SP-A was more susceptible to proteolysis. A conformational change of the protein could underlie this phenomenon. Surfactant isolated from ozone-exposed rats (0.4 ppm ozone for 12 h) was also less able to stimulate superoxide anion production by alveolar macrophages than surfactant from control rats, which suggested that SP-A in vivo was also susceptible to ozone. The results of this study suggest that SP-A-alveolar cell interactions can be inhibited by ozone exposure, which may contribute to the toxicity of ozone in the lungs.

  17. Critical Role of Arg/Lys343 in the Species-Dependent Recognition of Phosphatidylinositol by Plumonary Surfactant Protein D

    SciTech Connect

    Crouch,E.; McDonald, B.; Smith, K.; Roberts, M.; Mealy, T.; Seaton, B.; Head, J.

    2007-01-01

    Surfactant protein D (SP-D) plays important roles in lung host defense. However, it can also recognize specific host molecules and contributes to surfactant homeostasis. The major known surfactant-associated ligand is phosphatidylinositol (PI). Trimeric neck-carbohydrate recognition domains (NCRDs) of rat and human SP-D exhibited dose-dependent, calcium-dependent, and inositol-sensitive binding to solid-phase PI and to multilamellar PI liposomes. However, the rat protein exhibited a >5-fold higher affinity for solid-phase PI than the human NCRD. In addition, human dodecamers, but not full-length human trimers, efficiently coprecipitated with multilamellar PI liposomes in the presence of calcium. A human NCRD mutant resembling the rat and mouse proteins at position 343 (hR343K) showed much stronger binding to PI. A reciprocal rat mutant with arginine at the position of lysine 343 (rK343R) showed weak binding to PI, even weaker than that of the wild-type human protein. Crystal complexes of the human trimeric NCRD with myoinositol and inositol 1-phosphate showed binding of the equatorial OH groups of the cyclitol ring of the inositol to calcium at the carbohydrate binding site. Myoinositol binding occurred in two major orientations, while inositol 1-phosphate appeared primarily constrained to a single, different orientation. Our studies directly implicate the CRD in PI binding and reveal unexpected species differences in PI recognition that can be largely attributed to the side chain of residue 343. In addition, the studies indicate that oligomerization of trimeric subunits is an important determinant of recognition of PI by human SP-D.

  18. Stability of purple membranes from Halobacterium salinarum toward surfactants: inkjet printing of a retinal protein.

    PubMed

    Imhof, Martin; Pudewills, Jens; Rhinow, Daniel; Chizhik, Ivan; Hampp, Norbert

    2012-08-16

    Inkjet printing is a versatile technique widely applied in biological microarray technology. Because of its photochemical and photophysical properties, bacteriorhodopsin (BR) from Halobacterium salinarum holds promise for applications in nanotechnology, and inkjet printing would simplify the transfer of BR to suitable substrates. Surfactants are essential parts of inkjet formulations tuning viscosity, rheology, and spreading behavior of the solution. However, many surfactants destabilize the structure of proteins and often cause denaturation accompanied by a complete loss of function. Inkjet printing of membrane proteins is particularly challenging and special care must be taken in the choice of the surfactant. For BR, the situation is complicated by the fact that the structural integrity of BR depends on its native membrane environment, the so-called purple membrane (PM). PM contains 10 lipid molecules per BR monomer and is very sensitive toward surfactants. In this work, we identified surfactants suitable for inkjet formulations containing PM. Initially, we screened a variety of technically relevant surfactants for compatibility with PM using the UV-vis absorption of the retinal chromophore as a natural probe. Promising candidates were selected, and their impact on the structure of PM and BR was analyzed using UV-vis spectroscopy, CD spectroscopy, and small-angle X-ray scattering (SAXS). We identified two surfactants compatible with PM and suitable for inkjet formulations. An inkjet formulation containing PM as dye component was developed. We demonstrate that the photochromic properties of BR are maintained upon inkjet printing.

  19. Characteristics of sugar surfactants in stabilizing proteins during freeze-thawing and freeze-drying.

    PubMed

    Imamura, Koreyoshi; Murai, Katsuyuki; Korehisa, Tamayo; Shimizu, Noriyuki; Yamahira, Ryo; Matsuura, Tsutashi; Tada, Hiroko; Imanaka, Hiroyuki; Ishida, Naoyuki; Nakanishi, Kazuhiro

    2014-06-01

    Sugar surfactants with different alkyl chain lengths and sugar head groups were compared for their protein-stabilizing effect during freeze-thawing and freeze-drying. Six enzymes, different in terms of tolerance against inactivation because of freeze-thawing and freeze-drying, were used as model proteins. The enzyme activities that remained after freeze-thawing and freeze-drying in the presence of a sugar surfactant were measured for different types and concentrations of sugar surfactants. Sugar surfactants stabilized all of the tested enzymes both during freeze-thawing and freeze-drying, and a one or two order higher amount of added sugar surfactant was required for achieving protein stabilization during freeze-drying than for the cryoprotection. The comprehensive comparison showed that the C10-C12 esters of sucrose or trehalose were the most effective through the freeze-drying process: the remaining enzyme activities after freeze-thawing and freeze-drying increased at the sugar ester concentrations of 1-10 and 10-100 μM, respectively, and increased to a greater extent than for the other surfactants at higher concentrations. Results also indicate that, when a decent amount of sugar was also added, the protein-stabilizing effect of a small amount of sugar ester through the freeze-drying process could be enhanced.

  20. Phase Behavior and Phase Structure of Protein-Surfactant-Water Systems.

    PubMed

    Morén; Khan

    1999-10-15

    Phase behavior of oppositely charged ovalbumin-DOTAC and BSA-DOTAC, and similarly charged ovalbumin-SDS, BSA-SDS, lysozyme-DOTAC, and BLG-SDS systems within the concentration range of 20 wt% of both protein and surfactant are examined in water. Aqueous solutions of ovalbumin yield, in succession, precipitation, gel, and solution with increased addition of the surfactant dodecyltrimethylammonium chloride (DOTAC). The stability range of each region is determined. Both isotropic and anisotropic gels are detected. Solutions of bovine serum albumin (BSA) form only a solution phase with oppositely charged DOTAC. One solution phase is also obtained with all similarly charged protein-surfactant systems except the BLG-SDS-water system, which produces a gel phase in addition to a large solution phase. (2)H NMR longitudinal (R(1)) and transverse (R(2)) relaxation rates are determined in solution and gel by following the behavior of selectively deuterated surfactant at the alpha-methylene group next to the surfactant head group for the oppositely charged systems ovalbumin-DOTAC and BSA-DOTAC. Large R(2)-values proved the existence of large protein-surfactant aggregates in both systems. Copyright 1999 Academic Press.

  1. Determination of nanograms of proteins based on decreased resonance light scattering of zwitterionic gemini surfactant.

    PubMed

    Chen, Zhanguang; Liu, Guoliang; Chen, Maohuai; Peng, Yurui; Wu, Mingyao

    2009-01-15

    A new high-sensitivity detection of protein assay at the nanogram level is proposed based on the decreased resonance light scattering (RLS) signals of zwitterionic gemini surfactant (phosphodiesters quaternary ammonium salt [PQAS]). It was found that PQAS self-assembled into nanometer-scale PQAS aggregates, which induced intense RLS signal in Britton-Robinson (BR) buffer solution (pH 10.5). Under the optimum conditions, the RLS intensity quenching extent of PQAS aggregation was in proportion to the concentration of proteins in the range of 0.0012-1.08 microg/ml for bovine serum albumin, 0.0015-0.95 microg/ml for human serum albumin, and 0.0025-1.3 microg/ml for gamma-globulin. The detection limits were 0.8, 1.2, and 2.0 ng/ml, respectively. The proposed method was successfully applied to determine total protein in human serum samples, and the results were identical to those obtained by the Bradford assay. The mechanism of interaction between PQAS and protein was studied using RLS, fluorescence, and time-resolved fluorescence, which indicated that the new complex formed between them, disaggregating self-aggregation of PQAS, resulted in the dominated quenching of RLS signal of the assay system.

  2. Dimeric N-terminal segment of human surfactant protein B (dSP-B(1-25)) has enhanced surface properties compared to monomeric SP-B(1-25).

    PubMed Central

    Veldhuizen, E J; Waring, A J; Walther, F J; Batenburg, J J; van Golde, L M; Haagsman, H P

    2000-01-01

    Surfactant protein B (SP-B) is a 17-kDa dimeric protein produced by alveolar type II cells. Its main function is to lower the surface tension by inserting lipids into the air/liquid interface of the lung. SP-B's function can be mimicked by a 25-amino acid peptide, SP-B(1-25), which is based on the N-terminal sequence of SP-B. We synthesized a dimeric version of this peptide, dSP-B(1-25), and the two peptides were tested for their surface activity. Both SP-B(1-25) and dSP-B(1-25) showed good lipid mixing and adsorption activities. The dimeric peptide showed activity comparable to that of native SP-B in the pressure-driven captive bubble surfactometer. Spread surface films led to stable near-zero minimum surface tensions during cycling while protein free, and films containing SP-B(1-25) lost material from the interface during compression. We propose that dimerization of the peptide is required to create a lipid reservoir attached to the monolayer from which new material can enter the surface film upon expansion of the air/liquid interface. The dimeric state of SP-B can fulfill the same function in vivo. PMID:10866963

  3. Glucocorticoids regulate surfactant protein synthesis in a pulmonary adenocarcinoma cell line

    SciTech Connect

    O'Reilly, M.A.; Gazdar, A.F.; Clark, J.C.; Pilot-Matias, T.J.; Wert, S.E.; Hull, W.M.; Whitsett, J.A. )

    1989-12-01

    Synthesis of pulmonary surfactant proteins SP-A, SP-B, and SP-C was demonstrated in a cell line derived from a human adenocarcinoma of the lung. The cells contained numerous lamellar inclusion bodies and formed organized groups of cells containing well-developed junctional complexes and apical microvillous membranes. Synthesis of SP-A was detected in the cells by enzyme-linked immunoabsorbent assay and by immunoprecipitation of (35S)methionine-labeled protein. SP-A was identified as an Mr 31,000-36,000 polypeptide containing asparagine-linked carbohydrate. Northern blot analysis detected SP-A mRNA of 2.2 kb. Dexamethasone (1-10 nM) enhanced the relative abundance of SP-A mRNA. Despite stimulation of SP-A mRNA, intracellular SP-A content was unaltered or inhibited by dexamethasone. SP-B and SP-C mRNAs and synthesis of the SP-B and SP-C precursors were markedly induced by dexamethasone. ProSP-B was synthesized and secreted primarily as an Mr 42,000-46,000 polypeptide. Proteolysis of the proSP-B resulted in the generation of endoglycosidase F-sensitive Mr = 19,000-21,000 and 25,000-27,000 peptides, which were detected both intra- and extracellularly. SP-C proprotein of Mr = 22,000 and smaller SP-C fragments were detected intracellularly but were not detected in the media. Mature forms of SP-B (Mr = 8,000) and SP-C (Mr = 4,000) were not detected. Glucocorticoids directly enhance the relative synthesis and mRNA of the surfactant proteins SP-A, SP-B, and SP-C. Discrepancies among SP-A mRNA, its de novo synthesis, and cell content suggest that glucocorticoid may alter both pre- and posttranslational factors modulating SP-A expression.

  4. Protein kinase A activation of the surfactant protein B gene is mediated by phosphorylation of thyroid transcription factor 1.

    PubMed

    Yan, C; Whitsett, J A

    1997-07-11

    Thyroid transcription factor 1 (TTF-1) is a homeodomain-containing nuclear transcription factor expressed in epithelial cells of the lung and thyroid. TTF-1 binds to and activates the transcription of genes expressed selectively in the respiratory epithelium including pulmonary surfactant A, B, C and Clara cell secretory protein. Transfection with a plasmid encoding the cyclic AMP-dependent protein kinase (protein kinase A; PKA) catalytic subunit, Cat-beta, stimulated the phosphorylation of a TTF-1-flag fusion protein 6-7-fold in H441 pulmonary adenocarcinoma cells. Recombinant TTF-1 was phosphorylated by purified PKA catalytic subunit in the presence of [gamma-32P]ATP. PKA catalytic subunit family members, Cat-alpha and Cat-beta, markedly enhanced the transcriptional activation of surfactant B gene promoters by TTF-1 in vitro. Peptide mapping was used to identify a PKA phosphorylation site at the NH2 terminus of TTF-1. A 17-amino acid synthetic peptide comprising this site completely inhibited the PKA-dependent phosphorylation of TTF-1 in vitro. A substitution mutation of TTF-1 (Thr9 two head right arrow Ala) abolished phosphorylation by PKA and reduced transactivation of the surfactant B gene promoter. Transfection with a plasmid encoding the cAMP regulatory element binding factor inhibited transcriptional activity of the surfactant protein B gene promoter. Phosphorylation of TTF-1 mediates PKA-dependent activation of surfactant protein B gene transcription.

  5. Anionic surfactants enhance click reaction-mediated protein conjugation with ubiquitin.

    PubMed

    Schneider, Daniel; Schneider, Tatjana; Aschenbrenner, Joos; Mortensen, Franziska; Scheffner, Martin; Marx, Andreas

    2016-03-01

    The Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) has become increasingly important in the conjugation chemistry of biomolecules. For example, it is an efficient and convenient method to generate defined ubiquitin-protein conjugates. Here, we investigate the effect of surfactants on the efficiency of CuAAC for chemical protein ubiquitylation. We found that anionic surfactants enhance conjugate formation by up to 10-fold resulting in high yields even at low (i.e., micromolar) concentrations of the reactants. Notably, the herein investigated conjugates are functional and thus properly folded.

  6. Solubilizing and Stabilizing Proteins in Anhydrous Ionic Liquids through Formation of Protein-Polymer Surfactant Nanoconstructs.

    PubMed

    Brogan, Alex P S; Hallett, Jason P

    2016-04-06

    Nonaqueous biocatalysis is rapidly becoming a desirable tool for chemical and fuel synthesis in both the laboratory and industry. Similarly, ionic liquids are increasingly popular anhydrous reaction media for a number of industrial processes. Consequently, the use of enzymes in ionic liquids as efficient, environment-friendly, commercial biocatalysts is highly attractive. However, issues surrounding the poor solubility and low stability of enzymes in truly anhydrous media remain a significant challenge. Here, we demonstrate for the first time that engineering the surface of a protein to yield protein-polymer surfactant nanoconstructs allows for dissolution of dry protein into dry ionic liquids. Using myoglobin as a model protein, we show that this method can deliver protein molecules with near native structure into both hydrophilic and hydrophobic anhydrous ionic liquids. Remarkably, using temperature-dependent synchrotron radiation circular dichroism spectroscopy to measure half-denaturation temperatures, our results show that protein stability increases by 55 °C in the ionic liquid as compared to aqueous solution, pushing the solution thermal denaturation beyond the boiling point of water. Therefore, the work presented herein could provide a platform for the realization of biocatalysis at high temperatures or in anhydrous solvent systems.

  7. Capillary electromigration separation of proteins and microorganisms dynamically modified by chromophoric nonionogenic surfactant.

    PubMed

    Horká, Marie; Růzicka, Filip; Holá, Veronika; Kahle, Vladislav; Moravcová, Dana; Slais, Karel

    2009-08-15

    A chromophoric nonionogenic surfactant poly(ethylene glycol) 3-(2-hydroxy-5-n-octylphenylazo)-benzoate, HOPAB, has been prepared and used as a buffer additive for a dynamic modification of proteins and/or microorganisms including Escherichia coli , Staphylococcus epidermidis (biofilm-positive and biofilm-negative), and the strains of yeast cells Candida albicans and Candida parapsilosis (biofilm-positive and biofilm-negative) during a capillary electrophoresis and a capillary isoelectric focusing (CIEF) with UV detection at 326 nm. Values of isoelectric points of labeled proteins and microorganisms have been calculated using UV-detectable pI markers and have been found comparable with pI of the native compounds. Minimum detectable amount has been assessed lower than picograms of proteins and lower than a hundred cells injected into a separation capillary. The introduced labeling method facilitates CIEF separation of microorganisms from the clinical sample of the infected urine at their clinically important levels in the pH gradient pH range of 2-5 and their subsequent cultivation. At the same time, it has enabled the determination of albumin in human urine as a major clinical marker of urinary tract infections and kidney diseases.

  8. Photoinduced electron transfer in a protein-surfactant complex: probing the interaction of SDS with BSA.

    PubMed

    Chakraborty, Anjan; Seth, Debabrata; Setua, Palash; Sarkar, Nilmoni

    2006-08-24

    Photoinduced fluorescence quenching electron transfer from N,N-dimethyl aniline to different 7-amino coumarin dyes has been investigated in sodium dodecyl sulfate (SDS) micelles and in bovine serum albumin (BSA)-SDS protein-surfactant complexes using steady state and picosecond time resolved fluorescence spectroscopy. The electron transfer rate has been found to be slower in BSA-SDS protein-surfactant complexes compared to that in SDS micelles. This observation has been explained with the help of the "necklace-and-bead" structure formed by the protein-surfactant complex due to coiling of protein molecules around the micelles. In the correlation of free energy change to the fluorescence quenching electron transfer rate, we have observed that coumarin 151 deviates from the normal Marcus region, showing retardation in the electron transfer rate at higher negative free energy region. We endeavored to establish that the retardation in the fluorescence quenching electron transfer rate for coumarin 151 at higher free energy region is a result of slower rotational relaxation and slower translational diffusion of coumarin 151 (C-151) compared to its analogues coumarin 152 and coumarin 481 in micelles and in protein-surfactant complexes. The slower rotational relaxation and translational diffusion of C-151 are supposed to be arising from the different location of coumarin 151 compared to coumarin 152 and coumarin 481.

  9. Human Mitochondrial Protein Database

    National Institute of Standards and Technology Data Gateway

    SRD 131 Human Mitochondrial Protein Database (Web, free access)   The Human Mitochondrial Protein Database (HMPDb) provides comprehensive data on mitochondrial and human nuclear encoded proteins involved in mitochondrial biogenesis and function. This database consolidates information from SwissProt, LocusLink, Protein Data Bank (PDB), GenBank, Genome Database (GDB), Online Mendelian Inheritance in Man (OMIM), Human Mitochondrial Genome Database (mtDB), MITOMAP, Neuromuscular Disease Center and Human 2-D PAGE Databases. This database is intended as a tool not only to aid in studying the mitochondrion but in studying the associated diseases.

  10. A polymer surfactant corona dynamically replaces water in solvent-free protein liquids and ensures macromolecular flexibility and activity.

    PubMed

    Gallat, François-Xavier; Brogan, Alex P S; Fichou, Yann; McGrath, Nina; Moulin, Martine; Härtlein, Michael; Combet, Jérôme; Wuttke, Joachim; Mann, Stephen; Zaccai, Giuseppe; Jackson, Colin J; Perriman, Adam W; Weik, Martin

    2012-08-15

    The observation of biological activity in solvent-free protein-polymer surfactant hybrids challenges the view of aqueous and nonaqueous solvents being unique promoters of protein dynamics linked to function. Here, we combine elastic incoherent neutron scattering and specific deuterium labeling to separately study protein and polymer motions in solvent-free hybrids. Myoglobin motions within the hybrid are found to closely resemble those of a hydrated protein, and motions of the polymer surfactant coating are similar to those of the hydration water, leading to the conclusion that the polymer surfactant coating plasticizes protein structures in a way similar to hydration water.

  11. Inactivation of surfactant in rat lungs.

    PubMed

    Bruni, R; Fan, B R; David-Cu, R; Taeusch, H W; Walther, F J

    1996-02-01

    Although surfactant replacement therapy has dramatically improved the outcome of premature infants with respiratory distress syndrome, approximately 30% of treated infants show a transient or no response. Nonresponse to surfactant replacement therapy may be due to extreme lung immaturity and possibly surfactant inactivation. Surfactant inactivation involves aspecific biophysical events, such as interference with the formation or activity of an alveolar monolayer, and specific interactions with serum proteins, including antibodies, leaking into the alveolar space. As formulations containing surfactant proteins appear to better tolerate serum inactivation, we used an excised rat lung model to compare the susceptibility to serum inactivation of a mixture of synthetic phospholipids selected from surfactant lipid constituents, Exosurf (a protein-free synthetic surfactant), Survanta [containing surfactant proteins B and C (SP-B and -C)], and a porcine surfactant (containing SP-A, -B, and -C). For each of these preparations, we used pressure/volume determinations as an in situ measure of surfactant activity and retested the same preparations after mixing with human serum, a nonspecific surfactant inactivator. Human serum inactivated porcine surfactant to a lesser extent than Survanta, Exosurf, or synthetic phospholipids. Temperature exerted a significant effect on deflation stability, as shown by a greater lung compliance in untreated, normal lungs and a larger improvement in compliance after treating lavaged lungs with synthetic phospholipids at 37 degrees C than at 22 degrees C. We conclude that surfactant containing SP-A, -B, and -C is only moderately susceptible to inactivation with whole serum and may therefore exert a greater clinical response than protein-free surfactants or those containing only SP-B and -C.

  12. Effects of lung surfactant proteins, SP-B and SP-C, and palmitic acid on monolayer stability.

    PubMed Central

    Ding, J; Takamoto, D Y; von Nahmen, A; Lipp, M M; Lee, K Y; Waring, A J; Zasadzinski, J A

    2001-01-01

    Langmuir isotherms and fluorescence and atomic force microscopy images of synthetic model lung surfactants were used to determine the influence of palmitic acid and synthetic peptides based on the surfactant-specific proteins SP-B and SP-C on the morphology and function of surfactant monolayers. Lung surfactant-specific protein SP-C and peptides based on SP-C eliminate the loss to the subphase of unsaturated lipids necessary for good adsorption and respreading by inducing a transition between monolayers and multilayers within the fluid phase domains of the monolayer. The morphology and thickness of the multilayer phase depends on the lipid composition of the monolayer and the concentration of SP-C or SP-C peptide. Lung surfactant protein SP-B and peptides based on SP-B induce a reversible folding transition at monolayer collapse that allows all components of surfactant to be retained at the interface during respreading. Supplementing Survanta, a clinically used replacement lung surfactant, with a peptide based on the first 25 amino acids of SP-B also induces a similar folding transition at monolayer collapse. Palmitic acid makes the monolayer rigid at low surface tension and fluid at high surface tension and modifies SP-C function. Identifying the function of lung surfactant proteins and lipids is essential to the rational design of replacement surfactants for treatment of respiratory distress syndrome. PMID:11325728

  13. Surfactant Protein A integrates activation signal strength to differentially modulate T cell proliferation

    PubMed Central

    Mukherjee, Sambuddho; Giamberardino, Charles; Thomas, Joseph; Evans, Kathy; Goto, Hisatsugu; Ledford, Julie G; Hsia, Bethany; Pastva, Amy M; Wright, Jo Rae

    2011-01-01

    Pulmonary surfactant lipoproteins lower the surface tension at the alveolar:airway interface of the lung and participate in host defense. Previous studies reported that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that SP-A mediated modulation of T cell activation depends upon the strength, duration and type of lymphocyte activating signals. Modulation of T cell signal strength imparted by different activating agents ex and in vivo in different mouse models, and in vitro with human T cells show a strong correlation between strength of signal (SoS) and functional effects of SP-A interactions. T cell proliferation is enhanced in the presence of SP-A at low SoS imparted by exogenous mitogens, specific antibodies, APCs or in homeostatic proliferation. Proliferation is inhibited at higher SoS imparted by different doses of the same T cell mitogens, or indirect stimuli such as LPS. Importantly, reconstitution with exogenous SP-A into the lungs of SP-A-/- mice stimulated with a strong signal also resulted in suppression of T cell proliferation, while elevating baseline proliferation in unstimulated T cells. These signal strength and SP-A dependent effects are mediated by changes in intracellular Ca2+ levels over time, involving extrinsic Ca2+ activated channels late during activation. These effects are intrinsic to the global T cell population, and are manifested in vivo in naïve as well as memory phenotype T cells. Thus, SP-A appears to integrate signal thresholds to control T cell proliferation. PMID:22219327

  14. Rapid and sensitive determination of proteins with Eriochrome Red in the presence of anionic surfactant by multi-spectroscopic methods

    NASA Astrophysics Data System (ADS)

    Bian, Yongru; Cai, Changqun; Gong, Hang; Chen, Xiaoming

    2010-10-01

    A new high-sensitivity detection of protein assay at the nanogram level is proposed based on multi-spectroscopic methods including resonance light scattering (RLS), atomic force microscopy (AFM), ultraviolet spectra (UV) and fluorescence spectra etc. Under the optimum conditions, the amplified RLS signals of anionic azo dyes Eriochrome Red B (ERB) in the presence of anionic surfactant sodium dodecyl sulphonate (SDS) was in proportion to the concentration of proteins in the range of 0.0-3.5 mg L -1 and 3.5-12.5 mg L -1 for bovine serum albumin (BSA), 0.0-2.0 mg L -1 and 2.0-8.0 mg L -1 for human serum albumin (HSA). The detection limits were 4.2 ng mL -1 and 2.7 ng mL -1, respectively. The method was satisfactorily applied to the measurement of total protein in human serum samples and a high-sensitivity was achieved.

  15. Interaction of Moringa oleifera seed protein with a mineral surface and the influence of surfactants.

    PubMed

    Kwaambwa, Habauka M; Hellsing, Maja S; Rennie, Adrian R; Barker, Robert

    2015-06-15

    The paper describes the adsorption of purified protein from seeds of Moringa oleifera to a sapphire interface and the effects of addition of the anionic surfactant sodium dodecylsulfate (SDS) and the cationic surfactant hexadecyltrimethylammonium bromide (CTAB). Neutron reflection was used to determine the structure and composition of interfacial layers adsorbed at the solid/solution interface. The maximum surface excess of protein was found to be about 5.3 mg m(-2). The protein does not desorb from the solid/liquid interface when rinsed with water. Addition of SDS increases the reflectivity indicating co-adsorption. It was observed that CTAB is able to remove the protein from the interface. The distinct differences to the behavior observed previously for the protein at the silica/water interface are identified. The adsorption of the protein to alumina in addition to other surfaces has shown why it is an effective flocculating agent for the range of impurities found in water supplies. The ability to tailor different surface layers in combination with various surfactants also offers the potential for adsorbed protein to be used in separation technologies.

  16. Protein-nanoparticle interactions evaluation by immunomethods: Surfactants can disturb quantitative determinations.

    PubMed

    Fornaguera, Cristina; Calderó, Gabriela; Solans, Conxita; Vauthier, Christine

    2015-08-01

    The adsorption of proteins on nanoparticle surface is one of the first events that occur when nanoparticles enter in the blood stream, which influences nanoparticles lifetime and further biodistribution. Albumin, which is the most abundant protein in serum and which has been deeply characterized, is an interesting model protein to investigate nanoparticle-protein interactions. Therefore, the interaction of nanoparticles with serum albumin has been widely studied. Immunomethods were suggested for the investigation of adsorption isotherms because of their ease to quantify the non-adsorbed bovine serum albumin without the need of applying separation methods that could modify the balance between the adsorbed and non-adsorbed proteins. The present work revealed that this method should be applied with caution. Artifacts in the determination of free protein can be generated by the presence of surfactants such as polysorbate 80, widely used in the pharmaceutical and biomedical field, that are needed to preserve the stability of nanoparticle dispersions. It was shown that the presence of traces of polysorbate 80 in the dispersion leads to an overestimation of the amount of bovine serum albumin remaining free in the dispersion medium when determined by both radial immunodiffusion and rocket immunoelectrophoresis. However, traces of poloxamer 188 did not result in clear perturbed migrations. These methods are not appropriate to perform adsorption isotherms of proteins on nanoparticle dispersions containing traces of remaining free surfactant. They should only be applied on dispersions that are free of surfactant that is not associated with nanoparticles.

  17. Pulmonary surfactant protein A interacts with gel-like regions in monolayers of pulmonary surfactant lipid extract.

    PubMed Central

    Worthman, L A; Nag, K; Rich, N; Ruano, M L; Casals, C; Pérez-Gil, J; Keough, K M

    2000-01-01

    Epifluorescence microscopy was used to investigate the interaction of pulmonary surfactant protein A (SP-A) with spread monolayers of porcine surfactant lipid extract (PSLE) containing 1 mol % fluorescent probe (NBD-PC) spread on a saline subphase (145 mM NaCl, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, or 0.16 microg/ml SP-A and 0, 1.64, or 5 mM CaCl(2). In the absence of SP-A, no differences were noted in PSLE monolayers in the absence or presence of Ca(2+). Circular probe-excluded (dark) domains were observed against a fluorescent background at low surface pressures (pi approximately 5 mN/m) and the domains grew in size with increasing pi. Above 25 mN/m, the domain size decreased with increasing pi. The amount of observable dark phase was maximal at 18% of the total film area at pi approximately 25 mN/m, then decreased to approximately 3% at pi approximately 40 mN/m. The addition of 0.16 microg/ml SP-A with 0 or 1.64 mM Ca(2+) in the subphase caused an aggregation of dark domains into a loose network, and the total amount of dark phase was increased to approximately 25% between pi of 10-28 mN/m. Monolayer features in the presence of 5 mM Ca(2+) and SP-A were not substantially different from those spread in the absence of SP-A, likely due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca(2+). PSLE films were spread on a subphase containing 0.16 microg/ml SP-A with covalently bound Texas Red (TR-SP-A). In the absence of Ca(2+), TR-SP-A associated with the reorganized dark phase (as seen with the lipid probe). The presence of 5 mM Ca(2+) resulted in an appearance of TR-SP-A in the fluid phase and of aggregates at the fluid/gel phase boundaries of the monolayers. This study suggests that SP-A associates with PSLE monolayers, particularly with condensed or solid phase lipid, and results in some reorganization of rigid phase lipid in surfactant monolayers. PMID:11053138

  18. Human Plasma Protein C

    PubMed Central

    Kisiel, Walter

    1979-01-01

    Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (Mr = 62,000) contains 23% carbohydrate and is composed of a light chain (Mr = 21,000) and a heavy chain (Mr = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human α-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity. Images PMID:468991

  19. Crystal Structure of Trimeric Carbohydrate Recognition and Neck Domains of Surfactant Protein A

    SciTech Connect

    Head,J.; Mealy, T.; McCormack, F.; Seaton, B.

    2003-01-01

    Surfactant protein A (SP-A), one of four proteins associated with pulmonary surfactant, binds with high affinity to alveolar phospholipid membranes, positioning the protein at the first line of defense against inhaled pathogens. SP-A exhibits both calcium-dependent carbohydrate binding, a characteristic of the collectin family, and specific interactions with lipid membrane components. The crystal structure of the trimeric carbohydrate recognition domain and neck domain of SP-A was solved to 2.1-{angstrom} resolution with multiwavelength anomalous dispersion phasing from samarium. Two metalbinding sites were identified, one in the highly conserved lectin site and the other 8.5 {angstrom} away. The interdomain carbohydrate recognition domain-neck angle is significantly less in SP-A than in the homologous collectins, surfactant protein D, and mannose-binding protein. This conformational difference may endow the SP-A trimer with a more extensive hydrophobic surface capable of binding lipophilic membrane components. The appearance of this surface suggests a putative binding region for membrane-derived SP-A ligands such as phosphatidylcholine and lipid A, the endotoxic lipid component of bacterial lipopolysaccharide that mediates the potentially lethal effects of Gram-negative bacterial infection.

  20. Pulmonary surfactant mitigates silver nanoparticle toxicity in human alveolar type-I-like epithelial cells.

    PubMed

    Sweeney, Sinbad; Leo, Bey Fen; Chen, Shu; Abraham-Thomas, Nisha; Thorley, Andrew J; Gow, Andrew; Schwander, Stephan; Zhang, Junfeng Jim; Shaffer, Milo S P; Chung, Kian Fan; Ryan, Mary P; Porter, Alexandra E; Tetley, Teresa D

    2016-09-01

    Accompanying increased commercial applications and production of silver nanomaterials is an increased probability of human exposure, with inhalation a key route. Nanomaterials that deposit in the pulmonary alveolar region following inhalation will interact firstly with pulmonary surfactant before they interact with the alveolar epithelium. It is therefore critical to understand the effects of human pulmonary surfactant when evaluating the inhalation toxicity of silver nanoparticles. In this study, we evaluated the toxicity of AgNPs on human alveolar type-I-like epithelial (TT1) cells in the absence and presence of Curosurf(®) (a natural pulmonary surfactant substitute), hypothesising that the pulmonary surfactant would act to modify toxicity. We demonstrated that 20nm citrate-capped AgNPs induce toxicity in human alveolar type I-like epithelial cells and, in agreement with our hypothesis, that pulmonary surfactant acts to mitigate this toxicity, possibly through reducing AgNP dissolution into cytotoxic Ag(+) ions. For example, IL-6 and IL-8 release by TT1 cells significantly increased 10.7- and 35-fold, respectively (P<0.01), 24h after treatment with 25μg/ml AgNPs. In contrast, following pre-incubation of AgNPs with Curosurf(®), this effect was almost completely abolished. We further determined that the mechanism of this toxicity is likely associated with Ag(+) ion release and lysosomal disruption, but not with increased reactive oxygen species generation. This study provides a critical understanding of the toxicity of AgNPs in target human alveolar type-I-like epithelial cells and the role of pulmonary surfactant in mitigating this toxicity. The observations reported have important implications for the manufacture and application of AgNPs, in particular for applications involving use of aerosolised AgNPs.

  1. Implication of antigenic conversion of Helicobacter pylori lipopolysaccharides that involve interaction with surfactant protein D.

    PubMed

    Yokota, Shin-ichi; Amano, Ken-ichi; Nishitani, Chiaki; Ariki, Shigeru; Kuroki, Yoshio; Fujii, Nobuhiro

    2012-08-01

    We propose two antigenic types of Helicobacter pylori lipopolysaccharides (LPS): highly antigenic epitope-carrying LPS (HA-LPS) and weakly antigenic epitope-carrying LPS (WA-LPS) based on human serum reactivity. Strains carrying WA-LPS are highly prevalent in isolates from gastric cancer patients. WA-LPS exhibits more potent biological activities compared to HA-LPS, namely, upregulation of Toll-like receptor 4 (TLR4) expression and induction of enhanced epithelial cell proliferation. The results of competitive binding assays using monosaccharides and methylglycosides, as well as binding assays using glycosidase-treated LPS, suggested that β-linked N-acetyl-D-glucosamine and β-linked D-galactose residues largely contributed to the highly antigenic epitope and the weakly antigenic epitope, respectively. WA-LPS exhibited greater binding activity to surfactant protein D (SP-D) in a Ca(2+)-dependent manner, and this interaction was inhibited by methyl-β-D-galactoside. The biological activities of WA-LPS were markedly enhanced by the addition of SP-D. Lines of evidence suggested that removal of β-N-acetyl-D-glucosamine residue, which comprises the highly antigenic epitope, results in exposure of the weakly antigenic epitope. The weakly antigenic epitope interacted preferentially with SP-D, and SP-D enhanced the biological activity of WA-LPS.

  2. Structure and dynamics of a protein-surfactant assembly studied by ion-mobility mass spectrometry and molecular dynamics simulations.

    PubMed

    Borysik, Antoni J

    2015-09-01

    The structure and dynamics of a protein-surfactant assembly studied by ion-mobility mass spectrometry (IMS) and vacuum molecular dynamics (MD) simulations is reported. Direct evidence is provided for the ability of the surfactant dodecyl-β-D-maltoside (DDM) to prevent charge-induced unfolding of the membrane protein (PagP) in the gas-phase. Restraints obtained by IMS are used to map the surfactant positions onto the protein surface. Surfactants occupying more exposed positions at the apexes of the β-barrel structure are most in-line with the experimental observations. MD simulations provide additional evidence for this assembly organization through surfactant inversion and migration on the protein structure in the absence of solvent. Surfactant migration entails a net shift from apolar membrane spanning regions to more polar regions of the protein structure with the DDM molecule remaining attached to the protein via headgroup interactions. These data provide evidence for the role of protein-DDM headgroup interactions in stabilizing membrane protein structure from gas-phase unfolding.

  3. Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells cultured on plastic.

    PubMed

    Gonzales, L W; Angampalli, S; Guttentag, S H; Beers, M F; Feinstein, S I; Matlapudi, A; Ballard, P L

    2001-01-01

    We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18-22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day) 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively). Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PC) synthesis (2-fold) and higher saturation of PC (approximately 34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.

  4. Latherin: A Surfactant Protein of Horse Sweat and Saliva

    PubMed Central

    Beeley, John G.; Bovell, Douglas L.; Lu, Jian R.; Zhao, Xiubo; Cooper, Alan; Kennedy, Malcolm W.

    2009-01-01

    Horses are unusual in producing protein-rich sweat for thermoregulation, a major component of which is latherin, a highly surface-active, non-glycosylated protein. The amino acid sequence of latherin, determined from cDNA analysis, is highly conserved across four geographically dispersed equid species (horse, zebra, onager, ass), and is similar to a family of proteins only found previously in the oral cavity and associated tissues of mammals. Latherin produces a significant reduction in water surface tension at low concentrations (≤1 mg ml−1), and therefore probably acts as a wetting agent to facilitate evaporative cooling through a waterproofed pelt. Neutron reflection experiments indicate that this detergent-like activity is associated with the formation of a dense protein layer, about 10 Å thick, at the air-water interface. However, biophysical characterization (circular dichroism, differential scanning calorimetry) in solution shows that latherin behaves like a typical globular protein, although with unusual intrinsic fluorescence characteristics, suggesting that significant conformational change or unfolding of the protein is required for assembly of the air-water interfacial layer. RT-PCR screening revealed latherin transcripts in horse skin and salivary gland but in no other tissues. Recombinant latherin produced in bacteria was also found to be the target of IgE antibody from horse-allergic subjects. Equids therefore may have adapted an oral/salivary mucosal protein for two purposes peculiar to their lifestyle, namely their need for rapid and efficient heat dissipation and their specialisation for masticating and processing large quantities of dry food material. PMID:19478940

  5. Surfactant protein-C chromatin-bound green fluorescence protein reporter mice reveal heterogeneity of surfactant protein C-expressing lung cells.

    PubMed

    Lee, Joo-Hyeon; Kim, Jonghwan; Gludish, David; Roach, Rebecca R; Saunders, Arven H; Barrios, Juliana; Woo, Andrew Jonghan; Chen, Huaiyong; Conner, David A; Fujiwara, Yuko; Stripp, Barry R; Kim, Carla F

    2013-03-01

    The regeneration of alveolar epithelial cells is a critical aspect of alveolar reorganization after lung injury. Although alveolar Type II (AT2) cells have been described as progenitor cells for alveolar epithelia, more remains to be understood about how their progenitor cell properties are regulated. A nuclear, chromatin-bound green fluorescence protein reporter (H2B-GFP) was driven from the murine surfactant protein-C (SPC) promoter to generate SPC H2B-GFP transgenic mice. The SPC H2B-GFP allele allowed the FACS-based enrichment and gene expression profiling of AT2 cells. Approximately 97% of AT2 cells were GFP-labeled on Postnatal Day 1, and the percentage of GFP-labeled AT2 cells decreased to approximately 63% at Postnatal Week 8. Isolated young adult SPC H2B-GFP(+) cells displayed proliferation, differentiation, and self-renewal capacity in the presence of lung fibroblasts in a Matrigel-based three-dimensional culture system. Heterogeneity within the GFP(+) population was revealed, because cells with distinct alveolar and bronchiolar gene expression arose in three-dimensional cultures. CD74, a surface marker highly enriched on GFP(+) cells, was identified as a positive selection marker, providing 3-fold enrichment for AT2 cells. In vivo, GFP expression was induced within other epithelial cell types during maturation of the distal lung. The utility of the SPC H2B-GFP murine model for the identification of AT2 cells was greatest in early postnatal lungs and more limited with age, when some discordance between SPC and GFP expression was observed. In adult mice, this allele may allow for the enrichment and future characterization of other SPC-expressing alveolar and bronchiolar cells, including putative stem/progenitor cell populations.

  6. Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2*♦

    PubMed Central

    Kuronuma, Koji; Mitsuzawa, Hiroaki; Takeda, Katsuyuki; Nishitani, Chiaki; Chan, Edward D.; Kuroki, Yoshio; Nakamura, Mari; Voelker, Dennis R.

    2009-01-01

    Lipopolysaccharide (LPS), derived from Gram-negative bacteria, is a major cause of acute lung injury and respiratory distress syndrome. Pulmonary surfactant is secreted as a complex mixture of lipids and proteins onto the alveolar surface of the lung. Surfactant phospholipids are essential in reducing surface tension at the air-liquid interface and preventing alveolar collapse at the end of the respiratory cycle. In the present study, we determined that palmitoyl-oleoyl-phosphatidylglycerol and phosphatidylinositol, which are minor components of pulmonary surfactant, and synthetic dimyristoylphosphatidylglycerol regulated the inflammatory response of alveolar macrophages. The anionic lipids significantly inhibited LPS-induced nitric oxide and tumor necrosis factor-α production from rat and human alveolar macrophages and a U937 cell line by reducing the LPS-elicited phosphorylation of multiple intracellular protein kinases. The anionic lipids were also effective at attenuating inflammation when administered intratracheally to mice challenged with LPS. Binding studies revealed high affinity interactions between the palmitoyl-oleoyl-phosphatidylglycerol and the Toll-like receptor 4-interacting proteins CD14 and MD-2. Our data clearly identify important anti-inflammatory properties of the minor surfactant phospholipids at the environmental interface of the lung. PMID:19584052

  7. Alterations in nanoparticle protein corona by biological surfactants: impact of bile salts on β-lactoglobulin-coated gold nanoparticles.

    PubMed

    Winuprasith, Thunnalin; Chantarak, Sirinya; Suphantharika, Manop; He, Lili; McClements, David Julian

    2014-07-15

    The impact of biological surfactants (bile salts) on the protein (β-lactoglobulin) corona surrounding gold nanoparticles (200 nm) was studied using a variety of analytical techniques at pH 7: dynamic light scattering (DLS); particle electrophoresis (ζ-potential); UV-visible (UV) spectroscopy; transmission electron microscopy (TEM); and surface-enhanced Raman scattering (SERS). The bile salts adsorbed to the protein-coated nanoparticle surfaces and altered their interfacial composition, charge, and structure. SERS spectra of protein-coated nanoparticles after bile salt addition contained bands from both protein and bile salts, indicating that the protein was not fully displaced by the bile salts. UV, DLS and TEM techniques also indicated that the protein coating was not fully displaced from the nanoparticle surfaces. The impact of bile salts could be described by an orogenic mechanism: mixed interfaces were formed that consisted of islands of aggregated proteins surrounded by a sea of bile salts. This knowledge is useful for understanding the interactions of bile salts with protein-coated colloidal particles, which may be important for controlling the fate of colloidal delivery systems in the human gastrointestinal tract, or the gastrointestinal fate of ingested inorganic nanoparticles.

  8. Biophysical mimicry of lung surfactant protein B by random nylon-3 copolymers.

    PubMed

    Dohm, Michelle T; Mowery, Brendan P; Czyzewski, Ann M; Stahl, Shannon S; Gellman, Samuel H; Barron, Annelise E

    2010-06-16

    Non-natural oligomers have recently shown promise as functional analogues of lung surfactant proteins B and C (SP-B and SP-C), two helical and amphiphilic proteins that are critical for normal respiration. The generation of non-natural mimics of SP-B and SP-C has previously been restricted to step-by-step, sequence-specific synthesis, which results in discrete oligomers that are intended to manifest specific structural attributes. Here we present an alternative approach to SP-B mimicry that is based on sequence-random copolymers containing cationic and lipophilic subunits. These materials, members of the nylon-3 family, are prepared by ring-opening polymerization of beta-lactams. The best of the nylon-3 polymers display promising in vitro surfactant activities in a mixed lipid film. Pulsating bubble surfactometry data indicate that films containing the most surface-active polymers attain adsorptive and dynamic-cycling properties that surpass those of discrete peptides intended to mimic SP-B. Attachment of an N-terminal octadecanoyl unit to the nylon-3 copolymers, inspired by the post-translational modifications found in SP-C, affords further improvements by reducing the percent surface area compression to reach low minimum surface tension. Cytotoxic effects of the copolymers are diminished relative to that of an SP-B-derived peptide and a peptoid-based mimic. The current study provides evidence that sequence-random copolymers can mimic the in vitro surface-active behavior of lung surfactant proteins in a mixed lipid film. These findings raise the possibility that random copolymers might be useful for developing a lung surfactant replacement, which is an attractive prospect given that such polymers are easier to prepare than are sequence-specific oligomers.

  9. Factors affecting protein transfer into surfactant-isooctane solution: a case study of extraction behavior of chemically modified cytochrome c.

    PubMed

    Ono, T; Goto, M

    1998-01-01

    The extraction mechanism of proteins by surfactant molecules in an organic solvent has been investigated using a chemically modified protein. We conducted guanidylation on lysine residues of cytochrome c by replacing their amino groups with homoarginine to enhance the protein-surfactant interaction. Results have shown that guanidylated cytochrome c readily forms a hydrophobic complex with dioleyl phosphoric acid (DOLPA) through hydrogen bonding between the phosphate moiety and the guanidinium groups. Although improved protein-surfactant interaction activated the formation of a hydrophobic complex at the interface, it could not improve the protein transfer in isooctane. It has been established that the protein extraction mechanism using surfactant molecules is mainly governed by two processes: formation of an interfacial complex at the oil-water interface and the subsequent solubilization of the complex into the organic phase. In addition, a kinetic study demonstrated that guanidylation of lysine accelerated the initial extraction rate of cytochrome c. This fact implies that the protein transferability from aqueous phase into organic phase depends on the protein-surfactant interaction which can be modified by protein surface engineering.

  10. Effect of silk protein surfactant on silk degumming and its properties.

    PubMed

    Wang, Fei; Cao, Ting-Ting; Zhang, Yu-Qing

    2015-10-01

    The silk protein surfactant (SPS) first used as a silk degumming agent in this study is an amino acid-type anionic surfactant that was synthesized using silk fibroin amino acids and lauroyl chloride. We studied it systematically in comparison with the traditional degumming methods such as sodium carbonate (Na2CO3) and neutral soap (NS). The experimental results showed that the sericin can be completely removed from the silk fibroin fiber after boiling the fibers three times for 30 min and using a bath ratio of 1:80 (g/mL) and a concentration of 0.2% SPS in an aqueous solution. The results of the tensile properties, thermal analysis, and SEM all show that SPS is similar to the NS, far superior to Na2CO3. In short, SPS may be used as an environmentally friendly silk degumming/refining agent in the silk textile industry and in the manufacture of silk floss quilts.

  11. Determination of Lipid-Protein Interactions in Lung Surfactants Using Computer Simulations and Structural Bioinformatics.

    NASA Astrophysics Data System (ADS)

    Kaznessis, Yiannis

    2001-06-01

    Proteins are the primary components of the networks that conduct the flows of mass, energy and information in living organisms. The discovery of the principles of protein structure and function allows the development of design rules for biological activities. The microscopic nature of the operating mechanisms of protein activity, and the vast complexity of the networks of interaction call for the employment of powerful computational methodologies that can decipher the physicochemical and evolutionary principles underlying protein structure and function. An example will be presented that reflects the strength of computational approaches. Atomistic molecular dynamics simulations and structural bioinformatics tools are employed to investigate the interactions between the first 25 N-terminal residues of surfactant protein B (SP-B 1-25) and the lipid components of the lung surfactant (LS). An understanding of the molecular level interactions between the LS components is essential for the establishment of design rules for the development of synthetic LS and the treatment of the neonatal respiratory distress syndrome, which results from deficiency or inactivation of LS.

  12. Surfactant protein D induces immune quiescence and apoptosis of mitogen-activated peripheral blood mononuclear cells.

    PubMed

    Pandit, Hrishikesh; Thakur, Gargi; Koippallil Gopalakrishnan, Aghila Rani; Dodagatta-Marri, Eswari; Patil, Anushree; Kishore, Uday; Madan, Taruna

    2016-02-01

    Surfactant protein D (SP-D) is an integral molecule of the innate immunity secreted by epithelial cells lining the mucosal surfaces. The C-type lectin domain of SP-D performs pattern recognition functions while it binds to putative receptors on immune cells to modify cellular functions. Activation of immune cells and increased serum SP-D is observed in a range of patho-physiological conditions including infections. We speculated if SP-D can modulate systemic immune response via direct interaction with activated PBMCs. In this study, we examined interaction of a recombinant fragment of human SP-D (rhSP-D) on PHA-activated PBMCs. We report a significant downregulation of activation receptors such as TLR2, TLR4, CD11c and CD69 upon rhSP-D treatment. rhSP-D inhibited production of Th1 (TNF-α and IFN-γ) and Th17 (IL-17A) cytokines along with IL-6. Interestingly, levels of IL-2, Th2 (IL-4) and regulatory (IL-10 and TGF-β) cytokines remained unaltered. Analysis of co-stimulatory CD28 and co-inhibitory CTLA4 receptors along with their ligands CD80 and CD86 revealed a selective up-regulation of CTLA4 in the lymphocyte subset. rhSP-D induced apoptosis in the activated but not in non-activated lymphocytes. Blockade of CTLA4 inhibited rhSP-D mediated apoptosis of activated lymphocytes, confirming involvement of CTLA4. We conclude that SP-D restores immune homeostasis. It regulates expression of immunomodulatory receptors and cytokines, which is followed by induction of apoptosis in activated lymphocytes. These findings suggest a critical role of SP-D in immune surveillance against activated immune cells.

  13. Biosurfactants and surfactants interacting with membranes and proteins: Same but different?

    PubMed

    Otzen, Daniel E

    2017-04-01

    Biosurfactants (BS) are surface-active molecules produced by microorganisms. For several decades they have attracted interest as promising alternatives to current petroleum-based surfactants. Aside from their green profile, they have remarkably low critical micelle concentrations, reduce the air/water surface tension to very low levels and are excellent emulsifiers, all of which make them comparable or superior to their synthetic counterparts. These remarkable physical properties derive from their more complex chemical structures in which hydrophilic and hydrophobic regions are not as clearly separated as chemical surfactants but have a more mosaic distribution of polarity as well as branched or circular structures. This allows the lipopeptide surfactin to adopt spherical structures to facilitate dense packing at interfaces. They are also more complex. Glycolipid BS, e.g. rhamnolipids (RL) and sophorolipids, are produced biologically as mixtures which vary in the size and saturation of the hydrophobic region as well as modifications in the hydrophilic headgroup, such as the number of sugar groups and different levels of acetylation, leading to variable surface-active properties. Their amphiphilicity allows RL to insert easily into membranes at sub-cmc concentrations to modulate membrane structure and extract lipopolysaccharides, leading to extensive biofilm remodeling in vivo, sometimes in collaboration with hydrophobic RL precursors. Thanks to their mosaicity, even anionic BS like RL only bind weakly to proteins and show much lower denaturing potency, even supporting membrane protein refolding. Nevertheless, they can promote protein degradation by proteases e.g. by neutralizing positive charges, which together with their biofilm-combating properties makes them very promising detergent surfactants. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

  14. Protein-surfactant interactions at hydrophobic interfaces studied with total internal reflection fluorescence correlation spectroscopy (TIR-FCS).

    PubMed

    Sonesson, Andreas W; Blom, Hans; Hassler, Kai; Elofsson, Ulla M; Callisen, Thomas H; Widengren, Jerker; Brismar, Hjalmar

    2008-01-15

    The aim of this work was to study the dynamics of proteins near solid surfaces in the presence or absence of competing surfactants by means of total internal reflection fluorescence correlation spectroscopy (TIR-FCS). Two different proteins were studied, bovine serum albumin (BSA) and Thermomyces lanuginosus lipase (TLL). A nonionic/anionic (C12E6/LAS) surfactant composition was used to mimic a detergent formulation and the surfaces used were C18 terminated glass. It was found that with increasing surfactant concentrations the term in the autocorrelation function (ACF) representing surface binding decreased. This suggested that the proteins were competed off the hydrophobic surface by the surfactant. When fitting the measured ACF to a model for surface kinetics, it was seen that with raised C12E6/LAS concentration, the surface interaction rate increased for both proteins. Under these experimental conditions this meant that the time the protein was bound to the surface decreased. At 10 microM C12E6/LAS the surface interaction was not visible for BSA, whereas it was still distinguishable in the ACF for TLL. This indicated that TLL had a higher affinity than BSA for the C18 surface. The study showed that TIR-FCS provides a useful tool to quantify the surfactant effect on proteins adsorption.

  15. Surfactant-free purification of membrane proteins with intact native membrane environment.

    PubMed

    Jamshad, Mohammed; Lin, Yu-Pin; Knowles, Timothy J; Parslow, Rosemary A; Harris, Craig; Wheatley, Mark; Poyner, David R; Bill, Roslyn M; Thomas, Owen R T; Overduin, Michael; Dafforn, Tim R

    2011-06-01

    In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another. It is therefore essential that the membrane is disrupted in order to allow separation and hence purification of membrane proteins. In the present review, we examine recent advances in the methods employed to separate membrane proteins before purification. These approaches move away from solubilization methods based on the use of small surfactants, which have been shown to suffer from significant practical problems. Instead, the present review focuses on methods that stem from the field of nanotechnology and use a range of reagents that fragment the membrane into nanometre-scale particles containing the protein complete with the local membrane environment. In particular, we examine a method employing the amphipathic polymer poly(styrene-co-maleic acid), which is able to reversibly encapsulate the membrane protein in a 10 nm disc-like structure ideally suited to purification and further biochemical study.

  16. Hybrid Fluorinated and Hydrogenated Double-Chain Surfactants for Handling Membrane Proteins.

    PubMed

    Legrand, Fréderic; Breyton, Cécile; Guillet, Pierre; Ebel, Christine; Durand, Grégory

    2016-01-15

    Two hybrid fluorinated double-chain surfactants with a diglucosylated polar head were synthesized. The apolar domain consists of a perfluorohexyl main chain and a butyl hydrogenated branch as a side chain. They were found to self-assemble into small micelles at low critical micellar concentrations, demonstrating that the short branch increases the overall hydrophobicity while keeping the length of the apolar domain short. They were both able to keep the membrane protein bacteriorhodopsin stable, one of them for at least 3 months.

  17. Numerical validation of IFT in the analysis of protein-surfactant complexes with SAXS and SANS.

    PubMed

    Franklin, J Matthew; Surampudi, Lalitanand N; Ashbaugh, Henry S; Pozzo, Danilo C

    2012-08-28

    The use of the indirect Fourier transform methods for evaluating structural parameters directly in real space with small-angle scattering measurements is validated for the analysis of protein-surfactant complexes. An efficient Monte Carlo approach rapidly generates in silico structures based on a realistic pearl-necklace model for denatured proteins decorated with surfactant micelles. Corresponding scattering profiles are calculated and averaged over a large number of possible configurations for each structure. IFT algorithms are then used to calculate the corresponding pair-distance distribution function, and structural information is extracted directly without model fitting. The extracted parameters are compared and correlated with the known structure of the simulated complexes to assess the quality of the information that can be reliably obtained from these systems. The average extension, nearest-neighbor micelle distance, and average number of associated micelles are all accurately extracted through IFT calculations. Improved and simple approaches to reliably extract the average extension of the complex and the total number of associated micelles are presented.

  18. Low Levels of Exhaled Surfactant Protein A Associated With BOS After Lung Transplantation

    PubMed Central

    Ericson, Petrea A.; Mirgorodskaya, Ekaterina; Hammar, Oscar S.; Viklund, Emilia A.; Almstrand, Ann-Charlotte R.; Larsson, Per J-W.; Riise, Gerdt C.; Olin, Anna-Carin

    2016-01-01

    Background There is no clinically available marker for early detection or monitoring of chronic rejection in the form of bronchiolitis obliterans syndrome (BOS), the main long-term complication after lung transplantation. Sampling and analysis of particles in exhaled air is a valid, noninvasive method for monitoring surfactant protein A (SP-A) and albumin in the distal airways. Methods We asked whether differences in composition of exhaled particles can be detected when comparing stable lung transplant recipients (LTRs) (n = 26) with LTRs who develop BOS (n = 7). A comparison between LTRs and a matching group of healthy controls (n = 33) was also conducted. Using a system developed in-house, particles were collected from exhaled air by the principal of inertial impaction before chemical analysis by immunoassays. Results Surfactant protein A in exhaled particles and the SP-A/albumin ratio were lower (P = 0.002 and P = 0.0001 respectively) in the BOS group compared to the BOS-free group. LTRs exhaled higher amount of particles (P < 0.0001) and had lower albumin content (P < 0.0001) than healthy controls. Conclusions We conclude that low levels of SP-A in exhaled particles are associated with increased risk of BOS in LTRs. The possibility that this noninvasive method can be used to predict BOS onset deserves further study with prospective and longitudinal approaches. PMID:27795995

  19. Proteomic and Lipidomic Analysis of Nanoparticle Corona upon Contact with Lung Surfactant Reveals Differences in Protein, but Not Lipid Composition.

    PubMed

    Raesch, Simon Sebastian; Tenzer, Stefan; Storck, Wiebke; Rurainski, Alexander; Selzer, Dominik; Ruge, Christian Arnold; Perez-Gil, Jesus; Schaefer, Ulrich Friedrich; Lehr, Claus-Michael

    2015-12-22

    Pulmonary surfactant (PS) constitutes the first line of host defense in the deep lung. Because of its high content of phospholipids and surfactant specific proteins, the interaction of inhaled nanoparticles (NPs) with the pulmonary surfactant layer is likely to form a corona that is different to the one formed in plasma. Here we present a detailed lipidomic and proteomic analysis of NP corona formation using native porcine surfactant as a model. We analyzed the adsorbed biomolecules in the corona of three NP with different surface properties (PEG-, PLGA-, and Lipid-NP) after incubation with native porcine surfactant. Using label-free shotgun analysis for protein and LC-MS for lipid analysis, we quantitatively determined the corona composition. Our results show a conserved lipid composition in the coronas of all investigated NPs regardless of their surface properties, with only hydrophilic PEG-NPs adsorbing fewer lipids in total. In contrast, the analyzed NP displayed a marked difference in the protein corona, consisting of up to 417 different proteins. Among the proteins showing significant differences between the NP coronas, there was a striking prevalence of molecules with a notoriously high lipid and surface binding, such as, e.g., SP-A, SP-D, DMBT1. Our data indicate that the selective adsorption of proteins mediates the relatively similar lipid pattern in the coronas of different NPs. On the basis of our lipidomic and proteomic analysis, we provide a detailed set of quantitative data on the composition of the surfactant corona formed upon NP inhalation, which is unique and markedly different to the plasma corona.

  20. Elevation of serum surfactant protein-A with exacerbation in canine eosinophilic pneumonia

    PubMed Central

    SONE, Katsuhito; AKIYOSHI, Hideo; HAYASHI, Akiyoshi; OHASHI, Fumihito

    2015-01-01

    A 7-year-old female spayed Labrador Retriever was admitted to our hospital, because of cough with sputum. She was diagnosed as having canine eosinophilic pneumonia (CEP) based on blood eosinophilia, bronchial pattern and infiltrative shadow observed on thoracic radiography, bronchiolar obstruction and air-space consolidation predominantly affecting the right caudal lung lobe, as revealed by computed tomography (CT), predominant eosinophils in CT-guided fine needle aspiration and the clinical course. She exhibited a good response to steroid therapy, and the cough disappeared. The serum surfactant protein (SP)-A level increased with the aggravated symptom and decreased markedly with improvement compared with the C-reactive protein level and the number of eosinophils. We propose that serum SP-A level is a good biomarker in CEP. PMID:26300438

  1. Micelle-mediated extraction of elderberry blossom by whey protein and naturally derived surfactants.

    PubMed

    Śliwa, Karolina; Tomaszkiewicz-Potępa, Anna; Sikora, Elżbieta; Ogonowski, Jan

    2013-01-01

    Classical methods of the extraction of active ingredients from the plant material are expensive, complicated and often environmentally unfriendly. The micelle-mediated extraction method (MME) seems to be a good alternative. In this work, extractions of elderberry blossoms (Flos Sambuci) were performed using MME methods. Several popular surfactants and whey protein concentrate (WPC) was applied in the process. The obtained results were compared with those obtained in extraction by means of water. Antioxidant properties of the extracts were analyzed by using two different methods: reaction with di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) reagent and Follin's method. Furthermore, the flavonoid content in the extracts was determined. The results confirmed that the MME method with using whey protein might be an alternative method for obtaining, rich in natural antioxidants, plant extracts.

  2. Small surfactant-like peptides can drive soluble proteins into active aggregates

    PubMed Central

    2012-01-01

    Background Inactive protein inclusion bodies occur commonly in Escherichia coli (E. coli) cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from Clostridium cellulovorans (CBDclos) when expressed in E. coli. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF) and a short beta structure peptide ELK16 (LELELKLKLELELKLK) have a similar property. Results In this work, we explored a third type of peptides, surfactant-like peptides, for performing such a "pulling-down" function. One or more of three such peptides (L6KD, L6K2, DKL6) were fused to the carboxyl termini of model proteins including Aspergillus fumigatus amadoriase II (AMA, all three peptides were used), Bacillus subtilis lipase A (LipA, only L6KD was used, hereinafter the same), Bacillus pumilus xylosidase (XynB), and green fluorescent protein (GFP), and expressed in E. coli. All fusions were found to predominantly accumulate in the insoluble fractions, with specific activities ranging from 25% to 92% of the native counterparts. Transmission electron microscopic (TEM) and confocal fluorescence microscopic analyses confirmed the formation of protein aggregates in the cell. Furthermore, binding assays with amyloid-specific dyes (thioflavin T and Cong red) to the AMA-L6KD aggregate and the TEM analysis of the aggregate following digestion with protease K suggested that the AMA-L6KD aggregate may contain structures reminiscent of amyloids, including a fibril-like structure core. Conclusions This study shows that the surfactant-like peptides L6KD and it derivatives can act as a pull-down handler for converting soluble proteins into active aggregates, much like 18A and ELK16. These peptide-mediated protein aggregations might have important implications for protein aggregation in vivo, and can be

  3. Adsorption of the natural protein surfactant Rsn-2 onto liquid interfaces.

    PubMed

    Brandani, Giovanni B; Vance, Steven J; Schor, Marieke; Cooper, Alan; Kennedy, Malcolm W; Smith, Brian O; MacPhee, Cait E; Cheung, David L

    2017-03-22

    To stabilize foams, droplets and films at liquid interfaces a range of protein biosurfactants have evolved in nature. Compared to synthetic surfactants, these combine surface activity with biocompatibility and low solution aggregation. One recently studied example is Rsn-2, a component of the foam nest of the frog Engystomops pustulosus, which has been predicted to undergo a clamshell-like opening transition at the air-water interface. Using atomistic molecular dynamics simulations and surface tension measurements we study the adsorption of Rsn-2 onto air-water and cyclohexane-water interfaces. The protein adsorbs readily at both interfaces, with adsorption mediated by the hydrophobic N-terminus. At the cyclohexane-water interface the clamshell opens, due to the favourable interaction between hydrophobic residues and cyclohexane molecules and the penetration of cyclohexane molecules into the protein core. Simulations of deletion mutants showed that removal of the N-terminus inhibits interfacial adsorption, which is consistent with the surface tension measurements. Deletion of the hydrophilic C-terminus also affects adsorption, suggesting that this plays a role in orienting the protein at the interface. The characterisation of the interfacial behaviour gives insight into the factors that control the interfacial adsorption of proteins, which may inform new applications of this and similar proteins in areas including drug delivery and food technology and may also be used in the design of synthetic molecules showing similar changes in conformation at interfaces.

  4. Nanoparticles modulate surfactant protein A and D mediated protection against influenza A infection in vitro

    PubMed Central

    McKenzie, Zofi; Kendall, Michaela; Mackay, Rose-Marie; Tetley, Teresa D.; Morgan, Cliff; Griffiths, Mark; Clark, Howard W.; Madsen, Jens

    2015-01-01

    Numerous epidemiological and toxicological studies have indicated that respiratory infections are exacerbated following enhanced exposure to airborne particulates. Surfactant protein A (SP-A) and SP-D form an important part of the innate immune response in the lung and can interact with nanoparticles to modulate the cellular uptake of these particles. We hypothesize that this interaction will also affect the ability of these proteins to combat infections. TT1, A549 and differentiated THP-1 cells, representing the predominant cell types found in the alveolus namely alveolar type I (ATI) epithelial cells, ATII cells and macrophages, were used to examine the effect of two model nanoparticles, 100 nm amine modified (A-PS) and unmodified polystyrene (U-PS), on the ability of SP-A and SP-D to neutralize influenza A infections in vitro. Pre-incubation of low concentrations of U-PS with SP-A resulted in a reduction of SP-A anti-influenza activity in A549 cells, whereas at higher concentrations there was an increase in SP-A antiviral activity. This differential pattern of U-PS concentration on surfactant protein mediated protection against IAV was also shown with SP-D in TT1 cells. On the other hand, low concentrations of A-PS particles resulted in a reduction of SP-A activity in TT1 cells and a reduction in SP-D activity in A549 cells. These results indicate that nanoparticles can modulate the ability of SP-A and SP-D to combat viral challenges. Furthermore, the nanoparticle concentration, surface chemistry and cell type under investigation are important factors in determining the extent of these modulations. PMID:25533100

  5. Surfactant changes during experimental pneumocystosis are related to Pneumocystis development.

    PubMed

    Aliouat, E M; Escamilla, R; Cariven, C; Vieu, C; Mullet, C; Dei-Cas, E; Prévost, M C

    1998-03-01

    Pneumocystosis-related surfactant changes have been reported in both humans and corticosteroid-treated experimental hosts. As corticosteroids induce an increase in pulmonary surfactant, some findings could be considered as controversial. The aim of this study was to investigate whether the surfactant composition changes during experimental pneumocystosis were related to the Pneumocystis development. In this work two corticosteroid-untreated animal models were used: rabbits, which develop spontaneous pneumocystosis at weaning; and severe combined immunodeficiency mice, which were intranasally inoculated with Pneumocystis carinii. Surfactant phospholipid and protein content was explored by bronchoalveolar lavage. The in vitro effect of surfactant on P. carinii growth was also explored. In the two models, the surfactant phospholipid/protein ratio was significantly increased, whereas parasite rates were low. This ratio decreases with the slope increase of the parasite growth curve. These early surfactant changes suggested that Pneumocystis proliferation requires alveolar lining fluid changes, and that normal surfactant is not suitable for parasite development. In this way, in vitro experiments presented here have revealed an inhibitory effect of synthetic or seminatural surfactants on the P. carinii growth. Further studies are needed to determine how Pneumocystis induces the reported early modifications of the surfactant, and why the parasite development is inhibited by pulmonary surfactant.

  6. Utilization of dairy byproduct proteins, surfactants, and enzymes in frozen dough.

    PubMed

    Asghar, Ali; Anjum, Faqir Muhammad; Allen, Jonathan C

    2011-04-01

    Use of natural additives is gaining popularity among the masses as they are becoming more conscious about their diet and health. Frozen dough products are one of the recent examples of value-added cereal products which face stability problems during extended storage periods of times. Dairy whey proteins, surfactants, and certain enzymes are considered important natural additives which could be used to control the water redistribution problem in the dough structure during the storage condition. They interact with the starch and gluten network in a dough system and thus behave as dough improvers and strengtheners. These natural additives not only help to bind extra moisture but also to improve texture and sensory attributes in frozen dough bakery products.

  7. Immunohistochemical investigation of pulmonary surfactant-associated protein A in fire victims.

    PubMed

    Zhu, B L; Ishida, K; Oritani, S; Quan, L; Taniguchi, M; Li, D R; Fujita, M Q; Maeda, H

    2001-03-01

    To evaluate the forensic pathological significance of the immunohistochemical distribution of pulmonary surfactant-associated protein A (SP-A) in determining the cause of death in fires, 57 fire victims were examined by scoring the staining intensity. The highest SP-A score with dense granular deposits (aggregates) in the intra-alveolar space was frequently observed in cases with a lower blood carboxyhemoglobin (COHb) level (<60%). The SP-A score was relatively low in carbon monoxide intoxication due to causes other than fires. High SP-A scores showed a relation to the serum level and in part related to the bloody exudate in the lower airway. These observations suggested that the increase in SP-A in fire victims may be mainly related to pulmonary alveolar injury due to the inhalation of hot air and/or irritant gases rather than hypoxia.

  8. Involvement of eicosanoids and surfactant protein D in extrinsic allergic alveolitis.

    PubMed

    Higashi, A; Higashi, N; Tsuburai, T; Takeuchi, Y; Taniguchi, M; Mita, H; Saito, A; Takatori, K; Arimura, K; Akiyama, K

    2005-12-01

    The pathophysiology of extrinsic allergic alveolitis (EAA) involves oxidative lung damage as well as interstitial and alveolar inflammation. Macrophages and mast cells are inflammatory components of EAA that produce both leukotrienes (LTs) and prostaglandin D2 (PGD2). In addition, PGD2 is also produced by the free-radical-catalysed peroxidation of arachidonic acid during oxidative stress. Urinary 8-iso prostaglandin F2alpha (8-isoPGF2alpha) and serum surfactant protein D (SP-D) are considered appropriate biomarkers of oxidative stress and interstitial lung disease activity, respectively. The present study aimed to assess the association of these biomarkers with the pathophysiology of EAA. Two cases of acute EAA caused by the inhalation of fungi spores were reported. Eight asthmatic patients and six healthy control subjects were also enrolled in the current study. The serum SP-D and urinary eicosanoid (LTE4, PGD2 metabolite (9alpha,11betaPGF2), 8-isoPGF2alpha) concentrations markedly increased during the acute exacerbation phase. These concentrations decreased following corticosteroid therapy in the EAA patients. There was a significant correlation between serum SP-D and urinary 9alpha,11betaPGF2 concentrations in the EAA patients. In conclusion, although the present study proposes that serum surfactant protein-D and urinary eicosanoids are new biomarkers involved in the various immunological responses in extrinsic allergic alveolitis, further large-scale studies are needed to investigate the role of these compounds, not just as biomarkers, but also as biological potentiators of extrinsic allergic alveolitis.

  9. Surfactant Proteins A and D Suppress Alveolar Macrophage Phagocytosis via Interaction with SIRPα

    PubMed Central

    Janssen, William J.; McPhillips, Kathleen A.; Dickinson, Matthew G.; Linderman, Derek J.; Morimoto, Konosuke; Xiao, Yi Qun; Oldham, Kelly M.; Vandivier, R. William; Henson, Peter M.; Gardai, Shyra J.

    2008-01-01

    Rationale: Efficient removal of apoptotic cells is essential for the resolution of acute pulmonary inflammation. Alveolar macrophages ingest apoptotic cells less avidly than other professional phagocytes at rest but overcome this defect during acute inflammation. Surfactant protein (SP)-A and SP-D are potent modulators of macrophage function and may suppress clearance of apoptotic cells through activation of the transmembrane receptor signal inhibitory regulatory protein α (SIRPα). Objectives: To investigate whether binding of SP-A and SP-D to SIRPα on alveolar macrophages suppresses apoptotic cell clearance. Methods: Phagocytosis of apoptotic cells was assessed using macrophages pretreated with SP-A, SP-D, or the collectin-like molecule C1q. Binding of SP-A and SP-D to SIRPα was confirmed in vitro using blocking antibodies and fibroblasts transfected with active and mutant SIRPα. The effects of downstream molecules SHP-1 and RhoA on phagocytosis were studied using SHP-1–deficient mice, sodium stibogluconate, and a Rho kinase inhibitor. Lipopolysaccharide was given to chimeric mice to study the effects of SP-A and SP-D binding on inflammatory macrophages. Measurements and Main Results: Preincubation of macrophages with SP-A or SP-D suppressed apoptotic cell clearance. Surfactant suppression of macrophage phagocytosis was reversed by blocking SIRPα and inhibiting downstream molecules SHP-1 and RhoA. Macrophages from inflamed lungs ingested apoptotic cells more efficiently than resting alveolar macrophages. Recruited mononuclear phagocytes with low levels of SP-A and SP-D mediated this effect. Conclusions: SP-A and SP-D tonically inhibit alveolar macrophage phagocytosis by binding SIRPα. During acute pulmonary inflammation, defects in apoptotic cell clearance are overcome by recruited mononuclear phagocytes. PMID:18420961

  10. A Unique Sugar-binding Site Mediates the Distinct Anti-influenza Activity of Pig Surfactant Protein D*

    PubMed Central

    van Eijk, Martin; Rynkiewicz, Michael J.; White, Mitchell R.; Hartshorn, Kevan L.; Zou, Xueqing; Schulten, Klaus; Luo, Dong; Crouch, Erika C.; Cafarella, Tanya R.; Head, James F.; Haagsman, Henk P.; Seaton, Barbara A.

    2012-01-01

    Pigs can act as intermediate hosts by which reassorted influenza A virus (IAV) strains can be transmitted to humans and cause pandemic influenza outbreaks. The innate host defense component surfactant protein D (SP-D) interacts with glycans on the hemagglutinin of IAV and contributes to protection against IAV infection in mammals. This study shows that a recombinant trimeric neck lectin fragment derived from porcine SP-D (pSP-D) exhibits profound inhibitory activity against IAV, in contrast to comparable fragments derived from human SP-D. Crystallographic analysis of the pSP-D fragment complexed with a viral sugar component shows that a unique tripeptide loop alters the lectin site conformation of pSP-D. Molecular dynamics simulations highlight the role of this flexible loop, which adopts a more stable conformation upon sugar binding and may facilitate binding to viral glycans through contact with distal portions of the branched mannoside. The combined data demonstrate that porcine-specific structural features of SP-D contribute significantly to its distinct anti-IAV activity. These findings could help explain why pigs serve as important reservoirs for newly emerging pathogenic IAV strains. PMID:22685299

  11. Surfactant protein D (SP-D) deficiency is attenuated in humanised mice expressing the Met(11)Thr short nucleotide polymorphism of SP-D: implications for surfactant metabolism in the lung

    PubMed Central

    Knudsen, Lars; Ochs, Katharina; Boxler, Laura; Tornoe, Ida; Lykke-Sorensen, Grith; Mackay, Rose-Marie; Clark, Howard W; Holmskov, Uffe; Ochs, Matthias; Madsen, Jens

    2013-01-01

    Surfactant protein D (SP-D) is part of the innate immune system involved in lung homeostasis. SP-D knockout mice show accumulations of foamy alveolar macrophages, alveolar lipoproteinosis and pulmonary emphysema. Three single nucleotide polymorphisms (SNPs) have been described in the coding sequence of the human SP-D gene SFTPD. Clinical studies showed that the SNP SFTPD with a nucleotide change from A to C resulting in a Met to Thr substitution at position 11 in the protein (Met(11)Thr), is relevant. This study set out to create a humanised mouse model of the Met(11)Thr SNP. Transgenic mice lines expressing either Met(11) or Thr(11) SP-D under the control of the ubiquitously expressed pROSA26 promoter in C57Bl/6 SP-D deficient mice (DKO) was created. Both Met(11) (142 ± 52 ng mL−1) and Thr(11) (228 ± 76 ng mL−1) mice lines expressed human SP-D at almost similar levels. According to the literature this was within the range of SP-D levels found in wildtype (WT) mice (253 ± 22 ng mL−1). Met(11) or Thr(11) SP-D in serum from transgenic mice bound maltose in a calcium-dependent manner, and binding was inhibited in the presence of EDTA or maltose. Bronchoalveolar lavage showed for both transgenic mice lines complementation of the DKO phenotype by restoring cell counts, phospholipid levels and protein content back to WT levels. Cytospins of BAL pellet cells showed a resemblance to WT but both mice lines showed some foamy alveolar macrophages. The stereological analysis showed for none of the mice lines a complete abrogation of emphysematous alterations. However, both Met(11) and Thr(11) mice lines were partially reverted back to a WT phenotype when compared with DKO mice, indicating important effects on surfactant metabolism in vivo. PMID:24111992

  12. Segregated ordered lipid phases and protein-promoted membrane cohesivity are required for pulmonary surfactant films to stabilize and protect the respiratory surface.

    PubMed

    Bernardino de la Serna, Jorge; Vargas, Rodolfo; Picardi, Victoria; Cruz, Antonio; Arranz, Rocío; Valpuesta, José M; Mateu, Leonardo; Pérez-Gil, Jesús

    2013-01-01

    Pulmonary surfactant is a lipid-protein complex essential to stabilize alveoli, by forming surface active films able to reach and sustain very low surface tensions (< 2 mN m(-1)) during the film compression that occurs at end-expiration. The particular lipid composition of surfactant, including a high proportion of dipalmitoylphosphatidylcholine (DPPC), induces segregation of fluid ordered and disordered phases in surfactant membranes and films at physiological temperatures. The segregation of DPPC-enriched ordered phase has been related with the ability of surfactant films to produce very low tensions, while the presence in surfactant of two specific hydrophobic polypeptides, SP-B and SP-C, is absolutely required to facilitate surfactant dynamics, including film formation and re-spreading during expansion at inspiration. In the present study, we have used X-ray scattering to analyze the structure of (1) whole native surfactant membranes purified from porcine lungs, (2) membranes reconstituted from the organic extract of surfactant containing the full lipid complement and the physiological proportion of SP-B and SP-C, and (3) membranes reconstituted from the lipid fraction of surfactant depleted of proteins. Small angle X-ray scattering data from whole surfactant or from membranes reconstituted from surfactant organic extract indicated the co-existence of two lamellar phases with different thicknesses. Such phase coexistence disappeared upon heating of the samples at temperatures above physiological values. When assessed in a captive bubble surfactometer, which mimics interfacial compression-expansion dynamics, the ability of surfactant films to produce very low tensions is only maintained at temperatures permitting the coexistence of the two lamellar phases. On the other hand, membranes reconstituted in the absence of proteins produced diffractograms indicative of the existence of a single dominant lamellar phase at all temperatures. These data suggest that SP

  13. Interaction of surfactant protein A with the intermediate filaments desmin and vimentin.

    PubMed

    Garcia-Verdugo, Ignacio; Synguelakis, Monique; Degrouard, Jeril; Franco, Claudio-Areias; Valot, Benoit; Zivy, Michel; Chaby, Richard; Tanfin, Zahra

    2008-05-06

    Surfactant protein A (SP-A), a member of the collectin family that modulates innate immunity, has recently been involved in the physiology of reproduction. Consistent with the activation of ERK-1/2 and COX-2 induced by SP-A in myometrial cells, we reported previously the presence of two major proteins recognized by SP-A in these cells. Here we identify by mass spectrometry one of these SP-A targets as the intermediate filament (IF) desmin. In myometrial preparations derived from desmin-deficient mice, the absence of binding of SP-A to any 50 kDa protein confirmed the identity of this SP-A-binding site as desmin. Our data based on partial chymotrypsin digestion of pure desmin suggested that SP-A recognizes especially its rod domain, which is known to play an important role during the assembly of desmin into filaments. In line with that, electron microscopy experiments showed that SP-A inhibits in vitro the polymerization of desmin filaments. SP-A also recognized in vitro polymerized filaments in a calcium-dependent manner at a physiological ionic strength but not the C1q receptor gC1qR. Furthermore, Texas Red-labeled SP-A colocalized with desmin filaments in myometrial cells. Interestingly, vimentin, the IF characteristic of leukocytes, is one of the major proteins recognized by SP-A in protein extracts of U937 cells after PMA-induced differentiation of this monocytic cell line. Interaction of SP-A with vimentin was further confirmed using recombinant vimentin in solid-phase binding assays. The ability of SP-A to interact with desmin and vimentin, and to prevent polymerization of desmin monomers, shed light on unexpected and wider biological roles of this collectin.

  14. Effect of surfactants on Ra-sHSPI - A small heat shock protein from the cattle tick Rhipicephalus annulatus

    NASA Astrophysics Data System (ADS)

    Siddiqi, Mohammad Khursheed; Shahein, Yasser E.; Hussein, Nahla; Khan, Rizwan H.

    2016-09-01

    Electrostatic interaction plays an important role in protein aggregation phenomenon. In this study, we have checked the effect of anionic - Sodium Dodecyl Sulfate (SDS) and cationic-Cetyltrimethyl Ammonium Bromide (CTAB) surfactant on aggregation behavior of Ra-sHSPI, a small heat shock protein purified from Rhipicephalus annulatus tick. To monitor the effect of these surfactants, we have employed several spectroscopic methods such as Rayleigh light scattering measurements, ANS (8-Anilinonaphthalene-1-sulfonic acid) fluorescence measurements, ThT (Thioflavin T) binding assays, Far-UV CD (Circular Dichroism) and dynamic light scattering measurements. In the presence of anionic surfactant-SDS, Ra-sHSPI forms amyloid fibrils, in contrast, no amyloid formation was observed in presence of cationic surfactant at low pH. Enhancement of ANS fluorescence intensity confirms the exposition of more hydrophobic patches during aggregation. ThT binding assay confirms the amyloid fibrillar nature of the SDS induced Ra-sHSPI aggregates and supported by PASTA 2.0 (prediction of amyloid structural aggregation) software. This study demonstrates the crucial role of charge during amyloid fibril formation at low pH in Ra-sHSPI.

  15. Clinical value of surfactant protein-A in serum and sputum for pulmonary tuberculosis diagnosis.

    PubMed

    Hu, H; Teng, G L; Gai, L Z; Yang, Y; Zhu, C J

    2013-10-24

    The aim of this study was to explore the diagnostic and differential diagnosis value of surfactant protein-A (SP-A) in the serum and sputum for pulmonary tuberculosis. A total of 101 patients with pulmonary tuberculosis, 85 healthy volunteers, and 30 chronic obstructive pulmonary disease (COPD) patients were divided into pulmonary tuberculosis group, healthy control group, and COPD group, respectively. SP-A was determined in the serum and sputum in the three groups by enzyme-linked immunosorbent assay. The expression of SP-A in serum was significantly higher (P < 0.05) in the pulmonary tuberculosis group than in the healthy control and COPD groups. There were no differences in the SP-A expression in the sputum among the three groups. There was no significant effect of gender, age, tubercle bacillus antibodies, tuberculin purified protein derivative trial, leukocyte count, neutrophilic granulocyte, lymphocyte percentage, or lung cavities on SP-A levels in serum or sputum for the pulmonary tuberculosis group (P > 0.05). The detection of SP-A in serum and sputum was shown to be of great value for the diagnosis and differential diagnosis of pulmonary tuberculosis, and therefore merits further investigation.

  16. Surfactant protein A regulates IgG-mediated phagocytosis in inflammatory neutrophils.

    PubMed

    Wofford, Jessica A; Wright, Jo Rae

    2007-12-01

    Surfactant proteins (SP)-A and SP-D have been shown to affect the functions of a variety of innate immune cells and to interact with various immune proteins such as complement and immunoglobulins. The goal of the current study is to test the hypothesis that SP-A regulates IgG-mediated phagocytosis by neutrophils, which are major effector cells of the innate immune response that remove invading pathogens by phagocytosis and by extracellular killing mediated by reactive oxygen and nitrogen. We have previously shown that SP-A stimulates chemotaxis by inflammatory, but not peripheral, neutrophils. To evaluate the ability of SP-A to modulate IgG-mediated phagocytosis, polystyrene beads were coated with BSA and treated with anti-BSA IgG. SP-A significantly and specifically enhanced IgG-mediated phagocytosis by inflammatory neutrophils, but it had no effect on beads not treated with IgG. SP-A bound to IgG-coated beads and enhanced their uptake via direct interactions with the beads as well as direct interactions with the neutrophils. SP-A did not affect reactive oxygen production or binding of IgG to neutrophils and had modest effects on polymerization of actin. These data suggest that SP-A plays an important role in mediating the phagocytic response of neutrophils to IgG-opsonized particles.

  17. Surfactant Proteins SP-A and SP-D Modulate Uterine Contractile Events in ULTR Myometrial Cell Line

    PubMed Central

    Sotiriadis, Georgios; Dodagatta-Marri, Eswari; Kouser, Lubna; Alhamlan, Fatimah S.; Kishore, Uday; Karteris, Emmanouil

    2015-01-01

    Pulmonary surfactant proteins SP-A and SP-D are pattern recognition innate immune molecules. However, there is extrapulmonary existence, especially in the amniotic fluid and at the feto-maternal interface. There is sufficient evidence to suggest that SP-A and SP-D are involved in the initiation of labour. This is of great importance given that preterm birth is associated with increased mortality and morbidity. In this study, we investigated the effects of recombinant forms of SP-A and SP-D (rhSP-A and rhSP-D, the comprising of trimeric lectin domain) on contractile events in vitro, using a human myometrial cell line (ULTR) as an experimental model. Treatment with rhSP-A or rhSP-D increased the cell velocity, distance travelled and displacement by ULTR cells. rhSP-A and rhSP-D also affected the contractile response of ULTRs when grown on collagen matrices showing reduced surface area. We investigated this effect further by measuring contractility-associated protein (CAP) genes. Treatment with rhSP-A and rhSP-D induced expression of oxytocin receptor (OXTR) and connexin 43 (CX43). In addition, rhSP-A and rhSP-D were able to induce secretion of GROα and IL-8. rhSP-D also induced the expression of IL-6 and IL-6 Ra. We provide evidence that SP-A and SP-D play a key role in modulating events prior to labour by reconditioning the human myometrium and in inducing CAP genes and pro-inflammatory cytokines thus shifting the uterus from a quiescent state to a contractile one. PMID:26641881

  18. Comparison of a phospholipid-based protein-free surfactant and a natural bovine surfactant (SURVANTA) during pressure and volume-controlled ventilation in an improved rabbit fetus model.

    PubMed

    Häfner, D; Kilian, U; Bühler, R; Beume, R; Habel, R

    1993-03-01

    During pressure- or volume-controlled ventilation different surfactant preparations were compared in an improved rabbit fetus model. Based on a self-designed software program, this model enables on-line registration of lung mechanics and heart rate in up to ten fetuses. Using a commercially available bovine lung surfactant (SURVANTA) as standard, we compared animals treated with a protein-free surfactant preparation containing only phospholipids, PL (dipalmitoylphosphatidylcholine:palmitoyloleoylphosphatidylglycerol++ +, DPPC:POPG 70:30) plus palmitic acid (PA) with an untreated ventilated control group. During pressure-controlled ventilation the insufflation pressure (IP) was decreased and increased stepwise with and without positive end-expiratory pressure (PEEP). SURVANTA was significantly more potent than PL plus PA and both differed significantly from the untreated controls. With additional PEEP the differences between SURVANTA and PL+PA disappeared but the differences to the controls were still present. We found that, with additional PEEP, active natural surfactants lead to ECG-irregularities, which indicates that PEEP influences pulmonary and cardiovascular function and compromises the benefits of surfactant therapy. Also during volume-controlled ventilation SURVANTA was superior to PL+PA and the untreated controls. In order to raise the level of activity of pure PL mixtures to that of natural bovine surfactants, we suggest that a surface active protein (probably SP-C) must be added to such mixtures.

  19. Surfactant protein A (SP-A) inhibits agglomeration and macrophage uptake of toxic amine modified nanoparticles

    PubMed Central

    McKenzie, Zofi; Kendall, Michaela; Mackay, Rose-Marie; Whitwell, Harry; Elgy, Christine; Ding, Ping; Mahajan, Sumeet; Morgan, Cliff; Griffiths, Mark; Clark, Howard; Madsen, Jens

    2015-01-01

    Abstract The lung provides the main route for nanomaterial exposure. Surfactant protein A (SP-A) is an important respiratory innate immune molecule with the ability to bind or opsonise pathogens to enhance phagocytic removal from the airways. We hypothesised that SP-A, like surfactant protein D, may interact with inhaled nanoparticulates, and that this interaction will be affected by nanoparticle (NP) surface characteristics. In this study, we characterise the interaction of SP-A with unmodified (U-PS) and amine-modified (A-PS) polystyrene particles of varying size and zeta potential using dynamic light scatter analysis. SP-A associated with both 100 nm U-PS and A-PS in a calcium-independent manner. SP-A induced significant calcium-dependent agglomeration of 100 nm U-PS NPs but resulted in calcium-independent inhibition of A-PS self agglomeration. SP-A enhanced uptake of 100 nm U-PS into macrophage-like RAW264.7 cells in a dose-dependent manner but in contrast inhibited A-PS uptake. Reduced association of A-PS particles in RAW264.7 cells following pre-incubation of SP-A was also observed with coherent anti-Stokes Raman spectroscopy. Consistent with these findings, alveolar macrophages (AMs) from SP-A−/− mice were more efficient at uptake of 100 nm A-PS compared with wild type C57Bl/6 macrophages. No difference in uptake was observed with 500 nm U-PS or A-PS particles. Pre-incubation with SP-A resulted in a significant decrease in uptake of 100 nm A-PS in macrophages isolated from both groups of mice. In contrast, increased uptake by AMs of U-PS was observed after pre-incubation with SP-A. Thus we have demonstrated that SP-A promotes uptake of non-toxic U-PS particles but inhibits the clearance of potentially toxic A-PS particles by blocking uptake into macrophages. PMID:25676620

  20. Protein aggregation with poly(vinyl) alcohol surfactant reduces double emulsion-encapsulated mammalian cell-free expression

    PubMed Central

    Ho, Kenneth K. Y.; Lee, Jin Woo; Durand, Grégory; Majumder, Sagardip

    2017-01-01

    Development of artificial cell models requires encapsulation of biomolecules within membrane-bound compartments. There have been limited studies of using mammalian cell-free expression (CFE) system as the ‘cytosol’ of artificial cells. We exploit glass capillary droplet microfluidics for the encapsulation of mammalian CFE within double emulsion templated vesicles. The complexity of the physicochemical properties of HeLa cell-free lysate poses a challenge compared with encapsulating simple buffer solutions. In particular, we discovered the formation of aggregates in double emulsion templated vesicles encapsulating mammalian HeLa CFE, but not with bacterial CFE. The aggregates did not arise from insolubility of the proteins made from CFE nor due to the interaction of mammalian CFE with the organic solvents in the middle phase of the double emulsions. We found that aggregation is dependent on the concentration of poly(vinyl) alcohol (PVA) surfactant, a critical double emulsion-stabilizing surfactant, and the lysate concentration in mammalian CFE. Despite vesicle instability and reduced protein expression, we demonstrate protein expression by encapsulating mammalian CFE system. Using mass spectrometry and Western blot, we identified and verified that actin is one of the proteins inside the mammalian CFE that aggregated with PVA surfactant. Our work establishes a baseline description of mammalian CFE system encapsulated in double emulsion templated vesicles as a platform for building artificial cells. PMID:28358875

  1. Ranaspumin-2: Structure and Function of a Surfactant Protein from the Foam Nests of a Tropical Frog

    PubMed Central

    Mackenzie, Cameron D.; Smith, Brian O.; Meister, Annette; Blume, Alfred; Zhao, Xiubo; Lu, Jian R.; Kennedy, Malcolm W.; Cooper, Alan

    2009-01-01

    Abstract Ranaspumin-2 (Rsn-2) is a monomeric, 11 kDa surfactant protein identified as one of the major foam nest components of the túngara frog (Engystomops pustulosus), with an amino acid sequence unlike any other protein described so far. We report here on its structure in solution as determined by high-resolution NMR analysis, together with investigations of its conformation and packing at the air-water interface using a combination of infrared and neutron reflectivity techniques. Despite the lack of any significant sequence similarity, Rsn-2 in solution adopts a compact globular fold characteristic of the cystatin family, comprising a single helix over a four-stranded sheet, in a motif not previously associated with surfactant activity. The NMR structure of Rsn-2 shows no obvious amphiphilicity that might be anticipated for a surfactant protein. This suggests that it must undergo a significant conformational change when incorporated into the air-water interface that may involve a hinge-bending, clamshell opening of the separate helix and sheet segments to expose hydrophobic faces to air while maintaining the highly polar surfaces in contact with the underlying water layer. This model is supported by direct observation of the relative orientations of secondary structure elements at the interface by infrared reflection absorption spectroscopy, and by protein packing densities determined from neutron reflectivity profiles. PMID:19527658

  2. [Surfactant protein and thyroid transcription factor 1 in pleuro-pulmonary neoplasia. Immunohistochemical study].

    PubMed

    Dessy, E; Falleni, M; Del Curto, B; Braidotti, P; Pietra, G G

    2000-12-01

    Aim of this work was to investigate the ability of the antibodies against Surfactant proteins (SP) and Thyroid transcription factor 1 (TTF-1) to distinguish primary neoplasms of the lung from metastatic carcinomas to the lung and pleural mesotheliomas. We evaluated the immunohistochemical expression of the antibodies anti SP-A, SP-B, pro SP-C, SP-D, and TTF-1 in a series of 56 primary lung carcinomas, 9 metastatic carcinomas to the lung, 5 pleural mesotheliomas and 8 non-pulmonary carcinomas. Among primary lung neoplasms, only adenocarcinomas immunostained for all SP (specificity = 1; total sensitivity = 0.52). TTF-1 had an excellent specificity (= 1), but a weak sensitivity (= 0.34) in recognizing primary lung carcinomas. TTF-1 was present in lung adenocarcinomas which were negative for SPs; however it failed to distinguish the subtypes. Pleural mesotheliomas, pulmonary metastases and non-pulmonary carcinomas were not immunoreactive for SP-A, SP-B, SP-D, and TTF-1. Pro SP-C was positive also in the adenocarcinomas of the large bowel and in their pulmonary and nodal metastases. These results demonstrate that the combined use of antibodies anti SP-A, SP-B and TTF-1 is the best association in distinguishing primary lung carcinomas from metastatic carcinomas to the lung and pleural mesotheliomas.

  3. Quantification of unadsorbed protein and surfactant emulsifiers in oil-in-water emulsions.

    PubMed

    Berton, Claire; Genot, Claude; Ropers, Marie-Hélène

    2011-02-15

    Unadsorbed emulsifiers affect the physical and chemical behaviour of oil-in-water (O/W) emulsions. A simple methodology to quantify unadsorbed emulsifiers in the aqueous phase of O/W emulsions has been developed. Emulsions were centrifuged and filtered to separate the aqueous phase from the oil droplets and the concentration of unadsorbed emulsifiers in the aqueous phase determined. The quantification of unadsorbed surfactants based on the direct transesterification of their fatty acids was validated for Tween 20, Tween 80, citric acid ester (Citrem), Span 20 and monolauroyl glycerol. To determine unadsorbed proteins, results obtained with Folin-Ciocalteu reagent or UV-spectrophotometry were compared on emulsions stabilized by β-lactoglobulin (BLG), β-casein (BCN) or bovine serum albumin (BSA). The first method gave more accurate results especially during aging of emulsions in oxidative conditions. The whole methodology was applied to emulsions stabilized with single or mixed emulsifiers. This approach enables optimization of emulsion formulations and could be useful to follow changes in the levels of unadsorbed emulsifiers during physical or chemical aging processes.

  4. Identification of the Surfactant Protein A Receptor 210 as the Unconventional Myosin 18A*

    PubMed Central

    Yang, Ching-Hui; Szeliga, Jacek; Jordan, Jeremy; Faske, Shawn; Sever-Chroneos, Zvjezdana; Dorsett, Bre; Christian, Robert E.; Settlage, Robert E.; Shabanowitz, Jeffrey; Hunt, Donald F.; Whitsett, Jeffrey A.; Chroneos, Zissis C.

    2006-01-01

    Mass spectrometric characterization of the surfactant protein A (SP-A) receptor 210 (SP-R210) led to the identification of myosin (Myo) XVIIIA and nonmuscle myosin IIA. Antibodies generated against the unique C-terminal tail of MyoXVIIIA revealed that MyoXVIIIA, MyoIIA, and SP-R210 have overlapping tissue distribution, all being highly expressed in myeloid cells, bone marrow, spleen, lymph nodes, and lung. Western blot analysis of COS-1 cells stably transfected with either MyoXVIIIA or MyoIIA indicated that SP-R210 antibodies recognize MyoXVIIIA. Furthermore, MyoXVIIIA but not MyoIIA localized to the surface of COS-1 cells, and most importantly, expression of MyoXVIIIA in COS-1 cells conferred SP-A binding. Western analysis of recombinant MyoXVIIIA domains expressed in bacteria mapped the epitopes of previously derived SP-R210 antibodies to the neck region of MyoXVIIIA. Antibodies raised against the neck domain of MyoXVIIIA blocked the binding of SP-A to macrophages. Together, these findings indicate that MyoXVIIIA constitutes a novel receptor for SP-A. PMID:16087679

  5. A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting

    PubMed Central

    Wang, Shan-Mei; He, Xian; Li, Nan; Yu, Feng; Hu, Yang; Wang, Liu-Sheng; Zhang, Peng; Du, Yu-Kui; Du, Shan-Shan; Yin, Zhao-Fang; Wei, Ya-Ru; Mulet, Xavier; Coia, Greg; Weng, Dong; He, Jian-Hua; Wu, Min; Li, Hui-Ping

    2015-01-01

    Lung-targeting drugs are thought to be potential therapies of refractory lung diseases by maximizing local drug concentrations in the lung to avoid systemic circulation. However, a major limitation in developing lung-targeted drugs is the acquirement of lung-specific ligands. Pulmonary surfactant protein A (SPA) is predominantly synthesized by type II alveolar epithelial cells, and may serve as a potential lung-targeting ligand. Here, we generated recombinant rat pulmonary SPA (rSPA) as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies) specific for rSPA, designated Nb6 and Nb17. To assess these nanobodies’ potential for lung targeting, we evaluated their specificity to lung tissue and toxicity in mice. Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity. Furthermore, we intravenously injected fluorescein isothiocyanate-Nb17 in nude mice and observed its preferential accumulation in the lung to other tissues, suggesting high affinity of the nanobody for the lung. Studying acute and chronic toxicity of Nb17 revealed its safety in rats without causing apparent histological alterations. Collectively, we have generated and characterized lung-specific nanobodies, which may be applicable for lung drug delivery. PMID:25926731

  6. Alcohol--Induced Polyelectrolyte-Surfactant Complex Coacervate Systems: Characterization and Applications in Enzyme and Protein Extraction

    NASA Astrophysics Data System (ADS)

    Nejati Moshtaghin, Mahboubeh

    The focus of this thesis is to achieve a better understanding of the newly discovered surfactant-polyelectrolyte complex coacervate (SPCC) systems induced by fluoroalcohol/acid as well as short chain aliphatic alcohol; and to elucidate their applications in extraction and enrichment of proteins and enzyme. We have discovered that fluoroalcohols and --acids induce complex coacervation and phase separation in the aqueous mixtures of oppositely charged anionic polyelectrolytes; specifically, sodium salts of polyacrylic acid and polymethacrylic acid and cationic surfactant (cetyltrimethylammonium bromide, CTAB) over a broad range of concentrations of mole fractions of the oppositely charged amphiphiles. Accordingly, these new classes of coacervators will significantly broaden the scope and facilitate engineering of new coacervate phases. Toward these goals, we have inspected the formation of surfactant-polyelectrolyte complex coacervates in the presence of fluoroalcohols namely hexafluoroisopropanol (HFIP) and Trifluoroethanol (TFE). Furthermore, the extent of coacervation as a function of concentrations the system components, and charge ratios of the oppositely charged amphiphiles has been investigated. Polyelectrolytes are considered to be milder reagents, as compared to surfactants, regarding proteins denaturation. This highlights the importance of a detailed investigation of the efficiency of our coacervate systems for extraction and preconcentration of proteins and enzymes, especially, when the biological activity of the extracted proteins needs to be maintained based on the objectives mentioned above, the results of the investigations have been organized in four chapters. In Chapter II, the phase behavior of the FA-SPCC will be investigated. The objective is to examine the phase behavior and phase properties with respect to the extent of coacervation in different solution conditions. In particular, the effects of different solution variables such as concentration

  7. Exploring the affinity binding of alkylmaltoside surfactants to bovine serum albumin and their effect on the protein stability: A spectroscopic approach.

    PubMed

    Hierrezuelo, J M; Carnero Ruiz, C

    2015-08-01

    Steady-state and time-resolved fluorescence together with circular dichroism (CD) spectroscopic studies was performed to examine the interactions between bovine serum albumin (BSA) and two alkylmaltoside surfactants, i.e. n-decyl-β-D-maltoside (β-C10G2) and n-dodecyl-β-D-maltoside (β-C12G2), having identical structures but different tail lengths. Changes in the intrinsic fluorescence of BSA from static as well as dynamic measurements revealed a weak protein-surfactant interaction and gave the corresponding binding curves, suggesting that the binding mechanism of surfactants to protein is essentially cooperative in nature. The behavior of both surfactants is similar, so that the differences detected were attributed to the more hydrophobic nature of β-C12G2, which favors the adsorption of micelle-like aggregates onto the protein surface. These observations were substantially demonstrated by data derived from synchronous, three-dimensional and anisotropy fluorescence experiments. Changes in the secondary structure of the protein induced by the interaction with surfactants were analyzed by CD to determine the contents of α-helix and β-strand. It was noted that whereas the addition of β-C10G2 appears to stabilize the secondary structure of the protein, β-C12G2 causes a marginal denaturation of BSA for a protein:surfactant molar ratio as high as 1 to 100.

  8. Interactions of pulmonary surfactant protein A with phospholipid monolayers change with pH.

    PubMed Central

    Ruano, M L; Nag, K; Casals, C; Pérez-Gil, J; Keough, K M

    1999-01-01

    The interaction of pulmonary surfactant protein A (SP-A) labeled with Texas Red (TR-SP-A) with monolayers containing zwitterionic and acidic phospholipids has been studied at pH 7.4 and 4.5 using epifluorescence microscopy. At pH 7.4, TR-SP-A expanded the pi-A isotherms of film of dipalmitoylphosphatidylcholine (DPPC). It interacted at high concentration at the edges of condensed-expanded phase domains, and distributed evenly at lower concentration into the fluid phase with increasing pressure. At pH 4.5, TR-SP-A expanded DPPC monolayers to a slightly lower extent than at pH 7.4. It interacted primarily at the phase boundaries but it did not distribute into the fluid phase with increasing pressure. Films of DPPC/dipalmitoylphosphatidylglycerol (DPPG) 7:3 mol/mol were somewhat expanded by TR-SP-A at pH 7.4. The protein was distributed in aggregates only at the condensed-expanded phase boundaries at all surface pressures. At pH 4.5 TR-SP-A caused no expansion of the pi-A isotherm of DPPC/DPPG, but its fluorescence was relatively homogeneously distributed throughout the expanded phase at all pressures studied. These observations can be explained by a combination of factors including the preference for SP-A aggregates to enter monolayers at packing dislocations and their disaggregation in the presence of lipid under increasing pressure, together with the influence of pH on the aggregation state of SP-A and the interaction of SP-A with zwitterionic and acidic lipid. PMID:10465757

  9. Conformational Stability of the NH2-Terminal Propeptide of the Precursor of Pulmonary Surfactant Protein SP-B.

    PubMed

    Bañares-Hidalgo, Ángeles; Pérez-Gil, Jesús; Estrada, Pilar

    2016-01-01

    Assembly of pulmonary surfactant lipid-protein complexes depends on conformational changes coupled with proteolytic maturation of proSP-B, the precursor of pulmonary surfactant protein B (SP-B), along the surfactant biogenesis pathway in pneumocytes. Conformational destabilization of the N-terminal propeptide of proSP-B (SP-BN) triggers exposure of the mature SP-B domain for insertion into surfactant lipids. We have studied the conformational stability during GdmCl- or urea-promoted unfolding of SP-BN with trp fluorescence and circular dichroism spectroscopies. Binding of the intermediate states to bis-ANS suggests their molten globule-like character. ΔG0H2O was ~ 12.7 kJ·mol-1 either with urea or GdmCl. None of the thermal transitions of SP-BN detected by CD correspond to protein unfolding. Differential scanning calorimetry of SP-BN evidenced two endothermic peaks involved in oligomer dissociation as confirmed with 2 M urea. Ionic strength was relevant since at 150 mM NaCl, the process originating the endotherm at the highest temperature was irreversible (Tm2 = 108.5°C) with an activation energy of 703.8 kJ·mol-1. At 500 mM NaCl the process became reversible (Tm2 = 114.4°C) and data were fitted to the Non-two States model with two subpeaks. No free thiols in the propeptide could be titrated by DTNB with or without 5.7 M GdmCl, indicating disulfide bonds establishment.

  10. Conformational Stability of the NH2-Terminal Propeptide of the Precursor of Pulmonary Surfactant Protein SP-B

    PubMed Central

    Bañares-Hidalgo, Ángeles; Estrada, Pilar

    2016-01-01

    Assembly of pulmonary surfactant lipid-protein complexes depends on conformational changes coupled with proteolytic maturation of proSP-B, the precursor of pulmonary surfactant protein B (SP-B), along the surfactant biogenesis pathway in pneumocytes. Conformational destabilization of the N-terminal propeptide of proSP-B (SP-BN) triggers exposure of the mature SP-B domain for insertion into surfactant lipids. We have studied the conformational stability during GdmCl- or urea-promoted unfolding of SP-BN with trp fluorescence and circular dichroism spectroscopies. Binding of the intermediate states to bis-ANS suggests their molten globule-like character. ΔG0H2O was ~ 12.7 kJ·mol-1 either with urea or GdmCl. None of the thermal transitions of SP-BN detected by CD correspond to protein unfolding. Differential scanning calorimetry of SP-BN evidenced two endothermic peaks involved in oligomer dissociation as confirmed with 2 M urea. Ionic strength was relevant since at 150 mM NaCl, the process originating the endotherm at the highest temperature was irreversible (Tm2 = 108.5°C) with an activation energy of 703.8 kJ·mol-1. At 500 mM NaCl the process became reversible (Tm2 = 114.4°C) and data were fitted to the Non-two States model with two subpeaks. No free thiols in the propeptide could be titrated by DTNB with or without 5.7 M GdmCl, indicating disulfide bonds establishment. PMID:27380171

  11. Surfactant protein D, Club cell protein 16, Pulmonary and activation-regulated chemokine, C-reactive protein, and Fibrinogen biomarker variation in chronic obstructive lung disease.

    PubMed

    Lock-Johansson, Sofie; Vestbo, Jørgen; Sorensen, Grith Lykke

    2014-11-25

    Chronic obstructive pulmonary disease (COPD) is a multifaceted condition that cannot be fully described by the severity of airway obstruction. The limitations of spirometry and clinical history have prompted researchers to investigate a multitude of surrogate biomarkers of disease for the assessment of patients, prediction of risk, and guidance of treatment. The aim of this review is to provide a comprehensive summary of observations for a selection of recently investigated pulmonary inflammatory biomarkers (Surfactant protein D (SP-D), Club cell protein 16 (CC-16), and Pulmonary and activation-regulated chemokine (PARC/CCL-18)) and systemic inflammatory biomarkers (C-reactive protein (CRP) and fibrinogen) with COPD. The relevance of these biomarkers for COPD is discussed in terms of their biological plausibility, their independent association to disease and hard clinical outcomes, their modification by interventions, and whether changes in clinical outcomes are reflected by changes in the biomarker.

  12. Protein Analysis with Human Hair

    ScienceCinema

    Hart, Brad; Anex, Deon; Parker, Glendon

    2016-09-09

    In an important breakthrough for the forensic science community, researchers have developed the first-ever biological identification method that exploits the information encoded in proteins of human hair.

  13. Protein Analysis with Human Hair

    SciTech Connect

    Hart, Brad; Anex, Deon; Parker, Glendon

    2016-09-07

    In an important breakthrough for the forensic science community, researchers have developed the first-ever biological identification method that exploits the information encoded in proteins of human hair.

  14. Deficiency in pulmonary surfactant proteins in mice with fatty acid binding protein 4-Cre-mediated knockout of the tuberous sclerosis complex 1 gene.

    PubMed

    Xiang, Xinxin; Yuan, Fang; Zhao, Jing; Li, Ziru; Wang, Xian; Guan, Youfei; Tang, Chaoshu; Sun, Guang; Li, Yin; Zhang, Weizhen

    2013-03-01

    Tuberous sclerosis complex 1 (TSC1) forms a heterodimmer with tuberous sclerosis complex 2, to inhibit signalling by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). The mTORC1 stimulates cell growth by promoting anabolic cellular processes, such as gene transcription and protein translation, in response to growth factors and nutrient signals. Originally designed to test the role of TSC1 in adipocyte function, mice in which the gene for TSC1 was specifically deleted by the fatty acid binding protein 4 (FABP4)-Cre (Fabp4-Tsc1cKO mice) died prematurely within 48 h after birth. The Fabp4-Tsc1cKO mouse revealed a much smaller phenotype relative to the wild-type littermates. Maternal administration of rapamycin, a classical mTOR inhibitor, significantly increased the survival time of Fabp4-Tsc1cKO mice for up to 23 days. Both macroscopic and microscopic haemorrhages were observed in the lungs of Fabp4-Tsc1cKO mice, while other tissues showed no significant changes. Levels of surfactant proteins A and B demonstrated a significant decrease in the Fabp4-Tsc1cKO mice, which was rescued by maternal injection of rapamycin. Co-localization of FABP4 or TSC1 with surfactant protein B was also detected in neonatal pulmonary tissues. Our study suggests that TSC1-mTORC1 may be critical for the synthesis of surfactant proteins A and B.

  15. A human surfactant peptide-elastase inhibitor construct as a treatment for emphysema

    PubMed Central

    Guarnieri, Frank; Spencer, Jean L.; Lucey, Edgar C.; Nugent, Matthew A.; Stone, Phillip J.

    2010-01-01

    Two million Americans suffer from pulmonary emphysema, costing $2.5 billion/year and contributing to 100,000 deaths/year. Emphysema is thought to result from an imbalance between elastase and endogenous inhibitors of elastase, leading to tissue destruction and a loss of alveoli. Decades of research have still not resulted in an effective treatment other than stopping cigarette smoking, a highly addictive behavior. On the basis of our previous work, we hypothesize that small molecule inhibitors of human neutrophil elastase are ineffective because of rapid clearance from the lungs. To develop a long-acting elastase inhibitor with a lung pharmacodynamic profile that has minimal immunogenicity, we covalently linked an elastase inhibitor, similar to a trifluoro inhibitor that was used in clinical trials, to a 25-amino-acid fragment of human surfactant peptide B. We used this construct to prevent human neutrophil elastase-induced emphysema in a rodent model. The elastase inhibitor alone, although in a 70-fold molar excess to elastase in a mixture with <0.6% residual elastase activity, provided no protection from elastase-induced emphysema. Covalently combining an endogenous peptide from the target organ with a synthetic small molecule inhibitor is a unique way of endowing an active compound with the pharmacodynamic profile needed to create in vivo efficacy. PMID:20534582

  16. Weak and Saturable Protein-Surfactant Interactions in the Denaturation of Apo-α-Lactalbumin by Acidic and Lactonic Sophorolipid.

    PubMed

    Andersen, Kell K; Vad, Brian S; Roelants, Sophie; van Bogaert, Inge N A; Otzen, Daniel E

    2016-01-01

    Biosurfactants are of growing interest as sustainable alternatives to fossil-fuel-derived chemical surfactants, particularly for the detergent industry. To realize this potential, it is necessary to understand how they affect proteins which they may encounter in their applications. However, knowledge of such interactions is limited. Here, we present a study of the interactions between the model protein apo-α-lactalbumin (apo-aLA) and the biosurfactant sophorolipid (SL) produced by the yeast Starmerella bombicola. SL occurs both as an acidic and a lactonic form; the lactonic form (lactSL) is sparingly soluble and has a lower critical micelle concentration (cmc) than the acidic form [non-acetylated acidic sophorolipid (acidSL)]. We show that acidSL affects apo-aLA in a similar way to the related glycolipid biosurfactant rhamnolipid (RL), with the important difference that RL is also active below the cmc in contrast to acidSL. Using isothermal titration calorimetry data, we show that acidSL has weak and saturable interactions with apo-aLA at low concentrations; due to the relatively low cmc of acidSL (which means that the monomer concentration is limited to ca. 0-1 mM SL), it is only possible to observe interactions with monomeric acidSL at high apo-aLA concentrations. However, the denaturation kinetics of apo-aLA in the presence of acidSL are consistent with a collaboration between monomeric and micellar surfactant species, similar to RL and non-ionic or zwitterionic surfactants. Inclusion of diacetylated lactonic sophorolipid (lactSL) as mixed micelles with acidSL lowers the cmc and this effectively reduces the rate of unfolding, emphasizing that SL like other biosurfactants is a gentle anionic surfactant. Our data highlight the potential of these biosurfactants for future use in the detergent and pharmaceutical industry.

  17. In vitro analysis of the effect of alkyl-chain length of anionic surfactants on the skin by using a reconstructed human epidermal model.

    PubMed

    Yamaguchi, Fumiko; Watanabe, Shin-Ichi; Harada, Fusae; Miyake, Miyuki; Yoshida, Masaki; Okano, Tomomichi

    2014-01-01

    We investigated the effect of the alkyl-chain length of anionic surfactants on the skin using an in vitro model. The evaluated anionic surfactants were sodium alkyl sulfate (AS) and sodium fatty acid methyl ester sulfonate (MES), which had different alkyl-chain lengths (C8-C14). Skin tissue damage and permeability were examined using a reconstructed human epidermal model, LabCyte EPI-MODEL24. Skin tissue damage was examined by measuring cytotoxicity with an MTT assay. Liquid chromatography/tandem mass spectrometry (LC/MS-MS) and liquid chromatography/mass spectrometry (LC/MS) were used to detect surfactants that permeated into the assay medium through an epidermal model. To assess the permeation mechanism and cell damage caused by the surfactants through the epidermis, we evaluated the structural changes of Bovine Serum Albumin (BSA), used as a simple model protein, and the fluidity of 1,2-dipalmitoyl-sn-glycero-3-phosphpcholine (DPPC) liposome, which serves as one of the most abundant phospholipid models of living cell membranes in the epidermis. The effects of the surfactants on the proteins were measured using Circular Dichroism (CD) spectroscopy, while the effects on membrane fluidity were investigated by electron spin resonance (ESR) spectroscopy. ET50 (the 50% median effective time) increased as follows: C10 < C12 < C8 < C14 in AS and C8, C10 < C12 < C14 in MES. The order of permeation through the LabCyte EPI-MODEL24 was C10 > C12 > C14, for both AS and MES. For both AS and MES, the order parameter, which is the criteria for the microscopic viscosity of lipid bilayers, increased as follows: C10 < C12 < C14, which means the membrane fluidity is C10 > C12 > C14. It was determined that the difference in skin tissue damage in the LabCyte EPI-MODEL24 with C10 to C14 AS and MES was caused by the difference in permeation and cell membrane fluidity through the lipid bilayer path in the epidermis.

  18. Characterization of the Ca2+-dependent binding of annexin IV to surfactant protein A.

    PubMed Central

    Sohma, H; Creutz, C E; Saitoh, M; Sano, H; Kuroki, Y; Voelker, D R; Akino, T

    1999-01-01

    We have shown previously that surfactant protein A (SP-A) binds to annexin IV in a Ca2+-dependent manner [Sohma, Matsushima, Watanabe, Hattori, Kuroki and Akino (1995) Biochem. J. 312, 175-181]. Annexin IV is a member of the annexin family having four consensus repeats of about 70 amino acids and a unique N-terminal tail. In the present study, the functional site of both annexin IV and SP-A for the Ca2+-dependent binding was investigated using mutant proteins. SP-A bound in a Ca2+-dependent manner to an annexin-IV truncation mutant consisting of the N-terminal domain and the first three domains (T(N-1-2-3)). SP-A also bound to T3-4, but this interaction was not Ca2+-dependent. SP-A bound weakly to the other truncation mutants (T(N-1-2), T(2-3) and T(2-3-4)). Each consensus repeat of annexin IV possesses a conserved acidic amino acid residue (Glu70, Asp142, Glu226 and Asp301) that putatively ligates Ca2+. Using annexin-IV DE mutants in which one, two or three residues out of the four Asp/Glu were altered to Ala by site-directed mutagenesis [Nelson and Creutz (1995) Biochemistry 34, 3121-3132], it was revealed that Ca2+ binding in the third domain is more important than in the other Ca2+-binding sites. SP-A is a member of the animal lectin group homologous with mannose-binding protein A. The substitution of Arg197 of rat SP-A with Asp or Asn eliminated binding to annexin IV, whereas the substitution of Glu195 with Gln was silent. These results suggest that the Ca2+ binding to domain 3 of annexin IV is required for the Ca2+-dependent binding by SP-A and that Arg197 of SP-A is important in this binding. PMID:10377263

  19. Surfactant protein A genetic variants associate with severe respiratory insufficiency in pandemic influenza A virus infection

    PubMed Central

    2014-01-01

    Introduction Inherited variability in host immune responses influences susceptibility and outcome of Influenza A virus (IAV) infection, but these factors remain largely unknown. Components of the innate immune response may be crucial in the first days of the infection. The collectins surfactant protein (SP)-A1, -A2, and -D and mannose-binding lectin (MBL) neutralize IAV infectivity, although only SP-A2 can establish an efficient neutralization of poorly glycosylated pandemic IAV strains. Methods We studied the role of polymorphic variants at the genes of MBL (MBL2), SP-A1 (SFTPA1), SP-A2 (SFTPA2), and SP-D (SFTPD) in 93 patients with H1N1 pandemic 2009 (H1N1pdm) infection. Results Multivariate analysis showed that two frequent SFTPA2 missense alleles (rs1965708-C and rs1059046-A) and the SFTPA2 haplotype 1A0 were associated with a need for mechanical ventilation, acute respiratory failure, and acute respiratory distress syndrome. The SFTPA2 haplotype 1A1 was a protective variant. Kaplan-Meier analysis and Cox regression also showed that diplotypes not containing the 1A1 haplotype were associated with a significantly shorter time to ICU admission in hospitalized patients. In addition, rs1965708-C (P = 0.0007), rs1059046-A (P = 0.0007), and haplotype 1A0 (P = 0.0004) were associated, in a dose-dependent fashion, with lower PaO2/FiO2 ratio, whereas haplotype 1A1 was associated with a higher PaO2/FiO2 ratio (P = 0.001). Conclusions Our data suggest an effect of genetic variants of SFTPA2 on the severity of H1N1pdm infection and could pave the way for a potential treatment with haplotype-specific (1A1) SP-A2 for future IAV pandemics. PMID:24950659

  20. Human MSH2 protein

    DOEpatents

    de la Chapelle, Albert; Vogelstein, Bert; Kinzler, Kenneth W.

    1997-01-01

    The human MSH2 gene, responsible for hereditary non-polyposis colorectal cancer, was identified by virtue of its homology to the MutS class of genes, which are involved in DNA mismatch repair. The sequence of cDNA clones of the human gene are provided, and the sequence of the gene can be used to demonstrate the existence of germ line mutations in hereditary non-polyposis colorectal cancer (HNPCC) kindreds, as well as in replication error.sup.+ (RER.sup.+) tumor cells.

  1. Human MSH2 protein

    DOEpatents

    Chapelle, A. de la; Vogelstein, B.; Kinzler, K.W.

    1997-01-07

    The human MSH2 gene, responsible for hereditary non-polyposis colorectal cancer, was identified by virtue of its homology to the MutS class of genes, which are involved in DNA mismatch repair. The sequence of cDNA clones of the human gene are provided, and the sequence of the gene can be used to demonstrate the existence of germ line mutations in hereditary non-polyposis colorectal cancer (HNPCC) kindreds, as well as in replication error{sup +} (RER{sup +}) tumor cells. 19 figs.

  2. Integrin-dependent interaction of human vascular endothelial cells on biomimetic peptide surfactant polymers.

    PubMed

    Murugesan, Gurunathan; Ruegsegger, Mark A; Kligman, Faina; Marchant, Roger E; Kottke-Marchant, Kandice

    2002-01-01

    Biomimetic surfactant polymers designed by molecular grafting of pendant RGD peptides (Pep) and dextran oligosaccharides (Dex) in different ratios onto the backbone of poly(vinyl amine) (PVAm) were examined for their ability to promote endothelial cell (EC) growth. Adhesion, formation of focal contacts, and expression of integrin receptors were examined in EC seeded onto a series of novel surfactants containing 100% dextran (PVAm[Pep (0%)]) to 100% peptide (PVAm[Pep (100%)]) compared to fibronectin control. Interaction of EC on polymer was specific, as soluble GRGDSP, but not GRGESP, was able to inhibit both adhesion and spreading of EC. At three hours, EC attachment and spreading were rapid and comparable on fibronectin and PVAm[Pep (100%)], rounded on PVAm[Pep (0%)], and intermediate on PVAm[Pep (25%)], (PVAm[Pep (50%)], and PVAm[Pep (75%)], with increasing peptide ratio favoring more spreading, although all the substrates had similar hydrophilicity. Cells that spread well on fibronectin and PVAm[Pep (100%)] had sharp spikes of vinculin localized at the termination point of actin stress fibers. Formation of stress fibers and focal adhesions on other substrates were correlated with spreading pattern of EC and the peptide content. EC seeded on fibronectin expressed alpha5beta1 integrins all along the stress fibers and throughout the entire cytoskeleton, but this distribution pattern was less prominent on PVAm[Pep (100%)]. However, expression and distribution of vitronectin receptors (alpha(v)beta3) were similar on both fibronectin and PVAm[Pep (100%)], suggesting a strong cell adhesion on PVAm[Pep (100%)]. Viability of EC was also comparable on both fibronectin and PVAm[Pep (100%)] at 24 h. Substrates with high proportion of dextran limited cell adhesion, probably by decreasing protein adsorption. These results suggest that it may be possible to engineer substrates that promote cell adhesion in a receptor-dependent manner while blocking nonspecific protein

  3. Protein separation with surfactant-coated polystyrene involving Cibacron Blue 3GA-conjugated triton X-100.

    PubMed

    Saitoh, Tohru; Hattori, Naoto; Hiraide, Masataka

    2004-02-27

    Through mixing of porous polystyrene particles (Amberlite XAD-4), non-ionic surfactants, and surfactant-conjugated substrates (affinity ligand) in an aqueous solution led to the formation of a novel medium (affinity admicelle) for protein separation. The ligand (CB-Triton) was synthesized by mixing a triazine dye (Cibacron Blue 3GA (CB)) and a polyoxyethylene-type non-ionic surfactant (Triton X-100) in weakly alkaline solutions. Triton X-100 and CB-Triton were competitively sorbed onto XAD-4. Albumin (bovine serum), alcohol dehydrogenase (yeast), and lysozyme (chicken egg) having specific interaction to CB were collected onto the affinity admicelle. On the other hand, the collection of ovalubmin (chicken egg white), having no binding ability to CB, was negligibly small. Lysozyme in 100 microl of chicken egg white, diluted with 900 microl of 10 mM Tris-HCl (pH 7.4), was successfully collected on 18 mg of CB-Triton admicelles and, then, it was eluted with 1 ml of aqueous solution of 100 mM phosphate (pH 7.4). The recovery based on the activity for the lysis of micrococcus and the concentration factor were 60% and 40 (n = 3), respectively.

  4. Acidic pH triggers conformational changes at the NH2-terminal propeptide of the precursor of pulmonary surfactant protein B to form a coiled coil structure.

    PubMed

    Bañares-Hidalgo, A; Pérez-Gil, J; Estrada, P

    2014-07-01

    Pulmonary surfactant protein SP-B is synthesized as a larger precursor, proSP-B. We report that a recombinant form of human SP-BN forms a coiled coil structure at acidic pH. The protonation of a residue with pK=4.8±0.06 is the responsible of conformational changes detected by circular dichroism and intrinsic fluorescence emission. Sedimentation velocity analysis showed protein oligomerisation at any pH condition, with an enrichment of the species compatible with a tetramer at acidic pH. Low 2,2,2,-trifluoroethanol concentration promoted β-sheet structures in SP-BN, which bind Thioflavin T, at acidic pH, whereas it promoted coiled coil structures at neutral pH. The amino acid stretch predicted to form β-sheet parallel association in SP-BN overlaps with the sequence predicted by several programs to form coiled coil structure. A synthetic peptide ((60)W-E(85)) designed from the sequence of the amino acid stretch of SP-BN predicted to form coiled coil structure showed random coil conformation at neutral pH but concentration-dependent helical structure at acidic pH. Sedimentation velocity analysis of the peptide indicated monomeric state at neutral pH (s20, w=0.55S; Mr~3kDa) and peptide association (s20, w=1.735S; Mr=~14kDa) at acidic pH, with sedimentation equilibrium fitting to a Monomer-Nmer-Mmer model with N=6 and M=4 (Mr=14692Da). We propose that protein oligomerisation through coiled-coil motifs could then be a general feature in the assembly of functional units in saposin-like proteins in general and in the organization of SP-B in a functional surfactant, in particular.

  5. Enhanced removal of a human norovirus surrogate from fresh vegetables and fruits by a combination of surfactants and sanitizers.

    PubMed

    Predmore, Ashley; Li, Jianrong

    2011-07-01

    Fruits and vegetables are major vehicles for transmission of food-borne enteric viruses since they are easily contaminated at pre- and postharvest stages and they undergo little or no processing. However, commonly used sanitizers are relatively ineffective for removing human norovirus surrogates from fresh produce. In this study, we systematically evaluated the effectiveness of surfactants on removal of a human norovirus surrogate, murine norovirus 1 (MNV-1), from fresh produce. We showed that a panel of surfactants, including sodium dodecyl sulfate (SDS), Nonidet P-40 (NP-40), Triton X-100, and polysorbates, significantly enhanced the removal of viruses from fresh fruits and vegetables. While tap water alone and chlorine solution (200 ppm) gave only <1.2-log reductions in virus titer in all fresh produce, a solution containing 50 ppm of surfactant was able to achieve a 3-log reduction in virus titer in strawberries and an approximately 2-log reduction in virus titer in lettuce, cabbage, and raspberries. Moreover, a reduction of approximately 3 logs was observed in all the tested fresh produce after sanitization with a solution containing a combination of 50 ppm of each surfactant and 200 ppm of chlorine. Taken together, our results demonstrate that the combination of a surfactant with a commonly used sanitizer enhanced the efficiency in removing viruses from fresh produce by approximately 100 times. Since SDS is an FDA-approved food additive and polysorbates are recognized by the FDA as GRAS (generally recognized as safe) products, implementation of this novel sanitization strategy would be a feasible approach for efficient reduction of the virus load in fresh produce.

  6. Enhanced Removal of a Human Norovirus Surrogate from Fresh Vegetables and Fruits by a Combination of Surfactants and Sanitizers▿

    PubMed Central

    Predmore, Ashley; Li, Jianrong

    2011-01-01

    Fruits and vegetables are major vehicles for transmission of food-borne enteric viruses since they are easily contaminated at pre- and postharvest stages and they undergo little or no processing. However, commonly used sanitizers are relatively ineffective for removing human norovirus surrogates from fresh produce. In this study, we systematically evaluated the effectiveness of surfactants on removal of a human norovirus surrogate, murine norovirus 1 (MNV-1), from fresh produce. We showed that a panel of surfactants, including sodium dodecyl sulfate (SDS), Nonidet P-40 (NP-40), Triton X-100, and polysorbates, significantly enhanced the removal of viruses from fresh fruits and vegetables. While tap water alone and chlorine solution (200 ppm) gave only <1.2-log reductions in virus titer in all fresh produce, a solution containing 50 ppm of surfactant was able to achieve a 3-log reduction in virus titer in strawberries and an approximately 2-log reduction in virus titer in lettuce, cabbage, and raspberries. Moreover, a reduction of approximately 3 logs was observed in all the tested fresh produce after sanitization with a solution containing a combination of 50 ppm of each surfactant and 200 ppm of chlorine. Taken together, our results demonstrate that the combination of a surfactant with a commonly used sanitizer enhanced the efficiency in removing viruses from fresh produce by approximately 100 times. Since SDS is an FDA-approved food additive and polysorbates are recognized by the FDA as GRAS (generally recognized as safe) products, implementation of this novel sanitization strategy would be a feasible approach for efficient reduction of the virus load in fresh produce. PMID:21622782

  7. Inherited surfactant deficiency due to uniparental disomy of rare mutations in the surfactant protein-B and ATP binding cassette, subfamily A, member 3 genes

    PubMed Central

    Hamvas, Aaron; Nogee, Lawrence M.; Wegner, Daniel J.; DePass, Kelcey; Christodoulou, John; Bennetts, Bruce; McQuade, Leon R.; Gray, Peter H.; Deterding, Robin R.; Carroll, Travis R.; Kammesheidt, Anja; Kasch, Laura M.; Kulkarni, Shashikant; Cole, F. Sessions

    2009-01-01

    Objective To characterize inheritance of homozygous, rare, recessive loss-of-function mutations in the surfactant protein-B (SFTPB) or ATP binding cassette, subfamily A, member 3 (ABCA3) genes in newborns with lethal respiratory failure. Study design We resequenced parents whose infants were homozygous for mutations in SFTPB or ABCA3. For infants with only one heterozygous parent, we performed microsatellite analysis for chromosomes 2 (SFTPB) and 16 (ABCA3). Results We identified one infant homozygous for the c.1549C>GAA mutation (121ins2) in SFTPB for whom only the mother was heterozygous and 3 infants homozygous for mutations in ABCA3 (p.K914R, p.P147L, and c.806_7insGCT) for whom only the fathers were heterozygous. For the SP-B deficient infant, microsatellite markers confirmed maternal heterodisomy with segmental isodisomy. Microsatellite analysis confirmed paternal isodisomy for the three ABCA3 deficient infants. Two ABCA3 deficient infants underwent lung transplantation at 3 and 5 months of age, respectively, and two infants died. None exhibited any non-pulmonary phenotype. Conclusions Uniparental disomy should be suspected in infants with rare homozygous mutations in SFTPB or ABCA3. Confirmation of parental carrier status is important to provide recurrence risk and to monitor expression of other phenotypes that may emerge through reduction to homozygosity of recessive alleles. PMID:19647838

  8. Unique crystal structure of a novel surfactant protein from the foam nest of the frog Leptodactylus vastus.

    PubMed

    Cavalcante Hissa, Denise; Arruda Bezerra, Gustavo; Birner-Gruenberger, Ruth; Paulino Silva, Luciano; Usón, Isabel; Gruber, Karl; Maciel Melo, Vânia Maria

    2014-02-10

    Breeding by releasing eggs into stable biofoams ("foam nests") is a peculiar reproduction mode within anurans, fish, and tunicates; not much is known regarding the biochemistry or molecular mechanisms involved. Lv-ranaspumin (Lv-RSN-1) is the predominant protein from the foam nest of the frog Leptodactylus vastus. This protein shows natural surfactant activity, which is assumed to be crucial for stabilizing foam nests. We elucidated the amino acid sequence of Lv-RSN-1 by de novo sequencing with mass-spectrometry and determined the high-resolution X-ray structure of the protein. It has a unique fold mainly composed of a bundle of 11 α-helices and two small antiparallel β-strands. Lv-RSN-1 has a surface rich in hydrophilic residues and a lipophilic cavity in the region of the antiparallel β-sheet. It possesses intrinsic surface-active properties, reducing the surface tension of water from 73 to 61 mN m(-1) (15 μg mL(-1)). Lv-RSN-1 belongs to a new class of surfactants proteins for which little has been reported regarding structure or function.

  9. Engineering Halomonas spp. as A Low-Cost Production Host for Production of Bio-surfactant Protein PhaP.

    PubMed

    Lan, Lu-Hong; Zhao, Han; Chen, Jin-Chun; Chen, Guo-Qiang

    2016-12-01

    Halomonas spp. have been studied as a low cost production host for producing bulk materials such as polyhydroxyalkanoates (PHA) bioplastics, since they are able to grow at high pH and high NaCl concentration under unsterile and continuous conditions without microbial contamination. In this paper, Halomonas strain TD is used as a host to produce a protein named PHA phasin or PhaP which has a potential to be developed into a bio-surfactant. Four Halomonas TD expression strains are constructed based on a strong T7-family expression system. Of these, the strain with phaC deletion and chromosomal expression system resulted in the highest production of PhaP in soluble form, reaching 19% of total cellular soluble proteins and with a yield of 1.86 g/L in an open fed-batch fermentation process. A simple "heat lysis and salt precipitation" method is applied to allow rapid PhaP purification from a mixture of cellular proteins with a PhaP recovery rate of 63%. It clearly demonstrated that Halomonas TD could be used for high yield expression of a bio-surfactant protein PhaP for industrial application in an economical way.

  10. Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

    PubMed

    Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

    2014-01-01

    G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration.

  11. Pulmonary Surfactant Phosphatidylglycerol Inhibits Mycoplasma pneumoniae-stimulated Eicosanoid Production from Human and Mouse Macrophages*

    PubMed Central

    Kandasamy, Pitchaimani; Zarini, Simona; Chan, Edward D.; Leslie, Christina C.; Murphy, Robert C.; Voelker, Dennis R.

    2011-01-01

    Mycoplasma pneumoniae is a human pathogen causing respiratory infections that are also associated with serious exacerbations of chronic lung diseases. Membranes and lipoproteins from M. pneumoniae induced a 4-fold increase in arachidonic acid (AA) release from RAW264.7 and a 2-fold increase in AA release from primary human alveolar macrophages. The bacterial lipoprotein mimic and TLR2/1 agonist Pam3Cys and the TLR2/6 agonist MALP-2 produced effects similar to those elicited by M. pneumoniae in macrophages by inducing the phosphorylation of p38MAPK and p44/42ERK1/2 MAP kinases and cyclooxygenase-2 (COX-2) expression. M. pneumoniae induced the generation of prostaglandins PGD2 and PGE2 from RAW264.7 cells and thromboxane B2 (TXB2) from human alveolar macrophages. Anti-TLR2 antibody completely abolished M. pneumoniae-induced AA release and TNFα secretion from RAW264.7 cells and human alveolar macrophages. Disruption of the phosphorylation of p44/42ERK1/2 or inactivation of cytosolic phospholipase A2α (cPLA2α) completely inhibited M. pneumoniae-induced AA release from macrophages. The minor pulmonary surfactant phospholipid, palmitoyl-oleoyl-phosphatidylglycerol (POPG), antagonized the proinflammatory actions of M. pneumoniae, Pam3Cys, and MALP-2 by reducing the production of AA metabolites from macrophages. The effect of POPG was specific, insofar as saturated PG, and saturated and unsaturated phosphatidylcholines did not have significant effect on M. pneumoniae-induced AA release. Collectively, these data demonstrate that M. pneumoniae stimulates the production of eicosanoids from macrophages through TLR2, and POPG suppresses this pathogen-induced response. PMID:21205826

  12. Crystallization and preliminary X-ray diffraction of the surfactant protein Lv-ranaspumin from the frog Leptodactylus vastus.

    PubMed

    Hissa, Denise Cavalcante; Bezerra, Gustavo Arruda; Obrist, Britta; Birner-Grünberger, Ruth; Melo, Vânia Maria Maciel; Gruber, Karl

    2012-03-01

    Lv-ranaspumin is a natural surfactant protein with a molecular mass of 23.5 kDa which was isolated from the foam nest of the frog Leptodactylus vastus. Only a partial amino-acid sequence is available for this protein and it shows it to be distinct from any protein sequence reported to date. The protein was purified from the natural source by ion-exchange and size-exclusion chromatography and was crystallized by sitting-drop vapour diffusion using the PEG/Ion screen at 293 K. A complete data set was collected to 3.5 Å resolution. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 51.96, b = 89.99, c = 106.00 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be 54%.

  13. Human decidua-derived mesenchymal stem cells differentiate into functional alveolar type II-like cells that synthesize and secrete pulmonary surfactant complexes.

    PubMed

    Cerrada, Alejandro; de la Torre, Paz; Grande, Jesús; Haller, Thomas; Flores, Ana I; Pérez-Gil, Jesús

    2014-01-01

    Lung alveolar type II (ATII) cells are specialized in the synthesis and secretion of pulmonary surfactant, a lipid-protein complex that reduces surface tension to minimize the work of breathing. Surfactant synthesis, assembly and secretion are closely regulated and its impairment is associated with severe respiratory disorders. At present, well-established ATII cell culture models are not available. In this work, Decidua-derived Mesenchymal Stem Cells (DMSCs) have been differentiated into Alveolar Type II- Like Cells (ATII-LCs), which display membranous cytoplasmic organelles resembling lamellar bodies, the organelles involved in surfactant storage and secretion by native ATII cells, and accumulate disaturated phospholipid species, a surfactant hallmark. Expression of characteristic ATII cells markers was demonstrated in ATII-LCs at gene and protein level. Mimicking the response of ATII cells to secretagogues, ATII-LCs were able to exocytose lipid-rich assemblies, which displayed highly surface active capabilities, including faster interfacial adsorption kinetics than standard native surfactant, even in the presence of inhibitory agents. ATII-LCs could constitute a highly useful ex vivo model for the study of surfactant biogenesis and the mechanisms involved in protein processing and lipid trafficking, as well as the packing and storage of surfactant complexes.

  14. Proteins aggregation and human diseases

    NASA Astrophysics Data System (ADS)

    Hu, Chin-Kun

    2015-04-01

    Many human diseases and the death of most supercentenarians are related to protein aggregation. Neurodegenerative diseases include Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), frontotemporallobar degeneration, etc. Such diseases are due to progressive loss of structure or function of neurons caused by protein aggregation. For example, AD is considered to be related to aggregation of Aβ40 (peptide with 40 amino acids) and Aβ42 (peptide with 42 amino acids) and HD is considered to be related to aggregation of polyQ (polyglutamine) peptides. In this paper, we briefly review our recent discovery of key factors for protein aggregation. We used a lattice model to study the aggregation rates of proteins and found that the probability for a protein sequence to appear in the conformation of the aggregated state can be used to determine the temperature at which proteins can aggregate most quickly. We used molecular dynamics and simple models of polymer chains to study relaxation and aggregation of proteins under various conditions and found that when the bending-angle dependent and torsion-angle dependent interactions are zero or very small, then protein chains tend to aggregate at lower temperatures. All atom models were used to identify a key peptide chain for the aggregation of insulin chains and to find that two polyQ chains prefer anti-parallel conformation. It is pointed out that in many cases, protein aggregation does not result from protein mis-folding. A potential drug from Chinese medicine was found for Alzheimer's disease.

  15. Interactions of surfactants with a water treatment protein from Moringa oleifera seeds in solution studied by zeta-potential and light scattering measurements.

    PubMed

    Kwaambwa, Habauka M; Rennie, Adrian R

    2012-04-01

    Protein extracted from Moringa oleifera (MO) seeds has been advocated as a cheap and environmental friendly alternative to ionic flocculants for water purification. However, the nature and mechanism of its interaction with particles in water, as well as with dissolved surface-active molecules, are not well understood. In this article, we report studies of the protein and its interaction with four surfactants using dynamic light scattering (DLS), zeta-potential and turbidity measurements. Zeta-potential measurements identified points of charge reversal and the turbidity and DLS measurements were used to characterize the microstructure and size of protein-surfactant complexes. From the points of charge reversal, it was estimated that 7 anions are required to neutralize the positive charges of each protein molecule at pH 7. For protein mixtures with sodium dodecyl sulfate and dodecyl di-acid sodium salt, the peak in turbidity corresponds to concentrations with a large change in zeta-potential. No turbidity was observed for protein mixtures with either the nonionic surfactant Triton X-100 or the zwitterionic surfactant N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate. Changes of pH in the range 4-10 have little effect on the zeta-potential, turbidity, and the hydrodynamic radius reflecting the high isoelectric point of the protein. Addition of small amounts of salt has little effect on the size of protein in solution. These results are discussed in the context of the use of the MO protein in water treatment.

  16. Correlations of Ventricular Enlargement with Rheologically Active Surfactant Proteins in Cerebrospinal Fluid

    PubMed Central

    Schob, Stefan; Weiß, Alexander; Dieckow, Julia; Richter, Cindy; Pirlich, Mandy; Voigt, Peter; Surov, Alexey; Hoffmann, Karl-Titus; Quaeschling, Ulf; Preuß, Matthias

    2017-01-01

    Purpose: Surfactant proteins (SPs) are involved in the regulation of rheological properties of body fluids. Concentrations of SPs are altered in the cerebrospinal fluid (CSF) of hydrocephalus patients. The common hallmark of hydrocephalus is enlargement of the brain ventricles. The relationship of both phenomena has not yet been investigated. The aim of this study was to evaluate the association between SP concentrations in the CSF and enlargement of the brain ventricles. Procedures: Ninty-six individuals (41 healthy subjects and 55 hydrocephalus patients) were included in this retrospective analysis. CSF specimens were analyzed for SP-A, SP-B, SP-C and SP-D concentrations by use of enzyme linked immunosorbent assays (ELISA). Ventricular enlargement was quantified in T2 weighted (T2w) magnetic resonance imaging (MRI) sections using an uni-dimensional (Evans’ Index) and a two-dimensional approach (lateral ventricles area index, LVAI). Results: CSF-SP concentrations (mean ± standard deviation in ng/ml) were as follows: SP-A 0.71 ± 0.58, SP-B 0.18 ± 0.43, SP-C 0.89 ± 0.77 and SP-D 7.4 ± 5.4. Calculated values of Evans’ Index were 0.37 ± 0.11, a calculation of LVAI resulted in 0.18 ± 0.15 (each mean ± standard deviation). Significant correlations were identified for Evans’ Index with SP-A (r = 0.388, p < 0.001) and SP-C (r = 0.392, p < 0.001), LVAI with SP-A (r = 0.352, p = 0.001), SP-C (r = 0.471, p < 0.001) and SP-D (r = 0.233, p = 0.025). Furthermore, SP-C showed a clear inverse correlation with age (r = −0.357, p = 0.011). Conclusion: The present study confirmed significant correlations between SPs A, C and D in the CSF with enlargement of the inner CSF spaces. In conclusion, SPs clearly play an important role for CSF rheology. CSF rheology is profoundly altered in hydrocephalic diseases, however, diagnosis and therapy of hydrocephalic conditions are still almost exclusively based on ventricular enlargement. Until now it was unclear, whether the

  17. Protein Crystal Recombinant Human Insulin

    NASA Technical Reports Server (NTRS)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  18. Nanotribological effects of silicone type, silicone deposition level, and surfactant type on human hair using atomic force microscopy.

    PubMed

    La Torre, Carmen; Bhushan, Bharat

    2006-01-01

    The atomic/friction force microscope (AFM/FFM) has recently become an important tool for studying the micro/nanoscale structure and tribological properties of human hair. Of particular interest to hair and beauty care science is how common hair-care materials, such as conditioner, deposit onto and change hair's tribological properties, since these properties are closely tied to product performance. Since a conditioner is a complex network of many different ingredients (including silicones for lubrication and cationic surfactants for static control and gel network formulation), studying the effects of these individual components can give insight into the significance each has on hair properties. In this study, AFM/FFM is used to conduct nanotribological studies of surface roughness, friction force, and adhesive forces as a function of silicone type, silicone deposition level, and cationic surfactant type. Changes in the coefficient of friction as a result of soaking hair in de-ionized water are also discussed.

  19. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    NASA Astrophysics Data System (ADS)

    Paulin, Sarah; Jamshad, Mohammed; Dafforn, Timothy R.; Garcia-Lara, Jorge; Foster, Simon J.; Galley, Nicola F.; Roper, David I.; Rosado, Helena; Taylor, Peter W.

    2014-07-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

  20. Surfactant proteins, SP-A and SP-D, in respiratory fungal infections: their role in the inflammatory response.

    PubMed

    Carreto-Binaghi, Laura Elena; Aliouat, El Moukhtar; Taylor, Maria Lucia

    2016-06-01

    Pulmonary surfactant is a complex fluid that comprises phospholipids and four proteins (SP-A, SP-B, SP-C, and SP-D) with different biological functions. SP-B, SP-C, and SP-D are essential for the lungs' surface tension function and for the organization, stability and metabolism of lung parenchyma. SP-A and SP-D, which are also known as pulmonary collectins, have an important function in the host's lung immune response; they act as opsonins for different pathogens via a C-terminal carbohydrate recognition domain and enhance the attachment to phagocytic cells or show their own microbicidal activity by increasing the cellular membrane permeability. Interactions between the pulmonary collectins and bacteria or viruses have been extensively studied, but this is not the same for fungal pathogens. SP-A and SP-D bind glucan and mannose residues from fungal cell wall, but there is still a lack of information on their binding to other fungal carbohydrate residues. In addition, both their relation with immune cells for the clearance of these pathogens and the role of surfactant proteins' regulation during respiratory fungal infections remain unknown. Here we highlight the relevant findings associated with SP-A and SP-D in those respiratory mycoses where the fungal infective propagules reach the lungs by the airways.

  1. Changes in surfactant protein A mRNA levels in a rat model of insulin-treated diabetic pregnancy.

    PubMed

    Moglia, B B; Phelps, D S

    1996-02-01

    Maternal diabetes during pregnancy is associated with increased risk of neonatal respiratory distress syndrome (RDS). Previous studies using rat models for the diabetic pregnancy have documented decreased amounts of surfactant protein mRNA in the lungs of fetuses. In this study, we measured fetal lung surfactant-associated protein A (SP-A) mRNA from diabetic rats treated with insulin by daily injection or osmotic pump. Lungs were taken from fetuses on gestational d 20, and RNA was isolated and subjected to Northern blotting and densitometry to quantify SP-A mRNA. Fetal lung SP-A mRNA from untreated diabetic pregnancies was 34 +/- 2.9% of control. Insulin treatment increased levels to 55 +/- 4.2% of control values. Fetal lung SP-A mRNA levels were affected by the timing, length, and effectiveness of insulin treatment. Although levels from all treatment groups were still less than control values, insulin treatment during the last 5 or 10 d of pregnancy resulted in a substantial increase in SP-A mRNA levels over those of from untreated diabetic pregnancies. However, fetuses from the group with insulin treatment for the entire pregnancy showed decreases in fetal SP-A mRNA levels. Although the mechanism(s) responsible for the effects of diabetes and its treatment on fetal SP-A expression remain unclear, it appears unlikely that hyperglycemia is the principal cause.

  2. Diseases of Pulmonary Surfactant Homeostasis

    PubMed Central

    Whitsett, Jeffrey A.; Wert, Susan E.; Weaver, Timothy E.

    2015-01-01

    Advances in physiology and biochemistry have provided fundamental insights into the role of pulmonary surfactant in the pathogenesis and treatment of preterm infants with respiratory distress syndrome. Identification of the surfactant proteins, lipid transporters, and transcriptional networks regulating their expression has provided the tools and insights needed to discern the molecular and cellular processes regulating the production and function of pulmonary surfactant prior to and after birth. Mutations in genes regulating surfactant homeostasis have been associated with severe lung disease in neonates and older infants. Biophysical and transgenic mouse models have provided insight into the mechanisms underlying surfactant protein and alveolar homeostasis. These studies have provided the framework for understanding the structure and function of pulmonary surfactant, which has informed understanding of the pathogenesis of diverse pulmonary disorders previously considered idiopathic. This review considers the pulmonary surfactant system and the genetic causes of acute and chronic lung disease caused by disruption of alveolar homeostasis. PMID:25621661

  3. Pulmonary surfactant for neonatal respiratory disorders.

    PubMed

    Merrill, Jeffrey D; Ballard, Roberta A

    2003-04-01

    Surfactant therapy has revolutionized neonatal care and is used routinely for preterm infants with respiratory distress syndrome. Recent investigation has further elucidated the function of surfactant-associated proteins and their contribution toward surfactant and lung immune defense functions. As the field of neonatology moves away from intubation and mechanical ventilation of preterm infants at birth toward more aggressive use of nasal continuous positive airway pressure, the optimal timing of exogenous surfactant therapy remains unclear. Evidence suggests that preterm neonates with bronchopulmonary dysplasia and prolonged mechanical ventilation also experience surfactant dysfunction; however, exogenous surfactant therapy beyond the first week of life has not been well studied. Surfactant replacement therapy has been studied for use in other respiratory disorders, including meconium aspiration syndrome and pneumonia. Commercial surfactant preparations currently available are not optimal, given the variability of surfactant protein content and their susceptibility to inhibition. Further progress in the treatment of neonatal respiratory disorders may include the development of "designer" surfactant preparations.

  4. Identification of CD245 as myosin 18A, a receptor for surfactant A: A novel pathway for activating human NK lymphocytes

    PubMed Central

    De Masson, A.; Giustiniani, J.; Marie-Cardine, A.; Bouaziz, J. D.; Dulphy, N.; Gossot, D.; Validire, P.; Tazi, A.; Garbar, C.; Bagot, M.; Merrouche, Y.; Bensussan, A.

    2016-01-01

    ABSTRACT CD245 is a human surface antigen expressed on peripheral blood lymphocytes, initially delineated by two monoclonal antibodies DY12 and DY35. Until now, CD245 molecular and functional characteristics remained largely unknown. We combined immunological and proteomic approaches and identified CD245 as the unconventional myosin 18A, a highly conserved motor enzyme reported as a receptor for the surfactant protein A (SP-A), that plays a critical role in cytoskeleton organization and Golgi budding. We report that the recruitment of CD245 strongly enhanced NK cell cytotoxicity. Further, we show that the enhancement of the NK lymphocytes killing ability toward CD137-ligand expressing target cells could result from the induction of CD137 expression following CD245 engagement. The SP-A receptor could therefore represent a novel and promising target in cancer immunotherapy. PMID:27467939

  5. Comparative surfactant reactivity of canine and human stratum corneum: a plea for the use of the corneosurfametry bioassay.

    PubMed

    Goffin, V; Fontaine, J; Piérard, G E

    1999-01-01

    Comparative dermatology has paid little attention to the physiopathology of the stratum corneum. In this study, we investigated the responses of human and canine horny layers to marketed animal wash products by using the corneosurfametry bioassay. Previous work has shown that, with increasing surfactant aggressiveness to the stratum corneum, the colorimetric index of mildness (CIM) decreases, while both the corneosurfametry index (CSMI) and the overall difference in corneosurfametry (ODC) increase. In the present study, stratum corneum reactivity to wash products and inter-individual variability were significantly higher in humans than in dogs. For the three corneosurfametry variables, linear correlations were found between data gathered in the two panel groups. In conclusion, this pilot study suggests that mean stratum corneum reactivity to surfactants is stronger in humans than in dogs. Inter-individual variation, indicative of sensitive skin, also appears to be broader in humans. As a consequence, data gathered from dogs by using the corneosurfametry bioassay cannot be extrapolated to humans. Such variation between species could be important in the assessment of product safety and in supporting claims for mildness.

  6. FOLDING OF DIPHTHERIA TOXIN T-DOMAIN IN THE PRESENCE OF AMPHIPOLS AND FLUORINATED SURFACTANTS: TOWARD THERMODYNAMIC MEASUREMENTS OF MEMBRANE PROTEIN FOLDING

    PubMed Central

    Kyrychenko, Alexander; Rodnin, Mykola V.; Vargas-Uribe, Mauricio; Sharma, Shivaji K.; Durand, Grégory; Pucci, Bernard; Popot, Jean-Luc; Ladokhin, Alexey S.

    2011-01-01

    Solubilizing membrane proteins for functional, structural and thermodynamic studies is usually achieved with the help of detergents, which tend to destabilize them, however. Several classes of non-detergent surfactants have been designed as milder substitutes for detergents, most prominently amphipathic polymers called 'amphipols' and fluorinated surfactants. Here we test the potential usefulness of these compounds for thermodynamic studies by examining their effect on conformational transitions of the diphtheria toxin T-domain. The advantage of the T-domain as a model system is that it exists as a soluble globular protein at neutral pH yet is converted into a membrane-competent form by acidification and inserts into the lipid bilayer as part of its physiological action. We have examined the effects of various surfactants on two conformational transitions of the T-domain, thermal unfolding and pH-induced transition to a membrane-competent form. All tested detergent and non-detergent surfactants lowered the cooperativity of the thermal unfolding of the T-domain. The dependence of enthalpy of unfolding on surfactant concentration was found to be least for fluorinated surfactants, thus making them useful candidates for thermodynamic studies. Circular dichroism measurements demonstrate that non-ionic homo-polymeric amphipols (NAhPols), unlike any other surfactants, can actively cause a conformational change of the T-domain. NAhPol-induced structural rearrangements are different from those observed during thermal denaturation and are suggested to be related to the formation of the membrane-competent form of the T-domain. Measurements of vesicle content leakage indicate that interaction with NAhPols not only does not prevent the T-domain from inserting into the bilayer, but it can make bilayer permeabilization even more efficient, whereas the pH-dependence of membrane permeabilization becomes more cooperative. PMID:21945883

  7. Effects of the conjugation of whey proteins with gellan polysaccharides on surfactant-induced competitive displacement from the air-water interface.

    PubMed

    Cai, B; Ikeda, S

    2016-08-01

    Whey proteins can be used to stabilize foams and emulsions against coalescence because of their ability to form viscoelastic films at the interface that resist film rupture on collision between colloidal particles. However, whey proteins are competitively displaced from the interface if small-molecule surfactants are added, leading to destabilization of the entire system. This is because surfactants are more effective in molecular packing at the interface, and they lower interfacial tension to a greater degree than whey proteins do, but their interfacial films are poor in viscoelasticity. We hypothesized that whey proteins would become more resistant to surfactant-induced competitive displacement if they were conjugated with network-forming polysaccharides. The protein moiety of the conjugate would be expected to enable its adsorption to the interface, and the polysaccharide moiety would be expected to form self-assembled networks, strengthening the interfacial film as a whole. In this study, whey proteins were conjugated with gellan polysaccharides using the Maillard reaction. Atomic force microscopy images of interfacial films formed by the whey protein-gellan conjugate at the air-water interface and transferred onto mica sheets using the Langmuir-Blodgett method revealed that gellan did form self-assembled networks at the interface and that interfacial films also contained a large number of unconjugated whey protein molecules. Following the addition of a small-molecule surfactant (Tween 20) to the sub-phase, surface pressure increased, indicating spontaneous adsorption of surfactants to the interface. Atomic force microscopy images showed decreases in interfacial area coverage by whey proteins as surface pressure increased. At a given surface pressure, the interfacial area coverage by whey protein-gellan conjugates was greater than coverage by unconjugated whey proteins, confirming that whey proteins became more resistant to surfactant-induced displacement after

  8. Surfactant prevents quartz induced down-regulation of complement receptor 1 in human granulocytes.

    PubMed

    Zetterberg, G; Lundahl, J; Curstedt, T; Eklund, A

    1997-02-01

    Quartz is known to induce an inflammatory response in the alveolar space by recruitment of different effector cells. We investigated the interaction between granulocytes and quartz with respect to expression of complement receptor type 1 (CR1) and CR3, with and without the presence of surfactant. Granulocytes from hemolyzed blood were stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), which mobilize the intracellular pool of CR1 to the surface, and the mean fluorescence intensity (MFI) measured by cytofluorometry was 47.4 (46-63.6) (median; interquartile range). Quartz exposure reduced the CR1 expression to 23.2 (22.8-30.6) MFI units (P < 0.01), a porcine surfactant preparation added during quartz exposure abolished the down-regulation completely, 47.7 (43.2-62.3) MFI units (P < 0.001). Similar results were obtained after preincubation of the cells with surfactant followed by quartz exposure. No significant influence on CR1 expression was found by a synthetic lipid mixture, nor was the CR3 expression affected. In conclusion, this study demonstrates that the presence of surfactant inhibits quartz induced down-regulation of CR1 on activated granulocytes.

  9. Surfactant therapy for meconium aspiration syndrome: current status.

    PubMed

    Dargaville, Peter A; Mills, John F

    2005-01-01

    Meconium aspiration syndrome (MAS) is an important cause of respiratory distress in the term infant. Therapy for the disease remains problematic, and newer treatments such as high-frequency ventilation and inhaled nitric oxide are being applied with increasing frequency. There is a significant disturbance of the pulmonary surfactant system in MAS, with a wealth of experimental data indicating that inhibition of surfactant function in the alveolar space is an important element of the pathophysiology of the disease. This inhibition may be mediated by meconium, plasma proteins, haemoglobin and oedema fluid, and, at least in vitro, can be overcome by increasing surfactant phospholipid concentration. These observations have served as the rationale for administration of exogenous surfactant preparations in MAS, initially as standard bolus therapy and, more recently, in association with therapeutic lung lavage. Bolus surfactant therapy in ventilated infants with MAS has been found to improve oxygenation in most studies, although there are a significant proportion of nonresponders and in many cases the effect is transient. Pooled data from randomised controlled trials of surfactant therapy suggest a benefit in terms of a reduction in the requirement for extracorporeal membrane oxygenation (relative risk 0.48 in surfactant-treated infants) but no diminution of air leak or ventilator days. Current evidence would support the use of bolus surfactant therapy on a case by case basis in nurseries with a relatively high mortality associated with MAS, or the lack of availability of other forms of respiratory support such as high-frequency ventilation or nitric oxide. If used, bolus surfactant should be administered as early as practicable to infants who exhibit significant parenchymal disease, at a phospholipid dose of at least 100 mg/kg, rapidly instilled into the trachea. Natural surfactant or a third-generation synthetic surfactant should be used and the dosage repeated every 6

  10. Crystallization and preliminary X-ray diffraction of the surfactant protein Lv-ranaspumin from the frog Leptodactylus vastus

    PubMed Central

    Hissa, Denise Cavalcante; Bezerra, Gustavo Arruda; Obrist, Britta; Birner-Grünberger, Ruth; Melo, Vânia Maria Maciel; Gruber, Karl

    2012-01-01

    Lv-ranaspumin is a natural surfactant protein with a molecular mass of 23.5 kDa which was isolated from the foam nest of the frog Leptodactylus vastus. Only a partial amino-acid sequence is available for this protein and it shows it to be distinct from any protein sequence reported to date. The protein was purified from the natural source by ion-exchange and size-exclusion chromatography and was crystallized by sitting-drop vapour diffusion using the PEG/Ion screen at 293 K. A complete data set was collected to 3.5 Å resolution. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 51.96, b = 89.99, c = 106.00 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be 54%. PMID:22442233

  11. Possible influence of surfactants and proteins on the efficiency of contact agar microbiological surface sampling.

    PubMed

    Deckers, Sylvie M; Sindic, Marianne; Anceau, Christine; Brostaux, Yves; Detry, Jean G

    2010-11-01

    Agar contact microbiological sampling techniques, based on a transfer of the microorganisms present on a surface to a culture medium, are widely used to assess and control surface cleanliness and to evaluate microbial contamination levels. The effectiveness of these techniques depends on many environmental parameters that influence the strength of attachment of the bacteria to the surface. In the present study, stainless steel and high density polyethylene surfaces were inoculated with known concentrations of Staphylococcus epidermidis. Following an experimental design, the surfaces were sampled with different types of replicate organism direct agar contact plates and Petrifilm; results indicated that recovery rates were influenced by the presence of egg white albumin or Tween 80 in the inoculum solutions or by the introduction of surfactants into the contact agar of the microbiological sampling techniques. The techniques yielded significantly different results, depending on sampling conditions, underlining the need for a standardization of laboratory experiments to allow relevant comparisons of such techniques.

  12. Lung surfactant.

    PubMed Central

    Rooney, S A

    1984-01-01

    Aspects of pulmonary surfactant are reviewed from a biochemical perspective. The major emphasis is on the lipid components of surfactant. Topics reviewed include surfactant composition, cellular and subcellular sites as well as pathways of biosynthesis of phosphatidylcholine, disaturated phosphatidylcholine and phosphatidylglycerol. The surfactant system in the developing fetus and neonate is considered in terms of phospholipid content and composition, rates of precursor incorporation, activities of individual enzymes of phospholipid synthesis and glycogen content and metabolism. The influence of the following hormones and other factors on lung maturation and surfactant production is discussed: glucocorticoids, thyroid hormone, estrogen, prolactin, cyclic AMP, beta-adrenergic and cholinergic agonists, prostaglandins and growth factors. The influence of maternal diabetes, fetal sex, stress and labor are also considered. Nonphysiologic and toxic agents which influence surfactant in the fetus, newborn and adult are reviewed. PMID:6145585

  13. Adsorption of proteins at the solution/air interface influenced by added nonionic surfactants at very low concentrations for both components. 3. Dilational surface rheology.

    PubMed

    Fainerman, V B; Aksenenko, E V; Lylyk, S V; Lotfi, M; Miller, R

    2015-03-05

    The influence of the addition of the nonionic surfactants C12DMPO, C14DMPO, C10OH, and C10EO5 at concentrations between 10(-5) and 10(-1) mmol/L to solutions of β-casein (BCS) and β-lactoglobulin (BLG) at a fixed concentration of 10(-5) mmol/L on the dilational surface rheology is studied. A maximum in the viscoelasticity modulus |E| occurs at very low surfactant concentrations (10(-4) to 10(-3) mmol/L) for mixtures of BCS with C12DMPO and C14DMPO and for mixtures of BLG with C10EO5, while for mixture of BCS with C10EO5 the value of |E| only slightly increased. The |E| values calculated with a recently developed model, which assumes changes in the interfacial molar area of the protein molecules due to the interaction with the surfactants, are in satisfactory agreement with experimental data. A linear dependence exists between the ratio of the maximum modulus for the mixture to the modulus of the single protein solution and the coefficient reflecting the influence of the surfactants on the adsorption activity of the protein.

  14. Combinations of fluorescently labeled pulmonary surfactant proteins SP-B and SP-C in phospholipid films.

    PubMed Central

    Nag, K; Taneva, S G; Perez-Gil, J; Cruz, A; Keough, K M

    1997-01-01

    Hydrophobic pulmonary surfactant (PS) proteins B (SP-B) and C (SP-C) modulate the surface properties of PS lipids. Epifluorescence microscopy was performed on solvent-spread monolayers of fluorescently labeled porcine SP-B (R-SP-B, labeled with Texas Red) and SP-C (F-SP-C, labeled with fluorescein) in dipalmitoylphosphatidylcholine (DPPC) (at protein concentrations of 10 and 20 wt%, and 10 wt% of both) under conditions of cyclic compression and expansion. Matrix-assisted laser desorption/ionization (MALDI) spectroscopy of R-SP-B and F-SP-C indicated that the proteins were intact and labeled with the appropriate fluorescent probe. The monolayers were compressed and expanded for four cycles at an initial rate of 0.64 A2 x mol(-1) x s(-1) (333 mm2 x s x [-1]) up to a surface pressure pi approximately 65 mN/m, and pi-area per residue (pi-A) isotherms at 22 +/- 1 degrees C were obtained. The monolayers were microscopically observed for the fluorescence emission of the individual proteins present in the film lipid matrix, and their visual features were video recorded for image analysis. The pi-A isotherms of the DPPC/protein monolayers showed characteristic "squeeze out" effects at pi approximately 43 mN/m for R-SP-B and 55 mN/m for F-SP-C, as had previously been observed for monolayers of the native proteins in DPPC. Both proteins associated with the expanded (fluid) phase of DPPC monolayers remained in or associated with the monolayers at high pi (approximately 65 mN/m) and redispersed in the monolayer upon its reexpansion. At comparable pi and area/molecule of the lipid, the proteins reduced the amounts of condensed (gel-like) phase of DPPC monolayers, with F-SP-C having a greater effect on a weight basis than did R-SP-B. In any one of the lipid/protein monolayers the amounts of the DPPC in condensed phase were the same at equivalent pi during compression and expansion and from cycle to cycle. This indicated that only minor loss of components from these systems

  15. Surfactants and the Mechanics of Respiration

    NASA Astrophysics Data System (ADS)

    Jbaily, Abdulrahman; Szeri, Andrew J.

    2016-11-01

    Alveoli are small sacs found at the end of terminal bronchioles in human lungs with a mean diameter of 200 μm. A thin layer of fluid (hypophase) coats the inner face of an alveolus and is in contact with the air in the lungs. The thickness of this layer varies among alveoli, but is in the range of 0.1 to 0.5 μm for many portions of the alveolar network. The interfacial tension σ at the air-hypophase interface tends to favor collapse of the alveolus, and resists its expansion during inhalation. Type II alveolar cells synthesize and secrete a mixture of phospholipids and proteins called pulmonary surfactant. These surfactant molecules adsorb to the interface causing σ of water at body temperature is 70 mN/m and falls to an equilibrium value of 25 mN/m when surfactants are present. Also, in a dynamic sense, it is known that σ is reduced to near 0 during exhalation when the surfactant film compresses. In this work, the authors develop a mechanical and transport model of the alveolus to study the effect of surfactants on various aspects of respiration. The model is composed of three principal parts: (i) air movement into and out of the alveolus; (ii) a balance of linear momentum across the two-layered membrane of the alveolus (hypophase and elastic wall); and (iii) a pulmonary surfactant transport problem in the hypophase. The goal is to evaluate the influence of pulmonary surfactant on respiratory mechanics.

  16. Differential partitioning of pulmonary surfactant protein SP-A into regions of monolayers of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol.

    PubMed Central

    Ruano, M L; Nag, K; Worthman, L A; Casals, C; Pérez-Gil, J; Keough, K M

    1998-01-01

    The interaction of the pulmonary surfactant protein SP-A fluorescently labeled with Texas Red (TR-SP-A) with monolayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC/dipalmitoylphosphatidylglycerol 7:3 w/w has been investigated. The monolayers were spread on aqueous subphases containing TR-SP-A. TR-SP-A interacted with the monolayers of DPPC to accumulate at the boundary regions between liquid condensed (LC) and liquid expanded (LE) phases. Some TR-SP-A appeared in the LE phase but not in the LC phase. At intermediate surface pressures (10-20 mN/m), the protein caused the occurrence of more, smaller condensed domains, and it appeared to be excluded from the monolayers at surface pressure in the range of 30-40 mN/m. TR-SP-A interaction with DPPC/dipalmitoylphosphatidylglycerol monolayers was different. The protein did not appear in either LE or LC but only in large aggregates at the LC-LE boundary regions, a distribution visually similar to that of fluorescently labeled concanavalin A adsorbed onto monolayers of DPPC. The observations are consistent with a selectivity of interaction of SP-A with DPPC and for its accumulation in boundaries between LC and LE phase. PMID:9512012

  17. Impact of a Met(11)Thr single nucleotide polymorphism of surfactant protein D on allergic airway inflammation in a murine asthma model.

    PubMed

    Winkler, Carla; Bahlmann, Olaf; Viereck, Janika; Knudsen, Lars; Wedekind, Dirk; Hoymann, Heinz Gerd; Madsen, Jens; Thum, Thomas; Hohlfeld, Jens M; Ochs, Matthias

    2014-04-01

    The surfactant-associated proteins SP-A and D are pattern recognition molecules with collectin structure. A single nucleotide polymorphism (SNP) exchanging a methionine (Met) for a threonine (Thr) in the amino-terminal SP-D domain influences the oligomeric structure and function of the protein. In this study, we investigated the susceptibility of mice transgenic for the human SP-D Met(11)Thr SNP to allergic airway inflammation and consequences for microRNA (miRNA, miR) expression. Mice expressing either human Met or Thr SP-D were sensitized and challenged with ovalbumin (OVA) in an acute model of allergic asthma. The influence of the SP-D polymorphism on the allergic airway inflammation was evaluated by lung function measurement, pulmonary inflammation parameters, morphological analysis and miRNA expression. Airway hyperresponsiveness, allergic inflammation, and mucus metaplasia were not significantly different between mice expressing one or the other allelic variant of SP-D. OVA sensitization and challenge led to significant airway hyperresponsiveness in wildtype mice and significantly lower eosinophil numbers and interleukin 5 levels in Thr SP-D mice. OVA challenge induced an upregulation of miR-21 and 155 in Thr SP-D mice and a downregulation of miR-21 in Met SP-D mice. Our results show that murine expression of human polymorphic SP-D variants does not significantly influence the severity of allergic airway inflammation. MiR-21 and 155 are differentially regulated in transgenic mice in response to allergic inflammation. Further studies are required to elucidate the impact of this SNP on inflammatory conditions of the lung.

  18. Human Antimicrobial Peptides and Proteins

    PubMed Central

    Wang, Guangshun

    2014-01-01

    As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs) play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between −3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32) can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized medicine to

  19. Surfactant Protein A in Exhaled Endogenous Particles Is Decreased in Chronic Obstructive Pulmonary Disease (COPD) Patients: A Pilot Study

    PubMed Central

    Lärstad, Mona; Almstrand, Ann-Charlotte; Larsson, Per; Bake, Björn; Larsson, Sven; Ljungström, Evert; Mirgorodskaya, Ekaterina; Olin, Anna-Carin

    2015-01-01

    Background Exhaled, endogenous particles are formed from the epithelial lining fluid in small airways, where surfactant protein A (SP-A) plays an important role in pulmonary host defense. Based on the knowledge that chronic obstructive pulmonary disease (COPD) starts in the small airway epithelium, we hypothesized that chronic inflammation modulates peripheral exhaled particle SP-A and albumin levels. The main objective of this explorative study was to compare the SP-A and albumin contents in exhaled particles from patients with COPD and healthy subjects and to determine exhaled particle number concentrations. Methods Patients with stable COPD ranging from moderate to very severe (n = 13), and healthy non-smoking subjects (n = 12) were studied. Subjects performed repeated breath maneuvers allowing for airway closure and re-opening, and exhaled particles were optically counted and collected on a membrane using the novel PExA® instrument setup. Immunoassays were used to quantify SP-A and albumin. Results COPD patients exhibited significantly lower SP-A mass content of the exhaled particles (2.7 vs. 3.9 weight percent, p = 0.036) and lower particle number concentration (p<0.0001) than healthy subjects. Albumin mass contents were similar for both groups. Conclusions Decreased levels of SP-A may lead to impaired host defense functions of surfactant in the airways, contributing to increased susceptibility to COPD exacerbations. SP-A in exhaled particles from small airways may represent a promising non-invasive biomarker of disease in COPD patients. PMID:26656890

  20. Ion-exclusion/cation-exchange chromatographic determination of common inorganic ions in human saliva by using an eluent containing zwitterionic surfactant.

    PubMed

    Mori, Masanobu; Iwata, Tomotaka; Satori, Tatsuya; Ohira, Shin-Ichi; Itabashi, Hideyuki; Tanaka, Kazuhiko

    2008-12-12

    Ion-exclusion/cation-exchange chromatography with an eluent containing the bile salt-type zwitterionic surfactant CHAPS was performed in order to evaluate variations in anion (SO(4)(2-), NO(3)(-), and SCN(-)) and cation (Na(+), K(+), NH(4)(+), Mg(2+), and Ca(2+)) concentrations in human saliva. CHAPS prevents the adsorption of proteins to the stationary phase, i.e., weakly acidic cation-exchange resin, since it aggregates proteins without denaturing them. Addition of 1mM CHAPS to the eluent comprising 6mM tartaric acid and 7 mM 18-crown-6 yielded reproducible separations of anions and cations in protein-containing saliva. The resolutions of anions and cations were not significantly affected by the addition of CHAPS to the eluent. The concentrations of Na(+) and K(+) varied before and after meals; or that of SCN(-), upon smoking. The relative standard deviations of peak areas ranged from 0.3 to 5.1% in 1 day (n=20) and from 1.4 to 5.8% over 6 days (n=6).

  1. Aberrant processing forms of lung surfactant proteins SP-B and SP-C revealed by high-resolution mass spectrometry.

    PubMed

    Galetskiy, Dmitry; Woischnik, Markus; Ripper, Jan; Griese, Matthias; Przybylski, Michael

    2008-01-01

    The mutation (g.1286T>C) of the pulmonary surfactant-associated protein C gene (SFTPC) leads to the I73T substitution in the precursor protein (pro-SP-C) and results in interstitial lung disease with the histological pattern of non-specific interstitial pneumonia and pulmonary alveolar proteinosis. Central for the disease is the abnormal processing of the SP-C pro-protein to mature SP-C; however little is known about the nature of intermediates and processing products. We report here the application of high resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry to the characterization of processing intermediates of hydrophobic pulmonary surfactant proteins SP-B and SP-C in intra- alveolar surfactant material of a patient with I73T mutation. SP-C and SP-B processing forms were separated from broncho-alveolar lavage fluid using chloroform/methanol extraction and sodium dodecyl sulfate poly acrylamide gel electrophoreis, detected by Western blot and identified by electrospray- and matrix-assisted laser desorption/ionization-FT-ICR mass spectrometry. The mass spectrometric and immuno-analytical results show the intra-alveolar accumulation of an aberrant C-terminal SP-C processing products in which the mature SP-C protein part is missing and aberrant processing intermediates of SP-B.

  2. Cyclic AMP-responsive expression of the surfactant protein-A gene is mediated by increased DNA binding and transcriptional activity of thyroid transcription factor-1.

    PubMed

    Li, J; Gao, E; Mendelson, C R

    1998-02-20

    Surfactant protein (SP)-A gene transcription is stimulated by factors that increase cyclic AMP. In the present study, we observed that three thyroid transcription factor-1 (TTF-1) binding elements (TBEs) located within a 255 base pair region flanking the 5'-end of the baboon SP-A2 (bSP-A2) gene are required for maximal cyclic AMP induction of bSP-A2 promoter activity. We found that TTF-1 DNA binding activity was increased in nuclear extracts of pulmonary type II cells cultured in the presence of cyclic AMP. By contrast, the levels of immunoreactive TTF-1 protein were similar in nuclear extracts of control and cyclic AMP-treated type II cells. The incorporation of [32P]orthophosphate into immunoprecipitated TTF-1 protein also was markedly increased by cyclic AMP treatment. Moreover, exposure of nuclear extracts from cyclic AMP-treated type II cells either to potato acid phosphatase or alkaline phosphatase abolished the cyclic AMP-induced increase in TTF-1 DNA-binding activity. Interestingly, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), known to activate protein kinase C, also enhanced incorporation of [32P]orthophosphate into TTF-1 protein; however, the DNA binding activity of TTF-1 was decreased in nuclear extracts of TPA-treated type II cells. Expression vectors encoding TTF-1 and the catalytic subunit of protein kinase A (PKA-cat) were cotransfected into A549 lung adenocarcinoma cells together with an SPA:human growth hormone fusion gene (255 base pairs of 5'-flanking DNA from the baboon SP-A2 gene linked to human growth hormone, as reporter) containing TBEs, or with a reporter gene construct containing three tandem TBEs fused upstream of the bSP-A2 gene TATA box and the transcription initiation site. Coexpression of TTF-1 and PKA-cat increased fusion gene expression 3-4-fold as compared with expression of TTF-1 in the absence of PKA-cat. Moreover, the transcriptional activity of TTF-1 was suppressed by cotransfection of a dominant negative form

  3. Genetic variation in Surfactant Protein-A2 (SP-A2) leads to differential binding to Mycoplasma pneumoniae membranes and regulation of host responses

    PubMed Central

    Ledford, Julie G.; Voelker, Dennis R.; Addison, Kenneth J.; Wang, Ying; Nikam, Vinayak; Degan, Simone; Kandasamy, Pitachaimani; Tanyaratsrisakul, Sasipa; Fischer, Bernard M.; Kraft, Monica; Hollingsworth, John W.

    2015-01-01

    Mycoplasma pneumoniae (Mp) is an extracellular pathogen that colonizes mucosal surfaces of the respiratory tract and is associated with asthma exacerbations. Previous reports demonstrate that surfactant protein-A (SP-A) binds live Mp and mycoplasma membranes (MMF) with high affinity. Humans express a repertoire of single amino acid genetic variants of SP-A that may be associated with lung disease, and our findings demonstrate that allelic differences in SP-A2 (Gln223Lys) affect the binding to MMF. We show that SP-A−/− mice are more susceptible to MMF exposure and have significant increases in mucin production and neutrophil recruitment. Novel humanized-SP-A2 transgenic mice harboring the hSP-A2 223K allele exhibit reduced neutrophil influx and mucin production in the lungs, when challenged with MMF, compared to SP-A−/− mice. Conversely, mice expressing hSP-A2 223Q have increased neutrophil influx and mucin production that is similar to SP-A−/− mice. Using tracheal epithelial cell cultures, we show that enhanced mucin production to MMF occurs in the absence of SP-A, and is not dependent upon neutrophil recruitment. Increased phosphorylation of the epidermal growth factor receptor (EGFR) was evident in the lungs of MMF-challenged mice when SP-A was absent. Pharmacologic inhibition of EGFR prior to MMF challenge dramatically reduced mucin production in SP-A−/− mice. These findings suggest a protective role for SP-A in limiting MMF-stimulated mucin production that occurs through interference with EGFR mediated signaling. The SP-A interaction with the EGFR signaling pathway appears to occur in an allele specific manner that may have important implications for SP-A polymorphisms in human diseases. PMID:25957169

  4. Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability

    PubMed Central

    Wu, Huixing; Kuzmenko, Alexander; Wan, Sijue; Schaffer, Lyndsay; Weiss, Alison; Fisher, James H.; Kim, Kwang Sik; McCormack, Francis X.

    2003-01-01

    The pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), have been reported to bind lipopolysaccharide (LPS), opsonize microorganisms, and enhance the clearance of lung pathogens. In this study, we examined the effect of SP-A and SP-D on the growth and viability of Gram-negative bacteria. The pulmonary clearance of Escherichia coli K12 was reduced in SP-A–null mice and was increased in SP-D–overexpressing mice, compared with strain-matched wild-type controls. Purified SP-A and SP-D inhibited bacterial synthetic functions of several, but not all, strains of E. coli, Klebsiella pneumoniae, and Enterobacter aerogenes. In general, rough E. coli strains were more susceptible than smooth strains, and collectin-mediated growth inhibition was partially blocked by coincubation with rough LPS vesicles. Although both SP-A and SP-D agglutinated E. coli K12 in a calcium-dependent manner, microbial growth inhibition was independent of bacterial aggregation. At least part of the antimicrobial activity of SP-A and SP-D was localized to their C-terminal domains using truncated recombinant proteins. Incubation of E. coli K12 with SP-A or SP-D increased bacterial permeability. Deletion of the E. coli OmpA gene from a collectin-resistant smooth E. coli strain enhanced SP-A and SP-D–mediated growth inhibition. These data indicate that SP-A and SP-D are antimicrobial proteins that directly inhibit the proliferation of Gram-negative bacteria in a macrophage- and aggregation-independent manner by increasing the permeability of the microbial cell membrane. PMID:12750409

  5. Surfactant-free poly(styrene-co-glycidyl methacrylate) particles with surface-bound antibodies for activation and proliferation of human T cells.

    PubMed

    Thümmler, Katja; Häntzschel, Nadine; Skapenko, Alla; Schulze-Koops, Hendrik; Pich, Andrij

    2010-05-19

    In this article, we present our results on the design of new polymeric carriers for antibodies. Polymer colloids based on poly(styrene-co-glycidyl methacrylate) were synthesized by surfactant-free emulsion polymerization. Obtained polymer particles stabilized by grafted poly(ethylene glycol) (PEG) chains and carrying active epoxy groups were used for the covalent immobilization of activating antibodies against the human surface proteins CD (cluster of differentiation) 3 and CD28. The particle-antibody conjugates were employed for the stimulation of human CD4 memory T cells. This was analyzed by the up-regulation of the activation markers CD69 and CD25 on T cells and T cell proliferation as assessed by the dilution of a fluorescent dye on dividing daughter T cells. The particle-antibody conjugates were able to stimulate T cells at least as efficiently as conventional methods, e.g., surface-immobilized antibodies. Furthermore, an increase of the PEG chain length of the particles decreased the efficiency of the particle-antibody conjugates to activate T cells.

  6. Surfactant Protein D Binds to Coxiella burnetii and Results in a Decrease in Interactions with Murine Alveolar Macrophages.

    PubMed

    Soltysiak, Kelly A; van Schaik, Erin J; Samuel, James E

    2015-01-01

    Coxiella burnetii is a Gram-negative, obligate intracellular bacterium and the causative agent of Q fever. Infections are usually acquired after inhalation of contaminated particles, where C. burnetii infects its cellular target cells, alveolar macrophages. Respiratory pathogens encounter the C-type lectin surfactant protein D (SP-D) during the course of natural infection. SP-D is a component of the innate immune response in the lungs and other mucosal surfaces. Many Gram-negative pulmonary pathogens interact with SP-D, which can cause aggregation, bactericidal effects and aid in bacterial clearance. Here we show that SP-D binds to C. burnetii in a calcium-dependent manner with no detectable bacterial aggregation or bactericidal effects. Since SP-D interactions with bacteria often alter macrophage interactions, it was determined that SP-D treatment resulted in a significant decrease in C. burnetii interactions to a mouse alveolar macrophage model cell line MH-S indicating SP-D causes a significant decrease in phagocytosis. The ability of SP-D to modulate macrophage activation by C. burnetii was tested and it was determined that SP-D does not alter the correlates measured for macrophage activation. Taken together these studies support those demonstrating limited activation of alveolar macrophages with C. burnetii and demonstrate interactions with SP-D participate in reduction of phagocyte attachment and phagocytosis.

  7. The Ability of Pandemic Influenza Virus Hemagglutinins to Induce Lower Respiratory Pathology is Associated with Decreased Surfactant Protein D Binding

    PubMed Central

    Qi, Li; Kash, John C.; Dugan, Vivien G.; Jagger, Brett W.; Lau, Yuk-Fai; Sheng, Zhong-Mei; Crouch, Erika C.; Hartshorn, Kevan L.; Taubenberger, Jeffery K.

    2011-01-01

    Pandemic influenza viral infections have been associated with viral pneumonia. Chimeric influenza viruses with the hemagglutinin segment of the 1918, 1957, 1968 or 2009 pandemic influenza viruses in the context of a seasonal H1N1 influenza genome were constructed to analyze the role of hemagglutinin (HA) in pathogenesis and cell tropism in a mouse model. We also explored whether there was an association between the ability of lung surfactant protein D (SP-D) to bind to the HA and the ability of the corresponding chimeric virus to infect bronchiolar and alveolar epithelial cells of the lower respiratory tract. Viruses expressing the hemagglutinin of pandemic viruses were associated with significant pathology in the lower respiratory tract, including acute inflammation, and showed low binding activity for SP-D. In contrast, the virus expressing the HA of a seasonal influenza strain induced only mild disease with little lung pathology in infected mice and exhibited strong in vitro binding to SP-D. PMID:21334038

  8. A mutation in the surfactant protein B gene responsible for fatal neonatal respiratory disease in multiple kindreds.

    PubMed Central

    Nogee, L M; Garnier, G; Dietz, H C; Singer, L; Murphy, A M; deMello, D E; Colten, H R

    1994-01-01

    To determine the molecular defect accounting for the deficiency of pulmonary surfactant protein B (SP-B) in full-term neonates who died from respiratory failure associated with alveolar proteinosis, the sequence of the SP-B transcript in affected infants was ascertained. A frameshift mutation consisting of a substitution of GAA for C in codon 121 of the SP-B cDNA was identified. The three affected infants in the index family were homozygous for this mutation, which segregated in a fashion consistent with autosomal recessive inheritance of disease. The same mutation was found in two other unrelated infants who died from alveolar proteinosis, one of whom was also homozygous, and in the parents of an additional unrelated, affected infant, but was not observed in 50 control subjects. We conclude that this mutation is responsible for SP-B deficiency and neonatal alveolar proteinosis in multiple families and speculate that the disorder is more common than was recognized previously. Images PMID:8163685

  9. Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH(4)Cl.

    PubMed

    Tomanee, Panarat; Hsu, James T; Ito, Yoichiro

    2004-10-05

    Centrifugal precipitation chromatography (CPC) is a separation system that mainly employs a moving concentration gradient of precipitating agent along a channel and solutes of interest undergo repetitive precipitation-dissolution, fractionate at different locations, and elute out from the channel according to their solubility in the precipitating agent solution. We report here for the first time the use of a CPC system for fractionation of protein, RNA, and plasmid DNA in clarified lysate produced from bacterial culture. The cationic surfactant cetyltrimethylammonium bromide (CTAB) was initially used as a precipitating agent; however, all biomolecules showed no differential solubility in the moving concentration gradient of this surfactant and, as a result, no separation of protein, RNA, and plasmid DNA occurred. To overcome this problem, inorganic salts such as NaCl and NH(4)Cl were introduced into solution of CTAB. The protein and RNA were found to have higher solubility with the addition of these salts and separated from the plasmid DNA. Decreasing surface charge density of CTAB upon addition of NaCl and NH(4)Cl was believed to lead to lower surfactant complexation, and therefore caused differential solubility and fractionation of these biomolecules. Addition of CaCl(2) did not improve solubility and separation of RNA from plasmid DNA.

  10. Reverse micellar extraction of bovine serum albumin - a comparison between the effects of gemini surfactant and its corresponding monomeric surfactant.

    PubMed

    Xiao, Jing; Cai, Juan; Guo, Xia

    2013-01-15

    Gemini surfactant displayed distinct advantages over monomeric surfactant in the liquid-liquid reverse micellar extraction process. First, less amount of gemini surfactant than monomeric surfactant was needed for transferring almost complete bovine serum albumin (BSA) into organic phase from aqueous phase. Second, the loading capacity of gemini surfactant reverse micelle phase was much higher than that of the corresponding monomeric surfactant reverse micelle. Third, efficient backward extraction (75-92%) of BSA could be effected in a wide pH range from 4 to 9 with gemini surfactant reverse micelle while a pH of ca. 4.3 is prerequisite to the recovery of BSA from monomeric surfactant reverse micelle. So far, the reports about the effect of surfactant structure on protein extraction have been limited. This study indicates the important role of the spacer of gemini surfactant in protein extraction process and may provide more knowledge on how to optimise surfactant structure.

  11. An antibody against the surfactant protein A (SP-A)-binding domain of the SP-A receptor inhibits T cell-mediated immune responses to Mycobacterium tuberculosis.

    PubMed

    Samten, Buka; Townsend, James C; Sever-Chroneos, Zvjezdana; Pasquinelli, Virginia; Barnes, Peter F; Chroneos, Zissis C

    2008-07-01

    Surfactant protein A (SP-A) suppresses lymphocyte proliferation and IL-2 secretion, in part, by binding to its receptor, SP-R210. However, the mechanisms underlying this effect are not well understood. Here, we studied the effect of antibodies against the SP-A-binding (neck) domain (alpha-SP-R210n) or nonbinding C-terminal domain (alpha-SP-R210ct) of SP-R210 on human peripheral blood T cell immune responses against Mycobacterium tuberculosis. We demonstrated that both antibodies bind to more than 90% of monocytes and 5-10% of CD3+ T cells in freshly isolated PBMC. Stimulation of PBMC from healthy tuberculin reactors [purified protein derivative-positive (PPD+)] with heat-killed M. tuberculosis induced increased antibody binding to CD3+ cells. Increased antibody binding suggested enhanced expression of SP-R210, and this was confirmed by Western blotting. The antibodies (alpha-SP-R210n) cross-linking the SP-R210 through the SP-A-binding domain markedly inhibited cell proliferation and IFN-gamma secretion by PBMC from PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and antibodies (alpha-SP-R210ct) cross-linking SP-R210 through the non-SP-A-binding, C-terminal domain had no effect. Anti-SP-R210n also decreased M. tuberculosis-induced production of TNF-alpha but increased production of IL-10. Inhibition of IFN-gamma production by alpha-SP-R210n was abrogated by the combination of neutralizing antibodies to IL-10 and TGF-beta1. Together, these findings support the hypothesis that SP-A, via SP-R210, suppresses cell-mediated immunity against M. tuberculosis via a mechanism that up-regulates secretion of IL-10 and TGF-beta1.

  12. Natural Anti-Infective Pulmonary Proteins: In Vivo Cooperative Action of Surfactant Protein SP-A and the Lung Antimicrobial Peptide SP-BN.

    PubMed

    Coya, Juan Manuel; Akinbi, Henry T; Sáenz, Alejandra; Yang, Li; Weaver, Timothy E; Casals, Cristina

    2015-08-15

    The anionic antimicrobial peptide SP-B(N), derived from the N-terminal saposin-like domain of the surfactant protein (SP)-B proprotein, and SP-A are lung anti-infective proteins. SP-A-deficient mice are more susceptible than wild-type mice to lung infections, and bacterial killing is enhanced in transgenic mice overexpressing SP-B(N). Despite their potential anti-infective action, in vitro studies indicate that several microorganisms are resistant to SP-A and SP-B(N). In this study, we test the hypothesis that these proteins act synergistically or cooperatively to strengthen each other's microbicidal activity. The results indicate that the proteins acted synergistically in vitro against SP-A- and SP-B(N)-resistant capsulated Klebsiella pneumoniae (serotype K2) at neutral pH. SP-A and SP-B(N) were able to interact in solution (Kd = 0.4 μM), which enabled their binding to bacteria with which SP-A or SP-B(N) alone could not interact. In vivo, we found that treatment of K. pneumoniae-infected mice with SP-A and SP-B(N) conferred more protection against K. pneumoniae infection than each protein individually. SP-A/SP-B(N)-treated infected mice showed significant reduction of bacterial burden, enhanced neutrophil recruitment, and ameliorated lung histopathology with respect to untreated infected mice. In addition, the concentrations of inflammatory mediators in lung homogenates increased early in infection in contrast with the weak inflammatory response of untreated K. pneumoniae-infected mice. Finally, we found that therapeutic treatment with SP-A and SP-B(N) 6 or 24 h after bacterial challenge conferred significant protection against K. pneumoniae infection. These studies show novel anti-infective pathways that could drive development of new strategies against pulmonary infections.

  13. Interdependent TTF1 - ErbB4 interactions are critical for surfactant protein-B homeostasis in primary mouse lung alveolar type II cells.

    PubMed

    Marten, Elger; Nielsen, Heber C; Dammann, Christiane E L

    2015-09-01

    ErbB4 receptor and thyroid transcription factor (TTF)-1 are important modulators of fetal alveolar type II (ATII) cell development and injury. ErbB4 is an upstream regulator of TTF-1, promoting its expression in MLE-12 cells, an ATII cell line. Both proteins are known to promote surfactant protein-B gene (SftpB) and protein (SP-B) expression, but their feedback interactions on each other are not known. We hypothesized that TTF-1 expression has a feedback effect on ErbB4 expression in an in-vitro model of isolated mouse ATII cells. We tested this hypothesis by analyzing the effects of overexpressing HER4 and Nkx2.1, the genes of ErbB4 and TTF-1 on TTF-1 and ErbB4 protein expression, respectively, as well as SP-B protein expression in primary fetal mouse lung ATII cells. Transient ErbB4 protein overexpression upregulated TTF-1 protein expression in primary fetal ATII cells, similarly to results previously shown in MLE-12 cells. Transient TTF-1 protein overexpression down regulated ErbB4 protein expression in both cell types. TTF-1 protein was upregulated in primary transgenic ErbB4-depleted adult ATII cells, however SP-B protein expression in these adult transgenic ATII cells was not affected by the absence of ErbB4. The observation that TTF-1 is upregulated in fetal ATII cells by ErbB4 overexpression and also in ErbB4-deleted adult ATII cells suggests additional factors interact with ErbB4 to regulate TTF-1 levels. We conclude that the interdependency of TTF-1 and ErbB4 is important for surfactant protein levels. The interactive regulation of ErbB4 and TTF-1 needs further elucidation.

  14. Study of the binding between lysozyme and C10-TAB: determination and interpretation of the partial properties of protein and surfactant at infinite dilution.

    PubMed

    Morgado, Jorge; Aquino-Olivos, Marco Antonio; Martínez-Hernández, Ranulfo; Corea, Mónica; Grolier, Jean Pierre E; del Río, José Manuel

    2008-06-01

    This work examines the binding in aqueous solution, through the experimental determination of specific volumes and specific adiabatic compressibility coefficients, of decyltrimethylammonium bromide to lysozyme and to non-charged polymeric particles (which have been specially synthesized by emulsion polymerization). A method was developed to calculate the specific partial properties at infinite dilution and it was shown that a Gibbs-Duhem type equation holds at this limit for two solutes. With this equation, it is possible to relate the behavior of the partial properties along different binding types at a constant temperature. It was found that the first binding type, specific with high affinity, is related to a significant reduction of surfactant compressibility. The second binding type is accompanied by the unfolding of the protein and the third one is qualitatively identical to the binding of the surfactant to non-charged polymeric particles.

  15. Nuclear Matrix Proteins in Human Colon Cancer

    NASA Astrophysics Data System (ADS)

    Keesee, Susan K.; Meneghini, Marc D.; Szaro, Robert P.; Wu, Ying-Jye

    1994-03-01

    The nuclear matrix is the nonchromatin scaffolding of the nucleus. This structure confers nuclear shape, organizes chromatin, and appears to contain important regulatory proteins. Tissue specific nuclear matrix proteins have been found in the rat, mouse, and human. In this study we compared high-resolution two-dimensional gel electropherograms of nuclear matrix protein patterns found in human colon tumors with those from normal colon epithelia. Tumors were obtained from 18 patients undergoing partial colectomy for adenocarcinoma of the colon and compared with tissue from 10 normal colons. We have identified at least six proteins which were present in 18 of 18 colon tumors and 0 of 10 normal tissues, as well as four proteins present in 0 of 18 tumors and in 10 of 10 normal tissues. These data, which corroborate similar findings of cancer-specific nuclear matrix proteins in prostate and breast, suggest that nuclear matrix proteins may serve as important markers for at least some types of cancer.

  16. The role of inducible nitric oxide synthase for interstitial remodeling of alveolar septa in surfactant protein D-deficient mice

    PubMed Central

    Atochina-Vasserman, Elena N.; Massa, Christopher B.; Birkelbach, Bastian; Guo, Chang-Jiang; Scott, Pamela; Haenni, Beat; Beers, Michael F.; Ochs, Matthias; Gow, Andrew J.

    2015-01-01

    Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd−/−) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd−/− mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd−/− mice. These changes were reduced in DiNOS, and compared with Sftpd−/− mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd−/−. Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces. PMID:26320150

  17. The role of inducible nitric oxide synthase for interstitial remodeling of alveolar septa in surfactant protein D-deficient mice.

    PubMed

    Knudsen, Lars; Atochina-Vasserman, Elena N; Massa, Christopher B; Birkelbach, Bastian; Guo, Chang-Jiang; Scott, Pamela; Haenni, Beat; Beers, Michael F; Ochs, Matthias; Gow, Andrew J

    2015-11-01

    Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd(-/-)) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd(-/-) mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd(-/-) mice. These changes were reduced in DiNOS, and compared with Sftpd(-/-) mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd(-/-). Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces.

  18. Type IV pilus glycosylation mediates resistance of Pseudomonas aeruginosa to opsonic activities of the pulmonary surfactant protein A.

    PubMed

    Tan, Rommel M; Kuang, Zhizhou; Hao, Yonghua; Lee, Francis; Lee, Timothy; Lee, Ryan J; Lau, Gee W

    2015-04-01

    Pseudomonas aeruginosa is a major bacterial pathogen commonly associated with chronic lung infections in cystic fibrosis (CF). Previously, we have demonstrated that the type IV pilus (Tfp) of P. aeruginosa mediates resistance to antibacterial effects of pulmonary surfactant protein A (SP-A). Interestingly, P. aeruginosa strains with group I pilins are O-glycosylated through the TfpO glycosyltransferase with a single subunit of O-antigen (O-ag). Importantly, TfpO-mediated O-glycosylation is important for virulence in mouse lungs, exemplified by more frequent lung infection in CF with TfpO-expressing P. aeruginosa strains. However, the mechanism underlying the importance of Tfp glycosylation in P. aeruginosa pathogenesis is not fully understood. Here, we demonstrated one mechanism of increased fitness mediated by O-glycosylation of group 1 pilins on Tfp in the P. aeruginosa clinical isolate 1244. Using an acute pneumonia model in SP-A+/+ versus SP-A-/- mice, the O-glycosylation-deficient ΔtfpO mutant was found to be attenuated in lung infection. Both 1244 and ΔtfpO strains showed equal levels of susceptibility to SP-A-mediated membrane permeability. In contrast, the ΔtfpO mutant was more susceptible to opsonization by SP-A and by other pulmonary and circulating opsonins, SP-D and mannose binding lectin 2, respectively. Importantly, the increased susceptibility to phagocytosis was abrogated in the absence of opsonins. These results indicate that O-glycosylation of Tfp with O-ag specifically confers resistance to opsonization during host-mediated phagocytosis.

  19. Non-ionic Surfactants and Non-Catalytic Protein Treatment on Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass

    NASA Astrophysics Data System (ADS)

    Zheng, Yi; Pan, Zhongli; Zhang, Ruihong; Wang, Donghai; Jenkins, Bryan

    Our previous research has shown that saline Creeping Wild Ryegrass (CWR), Leymus triticoides, has a great potential to be used for bioethanol production because of its high fermentable sugar yield, up to 85% cellulose conversion of pretreated CWR. However, the high cost of enzyme is still one of the obstacles making large-scale lignocellulosic bioethanol production economically difficult. It is desirable to use reduced enzyme loading to produce fermentable sugars with high yield and low cost. To reduce the enzyme loading, the effect of addition of non-ionic surfactants and non-catalytic protein on the enzymatic hydrolysis of pretreated CWR was investigated in this study. Tween 20, Tween 80, and bovine serum albumin (BSA) were used as additives to improve the enzymatic hydrolysis of dilute sulfuric-acid-pretreated CWR. Under the loading of 0.1 g additives/g dry solid, Tween 20 was the most effective additive, followed by Tween 80 and BSA. With the addition of Tween 20 mixed with cellulase loading of 15 FPU/g cellulose, the cellulose conversion increased 14% (from 75 to 89%), which was similar to that with cellulase loading of 30 FPU/g cellulose and without additive addition. The results of cellulase and BSA adsorption on the Avicel PH101, pretreated CWR, and lignaceous residue of pretreated CWR support the theory that the primary mechanism behind the additives is prevention of non-productive adsorption of enzymes on lignaceous material of pretreated CWR. The addition of additives could be a promising technology to improve the enzymatic hydrolysis by reducing the enzyme activity loss caused by non-productive adsorption.

  20. Comparison of diffusion by anionic surfactants through cellulose acetate and collagen membranes.

    PubMed

    García Ramón, M T; Ribosa, I; Leal, J S; Parra, J L

    1989-06-01

    Synopsis From a dermatological point of view, it is important to know what is the irritation potential of surfactants on human skin. Recent research trends have been oriented towards the establishment of new 'in vitro' techniques that will avoid animal experimentation. In this paper, some results on the rate of diffusion of different anionic surfactants through both cellulose acetate and collagen membranes are described. A correlation between results of diffusion through the protein membrane and results published on the same surfactants and their irritation potential during 'in vivo' experiments appears possible.

  1. How to overcome surfactant dysfunction in meconium aspiration syndrome?

    PubMed

    Mokra, Daniela; Calkovska, Andrea

    2013-06-01

    Surfactant dysfunction in meconium aspiration syndrome (MAS) is caused by meconium components, by plasma proteins leaking through the injured alveolocapillary membrane and by substances originated in meconium-induced inflammation. Surfactant inactivation in MAS may be diminished by several ways. Firstly, aspirated meconium should be removed from the lungs to decrease concentrations of meconium inhibitors coming into the contact with surfactant in the alveolar compartment. Once the endogenous surfactant becomes inactivated, components of surfactant should be substituted by exogenous surfactant at a sufficient dose, and surfactant administration should be repeated, if oxygenation remains compromised. To delay the inactivation by inhibitors, exogenous surfactants may be enriched with surfactant proteins, phospholipids, or other substances such as polymers. Finally, to diminish an adverse action of products of meconium-induced inflammation on both endogenous and exogenously delivered surfactant, anti-inflammatory drugs may be administered. A combined therapeutic approach may result in better outcome in patients with MAS and in lower costs of treatment.

  2. Regulators of G protein signalling proteins in the human myometrium.

    PubMed

    Ladds, Graham; Zervou, Sevasti; Vatish, Manu; Thornton, Steven; Davey, John

    2009-05-21

    The contractile state of the human myometrium is controlled by extracellular signals that promote relaxation or contraction. Many of these signals function through G protein-coupled receptors at the cell surface, stimulating heterotrimeric G proteins and leading to changes in the activity of effector proteins responsible for bringing about the response. G proteins can interact with multiple receptors and many different effectors and are key players in the response. Regulators of G protein signalling (RGS) proteins are GTPase activating proteins for heterotrimeric G proteins and help terminate the signal. Little is known about the function of RGS proteins in human myometrium and we have therefore analysed transcript levels for RGS proteins at various stages of pregnancy (non-pregnant, preterm, term non-labouring, term labouring). RGS2 and RGS5 were the most abundantly expressed isolates in each of the patient groups. The levels of RGS4 and RGS16 (and to a lesser extent RGS2 and RGS14) increased in term labouring samples relative to the other groups. Yeast two-hybrid analysis and co-immunoprecipitation in myometrial cells revealed that both RGS2 and RGS5 interact directly with the cytoplasmic tail of the oxytocin receptor, suggesting they might help regulate signalling through this receptor.

  3. Tyrosine fluorescence probing of the surfactant-induced conformational changes of albumin.

    PubMed

    Zhdanova, Nadezda G; Shirshin, Evgeny A; Maksimov, Eugene G; Panchishin, Ivan M; Saletsky, Alexander M; Fadeev, Victor V

    2015-05-01

    Tyrosine fluorescence in native proteins is known to be effectively quenched, whereas its emission increases upon proteins' unfolding. This suggests that tyrosine fluorescence could be exploited for probing structural rearrangements of proteins in addition to the extensively used tryptophan emission. We studied the possibility of using tyrosine fluorescence as an indicator of surfactant-induced conformational changes in albumins. It was shown that fluorescence of tyrosine residues, which are uniformly distributed all over the protein molecules, allows the detection of subtle structural rearrangements of proteins upon surfactant binding, which do not influence the properties of a single tryptophan residue buried in the inner hydrophobic region of human serum albumin. Tyrosine fluorescence properties, including its fluorescence lifetime, revealed the multistage character of surfactant binding to albumin, consistent with the data provided by other methods. The obtained results demonstrate the possibility of probing conformational changes in proteins using tyrosine photophysical parameters as indicators.

  4. Surfactant as a critical factor when tuning the hydrophilicity in three-dimensional polyester-based scaffolds: impact of hydrophilicity on their mechanical properties and the cellular response of human osteoblast-like cells.

    PubMed

    Sun, Yang; Xing, Zhe; Xue, Ying; Mustafa, Kamal; Finne-Wistrand, Anna; Albertsson, Ann-Christine

    2014-04-14

    In tissue engineering, the hydrophilicity of porous scaffolds is essential and influences protein and cell adhesion as well as nutrient diffusion into the scaffold. The relative low hydrophilicity of degradable polyesters, which limits diffusion of nutrients, is a major drawback in large porous scaffolds of these materials when used for bone tissue engineering and repair of critical size defects. Designing porous biodegradable polymer scaffolds with improved hydrophilicity, while maintaining their mechanical, thermal, and degradation properties is therefore of clinical interest. Here, surfactants were used to tune the hydrophilicity and material properties. A total of 3-20% (w/w) of surfactant, polysorbate 80 (Tween 80), was used as an additive in poly(l-lactide-co-1,5-dioxepan-2-one) [poly(LLA-co-DXO)] and poly(l-lactide)-co-(ε-caprolactone) [poly(LLA-co-CL)] scaffolds. A significantly decreased water contact angle was recorded for all the blends and the crystallinity, glass transition temperature and crystallization temperature were reduced with increased amounts of surfactant. Copolymers with the addition of 3% Tween 80 had comparable mechanical properties as the pristine copolymers. However, the E-modulus and tensile stress of copolymers decreased significantly with the addition of 10 and 20% Tween 80. Initial cell response of the material was evaluated by seeding human osteoblast-like cells (HOB) on the scaffolds. The addition of 3% Tween 80 did not significantly influence cell attachment or proliferation, while 20% Tween 80 significantly inhibited osteoblast proliferation. RT-PCR results showed that 3% Tween 80 stimulated mRNA expression of alkaline phosphatase (ALP), osteoprotegerin (OPG), and bone morphogenetic protein-2 (BMP-2).

  5. Effect of exogenous surfactant on the development of surfactant synthesis in premature rabbit lung.

    PubMed

    Amato, Maurizio; Petit, Kevin; Fiore, Humberto H; Doyle, Cynthia A; Frantz, Ivan D; Nielsen, Heber C

    2003-04-01

    Surfactant replacement is an effective therapy for neonatal respiratory distress syndrome. Full recovery from respiratory distress syndrome requires development of endogenous surfactant synthesis and metabolism. The influence of exogenous surfactant on the development of surfactant synthesis in premature lungs is not known. We hypothesized that different exogenous surfactants have different effects on the development of endogenous surfactant production in the premature lung. We treated organ cultures of d 25 fetal rabbit lung for 3 d with 100 mg/kg body weight of natural rabbit surfactant, Survanta, and Exosurf and measured their effects on the development of surfactant synthesis. Additional experiments tested how these surfactants and Curosurf affected surfactant protein (SP) SP-A, SP-B, and SP-C mRNA expression. Surfactant synthesis was measured as the incorporation of 3H-choline and 14C-glycerol into disaturated phosphatidylcholine recovered from lamellar bodies. Randomized-block ANOVA showed significant differences among treatments for incorporation of both labels (p < 0.01), with natural rabbit surfactant less than control, Survanta greater than control, and Exosurf unchanged. Additional experiments with natural rabbit surfactant alone showed no significant effects in doses up to 1000 mg/kg. Survanta stimulated disaturated phosphatidylcholine synthesis (173 +/- 41% of control; p = 0.01), increased total lamellar body disaturated phosphatidylcholine by 22% (p < 0.05), and increased 14C-disat-PC specific activity by 35% (p < 0.05). The response to Survanta was dose-dependent up to 1000 mg/kg. Survanta did not affect surfactant release. No surfactant altered the expression of mRNA for SP-A, SP-B, or SP-C. We conclude that surfactant replacement therapy can enhance the maturation of surfactant synthesis, but this potential benefit differs with different surfactant preparations.

  6. Computational Prediction of Protein-Protein Interactions of Human Tyrosinase

    PubMed Central

    Wang, Su-Fang; Oh, Sangho; Si, Yue-Xiu; Wang, Zhi-Jiang; Han, Hong-Yan; Lee, Jinhyuk; Qian, Guo-Ying

    2012-01-01

    The various studies on tyrosinase have recently gained the attention of researchers due to their potential application values and the biological functions. In this study, we predicted the 3D structure of human tyrosinase and simulated the protein-protein interactions between tyrosinase and three binding partners, four and half LIM domains 2 (FHL2), cytochrome b-245 alpha polypeptide (CYBA), and RNA-binding motif protein 9 (RBM9). Our interaction simulations showed significant binding energy scores of −595.3 kcal/mol for FHL2, −859.1 kcal/mol for CYBA, and −821.3 kcal/mol for RBM9. We also investigated the residues of each protein facing toward the predicted site of interaction with tyrosinase. Our computational predictions will be useful for elucidating the protein-protein interactions of tyrosinase and studying its binding mechanisms. PMID:22577521

  7. A comparative human health risk assessment of p-dichlorobenzene-based toilet rimblock products versus fragrance/surfactant-based alternatives.

    PubMed

    Aronson, Dallas B; Bosch, Stephen; Gray, D Anthony; Howard, Philip H; Guiney, Patrick D

    2007-10-01

    A comparison of the human health risk to consumers using one of two types of toilet rimblock products, either a p-dichlorobenzene-based rimblock or two newer fragrance/surfactant-based alternatives, was conducted. Rimblock products are designed for global use by consumers worldwide and function by releasing volatile compounds into indoor air with subsequent exposure presumed to be mainly by inhalation of indoor air. Using the THERdbASE exposure model and experimentally determined emission data, indoor air concentrations and daily intake values were determined for both types of rimblock products. Modeled exposure concentrations from a representative p-dichlorobenzene rimblock product are an order of magnitude higher than those from the alternative rimblock products due to its nearly pure composition and high sublimation rate. Lifetime exposure to p-dichlorobenzene or the subset of fragrance components with available RfD values is not expected to lead to non-cancer-based adverse health effects based on the exposure concentrations estimated using the THERdbASE model. A similar comparison of cancer-based effects was not possible as insufficient data were available for the fragrance components.

  8. Comparison of in vitro eye irritation potential by bovine corneal opacity and permeability (BCOP) assay to erythema scores in human eye sting test of surfactant-based formulations.

    PubMed

    Cater, Kathleen C; Harbell, John W

    2008-01-01

    The bovine corneal opacity and permeability (BCOP) assay can be used to predict relative eye irritation potential of surfactant-based personal care formulations relative to a corporate benchmark. The human eye sting test is typically used to evaluate product claims of no tears/no stinging for children's bath products. A preliminary investigation was conducted to test a hypothesis that the BCOP assay could be used as a prediction model for relative ranking of human eye irritation responses under conditions of a standard human eye sting test to surfactant-based formulations. BCOP assays and human eye sting tests were conducted on 4 commercial and 1 prototype body wash (BW) developed specifically for children or as mild bath products. In the human eye sting test, 10 mul of a 10% dosing solution is instilled into one eye of each panelist (n = 20), and the contralateral eye is dosed with sterile water as a control. Bulbar conjunctival erythema responses of each eye are graded at 30 seconds by an ophthalmologist. The BCOP assay permeability values (optical density at 490 nm [OD(490)]) for the 5 BWs ranged from 0.438 to 1.252 (i.e., least to most irritating). By comparison, the number of panelists exhibiting erythema responses (mild to moderately pink) ranged from 3 of 20 panelists for the least irritating BW to 10 of 20 panelists for the most irritating BW tested. The relative ranking of eye irritation potential of the 5 BWs in the BCOP assay compares favorably with the relative ranking of the BWs in the human eye sting test. Based on these findings, the permeability endpoint of the BCOP assay, as described for surfactant-based formulations, showed promise as a prediction model for relative ranking of conjunctival erythema responses in the human eye. Consequently, screening of prototype formulations in the BCOP assay would allow for formula optimization of mild bath products prior to investment in a human eye sting test.

  9. Surfactant Protein A and B Gene Polymorphisms and Risk of Respiratory Distress Syndrome in Late-Preterm Neonates

    PubMed Central

    Tsitoura, Maria-Eleni I.; Stavrou, Eleana F.; Maraziotis, Ioannis A.; Sarafidis, Kosmas; Athanassiadou, Aglaia; Dimitriou, Gabriel

    2016-01-01

    Background and Objectives Newborns delivered late-preterm (between 340/7 and 366/7 weeks of gestation) are at increased risk of respiratory distress syndrome (RDS). Polymorphisms within the surfactant protein (SP) A and B gene have been shown to predispose to RDS in preterm neonates. The aim of this study was to investigate whether specific SP-A and/or SP-B genetic variants are also associated with RDS in infants born late-preterm. Methods This prospective cross-sectional study included 56 late-preterm infants with and 60 without RDS. Specific SP-A1/SP-A2 haplotypes and SP-B Ile131Thr polymorphic alleles were determined in blood specimens using polymerase-chain-reaction and DNA sequencing. Results The SP-A1 6A4 and the SP-A2 1A5 haplotypes were significantly overrepresented in newborns with RDS compared to controls (OR 2.86, 95%CI 1.20–6.83 and OR 4.68, 95%CI 1.28–17.1, respectively). The distribution of the SP-B Ile131Thr genotypes was similar between the two late-preterm groups. Overall, the SP-A1 6A4 or/and SP-A2 1A5 haplotype was present in 20 newborns with RDS (35.7%), resulting in a 4.2-fold (1.60–11.0) higher probability of RDS in carriers. Multivariable regression analysis revealed that the effect of SP-A1 6A4 and SP-A2 1A5 haplotypes was preserved when adjusting for known risk or protective factors, such as male gender, smaller gestational age, smaller weight, complications of pregnancy, and administration of antenatal corticosteroids. Conclusions Specific SP-A genetic variants may influence the susceptibility to RDS in late-preterm infants, independently of the effect of other perinatal factors. PMID:27835691

  10. In vivo effect of surfactant on inflammatory cytokines during endotoxin-induced lung injury in rodents.

    PubMed

    Mittal, Neha; Sanyal, Sankar Nath

    2011-01-01

    Lipopolysaccharide (LPS) is a known inducer of acute respiratory distress syndrome (ARDS) in humans and animals. In this study, ARDS was developed in rats by intratracheal instillation of LPS and the effect of two types of surfactant (natural vs. synthetic) was examined to determine their potential corrective roles in general, as well as to compare the two surfactants against one another in particular, in endotoxin-induced lung injury. Sprague-Dawley male rats were divided into four groups, i.e., rats given: buffer controls; 055:B5 E. coli LPS only; LPS and then porcine surfactant (P-SF); or, LPS and then synthetic surfactant (S-SF). In vivo administration of LPS led to an increase in expression of the cytokines tumor necrosis factor-α, interleukin (IL)-1β, IL-2, IL-4, interferon-γ, monocyte chemotactic protein-1, and macrophage inflammatory protein-1β in the lungs of rats. These effects were confirmed by immunofluorescence in lung tissue sections and/or by protein (Western immunoblot) and mRNA expression (reverse transcription polymerase chain reaction) analyses of tissue samples. Apart from IL-4, concentrations of each of these cytokines in bronchoalveolar lavage fluid recovered from the animals were significantly increased in the LPS-treated hosts. Instillation of either surfactant (70 h after the LPS) into the airways diminished the expression of each of the inducible-cytokines, with the porcine (natural) form seeming having the greater inhibitory effect. These data suggest that surfactant can play an important role in the treatment of endotoxin-induced lung injury and might possess robust anti-inflammatory effects. Further, it seems that both the natural and synthetic surfactants prevent inflammatory outcomes in the lungs by controlling cytokine(s) production by various inflammatory cells. Last, the studies here clearly indicated that in this aspect, natural surfactant appears to be more beneficial compared to synthetic surfactant.

  11. Surfactant compositions

    SciTech Connect

    Novakovic, M.; Abend, P.G.

    1987-09-29

    A surfactant composition is described for subsequent addition to a soap slurring comprising an acyloxy alkane sulfonate salt. The sulfonate salt is present in an amount by weight of about 44 percent of about 56 percent. The polyol is present in an amount by weight of about 2 percent to about 6 percent, and water is present in an amount by weight of 26 to 36 percent. The composition constituting a solid reversible solution at ambient temperature and having a solids content of about 58 to 72 percent, whereby subsequent addition of the surfactant composition to a soap slurry results in formation of a soap/detergent bar having a smooth texture, uniform wear properties and a lack of grittiness.

  12. Protein aggregation profile of the human kinome

    PubMed Central

    Graña-Montes, Ricardo; Sant'Anna de Oliveira, Ricardo; Ventura, Salvador

    2012-01-01

    Protein aggregation into amyloid fibrils is associated with the onset of an increasing number of human disorders, including Alzheimer's disease, diabetes, and some types of cancer. The ability to form toxic amyloids appears to be a property of most polypeptides. Accordingly, it has been proposed that reducing aggregation and its effect in cell fitness is a driving force in the evolution of proteins sequences. This control of protein solubility should be especially important for regulatory hubs in biological networks, like protein kinases. These enzymes are implicated in practically all processes in normal and abnormal cell physiology, and phosphorylation is one of the most frequent protein modifications used to control protein activity. Here, we use the AGGRESCAN algorithm to study the aggregation propensity of kinase sequences. We compared them with the rest of globular proteins to decipher whether they display differential aggregation properties. In addition, we compared the human kinase complement with the kinomes of other organisms to see if we can identify any evolutionary trend in the aggregational properties of this protein superfamily. Our analysis indicates that kinase domains display significant aggregation propensity, a property that decreases with increasing organism complexity. PMID:23181023

  13. (PCG) Protein Crystal Growth Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Human Serum Albumin. Contributes to many transport and regulatory processes and has multifunctional binding properties which range from various metals, to fatty acids, hormones, and a wide spectrum of therapeutic drugs. The most abundant protein of the circulatory system. It binds and transports an incredible variety of biological and pharmaceutical ligands throughout the blood stream. Principal Investigator on STS-26 was Larry DeLucas.

  14. New Anthocyanin-Human Salivary Protein Complexes.

    PubMed

    Ferrer-Gallego, Raúl; Soares, Susana; Mateus, Nuno; Rivas-Gonzalo, Julián; Escribano-Bailón, M Teresa; de Freitas, Victor

    2015-08-04

    The interaction between phenolic compounds and salivary proteins is considered the basis of the poorly understood phenomenon of astringency. Furthermore, this interaction is an important factor in relation to their bioavailability. In this work, interactions between anthocyanin and human salivary protein fraction were studied by mass spectrometry (MALDI-TOF-MS and FIA-ESI-MS) and saturation-transfer difference (STD) NMR spectroscopy. Anthocyanins were able to interact with saliva proteins. The dissociation constant (KD) between malvidin 3-glucoside and salivary proline-rich proteins was 1.92 mM for the hemiketal form (pH 3.4) and 1.83 mM for the flavylium cation (pH 1.0). New soluble complexes between these salivary proteins and malvidin 3-glucoside were identified for the first time.

  15. Nuclear matrix proteins in human colon cancer.

    PubMed Central

    Keesee, S K; Meneghini, M D; Szaro, R P; Wu, Y J

    1994-01-01

    The nuclear matrix is the nonchromatin scaffolding of the nucleus. This structure confers nuclear shape, organizes chromatin, and appears to contain important regulatory proteins. Tissue specific nuclear matrix proteins have been found in the rat, mouse, and human. In this study we compared high-resolution two-dimensional gel electropherograms of nuclear matrix protein patterns found in human colon tumors with those from normal colon epithelia. Tumors were obtained from 18 patients undergoing partial colectomy for adenocarcinoma of the colon and compared with tissue from 10 normal colons. We have identified at least six proteins which were present in 18 of 18 colon tumors and 0 of 10 normal tissues, as well as four proteins present in 0 of 18 tumors and in 10 of 10 normal tissues. These data, which corroborate similar findings of cancer-specific nuclear matrix proteins in prostate and breast, suggest that nuclear matrix proteins may serve as important markers for at least some types of cancer. Images PMID:8127905

  16. [Cow's milk protein allergy through human milk].

    PubMed

    Denis, M; Loras-Duclaux, I; Lachaux, A

    2012-03-01

    Cow's milk protein allergy (CMPA) is the first allergy that affects infants. In this population, the incidence rate reaches 7.5%. The multiplicity and aspecificity of the symptoms makes its diagnosis sometimes complicated, especially in the delayed type (gastrointestinal, dermatological, and cutaneous). CMPA symptoms can develop in exclusively breastfed infants with an incidence rate of 0.5%. It, therefore, raises questions about sensitization to cow's milk proteins through breast milk. Transfer of native bovine proteins such as β-lactoglobulin into the breast milk is controversial: some authors have found bovine proteins in human milk but others point to cross-reactivity between human milk proteins and cow's milk proteins. However, it seems that a small percentage of dietary proteins can resist digestion and become potentially allergenic. Moreover, some authors suspect the transfer of some of these dietary proteins from the maternal bloodstream to breast milk, but the mechanisms governing sensitization are still being studied. Theoretically, CMPA diagnosis is based on clinical observations, prick-test or patch-test results, and cow's milk-specific IgE antibody concentration. A positive food challenge test usually confirms the diagnosis. No laboratory test is available to make a certain diagnosis, but the detection of eosinophil cationic protein (ECP) in the mother's milk, for example, seems to be advantageous since it is linked to CMA. Excluding cow's milk from the mother's diet is the only cure when she still wants to breastfeed. Usually, cow's milk proteins are reintroduced after 6 months of exclusion. Indeed, the prognosis for infants is very good: 80% acquire a tolerance before the age of 3 or 4 years. Mothers should not avoid dairy products during pregnancy and breastfeeding as preventive measures against allergy.

  17. Surfactant lipids regulate LPS-induced interleukin-8 production in A549 lung epithelial cells by inhibiting translocation of TLR4 into lipid raft domains

    PubMed Central

    Abate, Wondwossen; Alghaithy, Abdulaziz A.; Parton, Joan; Jones, Kenneth P.; Jackson, Simon K.

    2010-01-01

    In addition to providing mechanical stability, growing evidence suggests that surfactant lipid components can modulate inflammatory responses in the lung. However, little is known of the molecular mechanisms involved in the immunomodulatory action of surfactant lipids. This study investigates the effect of the lipid-rich surfactant preparations Survanta®, Curosurf®, and the major surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) on interleukin-8 (IL-8) gene and protein expression in human A549 lung epithelial cells using immunoassay and PCR techniques. To examine potential mechanisms of the surfactant lipid effects, Toll-like receptor 4 (TLR4) expression was analyzed by flow cytometry, and membrane lipid raft domains were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The lipid-rich surfactant preparations Survanta®, Curosurf®, and DPPC, at physiological concentrations, significantly downregulated lipopolysaccharide (LPS)-induced IL-8 expression in A549 cells both at the mRNA and protein levels. The surfactant preparations did not affect the cell surface expression of TLR4 or the binding of LPS to the cells. However, LPS treatment induced translocation of TLR4 into membrane lipid raft microdomains, and this translocation was inhibited by incubation of the cells with the surfactant lipid. This study provides important mechanistic details of the immune-modulating action of pulmonary surfactant lipids. PMID:19648651

  18. Effect of irradiation/bone marrow transplantation on alveolar epithelial type II cells is aggravated in surfactant protein D deficient mice.

    PubMed

    Mühlfeld, Christian; Madsen, Jens; Mackay, Rose-Marie; Schneider, Jan Philipp; Schipke, Julia; Lutz, Dennis; Birkelbach, Bastian; Knudsen, Lars; Botto, Marina; Ochs, Matthias; Clark, Howard

    2017-01-01

    Irradiation followed by bone marrow transplantation (BM-Tx) is a frequent therapeutic intervention causing pathology to the lung. Although alveolar epithelial type II (AE2) cells are essential for lung function and are damaged by irradiation, the long-term consequences of irradiation and BM-Tx are not well characterized. In addition, it is unknown whether surfactant protein D (SP-D) influences the response of AE2 cells to the injurious events. Therefore, wildtype (WT) and SP-D(-/-) mice were subjected to a myeloablative whole body irradiation dose of 8 Gy and subsequent BM-Tx and compared with age- and sex-matched untreated controls. AE2 cell changes were investigated quantitatively by design-based stereology. Compared with WT, untreated SP-D(-/-) mice showed a higher number of larger sized AE2 cells and a greater amount of surfactant-storing lamellar bodies. Irradiation and BM-Tx induced hyperplasia and hypertrophy in WT and SP-D(-/-) mice as well as the formation of giant lamellar bodies. The experimentally induced alterations were more severe in the SP-D(-/-) than in the WT mice, particularly with respect to the surfactant-storing lamellar bodies which were sometimes extremely enlarged in SP-D(-/-) mice. In conclusion, irradiation and BM-Tx have profound long-term effects on AE2 cells and their lamellar bodies. These data may explain some of the clinical pulmonary consequences of this procedure. The data should also be taken into account when BM-Tx is used as an experimental procedure to investigate the impact of bone marrow-derived cells for the phenotype of a specific genotype in the mouse.

  19. Protein Phosphatase 1α Interacting Proteins in the Human Brain

    PubMed Central

    Esteves, Sara L.C.; Domingues, Sara C.; da Cruz e Silva, Odete A.B.; da Cruz e Silva, Edgar F.

    2012-01-01

    Abstract Protein Phosphatase 1 (PP1) is a major serine/threonine-phosphatase whose activity is dependent on its binding to regulatory subunits known as PP1 interacting proteins (PIPs), responsible for targeting PP1 to a specific cellular location, specifying its substrate or regulating its action. Today, more than 200 PIPs have been described involving PP1 in panoply of cellular mechanisms. Moreover, several PIPs have been identified that are tissue and event specific. In addition, the diversity of PP1/PIP complexes can further be achieved by the existence of several PP1 isoforms that can bind preferentially to a certain PIP. Thus, PP1/PIP complexes are highly specific for a particular function in the cell, and as such, they are excellent pharmacological targets. Hence, an in-depth survey was taken to identify specific PP1α PIPs in human brain by a high-throughput Yeast Two-Hybrid approach. Sixty-six proteins were recognized to bind PP1α, 39 being novel PIPs. A large protein interaction databases search was also performed to integrate with the results of the PP1α Human Brain Yeast Two-Hybrid and a total of 246 interactions were retrieved. PMID:22321011

  20. Absorption-enhancing effects of gemini surfactant on the intestinal absorption of poorly absorbed hydrophilic drugs including peptide and protein drugs in rats.

    PubMed

    Alama, Tammam; Kusamori, Kosuke; Katsumi, Hidemasa; Sakane, Toshiyasu; Yamamoto, Akira

    2016-02-29

    In general, the intestinal absorption of small hydrophilic molecules and macromolecules like peptides, after oral administration is very poor. Absorption enhancers are considered to be one of the most promising agents to enhance the intestinal absorption of drugs. In this research, we focused on a gemini surfactant, a new type of absorption enhancer. The intestinal absorption of drugs, with or without sodium dilauramidoglutamide lysine (SLG-30), a gemini surfactant, was examined by an in situ closed-loop method in rats. The intestinal absorption of 5(6)-carboxyfluorescein (CF) and fluorescein isothiocyanate-dextrans (FDs) was significantly enhanced in the presence of SLG-30, such effect being reversible. Furthermore, the calcium levels in the plasma significantly decreased when calcitonin was co-administered with SLG-30, suggestive of the increased intestinal absorption of calcitonin. In addition, no significant increase in the of lactate dehydrogenase (LDH) activity or in protein release from the intestinal epithelium was observed in the presence of SLG-30, suggestive of the safety of this compound. These findings indicate that SLG-30 is an effective absorption-enhancer for improving the intestinal absorption of poorly absorbed drugs, without causing serious damage to the intestinal epithelium.

  1. Surfactants in the management of rhinopathologies

    PubMed Central

    Rosen, Philip L.; Palmer, James N.; O'Malley, Bert W.

    2013-01-01

    Background: Surfactants are a class of amphiphilic surface active compounds that show several unique physical properties at liquid–liquid or liquid–solid surface interfaces including the ability to increase the solubility of substances, lower the surface tension of a liquid, and decrease friction between two mediums. Because of these unique physical properties several in vitro, ex vivo, and human trials have examined the role of surfactants as stand-alone or adjunct therapy in recalcitrant chronic rhinosinusitis (CRS). Methods: A review of the literature was performed. Results: The data from three different surfactants have been examined in this review: citric acid zwitterionic surfactant (CAZS; Medtronic ENT, Jacksonville FL), Johnson's Baby Shampoo (Johnson & Johnson, New Brunswick NJ), and SinuSurf (NeilMed Pharmaceuticals, Santa Rosa, CA). Dilute surfactant therapy shows in vitro antimicrobial effects with modest inhibition of bacterial biofilm formation. In patients with CRS, surfactants may improve symptoms, most likely through its mucolytic effects. In addition, surfactants have several distinct potential benefits including their ability to improve an irrigant's penetration of the nonoperated sinus and their synergistic effects with antibiotics. However, surfactants potential for nasal irritation and possible transient ciliotoxicity may limit their use. Conclusion: Recent data suggest a possible therapeutic role of surfactants in treating rhinopathologies associated with mucostasis. Further investigation, including a standardization of surfactant formulations, is warranted to further elucidate the potential benefits and drawbacks of this therapy. PMID:23710951

  2. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  3. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Plasma Protein Fraction (Human). 640.90 Section 640...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  4. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  5. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  6. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  7. Antibiotic proteins of human polymorphonuclear leukocytes.

    PubMed Central

    Gabay, J E; Scott, R W; Campanelli, D; Griffith, J; Wilde, C; Marra, M N; Seeger, M; Nathan, C F

    1989-01-01

    Nine polypeptide peaks with antibiotic activity were resolved from human polymorphonuclear leukocyte azurophil granule membranes. All but 1 of the 12 constituent polypeptides were identified by N-terminal sequence analysis. Near quantitative recovery of protein and activity permitted an assessment of the contribution of each species to the overall respiratory-burst-independent antimicrobial capacity of the cell. Three uncharacterized polypeptides were discovered, including two broad-spectrum antibiotics. One of these, a defensin that we have designated human neutrophil antimicrobial peptide 4, was more potent than previously described defensins but represented less than 1% of the total protein. The other, named azurocidin, was abundant and comparable to bactericidal permeability-increasing factor in its contribution to the killing of Escherichia coli. Images PMID:2501794

  8. Separation of human, bovine, and porcine insulins, three very closely related proteins, by micellar electrokinetic chromatography.

    PubMed

    Lamalle, Caroline; Roland, Diane; Crommen, Jacques; Servais, Anne-Catherine; Fillet, Marianne

    2015-10-01

    Human, bovine, and porcine insulins are small proteins with very closely related amino acid sequences, which makes their separation challenging. In this study, we took advantage of the high-resolution power of CE, and more particularly of micellar electrokinetic chromatography, to separate those biomolecules. Among several surfactants, perfluorooctanoic acid ammonium salt was selected. Then, using a design of experiments approach, the optimal BGE composition was found to consist of 50 mM ammonium acetate pH 9.0, 65 mM perfluorooctanoic acid ammonium salt, and 4% MeOH. The three insulins could be separated within 12 min with a satisfactory resolution. This method could be useful to detect possible counterfeit pharmaceutical formulations. Indeed, it would be easy to determine if human insulin was replaced by bovine or porcine insulin.

  9. Cow's milk proteins in human milk.

    PubMed

    Coscia, A; Orrù, S; Di Nicola, P; Giuliani, F; Rovelli, I; Peila, C; Martano, C; Chiale, F; Bertino, E

    2012-01-01

    Cow's milk proteins (CMPs) are among the best characterized food allergens. Cow's milk contains more than twenty five different proteins, but only whey proteins alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin (BSA), and lactoferrin, as well as the four caseins, have been identified as allergens. Aim of this study was to investigate by proteomics techniques cow's milk allergens in human colostrum of term and preterm newborns' mothers, not previously detected, in order to understand if such allergens could be cause of sensitization during lactation. Term colostrum samples from 62 healthy mothers and preterm colostrum samples from 11 healthy mothers were collected for this purpose. The most relevant finding was the detection of the intact bovine alpha-S1-casein in both term and preterm colostrum. Using this method, which allows direct proteins identification, beta-lactoglobulin was not detected in any of colostrum samples. According to our results bovine alpha 1 casein that is considered a major cow's milk allergen is readily secreted in human milk: further investigations are needed in order to clarify if alpha-1-casein has a major role in sensitization or tolerance to cow's milk of exclusively breastfed predisposed infants.

  10. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts.

    PubMed

    Carmona-Rodríguez, Bruno; Alvarez-Pérez, Marco Antonio; Narayanan, A Sampath; Zeichner-David, Margarita; Reyes-Gasga, José; Molina-Guarneros, Juan; García-Hernández, Ana Lilia; Suárez-Franco, José Luis; Chavarría, Ivet Gil; Villarreal-Ramírez, Eduardo; Arzate, Higinio

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  11. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    SciTech Connect

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio . E-mail: harzate@servidor.unam.mx

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  12. Neuroproteomic profiling of human brain tissue using multidimensional separation techniques and selective enrichment of membrane proteins.

    PubMed

    Musunuri, Sravani; Shevchenko, Ganna; Bergquist, Jonas

    2012-12-01

    Hydrophobic membrane proteins (MPs) occupy a unique niche in the brain proteome research due to their important physiological roles. Therefore, the extraction, separation, and identification of MPs are of great interest in proteomic analysis. We applied various proteomic techniques to enrich, separate, and analyze the human brain proteome, including membrane proteome. Temperature-induced phase fractionation with the nonionic surfactant Triton X-114 was used to simultaneously extract, separate, and concentrate low abundant hydrophobic and high abundant hydrophilic proteins from human brain tissue. The extracted and delipidated proteins were analyzed by two-dimensional gel electrophoresis (2DE). Approximately 600 spots were detected in the gels. In-solution digestion was performed on 3 kDa spin filters. Tryptic peptides were separated using RP nano-LC and analyzed using two different high performance mass spectrometers, linear ion trap-Fourier transform and a linear ion trap-Orbitrap to reveal the low abundant MPs. In total, 837 and 780 unique proteins were identified by using linear ion trap-Fourier transform and linear ion trap-Orbitrap mass spectrometers, respectively. More than 29% of the identified proteins were classified as MPs with significant biological functions such as ion channels and transporters. Our study establishes a simple and rapid shotgun approach for the characterization of the brain proteome, and allows comprehensive analysis of brain membrane proteomes.

  13. Luminescent probe in the study of surfactant-induced structural changes in serum albumin in human blood plasma

    NASA Astrophysics Data System (ADS)

    Melnikov, A. G.; Pravdin, A. B.; Kochubey, V. I.; Melnikov, G. V.

    2005-06-01

    The luminescence-kinetic technique of the monitoring of structural changes in albumins of human blood plasma that uses a luminescent probe-eosin is proposed. Phosphorescence of eosin bound to the globular proteins of blood plasma-albumins was recorded at room temperature. It is found that under the action of sodium dodecylsulfate on the albumins the rate constant of eosin phosphorescence decay grows and the intensity of eosin phosphorescence decreases. It is assumed that these changes are connected with the denaturing of blood plasma albumins by sodium dodecylsulfate.

  14. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

  15. Enhanced surfactant protein and defensin mRNA levels and reduced viral replication during parainfluenza virus type 3 pneumonia in neonatal lambs.

    PubMed

    Grubor, Branka; Gallup, Jack M; Meyerholz, David K; Crouch, Erika C; Evans, Richard B; Brogden, Kim A; Lehmkuhl, Howard D; Ackermann, Mark R

    2004-05-01

    Defensins and surfactant protein A (SP-A) and SP-D are antimicrobial components of the pulmonary innate immune system. The purpose of this study was to determine the extent to which parainfluenza type 3 virus infection in neonatal lambs alters expression of sheep beta-defensin 1 (SBD-1), SP-A, and SP-D, all of which are constitutively transcribed by respiratory epithelia. Parainfluenza type 3 viral antigen was detected by immunohistochemistry (IHC) in the bronchioles of all infected lambs 3 days postinoculation and at diminished levels 6 days postinoculation, but it was absent 17 days postinoculation. At all times postinoculation, lung homogenates from parainfluenza type 3 virus-inoculated animals had increased SBD-1, SP-A, and SP-D mRNA levels as detected by fluorogenic real-time reverse transcriptase PCR. Protein levels of SP-A in lung homogenates detected by quantitative-competitive enzyme-linked immunosorbent assay and protein antigen of SP-A detected by IHC were not altered. These studies demonstrate that parainfluenza type 3 virus infection results in enhanced expression of constitutively transcribed innate immune factors expressed by respiratory epithelia and that this increased expression occurs concurrently with decreased viral replication.

  16. Surfactant-aided precipitation/on-pellet-digestion (SOD) procedure provides robust and rapid sample preparation for reproducible, accurate and sensitive LC/MS quantification of therapeutic protein in plasma and tissues.

    PubMed

    An, Bo; Zhang, Ming; Johnson, Robert W; Qu, Jun

    2015-04-07

    For targeted protein quantification by liquid chromatography mass spectrometry (LC/MS), an optimal approach for efficient, robust and hi-throughput sample preparation is critical, but often remains elusive. Here we describe a straightforward surfactant-aided-precipitation/on-pellet-digestion (SOD) strategy that provides effective sample cleanup and enables high and constant peptide yields in various matrices, allowing reproducible, accurate and sensitive protein quantification. This strategy was developed using quantification of monocolnocal antibody in tissues and plasma as the model system. Surfactant treatment before precipitation substantially increased peptide recovery and reproducibility from plasma/tissue, likely because surfactant permits extensive denaturation/reduction/alkylation of proteins and inactivation of endogenous protease inhibitors, and facilitates removal of matrix components. The subsequent precipitation procedure effectively eliminates the surfactant and nonprotein matrix components, and the thorough denaturation by both surfactant and precipitation enabled very rapid on-pellet-digestion (45 min at 37 °C) with high peptide recovery. The performance of SOD was systematically compared against in-solution-digestion, in-gel-digestion and filter-aided-sample-preparation (FASP) in plasma/tissues, and then examined in a full pharmacokinetic study in rats. SOD achieved the best peptide recovery (∼21.0-700% higher than the other three methods across various matrices), reproducibility (3.75-10.9%) and sensitivity (28-30 ng/g across plasma and tissue matrices), and its performance was independent of matrix types. Finally, in validation and pharmacokinetic studies in rats, SOD outperformed other methods and provided highly accurate and precise quantification in all plasma samples without using stable isotope labeled (SIL)-protein internal standard (I.S.). In summary, the SOD method has proven to be highly robust, efficient and rapid, making it readily

  17. The Mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary C-type lectin surfactant protein A.

    PubMed

    Ragas, Aude; Roussel, Lucie; Puzo, Germain; Rivière, Michel

    2007-02-23

    Tuberculosis is still a major health problem, and understanding the mechanism by which Mycobacterium tuberculosis (Mtb) invades and colonizes its host target cells remains an important issue for the control of infection. The innate immune system C-type lectins (C-TLs), including the human pulmonary surfactant protein A (PSP-A), have been recently identified as determinant players in the early recognition of the invading pathogen and in mounting the host defense response. Although the antigenic lipoglycan mannosylated lipoarabinomannan is currently considered to be the major C-TL target on the mycobacterial surface, the recognition by some C-TLs of the only mycobacterial species composing the "Mtb complex" indicates that mannosylated lipoarabinomannan cannot account alone for this specificity. Thus, we searched for the mycobacterial molecules targeted by human PSP-A, focusing our attention on the Mtb surface glycoproteins. We developed an original functional proteomic approach based on a lectin blot assay using crude human bronchoalveolar lavage fluid as a source of physiological PSP-A. Combined with selective cell-surface protein extraction and mass spectrometry peptide mapping, this strategy allowed us to identify the Apa (alanine- and proline-rich antigenic) glycoprotein as new potential target for PSP-A. This result was supported by direct binding of PSP-A to purified Apa. Moreover, EDTA addition or deglycosylation of purified Apa samples completely abolished the interaction, demonstrating that the interaction is calcium- and mannose-dependent, as expected. Finally, we provide convincing evidence that Apa, formerly considered as mainly secreted, is associated with the cell wall for a sufficiently long time to aid in the attachment of PSP-A. Because, to date, Apa seems to be restricted to the Mtb complex strains, we propose that it may account for the selective recognition of those strains by PSP-A and other immune system C-TLs containing homologous functional

  18. Human skeletal muscle protein breakdown during spaceflight

    NASA Technical Reports Server (NTRS)

    Stein, T. P.; Schluter, M. D.

    1997-01-01

    Human spaceflight is associated with a loss of body protein. Excretion of 3-methylhistidine (3-MH) in the urine is a useful measurement of myofibrillar protein breakdown. Bed rest, particularly with 6 degrees head-down tilt, is an accepted ground-based model for human spaceflight. The objectives of this report were to compare 3-MH excretion from two Life Sciences shuttle missions (duration 9.5 and 15 days, n = 9) and from 17 days of bed rest (n = 7) with 6 degrees head-down tilt. The bed rest study was designed to mimic an actual Life Sciences spaceflight and so incorporated an extensive battery of physiological tests focused on the musculoskeletal system. Results showed that nitrogen retention, based on excretion of nitrogen in the urine, was reduced during both bed rest [from 22 +/- 1 to 1 +/- 5 mg N x kg(-1) x day(-1) (n = 7; P < 0.05)] and spaceflight [from 57 +/- 9 to 19 +/- 3 mg N x kg(-1) x day(-1) (n = 9; P < 0.05)]. 3-MH excretion was unchanged with either bed rest [pre-bed rest 5.30 +/- 0.29 vs. bed rest 5.71 +/- 0.30 micromol 3-MH x kg(-1) x day(-1), n = 7; P = not significant (NS)] or spaceflight [preflight 4.98 +/- 0.37 vs. 4.59 +/- 0.39 micromol 3-MH x kg(-1) x day(-1) in-flight, n = 9; P = NS]. We conclude that 1) 3-MH excretion was unaffected by spaceflight on the shuttle or with bed rest plus exercise, and 2) because protein breakdown (elevated 3-MH) was increased on Skylab but not on the shuttle, it follows that muscle protein breakdown is not an inevitable consequence of spaceflight.

  19. Application of Ultrasound-Assisted Surfactant-Enhanced Emulsification Microextraction Based on Solidification of Floating Organic Droplets and High Performance Liquid Chromatography for Preconcentration and Determination of Alprazolam and Chlordiazepoxide in Human Serum Samples.

    PubMed

    Goudarzi, Nasser; Amirnavaee, Monavar; Arab Chamjangali, Mansour; Farsimadan, Sahar

    2017-03-03

    An improved microextraction method is proposed on the basis of ultrasound-assisted surfactant-enhanced emulsification and solidification of a floating organic droplet procedure combined with high performance liquid chromatography for the preconcentration and quantification of alprazolam (ALP) and chlordiazepoxide (CHL) present in a number of human serum samples. Several parameters affecting the extraction efficiency were investigated by the Plackett -Burman factorial design as the screening design. Then the response surface methodology based on the Box-Behnken design was used to optimize the effective parameters in the proposed procedure. The limits of detection for the proposed method were found to be 3.0 and 3.1 ng mL-1 for CHL and ALP, respectively. The calibration curves obtained for the method were linear in the ranges of 10.0-3,500.0 and 10.0-3,000.0 ng mL-1 for CHL and ALP, respectively, with a good determination coefficient. The recoveries of the drugs in the spiked human serum samples were above 93.0%. The developed method was successfully applied to the analysis of these studied drugs in human serum samples. The pre-treatment of the serum samples was performed using acetonitrile to remove the proteins. The proposed procedure was an accurate and reliable one for the determination and preconcentration of these drugs in blood samples.

  20. Thermally cleavable surfactants

    DOEpatents

    McElhanon, James R.; Simmons, Blake A.; Zifer, Thomas; Jamison, Gregory M.; Loy, Douglas A.; Rahimian, Kamyar; Long, Timothy M.; Wheeler, David R.; Staiger, Chad L.

    2009-11-24

    Two new surfactant molecules are reported which contain thermally labile Diels-Alder adducts connecting the polar and non-polar sections of each molecule. The two surfactants possess identical non-polar dodecyl tail segments but exhibit different polar headgroups. The surfactants become soluble in water when anionic salts are formed through the deprotonation of the surfactant headgroups by the addition of potassium hydroxide. When either surfactant is exposed to temperature above about 60.degree. C., the retro Diels-Alder reaction occurs, yielding hydrophilic and hydrophobic fragments or the aqueous solutions of the surfactants subsequently exhibit loss of all surface-active behavior.

  1. Thermally cleavable surfactants

    DOEpatents

    McElhanon, James R.; Simmons, Blake A.; Zifer, Thomas; Jamison, Gregory M.; Loy, Douglas A.; Rahimian, Kamyar; Long, Timothy M.; Wheeler, David R.; Staiger, Chad L.

    2006-04-04

    Two new surfactant molecules are reported which contain thermally labile Diels-Alder adducts connecting the polar and non-polar sections of each molecule. The two surfactants possess identical non-polar dodecyl tail segments but exhibit different polar headgroups. The surfactants become soluble in water when anionic salts are formed through the deprotonation of the surfactant headgroups by the addition of potassium hydroxide. When either surfactant is exposed to temperature above about 60.degree. C., the retro Diels-Alder reaction occurs, yielding hydrophilic and hydrophobic fragments and the aqueous solutions of the surfactants subsequently exhibit loss of all surface-active behavior.

  2. Thermally cleavable surfactants

    DOEpatents

    McElhanon, James R.; Simmons, Blake A.; Zifer, Thomas; Jamison, Gregory M.; Loy, Douglas A.; Rahimian, Kamyar; Long, Timothy M.; Wheeler, David R.; Staiger, Chad L.

    2009-09-29

    Two new surfactant molecules are reported which contain thermally labile Diels-Alder adducts connecting the polar and non-polar sections of each molecule. The two surfactants possess identical non-polar dodecyl tail segments but exhibit different polar headgroups. The surfactants become soluble in water when anionic salts are formed through the deprotonation of the surfactant headgroups by the addition of potassium hydroxide. When either surfactant is exposed to temperature above about 60.degree. C., the retro Diels-Alder reaction occurs, yielding hydrophilic and hydrophobic fragments or the aqueous solutions of the surfactants subsequently exhibit loss of all surface-active behavior.

  3. Role of surfactant protein-A (SP-A) in lung injury in response to acute ozone exposure of SP-A deficient mice

    SciTech Connect

    Haque, Rizwanul; Umstead, Todd M.; Ponnuru, Padmavathi; Guo Xiaoxuan; Hawgood, Samuel; Phelps, David S.; Floros, Joanna . E-mail: jfloros@psu.edu

    2007-04-01

    Millions are exposed to ozone levels above recommended limits, impairing lung function, causing epithelial damage and inflammation, and predisposing some individuals to pneumonia, asthma, and other lung conditions. Surfactant protein-A (SP-A) plays a role in host defense, the regulation of inflammation, and repair of tissue damage. We tested the hypothesis that the lungs of SP-A(-/-) (KO) mice are more susceptible to ozone-induced damage. We compared the effects of ozone on KO and wild type (WT) mice on the C57BL/6 genetic background by exposing them to 2 parts/million of ozone for 3 or 6 h and sacrificing them 0, 4, and 24 h later. Lungs were subject to bronchoalveolar lavage (BAL) or used to measure endpoints of oxidative stress and inflammation. Despite more total protein in BAL of KO mice after a 3 h ozone exposure, WT mice had increased oxidation of protein and had oxidized SP-A dimers. In KO mice there was epithelial damage as assessed by increased LDH activity and there was increased phospholipid content. In WT mice there were more BAL PMNs and elevated macrophage inflammatory protein (MIP)-2 and monocyte chemoattractant protein (MCP)-1. Changes in MIP-2 and MCP-1 were observed in both KO and WT, however mRNA levels differed. In KO mice MIP-2 mRNA levels changed little with ozone, but in WT levels they were significantly increased. In summary, several aspects of the inflammatory response differ between WT and KO mice. These in vivo findings appear to implicate SP-A in regulating inflammation and limiting epithelial damage in response to ozone exposure.

  4. Polymer-surfactant treatment of meconium-induced acute lung injury.

    PubMed

    Lu, K W; William Taeusch, H; Robertson, B; Goerke, J; Clements, J A

    2000-08-01

    Substances (for example, serum proteins or meconium) that interfere with the activity of pulmonary surfactant in vitro may also be important in the pathogenesis or progression of acute lung injury. Addition of polymers such as dextran or polyethylene glycol (PEG) to surfactants prevents and reverses surfactant inactivation. The purpose of this study was to find out whether surfactant/polymer mixtures are more effective for treating one form of acute lung injury than is surfactant alone. Acute lung injury in adult rats was created by tracheal instillation of human meconium. Injured animals, which were anesthetized, paralyzed, and ventilated with 100% oxygen and not treated with surfactant mixtures, remained hypoxic and required high ventilator pressures to maintain Pa(CO(2)) in the normal range over the 3 h of the experiment. Uninjured animals maintained normal values for oxygen and compliance of the respiratory system. The greatest improvement in both oxygenation (178%) and compliance (42%) occurred in animals with lung injury that were treated with Survanta and PEG (versus untreated control animals; p < 0.01), whereas little improvement was found after treatment with Survanta alone. Similar results were found when postmortem pulmonary pressure-volume curves and histology were examined. We conclude that adding PEG to Survanta improves gas exchange, pulmonary mechanics, and histologic appearance of the lungs in a rat model of acute lung injury caused by meconium.

  5. Proteins of human milk. I. Identification of major components

    SciTech Connect

    Anderson, N.G.; Powers, M.T.; Tollaksen, S.L.

    1982-04-01

    Traditionally, human milk proteins are identified largely by reference to bovine milk. Hence, to identify the major proteins in human milk, we subjected human and bovine milk, in parallel, to high-resolution two-dimensional electrophoresis. Isoelectric precipitation at pH 4.6 was our criterion for distinguishing whey proteins from those of the casein complex. The ..cap alpha..- and..beta..-caseins were identified on the basis of relative abundance, relative molecular mass, and relative isoelectric points. No protein disappeared from ISO-DALT patterns of human milk after rennin treatment, and no new protein comparable to bovine para K-casein appeared in the BASO-DALT patterns; this suggests that K-casein is absent from human milk. The proteins identified in human milk patterns include the ..cap alpha.. and ..beta.. casein families, lactalbumin, albumin, transferrin, IgA, and lactoferrin. Numerous additional proteins seen in patterns for human milk remain to be identified.

  6. UV-vis spectra as an alternative to the Lowry method for quantify hair damage induced by surfactants.

    PubMed

    Pires-Oliveira, Rafael; Joekes, Inés

    2014-11-01

    It is well known that long term use of shampoo causes damage to human hair. Although the Lowry method has been widely used to quantify hair damage, it is unsuitable to determine this in the presence of some surfactants and there is no other method proposed in literature. In this work, a different method is used to investigate and compare the hair damage induced by four types of surfactants (including three commercial-grade surfactants) and water. Hair samples were immersed in aqueous solution of surfactants under conditions that resemble a shower (38 °C, constant shaking). These solutions become colored with time of contact with hair and its UV-vis spectra were recorded. For comparison, the amount of extracted proteins from hair by sodium dodecyl sulfate (SDS) and by water were estimated by the Lowry method. Additionally, non-pigmented vs. pigmented hair and also sepia melanin were used to understand the washing solution color and their spectra. The results presented herein show that hair degradation is mostly caused by the extraction of proteins, cuticle fragments and melanin granules from hair fiber. It was found that the intensity of solution color varies with the charge density of the surfactants. Furthermore, the intensity of solution color can be correlated to the amount of proteins quantified by the Lowry method as well as to the degree of hair damage. UV-vis spectrum of hair washing solutions is a simple and straightforward method to quantify and compare hair damages induced by different commercial surfactants.

  7. Angiotensin I-Converting Enzyme inhibitory and antioxidant activities and surfactant properties of protein hydrolysates as obtained of Amaranthus hypochondriacus L. grain.

    PubMed

    Soriano-Santos, J; Escalona-Buendía, H

    2015-04-01

    Even though some research has been carried out on surfactant properties of amaranth protein hydrolysates, their bio-functionality has not been studied yet. In this work amaranth grain Alb 1 and Glob were hydrolyzed (Alb 1H, Glob H) and foams and emulsions at optimal conditions (t, E/S, pH5) were prepared in order to assess techno-functional properties such as foaming (F) and emulsifying (E) (capacity (C) and stability (S)). FC and EC were much better for Glob H than for Alb H. Angiotensin I-converting enzyme-inhibitory activity was higher for Alb 1H (roughly 50 %) than that of Glob H (roughly 30 %). Scavenging of radicals activity (DPPH· or ABTS· (+) ) of Alb 1H and Glob H, at 2 mg/mL, was similar (approx. 40 %), but lower than Alb 1 (approx. 70 %), which was the best antioxidant. The low reducing power showed that hydrolysates barely donate an electron or hydrogen. Chelating activity on Cu(2+) was lower than that exhibited by Fe(2+,) which was remarkable, approx. 80 % as long as DH% > 10 %, where hydrolysates displayed high solubility (Alb 1H = 85 %, Glob H = 70 %) because of occurrence of 1-10 kDa peptides. Amaranth foams and emulsions prepared with protein hydrolysates have a potential as a nutraceutical food.

  8. Role of CBP/p300 and SRC-1 in transcriptional regulation of the pulmonary surfactant protein-A (SP-A) gene by thyroid transcription factor-1 (TTF-1).

    PubMed

    Yi, Ming; Tong, Guo-Xia; Murry, Barbara; Mendelson, Carole R

    2002-01-25

    Surfactant protein-A (SP-A) gene expression is developmentally regulated in fetal lung type II cells and is enhanced by cAMP. cAMP stimulation of SP-A gene expression is mediated by protein kinase A (PKA) phosphorylation of thyroid transcription factor 1 (TTF-1), expressed selectively in developing lung epithelium. In this study, we analyzed roles of CREB-binding protein (CBP) and steroid receptor coactivator-1 (SRC-1) in TTF-1 regulation of SP-A expression. Upon differentiation of human fetal lung in culture, nuclear localization of CBP, SRC-1, and TTF-1 increased in ductular epithelium in association with type II cell differentiation and induction of SP-A expression. In transient transfections, CBP and SRC-1 acted synergistically with TTF-1 to increase SP-A promoter activity. Overexpression of PKA catalytic subunit enhanced hSP-A promoter activation by SRC-1 plus TTF-1. Adenoviral E1A overexpression reduced TTF-1 +/- SRC-1 induction of SP-A promoter activity, suggesting a role of endogenous CBP/p300. TTF-1 interacted with SRC-1 and CBP in vitro. SRC-1 immunodepletion from type II cell nuclear extracts reduced binding to the TTF-1 binding element upstream of SP-A gene. In cultured type II cells, cAMP increased TTF-1 acetylation. This suggests that cAMP-mediated TTF-1 phosphorylation facilitates interaction with CBP and SRC-1, resulting in its hyperacetylation, further enhancing TTF-1 DNA-binding and transcriptional activity.

  9. Major house dust mite allergens Dermatophagoides pteronyssinus 1 and Dermatophagoides farinae 1 degrade and inactivate lung surfactant proteins A and D.

    PubMed

    Deb, Roona; Shakib, Farouk; Reid, Kenneth; Clark, Howard

    2007-12-21

    Lung surfactant proteins (SP) A and D are calcium-dependent carbohydrate-binding proteins. In addition to playing multiple roles in innate immune defense such as bacterial aggregation and modulation of leukocyte function, SP-A and SP-D have also been implicated in the allergic response. They interact with a wide range of inhaled allergens, competing with their binding to cell-sequestered IgE resulting in inhibition of mast cell degranulation, and exogenous administration of SP-A and SP-D diminishes allergic hypersensitivity in vivo. House dust mite allergens are a major cause of allergic asthma in the western world, and here we confirm the interaction of SP-A and SP-D with two major mite allergens, Dermatophagoides pteronyssinus 1 and Dermatophagoides farinae 1, and show that the cysteine protease activity of these allergens results in the degradation of SP-A and SP-D under physiological conditions, with multiple sites of cleavage. A recombinant fragment of SP-D that is effective in diminishing allergic hypersensitivity in mouse models of dust mite allergy was more susceptible to degradation than the native full-length protein. Degradation was enhanced in the absence of calcium, with different sites of cleavage, indicating that the calcium associated with SP-A and SP-D influences accessibility to the allergens. Degradation of SP-A and SP-D was associated with diminished binding to carbohydrates and to D. pteronyssinus 1 itself and diminished capacity to agglutinate bacteria. Thus, the degradation and consequent inactivation of SP-A and SP-D may be a novel mechanism to account for the potent allergenicity of these common dust mite allergens.

  10. The Proteins of Human Chromosome 21

    PubMed Central

    Gardiner, Katheleen; Costa, Alberto C. S.

    2009-01-01

    Recent genomic sequence annotation suggests that the long arm of human chromosome 21 encodes more than 400 genes. Because there is no evidence to exclude any significant segment of 21q from containing genes relevant to the Down syndrome cognitive phenotype, all genes in this entire set must be considered as candidates. Only a subset, however, is likely to make critical contributions. Determining which these are is both a major focus in biology and a critical step in efficient development of therapeutics. The subtle molecular abnormality in Down syndrome, the 50% increase in chromosome 21 gene expression, presents significant challenges for researchers in detection and quantitation. Another challenge is the current limitation in understanding gene functions and in interpreting biological characteristics. Here, we review information on chromosome 21-encoded proteins compiled from the literature and from genomics and proteomics databases. For each protein, we summarize their evolutionary conservation, the complexity of their known protein interactions and their level of expression in brain, and discuss the implications and limitations of these data. For a subset, we discuss neurologically relevant phenotypes of mouse models that include knockouts, mutations or overexpression. Lastly, we highlight a small number of genes for which recent evidence suggests a function in biochemical/cellular pathways that are relevant to cognition. Until knowledge deficits are overcome, we suggest that effective development of gene-phenotype correlations in Down syndrome requires a serious and continuous effort to assimilate broad categories of information on chromosome 21 genes, plus the creation of more versatile mouse models. PMID:17048356

  11. Fiber coating with surfactant solutions

    NASA Astrophysics Data System (ADS)

    Shen, Amy Q.; Gleason, Blake; McKinley, Gareth H.; Stone, Howard A.

    2002-11-01

    When a fiber is withdrawn at low speeds from a pure fluid, the variation in the thickness of the entrained film with imposed fiber velocity is well-predicted by the Landau-Levich-Derjaguin (LLD) equation. However, surfactant additives are known to alter this response. We study the film thickening properties of the protein BSA (bovine serum albumin), the nonionic surfactant Triton X-100, and the anionic surfactant SDS (sodium dodecyl sulfate). For each of these additives, the film thickening factor alpha (the ratio of the measured thickness to the LLD prediction) for a fixed fiber radius varies as a function of the ratio of the surfactant concentration c to the critical micelle concentration (CMC). In the case of BSA, which does not form micelles, the reference value is the concentration at which multilayers form. As a result of Marangoni effects, alpha reaches a maximum as c approaches the CMC from below. However, when the surfactant concentration c exceeds the CMC, the behavior of alpha varies as a consequence of the dynamic surface properties, owing for example to different sorption kinetics of these additives, or possibly surface or bulk rheological effects. For SDS, alpha begins to decrease when c exceeds the CMC and causes the surface to become partially or completely remobilized, which is consistent with the experimental and theoretical results published for studies of slug flows of bubbles and surfactant solutions in a capillary tube and the rise of bubbles in surfactant solutions. However, when the SDS or Triton X-100 surfactant concentration is well above the CMC, we observe that the film thickening parameter alpha increases once again. In the case of SDS we observe a second maximum in the film thickening factor. For all the experiments, transport of monomers to the interface is limited by diffusion and the second maximum in the film thickening factor may be explained as a result of a nonmonotonic change in the stability characteristics of suspended SDS

  12. The protein interaction landscape of the human CMGC kinase group.

    PubMed

    Varjosalo, Markku; Keskitalo, Salla; Van Drogen, Audrey; Nurkkala, Helka; Vichalkovski, Anton; Aebersold, Ruedi; Gstaiger, Matthias

    2013-04-25

    Cellular information processing via reversible protein phosphorylation requires tight control of the localization, activity, and substrate specificity of protein kinases, which to a large extent is accomplished by complex formation with other proteins. Despite their critical role in cellular regulation and pathogenesis, protein interaction information is available for only a subset of the 518 human protein kinases. Here we present a global proteomic analysis of complexes of the human CMGC kinase group. In addition to subgroup-specific functional enrichment and modularity, the identified 652 high-confidence kinase-protein interactions provide a specific biochemical context for many poorly studied CMGC kinases. Furthermore, the analysis revealed a kinase-kinase subnetwork and candidate substrates for CMGC kinases. Finally, the presented interaction proteome uncovered a large set of interactions with proteins genetically linked to a range of human diseases, including cancer, suggesting additional routes for analyzing the role of CMGC kinases in controlling human disease pathways.

  13. Addition of Surfactants and Non-Hydrolytic Proteins and Their Influence on Enzymatic Hydrolysis of Pretreated Sugarcane Bagasse.

    PubMed

    Méndez Arias, Johanna; de Oliveira Moraes, Anelize; Modesto, Luiz Felipe Amarante; de Castro, Aline Machado; Pereira, Nei

    2017-02-01

    Poly(ethylene glycol) (PEG 4000) and bovine serum albumin (BSA) were investigated with the purpose of evaluating their influence on enzymatic hydrolysis of sugarcane bagasse. Effects of these supplements were assayed for different enzymatic cocktails (Trichoderma harzianum and Penicillium funiculosum) that acted on lignocellulosic material submitted to different pretreatment methods with varying solid (25 and 100 g/L) and protein (7.5 and 20 mg/g cellulose) loadings. The highest levels of glucose release were achieved using partially delignified cellulignin as substrate, along with the T. harzianum cocktail: increases of 14 and 18 % for 25 g/L solid loadings and of 33 and 43 % for 100 g/L solid loadings were reached for BSA and PEG supplementation, respectively. Addition of these supplements could maintain hydrolysis yield even for higher solid loadings, but for higher enzymatic cocktail protein loadings, increases in glucose release were not observed. Results indicate that synergism might occur among these additives and cellulase and xylanases. The use of these supplements, besides depending on factors such as pretreatment method of sugarcane bagasse, enzymatic cocktails composition, and solid and protein loadings, may not always lead to positive effects on the hydrolysis of lignocellulosic material, making it necessary further statistical studies, according to process conditions.

  14. Mobile phone radiation might alter protein expression in human skin

    PubMed Central

    Karinen, Anu; Heinävaara, Sirpa; Nylund, Reetta; Leszczynski, Dariusz

    2008-01-01

    Background Earlier we have shown that the mobile phone radiation (radiofrequency modulated electromagnetic fields; RF-EMF) alters protein expression in human endothelial cell line. This does not mean that similar response will take place in human body exposed to this radiation. Therefore, in this pilot human volunteer study, using proteomics approach, we have examined whether a local exposure of human skin to RF-EMF will cause changes in protein expression in living people. Results Small area of forearm's skin in 10 female volunteers was exposed to RF-EMF (specific absorption rate SAR = 1.3 W/kg) and punch biopsies were collected from exposed and non-exposed areas of skin. Proteins extracted from biopsies were separated using 2-DE and protein expression changes were analyzed using PDQuest software. Analysis has identified 8 proteins that were statistically significantly affected (Anova and Wilcoxon tests). Two of the proteins were present in all 10 volunteers. This suggests that protein expression in human skin might be affected by the exposure to RF-EMF. The number of affected proteins was similar to the number of affected proteins observed in our earlier in vitro studies. Conclusion This is the first study showing that molecular level changes might take place in human volunteers in response to exposure to RF-EMF. Our study confirms that proteomics screening approach can identify protein targets of RF-EMF in human volunteers. PMID:18267023

  15. Surfactant therapy: the current practice and the future trends

    PubMed Central

    Altirkawi, Khalid

    2013-01-01

    The efficacy of surfactant preparations used in the prevention and treatment of respiratory distress syndrome (RDS) is a well known fact; however, many controversies remain. The debate over which surfactant to be used, when and what is the best mode of delivery is still raging. Currently, animal-derived surfactants are preferred and clearly recommended by various practice guidelines, but new synthetic surfactants containing peptides that mimic the action of surfactant proteins are emerging and they seem to have a comparable efficacy profile to the natural surfactants. It is hoped that with further improvements, they will outperform their natural counterparts in terms of reliability and cost-effectiveness. Early surfactant administration was shown to further reduce the risk of RDS and its complications. However, as nasal continuous positive airway pressure (nCPAP) is becoming increasingly the preferred first-line therapy for RDS, the less invasive approaches of respiratory support along with early selective surfactant administration (e.g. INSURE) appears to provide a better option. Although neonatal RDS is still the main indication of surfactant therapy, other pathological processes received considerable attention and major research has been dedicated to explore the role of surfactant in their management, Meconium aspiration syndrome (MAS) and congenital pneumonia are two worthy examples. The most updated practice guidelines do recommend the use of endotracheal instillation as the preferred mode of surfactant delivery. However, aerosolization and other non-invasive methods are being investigated with some success; nonetheless, further improvements are very much in need. PMID:27493353

  16. A method for whole protein isolation from human cranial bone.

    PubMed

    Lyon, Sarah M; Mayampurath, Anoop; Rogers, M Rose; Wolfgeher, Donald J; Fisher, Sean M; Volchenboum, Samuel L; He, Tong-Chuan; Reid, Russell R

    2016-12-15

    The presence of the dense hydroxyapatite matrix within human bone limits the applicability of conventional protocols for protein extraction. This has hindered the complete and accurate characterization of the human bone proteome thus far, leaving many bone-related disorders poorly understood. We sought to refine an existing method of protein extraction from mouse bone to extract whole proteins of varying molecular weights from human cranial bone. Whole protein was extracted from human cranial suture by mechanically processing samples using a method that limits protein degradation by minimizing heat introduction to proteins. The presence of whole protein was confirmed by western blotting. Mass spectrometry was used to sequence peptides and identify isolated proteins. The data have been deposited to the ProteomeXchange with identifier PXD003215. Extracted proteins were characterized as both intra- and extracellular and had molecular weights ranging from 9.4 to 629 kDa. High correlation scores among suture protein spectral counts support the reproducibility of the method. Ontology analytics revealed proteins of myriad functions including mediators of metabolic processes and cell organelles. These results demonstrate a reproducible method for isolation of whole protein from human cranial bone, representing a large range of molecular weights, origins and functions.

  17. Post-translational processing of surfactant protein-C proprotein: targeting motifs in the NH(2)-terminal flanking domain are cleaved in late compartments.

    PubMed

    Johnson, A L; Braidotti, P; Pietra, G G; Russo, S J; Kabore, A; Wang, W J; Beers, M F

    2001-03-01

    Rat surfactant protein (SP)-C is a 3.7-kD hydrophobic lung-specific protein generated from proteolytic processing of a 21-kD propeptide (SP-C(21)). We have demonstrated that initial post-translational processing of SP-C(21) involves two cleavages of the COOH-terminus (Beers and colleagues, J. Biol. Chem. 1994;269:20,318--20,328). The goal of the current study was to define processing and function of the NH(2)-terminal flanking domain. Epitope-specific antisera directed against spatially distinct regions of the NH(2) terminus, NPROSP-C(2-9) (epitope = D(2)-L(9)) and NPROSP-C(11-23) (= E(11)-Q(23)) were produced. By Western blotting, both antisera identified SP-C(21) in microsomes. A 6-kD form (SP-C(6)), enriched in lamellar bodies (LBs), was detected only by NPROSP-C(11-23) and not extractable with NaCO(3) treatment. Immunogold staining of ultrathin lung sections with NPROSP-C(11-23) identified proSP-C in both multivesicular bodies (mvb) and LBs whereas NPROSP-C(2-9) labeled only mvb. (35)S-pulse chase analysis demonstrated synthesis of SP-C(21) and three intermediate forms (SP-C(16), SP-C(7), and SP-C(6)). Complete processing involved four separate cleavages with a precursor- product relationship between the low molecular weight forms SP-C(7) and SP-C(6). Fluorescence microscopy of A549 cells expressing fusion proteins of enhanced green fluorescent protein (EGFP) and proSP-C NH(2)-terminal deletion mutants showed targeting of EGFP/SP-C(1-194) and EGFP/SP-C(10-194) to early endosomal antigen-1-negative, CD-63-positive cytoplasmic vesicles whereas EGFP/SP-C(19-194), EGFP/SP-C(Delta 10-18), and EGFP/SP-C(24-194) were restricted to the endoplasmic reticulum (ER). We conclude that synthetic processing includes a previously unrecognized cleavage of the proximal NH(2) terminus (M(1)-L(9)), which occurs after removal of COOH-flanking domains (H(59)-I(194)) but before packaging in LBs, and that the region M(10)-T(18) is required for targeting of proSP-C to post-ER vesicular

  18. Cellular responses of the late blight pathogen Phytophthora infestans to cyclic lipopeptide surfactants and their dependence on G proteins.

    PubMed

    van de Mortel, Judith E; Tran, Ha; Govers, Francine; Raaijmakers, Jos M

    2009-08-01

    Oomycete pathogens cause major yield losses for many crop plants, and their control depends heavily on agrochemicals. Cyclic lipopeptides (CLPs) were recently discovered as a new class of natural compounds with strong activities against oomycetes. The CLP massetolide A (Mass A), produced by Pseudomonas fluorescens, has zoosporicidal activity, induces systemic resistance, and reduces late blight in tomato. To gain further insight into the modes of action of CLPs, the effects of Mass A on pore formation, mycelial growth, sporangium formation, and zoospore behavior were investigated, as was the involvement of G proteins in the sensitivity of Phytophthora infestans to Mass A. The results showed that Mass A induced the formation of transmembrane pores with an estimated size of between 1.2 and 1.8 nm. Dose-response experiments revealed that zoospores were the most sensitive to Mass A, followed by mycelium and cysts. Mass A significantly reduced sporangium formation and caused increased branching and swelling of hyphae. At relatively low concentrations, Mass A induced encystment of zoospores. It had no effect on the chemotactic response of zoospores but did adversely affect zoospore autoaggregation. A loss-of-function transformant of P. infestans lacking the G-protein alpha subunit was more sensitive to Mass A, whereas a gain-of-function transformant required a higher Mass A concentration to interfere with zoospore aggregation. Results indicate that Mass A disturbs various developmental stages in the life cycle of P. infestans and suggest that the cellular responses of P. infestans to this CLP are, in part, dependent on G-protein signaling.

  19. TSTMP: target selection for structural genomics of human transmembrane proteins

    PubMed Central

    Varga, Julia; Dobson, László; Reményi, István; Tusnády, Gábor E.

    2017-01-01

    The TSTMP database is designed to help the target selection of human transmembrane proteins for structural genomics projects and structure modeling studies. Currently, there are only 60 known 3D structures among the polytopic human transmembrane proteins and about a further 600 could be modeled using existing structures. Although there are a great number of human transmembrane protein structures left to be determined, surprisingly only a small fraction of these proteins have ‘selected’ (or above) status according to the current version the TargetDB/TargetTrack database. This figure is even worse regarding those transmembrane proteins that would contribute the most to the structural coverage of the human transmembrane proteome. The database was built by sorting out proteins from the human transmembrane proteome with known structure and searching for suitable model structures for the remaining proteins by combining the results of a state-of-the-art transmembrane specific fold recognition algorithm and a sequence similarity search algorithm. Proteins were searched for homologues among the human transmembrane proteins in order to select targets whose successful structure determination would lead to the best structural coverage of the human transmembrane proteome. The pipeline constructed for creating the TSTMP database guarantees to keep the database up-to-date. The database is available at http://tstmp.enzim.ttk.mta.hu. PMID:27924015

  20. Adenovirus dodecahedron allows large multimeric protein transduction in human cells.

    PubMed

    Fender, P; Schoehn, G; Foucaud-Gamen, J; Gout, E; Garcel, A; Drouet, E; Chroboczek, J

    2003-04-01

    Adenovirus dodecahedron is a virus-like particle composed of only two viral proteins of human adenovirus serotype 3 that are responsible for virus attachment and internalization. We show here that this dodecameric particle, devoid of genetic information, efficiently penetrates human cells and can deliver large multimeric proteins such as immunoglobulins.

  1. Effect of natural porcine surfactant in Staphylococcus aureus induced pro-inflammatory cytokines and reactive oxygen species generation in monocytes and neutrophils from human blood.

    PubMed

    de Guevara, Yuliannis Lugones Ladrón; Hidalgo, Odalys Blanco; Santos, Sidnéia Sousa; Brunialti, Milena Karina Colo; Faure, Roberto; Salomao, Reinaldo

    2014-08-01

    Surfacen® is a clinical surfactant preparation of porcine origin. In the present study, we have evaluated the effect of Surfacen® in the modulation of oxidative burst in monocytes and neutrophils in human blood and pro-inflammatory cytokine production in peripheral blood mononuclear cells (PBMC). Reactive oxygen species (ROS) level was measured in monocytes and neutrophils by flow cytometry using 2,7-dichlorofluorescein diacetate (DCFH-DA) as substrate, while, tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were estimated in PBMC supernatant by enzyme-linked immunosorbent assays (ELISA). Our results show that Staphylococcus aureus-induced ROS level was slightly affected by Surfacen® added to whole blood monocytes and neutrophils. The time course experiments of pre-incubation with Surfacen® showed no significant increase of ROS level at 2h; however, the ROS level decreased when pre incubated for 4h and 6h with Surfacen®. Pre-incubation of PBMC cells with Surfacen® at 0.125 and 0.5mg/mL showed a dose-dependent suppression of TNF-α levels measured after 4h of S. aureus stimulation, an effect less impressive when cells were stimulated for 24h. A similar behavior was observed in IL-6 release. In summary, the present study provides experimental evidence supporting an anti-inflammatory role of Surfacen® in human monocytes and neutrophils in vitro.

  2. Surfactant Enhanced DNAPL Removal

    DTIC Science & Technology

    2001-08-01

    or the permeability contrast (i.e., degree of heterogeneity) that is present in the DNAPL zone. To solubilize DNAPL with surfactants, a sufficient...with respect to the effects of permeability and heterogeneity upon the costs of SEAR: as permeability decreases and/or the degree of heterogeneity...not be an issue for surfactant recovery at all sites. The degree to which MEUF will concentrate the calcium is a function of the surfactant itself

  3. Towards unravelling surfactant transport

    NASA Astrophysics Data System (ADS)

    Sellier, Mathieu; Panda, Satyananda

    2015-11-01

    Surfactant transport arises in many natural or industrial settings. Examples include lipid tear layers in the eye, pulmonary surfactant replacement therapy, or industrial coating flows. Flows driven by the surface tension gradient which arises as a consequence of surfactant concentration inhomogeneity, also known as Marangoni-driven flows, have attracted the attention of fluid dynamists for several decades and has led to the development of sophisticated models and the undeniable advancement of the understanding of such flows. Yet, experimental confirmation of these models has been hampered by the difficulty in reliably and accurately measuring the surfactant concentration and its temporal evolution. In this contribution, we propose a methodology which may help shed some light on surfactant transport at the surface of thin liquid films. The surface stress induced by surfactant concentration induces a flow at the free surface which is visible and measurable. In the context of thin film flows for which the lubrication approximation hold, we demonstrate how the knowledge of this free surface flow field provides sufficient information to reconstruct the surfactant tension field. From the surface tension and an assumed equation of state, the local surfactant concentration can also be calculated and other transport parameters such as the surfactant surface diffusivity indirectly inferred. In this contribution, the proposed methodology is tested with synthetic data generated by the forward solution of the governing partial differential equations in order to illustrate the feasibility of the algorithm and highlight numerical challenges.

  4. Surfactant phospholipid metabolism

    PubMed Central

    Agassandian, Marianna; Mallampalli, Rama K.

    2012-01-01

    Pulmonary surfactant is essential for life and is comprised of a complex lipoprotein-like mixture that lines the inner surface of the lung to prevent alveolar collapse at the end of expiration. The molecular composition of surfactant depends on highly integrated and regulated processes involving its biosynthesis, remodeling, degradation, and intracellular trafficking. Despite its multicomponent composition, the study of surfactant phospholipid metabolism has focused on two predominant components, disaturated phosphatidylcholine that confers surface-tension lowering activities, and phosphatidylglycerol, recently implicated in innate immune defense. Future studies providing a better understanding of the molecular control and physiological relevance of minor surfactant lipid components are needed. PMID:23026158

  5. Serum KL-6 and surfactant protein-D as monitoring and predictive markers of interstitial lung disease in patients with systemic sclerosis and mixed connective tissue disease

    PubMed Central

    Hagiwara, Eri; Kitamura, Hideya; Yamanaka, Yumie; Ikeda, Satoshi; Sekine, Akimasa; Baba, Tomohisa; Okudela, Koji; Iwasawa, Tae; Takemura, Tamiko; Kuwano, Kazuyoshi; Ogura, Takashi

    2017-01-01

    Background Interstitial lung disease (ILD) is frequent complication of systemic sclerosis (SSc) and mixed connective tissue disease (MCTD). The disease is heterogeneous, and its outcome is unpredictable. Some patients have severe and progressive deterioration of ILD, which is the leading cause of mortality. We aimed to determine whether serum levels of Krebs von den Lungen-6 (KL-6) and surfactant protein-D (SP-D) correlate with SSc/MCTD-associated ILD activity. Methods We retrospectively analyzed the medical records of 40 patients with SSc/MCTD-associated ILD: 29 patients with SSc and 11 patients with MCTD. Measurement of serum KL-6 and SP-D levels, pulmonary function tests, and high-resolution computed tomography (HRCT) performed in parallel were reviewed. Results Serum KL-6 correlated positively with diffusing capacity of the lung for carbon monoxide (DLCO) (% predicted) and disease extent on HRCT, and the changes in serum levels of KL-6 were significantly related to the changes in forced vital capacity (FVC) in SSc/MCTD-associated ILD. On the other hand, multivariate logistic regression analyses with calculation of the area under the curve of the receiver-operating characteristic curve suggested that a higher serum level of SP-D was a significant predictor of FVC decline in SSc/MCTD-associated ILD. Conclusions Our study suggests that serum KL-6 can be a useful monitoring tool of SSc/MCTD-associated ILD activity. In contrast, serum SP-D may be a significant predictor of potential FVC decline in the short term. PMID:28275485

  6. Can Serum Surfactant Protein D or CC-Chemokine Ligand 18 Predict Outcome of Interstitial Lung Disease in Patients with Early Systemic Sclerosis?

    PubMed Central

    Elhaj, Mona; Charles, Julio; Pedroza, Claudia; Liu, Xiaochun; Zhou, Xiaodong; Estrada-Y-Martin, Rosa M.; Gonzalez, Emilio B.; Lewis, Dorothy E.; Draeger, Hilda T.; Kim, Sarah; Arnett, Frank C.; Mayes, Maureen D.; Assassi, Shervin

    2013-01-01

    Objective To examine the predictive significance of 2 pneumoproteins, surfactant protein D (SP-D) and CC-chemokine ligand 18 (CCL18), for the course of systemic sclerosis (SSc)-related interstitial lung disease. Methods The pneumoproteins were determined in the baseline plasma samples of 266 patients with early SSc enrolled in the GENISOS observational cohort. They also were measured in 83 followup patient samples. Pulmonary function tests were obtained annually. The primary outcome was decline in forced vital capacity (FVC percentage predicted) over time. The predictive significance for longterm change in FVC was investigated by a joint analysis of longitudinal measurements (sequentially obtained FVC percentage predicted) and survival data. Results SP-D and CCL18 levels were both higher in patients with SSc than in matched controls (p < 0.001 and p = 0.015, respectively). Baseline SP-D levels correlated with lower concomitantly obtained FVC (r = −0.27, p < 0.001), but did not predict the short-term decline in FVC at 1 year followup visit or its longterm decline rate. CCL18 showed a significant correlation with steeper short-term decline in FVC (p = 0.049), but was not a predictor of its longterm decline rate. Similarly, a composite score of SP-D and CCL18 was a significant predictor of short-term decline in FVC but did not predict its longterm decline rate. Further, the longitudinal change in these 2 pneumoproteins did not correlate with the concomitant percentage change in FVC. Conclusion SP-D correlated with concomitantly obtained FVC, while CCL18 was a predictor of short-term decline in FVC. However, neither SP-D nor CCL18 was a longterm predictor of FVC course in patients with early SSc. PMID:23588945

  7. Protein-Protein Interactions Suggest Novel Activities of Human Cytomegalovirus Tegument Protein pUL103

    PubMed Central

    Ortiz, Daniel A.; Glassbrook, James E.

    2016-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is an enveloped double-stranded DNA virus that causes severe disease in newborns and immunocompromised patients. During infection, the host cell endosecretory system is remodeled to form the cytoplasmic virion assembly complex (cVAC). We and others previously identified the conserved, multifunctional HCMV virion tegument protein pUL103 as important for cVAC biogenesis and efficient secondary envelopment. To help define its mechanisms of action and predict additional functions, we used two complementary methods, coimmunoprecipitation (co-IP) and proximity biotinylation (BioID), to identify viral and cellular proteins that interact with pUL103. By using the two methods in parallel and applying stringent selection criteria, we identified potentially high-value interactions of pUL103 with 13 HCMV and 18 cellular proteins. Detection of the previously identified pUL103-pUL71 interaction, as well as verification of several interactions by reverse co-IP, supports the specificity of our screening process. As might be expected for a tegument protein, interactions were identified that suggest distinct roles for pUL103 across the arc of lytic infection, including interactions with proteins involved in cellular antiviral responses, nuclear activities, and biogenesis and transport of cytoplasmic vesicles. Further analysis of some of these interactions expands our understanding of the multifunctional repertoire of pUL103: we detected HCMV pUL103 in nuclei of infected cells and identified an ALIX-binding domain within the pUL103 sequence. IMPORTANCE Human cytomegalovirus (HCMV) is able to reconfigure the host cell machinery to establish a virion production factory, the cytoplasmic virion assembly complex (cVAC). cVAC biogenesis and operation represent targets for development of novel HCMV antivirals. We previously showed that the HCMV tegument protein pUL103 is required for cVAC biogenesis. Using pUL103 as bait, we investigated viral and

  8. Efficient expression and secretion of recombinant human growth hormone in the methylotrophic yeast Pichia pastoris: potential applications for other proteins.

    PubMed

    Apte-Deshpande, Anjali; Rewanwar, Sachin; Kotwal, Prakash; Raiker, Veena A; Padmanabhan, Sriram

    2009-11-13

    A simple high yielding process for the production of rhGH (recombinant human growth hormone) in the Pichia pastoris system is described. The approach adopted the addition of surfactants during fermentation along with methanol induction. A Pichia integrant harbouring multiple-copy, non-codon-optimized hGH showed poor expression in complex and defined media. Inclusion of the surfactants Tween 20 or Tween 80 during induction enhanced the expression levels significantly in shake flask studies. The combination of 2 litres of basal salt medium and Tween 20 in a bioreactor culminated in 3 x 10(4)-fold elevated expression of the protein (approximately 500 mg/l) as estimated by ELISA. SDS/PAGE and Western-blot analyses revealed that the Pichia-derived rhGH migrated as a single band with a molecular mass of approximately 22 kDa and had the same immunoreactivity as native commercial rhGH. Analysis of Pichia-derived purified rhGH and commercial rhGH on an Agilent 2100 Bioanalyzer revealed overlapping peaks displaying authentic N-terminal processing of Pichia-derived rhGH, which was further confirmed by N-terminal sequencing. In addition, matrix-assisted laser-desorption ionization-time of flight analysis of the protein confirmed its authenticity. These results indicate that the P. pastoris expression system can be effective in the production of rhGH at commercially relevant levels.

  9. Interferon-induced human protein with homology to protein Mx of influenza virus-resistant mice.

    PubMed Central

    Staeheli, P; Haller, O

    1985-01-01

    Polyclonal and monoclonal antibodies with specificity for protein Mx (a karyophilic 75,000-dalton protein induced by interferon [IFN] in mouse cells carrying the influenza virus resistance allele Mx+) detected an IFN-induced 80,000-dalton protein in peripheral blood lymphocytes and in fibroblasts of healthy human donors. The human protein, like protein Mx, was induced by IFN-alpha but not by IFN-gamma. Unlike the mouse protein, it was predominantly localized in the cell cytoplasm. Images PMID:3939324

  10. Gemini surfactants affect the structure, stability, and activity of ribonuclease Sa.

    PubMed

    Amiri, Razieh; Bordbar, Abdol-Khalegh; Laurents, Douglas V

    2014-09-11

    Gemini surfactants have important advantages, e.g., low micromolar CMCs and slow millisecond monomer ↔ micelle kinetics, for membrane mimetics and for delivering nucleic acids for gene therapy or RNA silencing. However, as a prerequisite, it is important to characterize interactions occurring between Gemini surfactants and proteins. Here NMR and CD spectroscopies are employed to investigate the interactions of cationic Gemini surfactants with RNase Sa, a negatively charged ribonuclease. We find that RNase Sa binds Gemini surfactant monomers and micelles at pH values above 4 to form aggregates. Below pH 4, where the protein is positively charged, these aggregates dissolve and interactions are undetectable. Thermal denaturation experiments show that surfactant lowers RNase Sa's conformational stability, suggesting that surfactant binds the protein's denatured state preferentially. Finally, Gemini surfactants were found to bind RNA, leading to the formation of large complexes. Interestingly, Gemini surfactant binding did not prevent RNase Sa from cleaving RNA.

  11. A unique story in neonatal research: the development of a porcine surfactant.

    PubMed

    Curstedt, Tore; Halliday, Henry L; Speer, Christian P

    2015-01-01

    proteins have been developed. After preclinical testing, CHF5633 (developed by Tore Curstedt and Jan Johansson in collaboration with Chiesi Farmaceutici) has undergone a preliminary first study in humans under the guidance of Christian Speer. If effective, this new surfactant preparation could revolutionize the treatment of preterm infants worldwide as it could be made consistently and safely in almost unlimited quantities. This story of a porcine surfactant preparation has been truly remarkable, and many thousands of preterm babies worldwide are now alive and well because of it.

  12. Attenuated allergic airway hyperresponsiveness in C57BL/6 mice is associated with enhanced surfactant protein (SP)-D production following allergic sensitization

    PubMed Central

    Atochina, Elena N; Beers, Michael F; Tomer, Yaniv; Scanlon, Seth T; Russo, Scott J; Panettieri, Reynold A; Haczku, Angela

    2003-01-01

    Background C57BL/6 mice have attenuated allergic airway hyperresponsiveness (AHR) when compared with Balb/c mice but the underlying mechanisms remain unclear. SP-D, an innate immune molecule with potent immunosuppressive activities may have an important modulatory role in the allergic airway response and the consequent physiological changes. We hypothesized that an elevated SP-D production is associated with the impaired ability of C57BL/6 mice to develop allergic AHR. Methods SP-D mRNA and protein expression was investigated during development of allergic airway changes in a model of Aspergillus fumigatus (Af)-induced allergic inflammation. To study whether strain dependency of allergic AHR is associated with different levels of SP-D in the lung, Balb/c and C57BL/6 mice were compared. Results Sensitization and exposure to Af induced significant airway inflammation in both mouse strains in comparison with naïve controls. AHR to acetylcholine however was significantly attenuated in C57BL/6 mice in spite of increased eosinophilia and serum IgE when compared with Balb/c mice (p < 0.05). Af challenge of sensitized C57BL/6 mice induced a markedly increased SP-D protein expression in the SA surfactant fraction (1,894 ± 170% of naïve controls) that was 1.5 fold greater than the increase in Balb/c mice (1,234 ± 121% p < 0.01). These changes were selective since levels of the hydrophobic SP-B and SP-C and the hydrophilic SP-A were significantly decreased following sensitization and challenge with Af in both strains. Further, sensitized and exposed C57BL/6 mice had significantly lower IL-4 and IL-5 in the BAL fluid than that of Balb/c mice (p < 0.05). Conclusions These results suggest that enhanced SP-D production in the lung of C57BL/6 mice may contribute to an attenuated AHR in response to allergic airway sensitization. SP-D may act by inhibiting synthesis of Th2 cytokines. PMID:14748931

  13. Bioactive proteins in human milk: mechanisms of action.

    PubMed

    Lönnerdal, Bo

    2010-02-01

    Human milk contains a multitude of bioactive proteins, with very diverse functions. Some of these proteins are involved in the synthesis and expression of milk, but the majority appears to have evolved to provide physiological activities in the breast-fed infant. These activities are exerted by a wide variety of mechanisms and have largely been unraveled by in vitro studies. To be active in the gastrointestinal tract, these proteins must be able to resist proteolytic degradation, at least for some time. We have evaluated the human milk proteins lactoferrin, haptocorrin, alpha(1)-antitrypsin, and transforming growth factor -beta in an in vitro digestion model, mimicking the conditions of the infant gastrointestinal milieu. These bioactive proteins are resistant against proteolysis and can remain intact or as larger fragments through passage of the gastrointestinal tract. In vitro digestibility assays can be helpful to assess which human milk proteins can resist proteolysis and to what extent.

  14. Human lipopolysaccharide-binding protein potentiates bactericidal activity of human bactericidal/permeability-increasing protein.

    PubMed Central

    Horwitz, A H; Williams, R E; Nowakowski, G

    1995-01-01

    Human bactericidal/permeability-increasing protein (BPI) from neutrophils and a recombinant amino-terminal fragment, rBPI23, bind to and are cytotoxic for gram-negative bacteria both in vitro and ex vivo in plasma or whole blood. To function in vivo as an extracellular bactericidal agent, rBPI23 must act in the presence of the lipopolysaccharide-binding protein (LBP), which also binds to but has no reported cytotoxicity for gram-negative bacteria. LBP, which is present at 5 to 10 micrograms/ml in healthy humans and at much higher levels in septic patients, mediates proinflammatory host responses to gram-negative infection. On the basis of these previous observations, we have examined the effect of recombinant LBP (rLBP) on the bactericidal activity of rBPI23 against Escherichia coli J5 in vitro. Physiological concentrations of rLBP (5 to 20 micrograms/ml) had little or no bactericidal activity but reduced by up to approximately 10,000-fold the concentration of BPI required for bactericidal or related activities in assays which measure (i) cell viability as CFUs on solid media or growth in broth culture and (ii) protein synthesis following treatment with BPI. LBP also potentiated BPI-mediated permeabilization of the E. coli outer membrane to actinomycin D by about 100-fold but had no permeabilizing activity of its own. Under optimal conditions for potentiation, fewer than 100 BPI molecules were required to kill a single E. coli J5 bacterium. PMID:7822017

  15. Global landscape of HIV-human protein complexes.

    PubMed

    Jäger, Stefanie; Cimermancic, Peter; Gulbahce, Natali; Johnson, Jeffrey R; McGovern, Kathryn E; Clarke, Starlynn C; Shales, Michael; Mercenne, Gaelle; Pache, Lars; Li, Kathy; Hernandez, Hilda; Jang, Gwendolyn M; Roth, Shoshannah L; Akiva, Eyal; Marlett, John; Stephens, Melanie; D'Orso, Iván; Fernandes, Jason; Fahey, Marie; Mahon, Cathal; O'Donoghue, Anthony J; Todorovic, Aleksandar; Morris, John H; Maltby, David A; Alber, Tom; Cagney, Gerard; Bushman, Frederic D; Young, John A; Chanda, Sumit K; Sundquist, Wesley I; Kortemme, Tanja; Hernandez, Ryan D; Craik, Charles S; Burlingame, Alma; Sali, Andrej; Frankel, Alan D; Krogan, Nevan J

    2011-12-21

    Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host's cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV-human protein-protein interactions involving 435 individual human proteins, with ∼40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.

  16. SURFACTANTS IN LUBRICATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Surfactants are one of the most widely applied materials by consumers and industry. The application areas for surfactants span from everyday mundane tasks such as cleaning, to highly complex processes involving the formulation of pharmaceuticals, foods, pesticides, lubricants, etc. Even though sur...

  17. SURFACTANTS AND SUBSURFACE REMEDIATION

    EPA Science Inventory

    Because of the limitations of pump-and-trat technology, attention is now focused on the feasibility of surfactant use to increase its efficiency. Surfactants have been studied for use in soil washing and enhanced oil recovery. Although similarities exist between the application...

  18. Exogenous surfactant restores lung function but not peripheral immunosuppression in ventilated surfactant-deficient rats.

    PubMed

    Vreugdenhil, Harriet A; Lachmann, Burkhard; Haitsma, Jack J; Zijlstra, Jitske; Heijnen, Cobi J; Jansen, Nicolaas J; van Vught, Adrianus J

    2006-01-01

    The authors have previously shown that mechanical ventilation can result in increased pulmonary inflammation and suppressed peripheral leukocyte function. In the present study the effect of surfactant therapy on pulmonary inflammation and peripheral immune function in ventilated surfactant-deficient rats was assessed. Surfactant deficiency was induced by repeated lung lavage, treated rats with surfactant or left them untreated, and ventilated the rats during 2 hours. Nonventilated rats served as healthy control group. Expression of macrophage inflammatory protein (MIP)-2 was measured in bronchoalveolar lavage (BAL), interleukin (IL)-1beta, and heat shock protein 70 (HSP70) were measured in total lung homogenates. Outside the lung phytohemagglutinin (PHA)-induced lymphocyte proliferation, interferon (IFN)-gamma and IL-10 production, and natural killer activity were measured in splenocytes. After 2 hours of mechanical ventilation, expression of MIP-2, IL-1beta, and HSP70 increased significantly in the lungs of surfactant-deficient rats. Outside the lung, mitogen-induced proliferation and production of IFN-gamma and IL-10 reduced significantly. Only natural killer cell activity remained unaffected. Surfactant treatment significantly improved lung function, but could not prevent increased pulmonary expression of MIP-2, IL-1beta, and HSP70 and decreased peripheral mitogen-induced lymphocyte proliferation and IFN-gamma and IL-10 production in vitro. In conclusion, 2 hours of mechanical ventilation resulted in increased lung inflammation and partial peripheral leukocyte suppression in surfactant-deficient rats. Surfactant therapy ameliorated lung function but could not prevent or restore peripheral immunosuppression. The authors postulate that peripheral immunosuppression may occur in ventilated surfactant deficient patients, which may enhance susceptibility for infections.

  19. Human carotid atherosclerotic plaque protein(s) change HDL protein(s) composition and impair HDL anti-oxidant activity.

    PubMed

    Cohen, Elad; Aviram, Michael; Khatib, Soliman; Volkova, Nina; Vaya, Jacob

    2016-01-01

    High density lipoprotein (HDL) anti-atherogenic functions are closely associated with cardiovascular disease risk factor, and are dictated by its composition, which is often affected by environmental factors. The present study investigates the effects of the human carotid plaque constituents on HDL composition and biological functions. To this end, human carotid plaques were homogenized and incubated with HDL. Results showed that after incubation, most of the apolipoprotein A1 (Apo A1) protein was released from the HDL, and HDL diameter increased by an average of approximately 2 nm. In parallel, HDL antioxidant activity was impaired. In response to homogenate treatment HDL could not prevent the accelerated oxidation of LDL caused by the homogenate. Boiling of the homogenate prior to its incubation with HDL abolished its effects on HDL composition changes. Moreover, tryptophan fluorescence quenching assay revealed an interaction between plaque component(s) and HDL, an interaction that was reduced by 50% upon using pre-boiled homogenate. These results led to hypothesize that plaque protein(s) interacted with HDL-associated Apo A1 and altered the HDL composition. Immuno-precipitation of Apo A1 that was released from the HDL after its incubation with the homogenate revealed a co-precipitation of three isomers of actin. However, beta-actin alone did not significantly affect the HDL composition, and yet the active protein within the plaque was elusive. In conclusion then, protein(s) in the homogenate interact with HDL protein(s), leading to release of Apo A1 from the HDL particle, a process that was associated with an increase in HDL diameter and with impaired HDL anti-oxidant activity.

  20. Identification of proteins binding coding and non-coding human RNAs using protein microarrays

    PubMed Central

    2012-01-01

    Background The regulation and function of mammalian RNAs has been increasingly appreciated to operate via RNA-protein interactions. With the recent discovery of thousands of novel human RNA molecules by high-throughput RNA sequencing, efficient methods to uncover RNA-protein interactions are urgently required. Existing methods to study proteins associated with a given RNA are laborious and require substantial amounts of cell-derived starting material. To overcome these limitations, we have developed a rapid and large-scale approach to characterize binding of in vitro transcribed labeled RNA to ~9,400 human recombinant proteins spotted on protein microarrays. Results We have optimized methodology to probe human protein microarrays with full-length RNA molecules and have identified 137 RNA-protein interactions specific for 10 coding and non-coding RNAs. Those proteins showed strong enrichment for common human RNA binding domains such as RRM, RBD, as well as K homology and CCCH type zinc finger motifs. Previously unknown RNA-protein interactions were discovered using this technique, and these interactions were biochemically verified between TP53 mRNA and Staufen1 protein as well as between HRAS mRNA and CNBP protein. Functional characterization of the interaction between Staufen 1 protein and TP53 mRNA revealed a novel role for Staufen 1 in preserving TP53 RNA stability. Conclusions Our approach demonstrates a scalable methodology, allowing rapid and efficient identification of novel human RNA-protein interactions using RNA hybridization to human protein microarrays. Biochemical validation of newly identified interactions between TP53-Stau1 and HRAS-CNBP using reciprocal pull-down experiments, both in vitro and in vivo, demonstrates the utility of this approach to study uncharacterized RNA-protein interactions. PMID:23157412

  1. Protein interactions in human genetic diseases

    PubMed Central

    Schuster-Böckler, Benjamin; Bateman, Alex

    2008-01-01

    We present a novel method that combines protein structure information with protein interaction data to identify residues that form part of an interaction interface. Our prediction method can retrieve interaction hotspots with an accuracy of 60% (at a 20% false positive rate). The method was applied to all mutations in the Online Mendelian Inheritance in Man (OMIM) database, predicting 1,428 mutations to be related to an interaction defect. Combining predicted and hand-curated sets, we discuss how mutations affect protein interactions in general. PMID:18199329

  2. Spatial and temporal control of surfactant systems.

    PubMed

    Liu, Xiaoyang; Abbott, Nicholas L

    2009-11-01

    This paper reviews some recent progress on approaches leading to spatial and temporal control of surfactant systems. The approaches revolve around the use of redox-active and light-sensitive surfactants. Perspectives are presented on experiments that have realized approaches for active control of interfacial properties of aqueous surfactant systems, reversible control of microstructures and nanostructures formed within bulk solutions, and in situ manipulation of the interactions of surfactants with polymers, DNA and proteins. A particular focus of this review is devoted to studies of amphiphiles that contain the redox-active group ferrocene - reversible control of the oxidation state of ferrocene leads to changes in the charge/hydrophobicity of these amphiphiles, resulting in substantial changes in their self-assembly. Light-sensitive surfactants containing azobenzene, which undergo changes in shape/polarity upon illumination with light, are a second focus of this review. Examples of both redox-active and light-sensitive surfactants that lead to large (>20mN/m) and spatially localized ( approximately mm) changes in surface tensions on a time scale of seconds are presented. Systems that permit reversible transformations of bulk solution nanostructures - such as micelle-to-vesicle transitions or monomer-to-micelle transitions - are also described. The broad potential utility of these emerging classes of amphiphiles are illustrated by the ability to drive changes in functional properties of surfactant systems, such as rheological properties and reversible solubilization of oils, as well as the ability to control interactions of surfactants with biomolecules to modulate their transport into cells.

  3. The evolution of human cells in terms of protein innovation.

    PubMed

    Sardar, Adam J; Oates, Matt E; Fang, Hai; Forrest, Alistair R R; Kawaji, Hideya; Gough, Julian; Rackham, Owen J L

    2014-06-01

    Humans are composed of hundreds of cell types. As the genomic DNA of each somatic cell is identical, cell type is determined by what is expressed and when. Until recently, little has been reported about the determinants of human cell identity, particularly from the joint perspective of gene evolution and expression. Here, we chart the evolutionary past of all documented human cell types via the collective histories of proteins, the principal product of gene expression. FANTOM5 data provide cell-type-specific digital expression of human protein-coding genes and the SUPERFAMILY resource is used to provide protein domain annotation. The evolutionary epoch in which each protein was created is inferred by comparison with domain annotation of all other completely sequenced genomes. Studying the distribution across epochs of genes expressed in each cell type reveals insights into human cellular evolution in terms of protein innovation. For each cell type, its history of protein innovation is charted based on the genes it expresses. Combining the histories of all cell types enables us to create a timeline of cell evolution. This timeline identifies the possibility that our common ancestor Coelomata (cavity-forming animals) provided the innovation required for the innate immune system, whereas cells which now form the brain of human have followed a trajectory of continually accumulating novel proteins since Opisthokonta (boundary of animals and fungi). We conclude that exaptation of existing domain architectures into new contexts is the dominant source of cell-type-specific domain architectures.

  4. Untapped Potential of Disordered Proteins in Current Druggable Human Proteome.

    PubMed

    Hu, Gang; Wu, Zhonghua; Wang, Kui; Uversky, Vladimir N; Kurgan, Lukasz

    2016-01-01

    Current efforts in design and characterization of drugs often rely on the structure of their protein targets. However, a large fraction of proteins lack unique 3-D structures and exist as highly dynamic structural ensembles. These intrinsically disordered proteins are involved in pathogenesis of various human diseases and are highly abundant in eukaryotes. Based on a comprehensive analysis of the current druggable human proteome covering 12 drug classes and 18 major classes of drug targets we show a significant bias toward high structural coverage and low abundance of intrinsic disorder. We review reasons for this bias including widespread use of the structural information in various stages of drug development and characterization process and difficulty with attaining structures for the intrinsically disordered proteins. We also discuss future of intrinsically disordered proteins as drug targets. Given the overall high disorder content of the human proteome and current bias of the druggable human proteome toward structural proteins, it is inevitable that disordered proteins will have to raise up on the list of prospective drug targets. The protein disorder-assisted drug design can draw from current rational drug design techniques and would also need novel approaches that no longer rely on a unique protein structure.

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

    PubMed

    Kopincova, Jana; Calkovska, Andrea

    2016-04-01

    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.

  6. Coacervation with surfactants: From single-chain surfactants to gemini surfactants.

    PubMed

    Zhao, Weiwei; Wang, Yilin

    2017-01-01

    Coacervation is a spontaneous process during which a colloidal dispersion separates into two immiscible liquid phases: a colloid-rich liquid phase in equilibrium with a diluted phase. Coacervation is usually divided into simple coacervation and complex coacervation according to the number of components. Surfactant-based coacervation normally contains traditional single-chain surfactants. With the development of surfactants, gemini surfactants with two amphiphilic moieties have been applied to form coacervation. This review summarizes the development of simple coacervation and complex coacervation in the systems of single-chain surfactants and gemini surfactants. Simple coacervation in surfactant solutions with additives or at elevated temperature and complex coacervation in surfactant/polymer mixtures by changing charge densities, molecular weight, ionic strength, pH, or temperature are reviewed. The comparison between gemini surfactants and corresponding monomeric single-chain surfactants reveals that the unique structures of gemini surfactants endow them with higher propensity to generate coacervation.

  7. Influence of genetic variability at the surfactant proteins A and D in community-acquired pneumonia: a prospective, observational, genetic study

    PubMed Central

    2011-01-01

    Introduction Genetic variability of the pulmonary surfactant proteins A and D may affect clearance of microorganisms and the extent of the inflammatory response. The genes of these collectins (SFTPA1, SFTPA2 and SFTPD) are located in a cluster at 10q21-24. The objective of this study was to evaluate the existence of linkage disequilibrium (LD) among these genes, and the association of variability at these genes with susceptibility and outcome of community-acquired pneumonia (CAP). We also studied the effect of genetic variability on SP-D serum levels. Methods Seven non-synonymous polymorphisms of SFTPA1, SFTPA2 and SFTPD were analyzed. For susceptibility, 682 CAP patients and 769 controls were studied in a case-control study. Severity and outcome were evaluated in a prospective study. Haplotypes were inferred and LD was characterized. SP-D serum levels were measured in healthy controls. Results The SFTPD aa11-C allele was significantly associated with lower SP-D serum levels, in a dose-dependent manner. We observed the existence of LD among the studied genes. Haplotypes SFTPA1 6A2 (P = 0.0009, odds ration (OR) = 0.78), SFTPA2 1A0 (P = 0.002, OR = 0.79), SFTPA1-SFTPA2 6A2-1A0 (P = 0.0005, OR = 0.77), and SFTPD-SFTPA1-SFTPA2 C-6A2-1A0 (P = 0.00001, OR = 0.62) were underrepresented in patients, whereas haplotypes SFTPA2 1A10 (P = 0.00007, OR = 6.58) and SFTPA1-SFTPA2 6A3-1A (P = 0.0007, OR = 3.92) were overrepresented. Similar results were observed in CAP due to pneumococcus, though no significant differences were now observed after Bonferroni corrections. 1A10 and 6A-1A were associated with higher 28-day and 90-day mortality, and with multi-organ dysfunction syndrome (MODS) and acute respiratory distress syndrome (ARDS) respectively. SFTPD aa11-C allele was associated with development of MODS and ARDS. Conclusions Our study indicates that missense single nucleotide polymorphisms and haplotypes of SFTPA1, SFTPA2 and SFTPD are associated with susceptibility to CAP, and

  8. Spaceflight and protein metabolism, with special reference to humans

    NASA Technical Reports Server (NTRS)

    Stein, T. P.; Gaprindashvili, T.

    1994-01-01

    Human space missions have shown that human spaceflight is associated with a loss of body protein. Specific changes include a loss of lean body mass, decreased muscle mass in the calves, decreased muscle strength, and changes in plasma proteins and amino acids. The major muscle loss is believed to be associated with the antigravity (postural) muscle. The most significant loss of protein appears to occur during the first month of flight. The etiology is believed to be multifactorial with contributions from disuse atrophy, undernutrition, and a stress type of response. This article reviews the results of American and Russian space missions to investigate this problem in humans, monkeys, and rats. The relationship of the flight results with ground-based models including bedrest for humans and hindlimb unweighting for rats is also discussed. The results suggest that humans adapt to spaceflight much better than either monkeys or rats.

  9. Mitochondrial protein import and human health and disease.

    PubMed

    MacKenzie, James A; Payne, R Mark

    2007-05-01

    The targeting and assembly of nuclear-encoded mitochondrial proteins are essential processes because the energy supply of humans is dependent upon the proper functioning of mitochondria. Defective import of mitochondrial proteins can arise from mutations in the targeting signals within precursor proteins, from mutations that disrupt the proper functioning of the import machinery, or from deficiencies in the chaperones involved in the proper folding and assembly of proteins once they are imported. Defects in these steps of import have been shown to lead to oxidative stress, neurodegenerative diseases, and metabolic disorders. In addition, protein import into mitochondria has been found to be a dynamically regulated process that varies in response to conditions such as oxidative stress, aging, drug treatment, and exercise. This review focuses on how mitochondrial protein import affects human health and disease.

  10. Human protein reference database as a discovery resource for proteomics

    PubMed Central

    Peri, Suraj; Navarro, J. Daniel; Kristiansen, Troels Z.; Amanchy, Ramars; Surendranath, Vineeth; Muthusamy, Babylakshmi; Gandhi, T. K. B.; Chandrika, K. N.; Deshpande, Nandan; Suresh, Shubha; Rashmi, B. P.; Shanker, K.; Padma, N.; Niranjan, Vidya; Harsha, H. C.; Talreja, Naveen; Vrushabendra, B. M.; Ramya, M. A.; Yatish, A. J.; Joy, Mary; Shivashankar, H. N.; Kavitha, M. P.; Menezes, Minal; Choudhury, Dipanwita Roy; Ghosh, Neelanjana; Saravana, R.; Chandran, Sreenath; Mohan, Sujatha; Jonnalagadda, Chandra Kiran; Prasad, C. K.; Kumar-Sinha, Chandan; Deshpande, Krishna S.; Pandey, Akhilesh

    2004-01-01

    The rapid pace at which genomic and proteomic data is being generated necessitates the development of tools and resources for managing data that allow integration of information from disparate sources. The Human Protein Reference Database (http://www.hprd.org) is a web-based resource based on open source technologies for protein information about several aspects of human proteins including protein–protein interactions, post-translational modifications, enzyme–substrate relationships and disease associations. This information was derived manually by a critical reading of the published literature by expert biologists and through bioinformatics analyses of the protein sequence. This database will assist in biomedical discoveries by serving as a resource of genomic and proteomic information and providing an integrated view of sequence, structure, function and protein networks in health and disease. PMID:14681466

  11. Surfactants in the environment.

    PubMed

    Ivanković, Tomislav; Hrenović, Jasna

    2010-03-01

    Surfactants are a diverse group of chemicals that are best known for their wide use in detergents and other cleaning products. After use, residual surfactants are discharged into sewage systems or directly into surface waters, and most of them end up dispersed in different environmental compartments such as soil, water or sediment. The toxic effects of surfactants on various aquatic organisms are well known. In general, surfactants are present in the environment at levels below toxicity and in Croatia below the national limit. Most surfactants are readily biodegradable and their amount is greatly reduced with secondary treatment in wastewater treatment plants. The highest concern is the release of untreated wastewater or wastewater that has undergone primary treatment alone. The discharge of wastewater polluted with massive quantities of surfactants could have serious effects on the ecosystem. Future studies of surfactant toxicities and biodegradation are necessary to withdraw highly toxic and non-biodegradable compounds from commercial use and replace them with more environmentally friendly ones.

  12. Identification of the human testis protein phosphatase 1 interactome.

    PubMed

    Fardilha, Margarida; Esteves, Sara L C; Korrodi-Gregório, Luís; Vintém, Ana Paula; Domingues, Sara C; Rebelo, Sandra; Morrice, Nick; Cohen, Patricia T W; da Cruz e Silva, Odete A B; da Cruz e Silva, Edgar F

    2011-11-15

    Protein phosphorylation is a critical regulatory mechanism in cellular signalling. To this end, PP1 is a major eukaryotic serine/threonine-specific phosphatase whose cellular functions, in turn, depend on complexes it forms with PP1 interacting proteins-PIPs. The importance of the testis/sperm-enriched variant, PP1γ2, in sperm motility and spermatogenesis has previously been shown. Given the key role of PIPs, it is imperative to identify the physiologically relevant PIPs in testis and sperm. Hence, we performed Yeast Two-Hybrid screens of a human testis cDNA library using as baits the different PP1 isoforms and also a proteomic approach aimed at identifying PP1γ2 binding proteins. To the best of our knowledge this is the largest data set of the human testis PP1 interactome. We report the identification of 77 proteins in human testis and 7 proteins in human sperm that bind PP1. The data obtained increased the known PP1 interactome by reporting 72 novel interactions. Confirmation of the interaction of PP1 with 5 different proteins was also further validated by co-immunoprecipitation or protein overlays. The data here presented provides important insights towards the function of these proteins and opens new possibilities for future research. In fact, such diversity in PP1 regulators makes them excellent targets for pharmacological intervention.

  13. The human protein index: a prospect for laboratory medicine

    SciTech Connect

    Anderson, N.G.; Anderson, N.L.

    1982-06-01

    A description is given of an annotated human protein index constructed with the use of new, high-resolution separation techniques combined with computerized image analysis, data reduction and data-base organization and management methods. Isoelectric focusing and gel electrophoresis have been combined in a two-dimensional procedure which should be capable of separating more than 10,000 proteins. Uses of the protein map in molecular pathology and biotechnology are discussed.

  14. Metathesis depolymerizable surfactants

    DOEpatents

    Jamison, Gregory M.; Wheeler, David R.; Loy, Douglas A.; Simmons, Blake A.; Long, Timothy M.; McElhanon, James R.; Rahimian, Kamyar; Staiger, Chad L.

    2008-04-15

    A class of surfactant molecules whose structure includes regularly spaced unsaturation in the tail group and thus, can be readily decomposed by ring-closing metathesis, and particularly by the action of a transition metal catalyst, to form small molecule products. These small molecules are designed to have increased volatility and/or enhanced solubility as compared to the original surfactant molecule and are thus easily removed by solvent extraction or vacuum extraction at low temperature. By producing easily removable decomposition products, the surfactant molecules become particularly desirable as template structures for preparing meso- and microstructural materials with tailored properties.

  15. Ki-67 acts as a biological surfactant to disperse mitotic chromosomes.

    PubMed

    Cuylen, Sara; Blaukopf, Claudia; Politi, Antonio Z; Müller-Reichert, Thomas; Neumann, Beate; Poser, Ina; Ellenberg, Jan; Hyman, Anthony A; Gerlich, Daniel W

    2016-07-14

    Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes and the discovery of proteins at the chromosome surface, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.

  16. Protein Dynamics in Individual Human Cells: Experiment and Theory

    PubMed Central

    Geva-Zatorsky, Naama; Danon, Tamar; Issaeva, Irina; Kopito, Ronen Benjamine; Perzov, Natalie; Milo, Ron; Sigal, Alex; Alon, Uri

    2009-01-01

    A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle–dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell–cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell–cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells. PMID:19381343

  17. Cell polarity proteins: common targets for tumorigenic human viruses

    PubMed Central

    Javier, RT

    2012-01-01

    Loss of polarity and disruption of cell junctions are common features of epithelial-derived cancer cells, and mounting evidence indicates that such defects have a direct function in the pathology of cancer. Supporting this idea, results with several different human tumor viruses indicate that their oncogenic potential depends in part on a common ability to inactivate key cell polarity proteins. For example, adenovirus (Ad) type 9 is unique among human Ads by causing exclusively estrogen-dependent mammary tumors in experimental animals and in having E4 region-encoded open reading frame 1 (E4-ORF1) as its primary oncogenic determinant. The 125-residue E4-ORF1 protein consists of two separate protein-interaction elements, one of which defines a PDZ domain-binding motif (PBM) required for E4-ORF1 to induce both cellular transformation in vitro and tumorigenesis in vivo. Most notably, the E4-ORF1 PBM mediates interactions with a selected group of cellular PDZ proteins, three of which include the cell polarity proteins Dlg1, PATJ and ZO-2. Data further indicate that these interactions promote disruption of cell junctions and a loss of cell polarity. In addition, one or more of the E4-ORF1-interacting cell polarity proteins, as well as the cell polarity protein Scribble, are common targets for the high-risk human papillomavirus (HPV) E6 or human T-cell leukemia virus type 1 (HTLV-1) Tax oncoproteins. Underscoring the significance of these observations, in humans, high-risk HPV and HTLV-1 are causative agents for cervical cancer and adult T-cell leukemia, respectively. Consequently, human tumor viruses should serve as powerful tools for deciphering mechanisms whereby disruption of cell junctions and loss of cell polarity contribute to the development of many human cancers. This review article discusses evidence supporting this hypothesis, with an emphasis on the human Ad E4-ORF1 oncoprotein. PMID:19029943

  18. Meat protein fractions enhance nonheme iron absorption in humans.

    PubMed

    Hurrell, Richard F; Reddy, Manju B; Juillerat, Marcel; Cook, James D

    2006-11-01

    The nature of the enhancing effect of muscle tissue on nonheme iron absorption in humans is unclear but thought to be related to muscle proteins. We conducted radioiron absorption studies to compare iron absorption from proteins isolated from beef and chicken muscle with that from freeze-dried beef and chicken muscle and from egg albumin. All meals contained an equivalent amount of protein as part of a semisynthetic liquid formula. Freeze-dried beef and chicken muscle increased iron absorption 180% (P < 0.001) and 100% (P < 0.001), respectively, relative to egg albumin. When added to the meal at an equivalent protein level (15 g), the isolated beef protein and the isolated heme-free beef protein with 94 and 98% protein content, respectively, increased iron absorption to the same extent as the native beef muscle. Similarly, when added to the meal at an equivalent protein level (30 g), isolated chicken muscle protein (94% protein) increased iron absorption similarly to native chicken muscle. Iron absorption from the meal containing the isolated heme-free chicken protein, however, was 120% (P < 0.01) greater than from the meal containing freeze-dried chicken muscle, indicating that a nonprotein component of muscle tissue with iron-binding potential may have been removed or concentrated by the protein extraction and separation procedures. Our results support the hypothesis that the enhancing effect of muscle tissue on iron absorption is mainly protein related but indicate that other factors may also play a role.

  19. Human SUMO fusion systems enhance protein expression and solubility.

    PubMed

    Wang, Zhongyuan; Li, Haolong; Guan, Wei; Ling, Haili; Wang, Zhiyong; Mu, Tianyang; Shuler, Franklin D; Fang, Xuexun

    2010-10-01

    A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins.

  20. Prediction of the Ebola virus infection related human genes using protein-protein interaction network.

    PubMed

    Cao, HuanHuan; Zhang, YuHang; Zhao, Jia; Zhu, Liucun; Wang, Yi; Li, JiaRui; Feng, Yuanming; Zhang, Ning

    2017-03-10

    Ebola hemorrhagic fever (EHF) is caused by Ebola virus (EBOV). It is reported that human could be infected by EBOV with a high fatality rate. However, association factors between EBOV and host still tend to be ambiguous. According to the "guilt by association" (GBA) principle, proteins interacting with each other are very likely to function similarly or the same. Based on this assumption, we tried to obtain EBOV infection-related human genes in a protein-protein interaction network using Dijkstra algorithm. We hope it could contribute to the discovery of novel effective treatments. Finally, 15 genes were selected as potential EBOV infection-related human genes.

  1. Converting Human Proteins into Precision Polymer Therapeutics.

    PubMed

    Boldt, Felix; Liu, Weina; Wu, Yuzhou; Weil, Tanja

    2016-01-01

    Cells as the smallest unit of life rely on precise macromolecules and programmable supramolecular interactions to accomplish the various vital functions. To translate such strategies to precisely control architectures and interactions into the synthetic world represents an exciting endeavor. Polymers with distinct structures, sequences and architectures are still challenging to achieve. However, in particular for biomedical applications, reproducible synthesis, narrow dispersities, tunable functionalities and additionally biocompatibility of the polymeric materials are crucial. Polymers derived from protein precursors provide many advantages of proteins such as precise monomer sequences and contour lengths, biodegradability and multiple functionalities, which can be synergistically combined with the valuable features of synthetic polymers e.g. stability, tunable solubility and molecular weights. The resulting polymeric biohybrid materials offer many applications ranging from drug delivery to biosensing and therapeutic hydrogels. This minireview summarizes the most recent advances in this field.

  2. Apoptosis induced by a human milk protein.

    PubMed

    Håkansson, A; Zhivotovsky, B; Orrenius, S; Sabharwal, H; Svanborg, C

    1995-08-15

    To the breast-fed infant, human milk is more than a source of nutrients; it furnishes a wide array of molecules that restrict microbes, such as antibodies, bactericidins, and inhibitors of bacterial adherence. However, it has rarely been considered that human milk may also contain substances bioactive toward host cells. While investigating the effect of human milk on bacterial adherence to a human lung cancer cell line, we were surprised to discover that the milk killed the cells. Analysis of this effect revealed that a component of milk in a particular physical state--multimeric alpha-lact-albumin--is a potent Ca(2+)-elevating and apoptosis-inducing agent with broad, yet selective, cytotoxic activity. Multimeric alpha-lactalbumin killed all transformed, embryonic, and lymphoid cells tested but spared mature epithelial elements. These findings raise the possibility that milk contributes to mucosal immunity not only by furnishing antimicrobial molecules but also by policing the function of lymphocytes and epithelium. Finally, analysis of the mechanism by which multimeric alpha-lactalbumin induces apoptosis in transformed epithelial cells could lead to the design of antitumor agents.

  3. Human enterovirus 71 protein interaction network prompts antiviral drug repositioning

    PubMed Central

    Han, Lu; Li, Kang; Jin, Chaozhi; Wang, Jian; Li, Qingjun; Zhang, Qiling; Cheng, Qiyue; Yang, Jing; Bo, Xiaochen; Wang, Shengqi

    2017-01-01

    As a predominant cause of human hand, foot, and mouth disease, enterovirus 71 (EV71) infection may lead to serious diseases and result in severe consequences that threaten public health and cause widespread panic. Although the systematic identification of physical interactions between viral proteins and host proteins provides initial information for the recognition of the cellular mechanism involved in viral infection and the development of new therapies, EV71-host protein interactions have not been explored. Here, we identified interactions between EV71 proteins and host cellular proteins and confirmed the functional relationships of EV71-interacting proteins (EIPs) with virus proliferation and infection by integrating a human protein interaction network and by functional annotation. We found that most EIPs had known interactions with other viruses. We also predicted ATP6V0C as a broad-spectrum essential host factor and validated its essentiality for EV71 infection in vitro. EIPs and their interacting proteins were more likely to be targets of anti-inflammatory and neurological drugs, indicating their potential to serve as host-oriented antiviral targets. Thus, we used a connectivity map to find drugs that inhibited EIP expression. We predicted tanespimycin as a candidate and demonstrated its antiviral efficiency in vitro. These findings provide the first systematic identification of EV71-host protein interactions, an analysis of EIP protein characteristics and a demonstration of their value in developing host-oriented antiviral therapies. PMID:28220872

  4. Human enterovirus 71 protein interaction network prompts antiviral drug repositioning.

    PubMed

    Han, Lu; Li, Kang; Jin, Chaozhi; Wang, Jian; Li, Qingjun; Zhang, Qiling; Cheng, Qiyue; Yang, Jing; Bo, Xiaochen; Wang, Shengqi

    2017-02-21

    As a predominant cause of human hand, foot, and mouth disease, enterovirus 71 (EV71) infection may lead to serious diseases and result in severe consequences that threaten public health and cause widespread panic. Although the systematic identification of physical interactions between viral proteins and host proteins provides initial information for the recognition of the cellular mechanism involved in viral infection and the development of new therapies, EV71-host protein interactions have not been explored. Here, we identified interactions between EV71 proteins and host cellular proteins and confirmed the functional relationships of EV71-interacting proteins (EIPs) with virus proliferation and infection by integrating a human protein interaction network and by functional annotation. We found that most EIPs had known interactions with other viruses. We also predicted ATP6V0C as a broad-spectrum essential host factor and validated its essentiality for EV71 infection in vitro. EIPs and their interacting proteins were more likely to be targets of anti-inflammatory and neurological drugs, indicating their potential to serve as host-oriented antiviral targets. Thus, we used a connectivity map to find drugs that inhibited EIP expression. We predicted tanespimycin as a candidate and demonstrated its antiviral efficiency in vitro. These findings provide the first systematic identification of EV71-host protein interactions, an analysis of EIP protein characteristics and a demonstration of their value in developing host-oriented antiviral therapies.

  5. Recent advances in gemini surfactants: oleic Acid-based gemini surfactants and polymerizable gemini surfactants.

    PubMed

    Sakai, Kenichi; Sakai, Hideki; Abe, Masahiko

    2011-01-01

    Gemini surfactants recently developed by our research group are introduced from the standpoints of their syntheses, aqueous solution properties, and potential applications. Two series of gemini surfactants are introduced in this short review, the first of which is the oleic acid-based gemini surfactants, and the second is the polymerizable gemini surfactants. These gemini surfactants have been developed not only as environmentally friendly materials (the use of gemini surfactants enables the reduction of the total consumption of surfactants in chemical products owing to their excellent adsorption and micellization capabilities at low concentrations) but also as functional organic materials.

  6. Phosphine oxide surfactants revisited.

    PubMed

    Stubenrauch, Cosima; Preisig, Natalie; Laughlin, Robert G

    2016-04-01

    This review summarizes everything we currently know about the nonionic surfactants alkyl dimethyl (C(n)DMPO) and alkyl diethyl (C(n)DEPO) phosphine oxide (PO surfactants). The review starts with the synthesis and the general properties (Section 2) of these compounds and continues with their interfacial properties (Section 3) such as surface tension, surface rheology, interfacial tension and adsorption at solid surfaces. We discuss studies on thin liquid films and foams stabilized by PO surfactants (Section 4) as well as studies on their self-assembly into lyotropic liquid crystals and microemulsions, respectively (Section 5). We aim at encouraging colleagues from both academia and industry to take on board PO surfactants whenever possible and feasible because of their broad variety of excellent properties.

  7. Deep conservation of human protein tandem repeats within the eukaryotes.

    PubMed

    Schaper, Elke; Gascuel, Olivier; Anisimova, Maria

    2014-05-01

    Tandem repeats (TRs) are a major element of protein sequences in all domains of life. They are particularly abundant in mammals, where by conservative estimates one in three proteins contain a TR. High generation-scale duplication and deletion rates were reported for nucleic TR units. However, it is not known whether protein TR units can also be frequently lost or gained providing a source of variation for rapid adaptation of protein function, or alternatively, tend to have conserved TR unit configurations over long evolutionary times. To obtain a systematic picture, we performed a proteome-wide analysis of the mode of evolution for human protein TRs. For this purpose, we propose a novel method for the detection of orthologous TRs based on circular profile hidden Markov models. For all detected TRs, we reconstructed bispecies TR unit phylogenies across 61 eukaryotes ranging from human to yeast. Moreover, we performed additional analyses to correlate functional and structural annotations of human TRs with their mode of evolution. Surprisingly, we find that the vast majority of human TRs are ancient, with TR unit number and order preserved intact since distant speciation events. For example, ≥ 61% of all human TRs have been strongly conserved at least since the root of all mammals, approximately 300 Ma. Further, we find no human protein TR that shows evidence for strong recent duplications and deletions. The results are in contrast to the high generation-scale mutability of nucleic TRs. Presumably, most protein TRs fold into stable and conserved structures that are indispensable for the function of the TR-containing protein. All of our data and results are available for download from http://www.atgc-montpellier.fr/TRE.

  8. Human Proteinpedia enables sharing of human protein data

    SciTech Connect

    Mathivanan, Suresh; Ahmed, Mukhtar; Ahn, Natalie G.; Alexandre, Hainard; Amanchy, Ramars; Andrews, Philip C.; Bader, Joel S.; Balgley, Brian M.; Bantscheff, Marcus; Bennett, Keiryn; Bjorling, Erik; Blagoev, Blagoy; Bose , Ron; Brahmachari, Samir K.; Burlingame, Alma S.; Bustelo, Xos R.; Cagney, Gerard; Cantin, Greg T; Cardasis, Helene L; Celis, Julio E; Chaerkady, Raghothama; Chu, Feixia; Cole, Phillip A.; Costello, Catherine E; Cotter , Robert J.; Crockett, David; DeLany , James P.; De Marzo, Angelo M; DeSouza, Leroi V; Deutsch, Eric W.; Dransfield , Eric; Drewes , Gerard; Droit , Arnaud; Dunn, Michael; Elenitoba-Johnson, Kojo; Ewing, Rob M.; Van Eyk , Jennifer; Faca , Vitor; Falkner , Jayson; Fang, Xiangming; Fenselau , Catherine; Figeys , Daniel; Gagne , Pierre; Gelfi , Cecilia; Gevaert , Kris; Gimble , Jeffrey; Gnad , Florian; Goel, Renu; Gromov , Pavel; Hanash, Samir M.; Hancock, William S.; Harsha , HC; Hart , Gerald; Faith , Hays; He , Fuchu; Hebbar , Prashantha; Helsens , Kenny; Hermeking , Heiko; Hide , Winston; Hjerno, Karin; Hochstrasser, Denis F.; Hofmann, Oliver; Horn , David M.; Hruban , Ralph H.; Ibarrola , Nieves; James , Peter; Jensen , Ole N.; Jensen, Pia H.; Jung , Peter; Kandasamy, Kumaran; Kheterpal , Indu; Kikuno , Reiko; Korf, Ulrike; Korner, Roman; Kuster, Bernhard; Kwon , Min-Seok; Lee , Hyoung-Joo; Lee , Young - Jin; Lefevre , Michael; Lehvaslaiho, Minna; Lescuyer, Pierre; Levander, Fredrik; Lim, Megan S.; Lobke, Christian; Loo, Joseph; Mann, Matthias; Martens , Lennart; Martinez-Heredia, Juan; McComb, Mark E.; McRedmond , James; Mehrle, Alexander; Menon, Rajasree; Miller, Christine A.; Mischak, Harald; Mohan, S Sujatha; Mohmood , Riaz; Molina , Henrik; Moran , Michael F.; Morgan, James D.; Moritz , Robert; Morzel, Martine; Muddiman, David C.; Nalli , Anuradha; Navarro, J. D.; Neubert , Thomas A.; Ohara , Osamu; Oliva, Rafael; Omenn, Gilbert; Oyama , Masaaki; Paik, Young-Ki; Pennington , Kyla; Pepperkok, Rainer; Periaswamy, Balamurugan; Petricoin, Emanuel F.; Poirier, Guy G.; Prasad, T S Keshava; Purvine, Samuel O.; Rahiman , B Abdul; Ramachandran, Prasanna; Ramachandra , Y L; Rice, Robert H.; Rick , Jens; Ronnholm , Ragna H.; Salonen , Johanna; Sanchez , Jean - Charles; Sayd , Thierry; Seshi, Beerelli; Shankari, Kripa; Sheng , Shi Jun; Shetty , Vivekananda; Shivakumar, K.; Simpson, Richard J.; Sirdeshmukh, Ravi; Siu , K W Michael; Smith, Jeffrey C.; Smith, Richard D.; States, David J.; Sugano, Sumio; Sullivan , Matthew; Superti - Furga, Giulio; Takatalo , Maarit; Thongboonkerd , Visith; Trinidad , Jonathan C.; Uhlen , Mathias; Vandekerckhove, Joel; Vasilescu , Julian; Veenstra, Timothy D.; Vidal - Taboada, Jose - Manuel; Vihinen, Mauno; Wait , Robin; Wang, Xiaoyue; Wiemann, Stefan; Wu , Billy; Xu, Tao; Yates, John R.; Zhong, Jun; Zhou, Ming; Zhu, Yunping; Zurbig, Petra; Pandey, Akhilesh

    2008-02-01

    Proteomic technologies, such as yeast twohybrid, mass spectrometry (MS), protein/ peptide arrays and fluorescence microscopy, yield multi-dimensional data sets, which are often quite large and either not published or published as supplementary information that is not easily searchable. Without a system in place for standardizing and sharing data, it is not fruitful for the biomedical community to contribute these types of data to centralized repositories. Even more difficult is the annotation and display of pertinent information in the context of the corresponding proteins. Wikipedia, an online encyclopedia that anyone can edit, has already proven quite successful1 and can be used as a model for sharing biological data. However, the need for experimental evidence, data standardization and ownership of data creates scientific obstacles.

  9. Development of a full-length human protein production pipeline

    PubMed Central

    Saul, Justin; Petritis, Brianne; Sau, Sujay; Rauf, Femina; Gaskin, Michael; Ober-Reynolds, Benjamin; Mineyev, Irina; Magee, Mitch; Chaput, John; Qiu, Ji; LaBaer, Joshua

    2014-01-01

    There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high-throughput (HT) methods, we transferred the genes of 31 full-length proteins into three expression vectors, and expressed the collection as N-terminal HaloTag fusion proteins in Escherichia coli and two commercial cell-free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip® GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from E. coli, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full-length human proteins in these three expression systems. PMID:24806540

  10. Canola Proteins for Human Consumption: Extraction, Profile, and Functional Properties

    PubMed Central

    Tan, Siong H; Mailer, Rodney J; Blanchard, Christopher L; Agboola, Samson O

    2011-01-01

    Canola protein isolate has been suggested as an alternative to other proteins for human food use due to a balanced amino acid profile and potential functional properties such as emulsifying, foaming, and gelling abilities. This is, therefore, a review of the studies on the utilization of canola protein in human food, comprising the extraction processes for protein isolates and fractions, the molecular character of the extracted proteins, as well as their food functional properties. A majority of studies were based on proteins extracted from the meal using alkaline solution, presumably due to its high nitrogen yield, followed by those utilizing salt extraction combined with ultrafiltration. Characteristics of canola and its predecessor rapeseed protein fractions such as nitrogen yield, molecular weight profile, isoelectric point, solubility, and thermal properties have been reported and were found to be largely related to the extraction methods. However, very little research has been carried out on the hydrophobicity and structure profiles of the protein extracts that are highly relevant to a proper understanding of food functional properties. Alkaline extracts were generally not very suitable as functional ingredients and contradictory results about many of the measured properties of canola proteins, especially their emulsification tendencies, have also been documented. Further research into improved extraction methods is recommended, as is a more systematic approach to the measurement of desired food functional properties for valid comparison between studies. PMID:21535703

  11. Molecular interactions of graphene oxide with human blood plasma proteins

    NASA Astrophysics Data System (ADS)

    Kenry, Affa Affb Affc; Loh, Kian Ping; Lim, Chwee Teck

    2016-04-01

    We investigate the molecular interactions between graphene oxide (GO) and human blood plasma proteins. To gain an insight into the bio-physico-chemical activity of GO in biological and biomedical applications, we performed a series of biophysical assays to quantify the molecular interactions between GO with different lateral size distributions and the three essential human blood plasma proteins. We elucidate the various aspects of the GO-protein interactions, particularly, the adsorption, binding kinetics and equilibrium, and conformational stability, through determination of quantitative parameters, such as GO-protein association constants, binding cooperativity, and the binding-driven protein structural changes. We demonstrate that the molecular interactions between GO and plasma proteins are significantly dependent on the lateral size distribution and mean lateral sizes of the GO nanosheets and their subtle variations may markedly influence the GO-protein interactions. Consequently, we propose the existence of size-dependent molecular interactions between GO nanosheets and plasma proteins, and importantly, the presence of specific critical mean lateral sizes of GO nanosheets in achieving very high association and fluorescence quenching efficiency of the plasma proteins. We anticipate that this work will provide a basis for the design of graphene-based and other related nanomaterials for a plethora of biological and biomedical applications.

  12. The nucleocapsid protein of human coronavirus NL63.

    PubMed

    Zuwała, Kaja; Golda, Anna; Kabala, Wojciech; Burmistrz, Michał; Zdzalik, Michal; Nowak, Paulina; Kedracka-Krok, Sylwia; Zarebski, Mirosław; Dobrucki, Jerzy; Florek, Dominik; Zeglen, Sławomir; Wojarski, Jacek; Potempa, Jan; Dubin, Grzegorz; Pyrc, Krzysztof

    2015-01-01

    Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the Coronavirinae subfamily, especially human coronavirus 229E (HCoV-229E). Detailed analysis, however, reveals several unique features of the pathogen. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV.

  13. Distribution of Tight Junction Proteins in Adult Human Salivary Glands

    PubMed Central

    Maria, Ola M.; Kim, Jung-Wan Martin; Gerstenhaber, Jonathan A.; Baum, Bruce J.; Tran, Simon D.

    2008-01-01

    Tight junctions (TJs) are an essential structure of fluid-secreting cells, such as those in salivary glands. Three major families of integral membrane proteins have been identified as components of the TJ: claudins, occludin, and junctional adhesion molecules (JAMs), plus the cytosolic protein zonula occludens (ZO). We have been working to develop an orally implantable artificial salivary gland that would be suitable for treating patients lacking salivary parenchymal tissue. To date, little is known about the distribution of TJ proteins in adult human salivary cells and thus what key molecular components might be desirable for the cellular component of an artificial salivary gland device. Therefore, the aim of this study was to determine the distribution of TJ proteins in human salivary glands. Salivary gland samples were obtained from 10 patients. Frozen and formalin-fixed paraffin-embedded sections were stained using IHC methods. Claudin-1 was expressed in ductal, endothelial, and ∼25% of serous cells. Claudins-2, -3, and -4 and JAM-A were expressed in both ductal and acinar cells, whereas claudin-5 was expressed only in endothelial cells. Occludin and ZO-1 were expressed in acinar, ductal, and endothelial cells. These results provide new information on TJ proteins in two major human salivary glands and should serve as a reference for future studies to assess the presence of appropriate TJ proteins in a tissue-engineered human salivary gland. (J Histochem Cytochem 56:1093–1098, 2008) PMID:18765838

  14. Exceptional overproduction of a functional human membrane protein.

    PubMed

    Nyblom, Maria; Oberg, Fredrik; Lindkvist-Petersson, Karin; Hallgren, Karin; Findlay, Heather; Wikström, Jennie; Karlsson, Anders; Hansson, Orjan; Booth, Paula J; Bill, Roslyn M; Neutze, Richard; Hedfalk, Kristina

    2007-11-01

    Eukaryotic--especially human--membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP1 was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQP1 in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization.

  15. Human CAP18: a novel antimicrobial lipopolysaccharide-binding protein.

    PubMed Central

    Larrick, J W; Hirata, M; Balint, R F; Lee, J; Zhong, J; Wright, S C

    1995-01-01

    CAP18 (18-kDa cationic antimicrobial protein) is a protein originally identified and purified from rabbit leukocytes on the basis of its capacity to bind and inhibit various activities of lipopolysaccharide (LPS). Here we report the cloning of human CAP18 and characterize the anti-LPS activity of the C-terminal fragment. Oligonucleotide probes designed from the rabbit CAP18 cDNA were used to identify human CAP18 from a bone marrow cDNA library. The cDNA encodes a protein composed of a 30-amino-acid signal peptide, a 103-amino-acid N-terminal domain of unknown function, and a C-terminal domain of 37 amino acids homologous to the LPS-binding antimicrobial domain of rabbit CAP18, designated CAP18(104-140). A human CAP18-specific antiserum was generated by using CAP18 expressed as a fusion protein with the maltose-binding protein. Western blots (immunoblots) with this antiserum showed specific expression of human CAP18 in granulocytes. Synthetic human CAP18(104-140) and a more active truncated fragment, CAP18(104-135), were shown to (i) bind to erythrocytes coated with diverse strains of LPS, (ii) inhibit LPS-induced release of nitric oxide from macrophages, (iii) inhibit LPS-induced generation of tissue factor, and (iv) protect mice from LPS lethality. CAP18(104-140) may have therapeutic utility for conditions associated with elevated concentrations of LPS. PMID:7890387

  16. Systems Proteomics View of the Endogenous Human Claudin Protein Family

    PubMed Central

    Liu, Fei; Koval, Michael; Ranganathan, Shoba; Fanayan, Susan; Hancock, William S.; Lundberg, Emma K.; Beavis, Ronald C.; Lane, Lydie; Duek, Paula; McQuade, Leon; Kelleher, Neil L.; Baker, Mark S.

    2016-01-01

    Claudins are the major transmembrane protein components of tight junctions in human endothelia and epithelia. Tissue-specific expression of claudin members suggests that this protein family is not only essential for sustaining the role of tight junctions in cell permeability control but also vital in organizing cell contact signaling by protein–protein interactions. How this protein family is collectively processed and regulated is key to understanding the role of junctional proteins in preserving cell identity and tissue integrity. The focus of this review is to first provide a brief overview of the functional context, on the basis of the extensive body of claudin biology research that has been thoroughly reviewed, for endogenous human claudin members and then ascertain existing and future proteomics techniques that may be applicable to systematically characterizing the chemical forms and interacting protein partners of this protein family in human. The ability to elucidate claudin-based signaling networks may provide new insight into cell development and differentiation programs that are crucial to tissue stability and manipulation. PMID:26680015

  17. Crystal cataracts: Human genetic cataract caused by protein crystallization

    NASA Astrophysics Data System (ADS)

    Pande, Ajay; Pande, Jayanti; Asherie, Neer; Lomakin, Aleksey; Ogun, Olutayo; King, Jonathan; Benedek, George B.

    2001-05-01

    Several human genetic cataracts have been linked recently to point mutations in the D crystallin gene. Here we provide a molecular basis for lens opacity in two genetic cataracts and suggest that the opacity occurs because of the spontaneous crystallization of the mutant proteins. Such crystallization of endogenous proteins leading to pathology is an unusual event. Measurements of the solubility curves of crystals of the Arg-58 to His and Arg-36 to Ser mutants of D crystallin show that the mutations dramatically lower the solubility of the protein. Furthermore, the crystal nucleation rate of the mutants is enhanced considerably relative to that of the wild-type protein. It should be noted that, although there is a marked difference in phase behavior, there is no significant difference in protein conformation among the three proteins.

  18. DNA helicase activity in purified human RECQL4 protein.

    PubMed

    Suzuki, Takahiro; Kohno, Toshiyuki; Ishimi, Yukio

    2009-09-01

    Human RECQL4 protein was expressed in insect cells using a baculovirus protein expression system and it was purified to near homogeneity. The protein sedimented at a position between catalase (230 kDa) and ferritin (440 kDa) in glycerol gradient centrifugation, suggesting that it forms homo-multimers. Activity to displace annealed 17-mer oligonucleotide in the presence of ATP was co-sedimented with hRECQL4 protein. In ion-exchange chromatography, both DNA helicase activity and single-stranded DNA-dependent ATPase activity were co-eluted with hRECQL4 protein. The requirements of ATP and Mg for the helicase activity were different from those for the ATPase activity. The data suggest that the helicase migrates on single-stranded DNA in a 3'-5' direction. These results suggest that the hRECQL4 protein exhibits DNA helicase activity.

  19. Development of human protein reference database as an initial platform for approaching systems biology in humans.

    PubMed

    Peri, Suraj; Navarro, J Daniel; Amanchy, Ramars; Kristiansen, Troels Z; Jonnalagadda, Chandra Kiran; Surendranath, Vineeth; Niranjan, Vidya; Muthusamy, Babylakshmi; Gandhi, T K B; Gronborg, Mads; Ibarrola, Nieves; Deshpande, Nandan; Shanker, K; Shivashankar, H N; Rashmi, B P; Ramya, M A; Zhao, Zhixing; Chandrika, K N; Padma, N; Harsha, H C; Yatish, A J; Kavitha, M P; Menezes, Minal; Choudhury, Dipanwita Roy; Suresh, Shubha; Ghosh, Neelanjana; Saravana, R; Chandran, Sreenath; Krishna, Subhalakshmi; Joy, Mary; Anand, Sanjeev K; Madavan, V; Joseph, Ansamma; Wong, Guang W; Schiemann, William P; Constantinescu, Stefan N; Huang, Lily; Khosravi-Far, Roya; Steen, Hanno; Tewari, Muneesh; Ghaffari, Saghi; Blobe, Gerard C; Dang, Chi V; Garcia, Joe G N; Pevsner, Jonathan; Jensen, Ole N; Roepstorff, Peter; Deshpande, Krishna S; Chinnaiyan, Arul M; Hamosh, Ada; Chakravarti, Aravinda; Pandey, Akhilesh

    2003-10-01

    Human Protein Reference Database (HPRD) is an object database that integrates a wealth of information relevant to the function of human proteins in health and disease. Data pertaining to thousands of protein-protein interactions, posttranslational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization were extracted from the literature for a nonredundant set of 2750 human proteins. Almost all the information was obtained manually by biologists who read and interpreted >300,000 published articles during the annotation process. This database, which has an intuitive query interface allowing easy access to all the features of proteins, was built by using open source technologies and will be freely available at http://www.hprd.org to the academic community. This unified bioinformatics platform will be useful in cataloging and mining the large number of proteomic interactions and alterations that will be discovered in the postgenomic era.

  20. Development of Human Protein Reference Database as an Initial Platform for Approaching Systems Biology in Humans

    PubMed Central

    Peri, Suraj; Navarro, J. Daniel; Amanchy, Ramars; Kristiansen, Troels Z.; Jonnalagadda, Chandra Kiran; Surendranath, Vineeth; Niranjan, Vidya; Muthusamy, Babylakshmi; Gandhi, T.K.B.; Gronborg, Mads; Ibarrola, Nieves; Deshpande, Nandan; Shanker, K.; Shivashankar, H.N.; Rashmi, B.P.; Ramya, M.A.; Zhao, Zhixing; Chandrika, K.N.; Padma, N.; Harsha, H.C.; Yatish, A.J.; Kavitha, M.P.; Menezes, Minal; Choudhury, Dipanwita Roy; Suresh, Shubha; Ghosh, Neelanjana; Saravana, R.; Chandran, Sreenath; Krishna, Subhalakshmi; Joy, Mary; Anand, Sanjeev K.; Madavan, V.; Joseph, Ansamma; Wong, Guang W.; Schiemann, William P.; Constantinescu, Stefan N.; Huang, Lily; Khosravi-Far, Roya; Steen, Hanno; Tewari, Muneesh; Ghaffari, Saghi; Blobe, Gerard C.; Dang, Chi V.; Garcia, Joe G.N.; Pevsner, Jonathan; Jensen, Ole N.; Roepstorff, Peter; Deshpande, Krishna S.; Chinnaiyan, Arul M.; Hamosh, Ada; Chakravarti, Aravinda; Pandey, Akhilesh

    2003-01-01

    Human Protein Reference Database (HPRD) is an object database that integrates a wealth of information relevant to the function of human proteins in health and disease. Data pertaining to thousands of protein-protein interactions, posttranslational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization were extracted from the literature for a nonredundant set of 2750 human proteins. Almost all the information was obtained manually by biologists who read and interpreted >300,000 published articles during the annotation process. This database, which has an intuitive query interface allowing easy access to all the features of proteins, was built by using open source technologies and will be freely available at http://www.hprd.org to the academic community. This unified bioinformatics platform will be useful in cataloging and mining the large number of proteomic interactions and alterations that will be discovered in the postgenomic era. PMID:14525934

  1. Application of peptide gemini surfactants as novel solubilization surfactants for photosystems I and II of cyanobacteria.

    PubMed

    Koeda, Shuhei; Umezaki, Katsunari; Noji, Tomoyasu; Ikeda, Atsushi; Kawakami, Keisuke; Kondo, Masaharu; Yamamoto, Yasushi; Shen, Jian-Ren; Taga, Keijiro; Dewa, Takehisa; Ito, Shigeru; Nango, Mamoru; Tanaka, Toshiki; Mizuno, Toshihisa

    2013-09-17

    We designed novel peptide gemini surfactants (PG-surfactants), DKDKC12K and DKDKC12D, which can solubilize Photosystem I (PSI) of Thermosynecoccus elongatus and Photosystem II (PSII) of Thermosynecoccus vulcanus in an aqueous buffer solution. To assess the detailed effects of PG-surfactants on the original supramolecular membrane protein complexes and functions of PSI and PSII, we applied the surfactant exchange method to the isolated PSI and PSII. Spectroscopic properties, light-induced electron transfer activity, and dynamic light scattering measurements showed that PSI and PSII could be solubilized not only with retention of the original supramolecular protein complexes and functions but also without forming aggregates. Furthermore, measurement of the lifetime of light-induced charge-separation state in PSI revealed that both surfactants, especially DKDKC12D, displayed slight improvement against thermal denaturation below 60 °C compared with that using β-DDM. This degree of improvement in thermal resistance still seems low, implying that the peptide moieties did not interact directly with membrane protein surfaces. By conjugating an electron mediator such as methyl viologen (MV(2+)) to DKDKC12K (denoted MV-DKDKC12K), we obtained derivatives that can trap the generated reductive electrons from the light-irradiated PSI. After immobilization onto an indium tin oxide electrode, a cathodic photocurrent from the electrode to the PSI/MV-DKDKC12K conjugate was observed in response to the interval of light irradiation. These findings indicate that the PG-surfactants DKDKC12K and DKDKC12D provide not only a new class of solubilization surfactants but also insights into designing other derivatives that confer new functions on PSI and PSII.

  2. [Proteins of human milk involved in immunological processes].

    PubMed

    Lis, Jolanta; Orczyk-Pawiłowicz, Magdalena; Kątnik-Prastowska, Iwona

    2013-05-31

    Human milk contains a lot of components (i.e. proteins, carbohydrates, lipids, inorganic elements) which provide basic nutrients for infants during the first period of their lives. Qualitative composition of milk components of healthy mothers is similar, but their levels change during lactation stages. Colostrum is the fluid secreted during the first days postpartum by mammary epithelial cells. Colostrum is replaced by transitional milk during 5-15 days postpartum and from 15 days postpartum mature milk is produced. Human milk, apart from nutritional components, is a source of biologically active molecules, i.e. immunoglobulins, growth factors, cytokines, acute phase proteins, antiviral and antibacterial proteins. Such components of human milk are responsible for specific biological activities of human milk. This secretion plays an important role in growth and development of newborns. Bioactive molecules present in the milk support the immature immune system of the newborn and also protect against the development of infection. In this article we describe the pathways involved in the production and secretion of human milk, the state of knowledge on the proteome of human milk, and the contents of components of milk during lactation. Moreover, some growth factors and proteins involved in innate and specific immunity, intercellular communication, immunomodulation, and inflammatory processes have been characterized.

  3. 2-DE using hemi-fluorinated surfactants.

    PubMed

    Starita-Geribaldi, Mireille; Thebault, Pascal; Taffin de Givenchy, Elisabeth; Guittard, Frederic; Geribaldi, Serge

    2007-07-01

    The synthesis of hemi-fluorinated zwitterionic surfactants was realized and assessed for 2-DE, a powerful separation method for proteomic analysis. These new fluorinated amidosulfobetaine (FASB-p,m) were compared to their hydrocarbon counterparts amidosulfobetaine (ASB-n) characterized by a hydrophilic polar head, a hydrophobic and lipophilic tail, and an amido group as connector. The tail of these FASB surfactants was in part fluorinated resulting in the modulation of its lipophilicity (or oleophobicity). Their effect on the red blood cell (RBC) membrane showed a specific solubilization depending on the length of the hydrophobic part. A large number of polypeptide spots appeared in the 2-DE patterns by using FASB-p,m. The oleophobic character of these surfactants was confirmed by the fact that Band 3, a highly hydrophobic transmembrane protein, was not solubilized by these fluorinated structures. The corresponding pellet was very rich in Band 3 and could then be solubilized by using a strong detergent such as amidosulfobetaine with an alkyl tail containing 14 carbon atoms (ASB-14). Thus, these hemi-fluorinated surfactants appeared as powerful tools when used at the first step of a two-step solubilization strategy using a hydrocarbon homologous surfactant in the second step.

  4. Mechanisms and Treatment of Lung Lesions and Associated Surfactant Damage in Shock.

    DTIC Science & Technology

    1978-03-17

    the role of proteins associated with the surfactant system...Isolation and characterization of proteins associated with the lung surfactant system. F— Rabbit lung washings and purified lung surf actan t were...the unfractionated lung washings confirming our previous study which indicated that there is no specific protein associated with sur— factant

  5. Crystal Structure of Human Retinoblastoma Binding Protein 9

    SciTech Connect

    Vorobiev, S.; Su, M; Seetharaman, J; Huang, Y; Chen, C; Maglaqui, M; Janjua, H; Montelione, G; Tong, L; et. al.

    2009-01-01

    As a step towards better integrating protein three-dimensional (3D) structural information in cancer systems biology, the Northeast Structural Genomics Consortium (NESG) (www.nesg.org) has constructed a Human Cancer Pathway Protein Interaction Network (HCPIN) by analysis of several classical cancer-associated signaling pathways and their physical protein-protein interactions. Many well-known cancer-associated proteins play central roles as hubs or bottlenecks in the HCPIN (http://nmr.cabm.rutgers.edu/hcpin). NESG has selected more than 1000 human proteins and protein domains from the HCPIN for sample production and 3D structure determination. The long-range goal of this effort is to provide a comprehensive 3D structure-function database for human cancer-associated proteins and protein complexes, in the context of their interaction networks. Human retinoblastoma binding protein 9 (RBBP9) is one of the HCPIN proteins targeted by NESG. RBBP9 was initially identified as the product of a new gene, Bog (for B5T over-expressed gene), in several transformed rat liver epithelial cell lines resistant to the growth-inhibitory effect of TGF-1 as well as in primary human liver tumors. RBBP9 contains the retinoblastoma (Rb) binding motif LxCxE in its sequence, and was shown to interact with Rb by yeast two-hybrid and coimmunoprecipitation experiments. Mutation of the Leu residue in this motif to Gln blocked the binding to Rb. RBBP9 can displace E2F1 from E2F1-Rb complexes, and over expression of RBBP9 overcomes TGF-1 induced growth arrest and results in transformation of rat liver epithelial cells leading to hepatoblastoma-like tumors in nude mice. RBBP9 may also play a role in cellular responses to chronic low dose radiation. A close homolog of RBBP9, sharing 93% amino acid sequence identity and also known as RBBP10, interacts with a protein with sua5-yciO-yrdC domains.

  6. Exploiting Bacterial Operons To Illuminate Human Iron-Sulfur Proteins.

    PubMed

    Andreini, Claudia; Banci, Lucia; Rosato, Antonio

    2016-04-01

    Organisms from all kingdoms of life use iron-sulfur proteins (FeS-Ps) in a multitude of functional processes. We applied a bioinformatics approach to investigate the human portfolio of FeS-Ps. Sixty-one percent of human FeS-Ps bind Fe4S4 clusters, whereas 39% bind Fe2S2 clusters. However, this relative ratio varies significantly depending on the specific cellular compartment. We compared the portfolio of human FeS-Ps to 12 other eukaryotes and to about 700 prokaryotes. The comparative analysis of the organization of the prokaryotic homologues of human FeS-Ps within operons allowed us to reconstruct the human functional networks involving the conserved FeS-Ps common to prokaryotes and eukaryotes. These functional networks have been maintained during evolution and thus presumably represent fundamental cellular processes. The respiratory chain and the ISC machinery for FeS-P biogenesis are the two conserved processes that involve the majority of human FeS-Ps. Purine metabolism is another process including several FeS-Ps, in which BOLA proteins possibly have a regulatory role. The analysis of the co-occurrence of human FeS-Ps with other proteins highlighted numerous links between the iron-sulfur cluster machinery and the response mechanisms to cell damage, from repair to apoptosis. This relationship probably relates to the production of reactive oxygen species within the biogenesis and degradation of FeS-Ps.

  7. Ribosomal protein gene mapping and human chromosomal disorders

    SciTech Connect

    Kenmochi, N.; Goodman, N.; Page, D.C.

    1994-09-01

    In Drosophila, the Minute phenotype (reduced body size, diminished viability and fertility, and short, thin bristles) results from heterozygous deficiencies (deletions) at any one of 50 loci scattered about the genome. A handful of these Minute loci have been molecularly characterized, and all have been found to encode ribosomal proteins. Thus, the Minute phenotype appears to result from reduced protein synthetic capacity in flies with one rather than two copies of a given ribosomal protein (rp) gene. We are pursuing the possibility that similar reductions in protein synthetic capacity--again resulting from rp gene deficiencies--might underlie phenotypes associated with certain chromosomal disorders in humans. We and our colleagues have reported findings consistent with a role for RPS4 deficiency in the etiology of certain features of Turner syndrome, a complex human disorder classically associated with an XO karyotype. We are intrigued by the possibility that deficiencies of other human rp genes might cause phenotypic abnormalities similar to those seen in Turner syndrome--just as deficiencies of any of a number of Drosophila rp genes cause the Minute phenotype. We must first learn the chromosomal map position of each of the estimated 83 human rp genes. The task of mapping the functional (intron-containing) rp genes is complicated by the existence of processed pseudogenes elsewhere in the genome. To date, we have assigned (or confirmed the previous assignment of) 38 rp genes to individual human chromosomes by PCR analysis of human-rodent somatic cell hybrids containing subsets of human chromosomes, with all but four chromosomes carrying at least one rp gene. We have also identified more than 100 large-insert human YAC (yeast artificial chromosome) clones that contain individual rp genes. Such screening of YAC libraries will result in precise positioning of the rp genes on the emerging physical map of the human genome.

  8. Tunable protein synthesis by transcript isoforms in human cells.

    PubMed

    Floor, Stephen N; Doudna, Jennifer A

    2016-01-06

    Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5' and 3' untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5' untranslated regions exert robust translational control between cell lines, while 3' untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels.

  9. Studies of alpha-granule proteins in cultured human megakaryocytes.

    PubMed

    Veljkovic, Dragoslava Kika; Cramer, Elisabeth M; Alimardani, Gulie; Fichelson, Serge; Massé, Jean-Marc; Hayward, Catherine P M

    2003-11-01

    alpha-Granule protein storage is important for producing platelets with normal haemostatic function. The low to undetectable levels of several megakaryocyte-synthesized alpha-granule proteins in normal plasma suggest megakaryocytes are important to sequester these proteins in vivo. alpha-Granule protein storage in vitro has been studied using other cell types, with differences observed in how some proteins are processed compared to platelets. Human megakaryocytes, cultured from cord blood CD34(+) cells and grown in serum-free media containing thrombopoietin, were investigated to determine if they could be used as a model for studying normal alpha-granule protein processing and storage. ELISA indicated that cultured megakaryocytes contained the alpha-granule proteins multimerin, von Willebrand factor, thrombospondin-1, beta-thromboglobulin and platelet factor 4, but no detectable fibrinogen and factor V. A significant proportion of the alpha-granule protein in megakaryocyte cultures was contained within the cells (averages: 41-71 %), consistent with storage. Detailed analyses of multimerin and von Willebrand factor confirmed that alpha-granule proteins were processed to mature forms and were predominantly located in the alpha-granules of cultured megakaryocytes.Thrombopoietin-stimulated cultured megakaryocytes provide a useful model for studying alpha-granule protein processing and storage.

  10. Informing the Human Plasma Protein Binding of ...

    EPA Pesticide Factsheets

    The free fraction of a xenobiotic in plasma (Fub) is an important determinant of chemical adsorption, distribution, metabolism, elimination, and toxicity, yet experimental plasma protein binding data is scarce for environmentally relevant chemicals. The presented work explores the merit of utilizing available pharmaceutical data to predict Fub for environmentally relevant chemicals via machine learning techniques. Quantitative structure-activity relationship (QSAR) models were constructed with k nearest neighbors (kNN), support vector machines (SVM), and random forest (RF) machine learning algorithms from a training set of 1045 pharmaceuticals. The models were then evaluated with independent test sets of pharmaceuticals (200 compounds) and environmentally relevant ToxCast chemicals (406 total, in two groups of 238 and 168 compounds). The selection of a minimal feature set of 10-15 2D molecular descriptors allowed for both informative feature interpretation and practical applicability domain assessment via a bounded box of descriptor ranges and principal component analysis. The diverse pharmaceutical and environmental chemical sets exhibit similarities in terms of chemical space (99-82% overlap), as well as comparable bias and variance in constructed learning curves. All the models exhibit significant predictability with mean absolute errors (MAE) in the range of 0.10-0.18 Fub. The models performed best for highly bound chemicals (MAE 0.07-0.12), neutrals (MAE 0

  11. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays

    PubMed Central

    Yu, Xiaobo; LaBaer, Joshua

    2015-01-01

    Summary AMPylation (adenylylation) has been recognized as an important post translational modification employed by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes and is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method to identify new substrates using protein microarrays, which can significantly expand the list of potential substrates. Here, we describe procedures to detect AMPylated and auto-AMPylated proteins in a sensitive, high throughput, and non-radioactive manner. The approach employs high-density protein microarrays fabricated using NAPPA (Nucleic Acid Programmable Protein Arrays) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide–alkyne cycloaddition. The assay can be accomplished within 11 hours. PMID:25881200

  12. Human immunodeficiency virus type 1, human protein interaction database at NCBI.

    PubMed

    Fu, William; Sanders-Beer, Brigitte E; Katz, Kenneth S; Maglott, Donna R; Pruitt, Kim D; Ptak, Roger G

    2009-01-01

    The 'Human Immunodeficiency Virus Type 1 (HIV-1), Human Protein Interaction Database', available through the National Library of Medicine at www.ncbi.nlm.nih.gov/RefSeq/HIVInteractions, was created to catalog all interactions between HIV-1 and human proteins published in the peer-reviewed literature. The database serves the scientific community exploring the discovery of novel HIV vaccine candidates and therapeutic targets. To facilitate this discovery approach, the following information for each HIV-1 human protein interaction is provided and can be retrieved without restriction by web-based downloads and ftp protocols: Reference Sequence (RefSeq) protein accession numbers, Entrez Gene identification numbers, brief descriptions of the interactions, searchable keywords for interactions and PubMed identification numbers (PMIDs) of journal articles describing the interactions. Currently, 2589 unique HIV-1 to human protein interactions and 5135 brief descriptions of the interactions, with a total of 14,312 PMID references to the original articles reporting the interactions, are stored in this growing database. In addition, all protein-protein interactions documented in the database are integrated into Entrez Gene records and listed in the 'HIV-1 protein interactions' section of Entrez Gene reports. The database is also tightly linked to other databases through Entrez Gene, enabling users to search for an abundance of information related to HIV pathogenesis and replication.

  13. Surfactant-cobalt(III) complexes: The impact of hydrophobicity on interaction with HSA and DNA - insights from experimental and theoretical approach.

    PubMed

    Veeralakshmi, Selvakumar; Sabapathi, Gopal; Nehru, Selvan; Venuvanalingam, Ponnambalam; Arunachalam, Sankaralingam

    2017-02-16

    To develop surfactant-based metallodrugs, it is very important to know about their hydrophobicity, micelle forming capacity, their interaction with biomacromolecules such as proteins and nucleic acids, and biological activities. Here, diethylenetriamine (dien) and tetradecylamine ligand (TA) based surfactant-cobalt(III) complexes with single chain domain, [Co(dien)(TA)Cl2]ClO4 (1) and double chain domain [Co(dien)(TA)2Cl](ClO4)2 (2) were chosen to study the effect of hydrophobicity on the interaction with human serum albumin and calf thymus DNA. The obtained results showed that (i) single chain surfactant-cobalt(III) complex (1) interact with HSA and DNA via electrostatic interaction and groove binding, respectively; (ii) double chain surfactant-cobalt(III) complex (2) interact with HSA and DNA via hydrophobic interaction and partial intercalation, respectively, due to the play of hydrophobicity by single and double chain domains. Further it is noted that, double chain surfactant-cobalt(III) complex interact strongly with HSA and DNA, compared single chain surfactant-cobalt(III) complex due to their more hydrophobicity nature. DFT and molecular docking studies offer insights into the mechanism and mode of binding towards the molecular target CT-DNA and HSA. Hence, the present findings will create new avenue towards the use of hydrophobic metallodrugs for various therapeutic applications.

  14. Biodegradation of phenanthrene in soils in the presence of surfactants

    SciTech Connect

    Jahan, K.

    1993-01-01

    This research addresses the effect of low surfactant concentrations on the biodegradation of slightly soluble organic compounds in the presence and absence of soil. Biodegradation of phenanthrene in excess of its aqueous solubility by an acclimated mixed culture was studied in the presence of nonionic surfactants. Nonionic surfactants were selected over other types of surfactants because of their higher hydrocarbon solubilizing power, weaker adsorption to charged sites, less toxicity to bacteria, and poor foaming properties. Surfactants were tested to measure their effectiveness for increasing the solubility of phenanthrene, their adsorption on the soil matrix, their biodegradability, their effect on the adsorption of phenanthrene and on the rates of biodegradation of phenanthrene. Solubility enhancement studies of phenanthrene by the surfactants indicated relatively small effects at sub-micellar surfactant concentrations. Batch biodegradation studies in which phenanthrene was available as particulates and as a surface coating on sand were carried out in closed BOD bottles in the Hach manometric system. Addition of surfactants at 25 mg/L enhanced biodegradation rates as measured by oxygen uptake, protein production and disappearance of phenanthrene. A dynamic model which couples dissolution and biodegradation processes could adequately represent the experimental batch data. Modelling studies suggest that biodegradation was accelerated because the dissolution rates of phenanthrene increased in presence of the surfactants. Continuous flow column studies with phenanthrene coated Jordan sand was carried out to simulate groundwater flow conditions. Sorption studies on Jordan aquifer sand indicated that this low-carbon aquifer material adsorbs small amounts of phenanthrene as well as surfactants. The tests show that low surfactant concentrations were marginally beneficial in washing phenanthrene from precoated sand.

  15. Structural studies of human glioma pathogenesis-related protein 1

    SciTech Connect

    Asojo, Oluwatoyin A.; Koski, Raymond A.; Bonafé, Nathalie

    2011-10-01

    Structural analysis of a truncated soluble domain of human glioma pathogenesis-related protein 1, a membrane protein implicated in the proliferation of aggressive brain cancer, is presented. Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn{sup 2+} complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn{sup 2+} similarly to snake-venom CRISPs, which are involved in Zn{sup 2+}-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.

  16. Viral proteins that bridge unconnected proteins and components in the human PPI network.

    PubMed

    Rachita, H R; Nagarajaram, H A

    2014-07-29

    Viruses, despite having small genomes and few proteins, make an array of interactions with host proteins as they solely depend on host machinery for their replication and reproduction. Hence, analysis of the Human-Virus Protein-Protein Interaction Network (Hu-Vir PPI network) helps us to gain certain insights into the molecular mechanisms underlying the hijacking of host cell machinery by viruses for their perpetuation. Here we report an analysis of the Human-Virus Bridged PPI Networks that has led us to identify viral articulation points (VAPs) which connect unconnected components of the Human-PPI (Hu-PPI) network. VAPs cross-link peripheral nodes to the giant component of the Hu-PPI network. VAPs interact with a number of relatively lower topologically central human proteins and are conserved among related viruses. The linked nodes comprise of those that are mostly expressed during viral infection, as well as those that are found exclusively in some metabolic pathways, indicating that the novel viral mediation of certain human protein-protein interactions may form the basis for virus-specific tuning of the host machinery. The functional importance of VAPs and their interaction partners in virus replication make them potential drug targets against viral infection. Our investigations also led to the discovery of an example of a Human Endogenous Retrovirus (HERV) encoded protein, syncytin, as an Articulation Point (AP) in the Hu-PPI network, suggesting that VAPs may be retained in a genome if they result in any beneficial function in the host.

  17. Transduction of human recombinant proteins into mitochondria as a protein therapeutic approach for mitochondrial disorders.

    PubMed

    Papadopoulou, Lefkothea C; Tsiftsoglou, Asterios S

    2011-11-01

    Protein therapy is considered an alternative approach to gene therapy for treatment of genetic-metabolic disorders. Human protein therapeutics (PTs), developed via recombinant DNA technology and used for the treatment of these illnesses, act upon membrane-bound receptors to achieve their pharmacological response. On the contrary, proteins that normally act inside the cells cannot be developed as PTs in the conventional way, since they are not able to "cross" the plasma membrane. Furthermore, in mitochondrial disorders, attributed either to depleted or malfunctioned mitochondrial proteins, PTs should also have to reach the subcellular mitochondria to exert their therapeutic potential. Nowadays, there is no effective therapy for mitochondrial disorders. The development of PTs, however, via the Protein Transduction Domain (PTD) technology offered new opportunities for the deliberate delivery of human recombinant proteins inside eukaryotic subcellular organelles. To this end, mitochondrial disorders could be clinically encountered with the delivery of human mitochondrial proteins (engineered via recombinant DNA and PTD technologies) at specific intramitochondrial sites to exert their function. Overall, PTD-mediated Protein Replacement Therapy emerges as a suitable model system for the therapeutic approach for mitochondrial disorders.

  18. Expansion of the Protein Repertoire in Newly Explored Environments: Human Gut Microbiome Specific Protein Families

    PubMed Central

    Ellrott, Kyle; Jaroszewski, Lukasz; Li, Weizhong; Wooley, John C.; Godzik, Adam

    2010-01-01

    The microbes that inhabit particular environments must be able to perform molecular functions that provide them with a competitive advantage to thrive in those environments. As most molecular functions are performed by proteins and are conserved between related proteins, we can expect that organisms successful in a given environmental niche would contain protein families that are specific for functions that are important in that environment. For instance, the human gut is rich in polysaccharides from the diet or secreted by the host, and is dominated by Bacteroides, whose genomes contain highly expanded repertoire of protein families involved in carbohydrate metabolism. To identify other protein families that are specific to this environment, we investigated the distribution of protein families in the currently available human gut genomic and metagenomic data. Using an automated procedure, we identified a group of protein families strongly overrepresented in the human gut. These not only include many families described previously but also, interestingly, a large group of previously unrecognized protein families, which suggests that we still have much to discover about this environment. The identification and analysis of these families could provide us with new information about an environment critical to our health and well being. PMID:20532204

  19. Beneficial effects of synthetic KL₄ surfactant in experimental lung transplantation.

    PubMed

    Sáenz, A; Alvarez, L; Santos, M; López-Sánchez, A; Castillo-Olivares, J L; Varela, A; Segal, R; Casals, C

    2011-04-01

    The aim of this study was to investigate whether intratracheal administration of a new synthetic surfactant that includes the cationic, hydrophobic 21-residue peptide KLLLLKLLLLKLLLLKLLLLK (KL₄), might be effective in reducing ischaemia-reperfusion injury after lung transplantation. Single left lung transplantation was performed in Landrace pigs 22 h post-harvest. KL₄ surfactant at a dose of 25 mg total phospholipid·kg body weight⁻¹ (2.5 mL·kg body weight⁻¹) was instilled at 37°C to the donor left lung (n = 8) prior to explantation. Saline (2.5 mL·kg body weight⁻¹; 37°C) was instilled into the donor left lung of the untreated group (n = 6). Lung function in recipients was measured during 2 h of reperfusion. Recipient left lung bronchoalveolar lavage (BAL) provided native cytometric, inflammatory marker and surfactant data. KL(4) surfactant treatment recovered oxygen levels in the recipient blood (mean ± sd arterial oxygen tension/inspiratory oxygen fraction 424 ± 60 versus 263 ± 101 mmHg in untreated group; p=0.01) and normalised alveolar-arterial oxygen tension difference. Surfactant biophysical function was also recovered in KL₄ surfactant-treated lungs. This was associated with decreased C-reactive protein levels in BAL, and recovery of surfactant protein A content, normalised protein/phospholipid ratios, and lower levels of both lipid peroxides and protein carbonyls in large surfactant aggregates. These findings suggest an important protective role for KL₄ surfactant treatment in lung transplantation.

  20. N-Linked glycoengineering for human therapeutic proteins in bacteria.

    PubMed

    Pandhal, Jagroop; Wright, Phillip C

    2010-09-01

    Approx. 70% of human therapeutic proteins are N-linked glycoproteins, and therefore host cells for production must contain the relevant protein modification machinery. The discovery and characterisation of the N-linked glycosylation pathway in the pathogenic bacterium Campylobacter jejuni, and subsequently its functional transfer to Escherichia coli, presents the opportunity of using prokaryotes as cell factories for therapeutic protein production. Not only could bacteria reduce costs and increase yields, but the improved feasibility to genetically control microorganisms means new and improved pharmacokinetics of therapeutics is an exciting possibility. This is a relatively new concept, and progress in bacterial N-glycosylation characterisation is reviewed and metabolic engineering targets revealed.

  1. Human muscle proteins: analysis by two-dimensional electrophoresis

    SciTech Connect

    Giometti, C.S.; Danon, M.J.; Anderson, N.G.

    1983-09-01

    Proteins from single frozen sections of human muscle were separated by two-dimensional gel electrophoresis and detected by fluorography or Coomassie Blue staining. The major proteins were identical in different normal muscles obtained from either sex at different ages, and in Duchenne and myotonic dystrophy samples. Congenital myopathy denervation atrophy, polymyositis, and Becker's muscular dystrophy samples, however, showed abnormal myosin light chain compositions, some with a decrease of fast-fiber myosin light chains and others with a decrease of slow-fiber light chains. These protein alterations did not correlate with any specific disease, and may be cause by generalized muscle-fiber damage.

  2. Comparison of rSP-C surfactant with natural and synthetic surfactants after late treatment in a rat model of the acute respiratory distress syndrome.

    PubMed

    Häfner, D; Germann, P G; Hauschke, D

    1998-07-01

    1. In a previous paper we showed that an SP-C containing surfactant preparation has similar activity as bovine-derived surfactants in a rat lung lavage model of the adult respiratory distress syndrome. In this study surfactant was given ten minutes after the last lavage (early treatment). In the present investigation we were interested how different surfactant preparations behave when they are administered 1 h after the last lavage (late treatment). 2. Four protein containing surfactants (rSP-C surfactant, bLES, Infasurf and Survanta) were compared with three protein-free surfactants (ALEC, Exosurf and the phospholipid (PL) mixture of the rSP-C surfactant termed PL surfactant) with respect to their ability to improve gas exchange in this more stringent model when surfactant is given one hour after the last lavage. For better comparison of the surfactants the doses were related to phospholipids. The surfactants were given at doses of 25, 50 and 100 mg kg(-1) body weight. The surfactants were compared to an untreated control group that was only ventilated for the whole experimental period. 3. Tracheotomized rats (8-12 per dose and surfactant) were pressure-controlled ventilated (Siemens Servo Ventilator 900C) with 100% oxygen at a respiratory rate of 30 breaths min(-1), inspiration expiration ratio of 1:2, peak inspiratory pressure of 28 cmH2O at positive endexpiratory pressure (PEEP) of 8 cmH2O. Animals were ventilated for one hour after the last lavage and thereafter the surfactants were intratracheally instilled. During the whole experimental period the ventilation was not changed. 4. Partial arterial oxygen pressures (PaO2, mmHg) at 30 min and 120 min after treatment were used for statistical comparison. All protein containing surfactants caused a dose-dependent increase of the reduced PaO2 values at 30 min after treatment. The protein-free surfactants showed only weak dose-dependent increase in PaO2 values at this time. This difference between the protein

  3. ABC proteins protect the human body and maintain optimal health.

    PubMed

    Ueda, Kazumitsu

    2011-01-01

    Human MDR1, a multi-drug transporter gene, was isolated as the first of the eukaryote ATP Binding Cassette (ABC) proteins from a multidrug-resistant carcinoma cell line in 1986. To date, over 25 years, many ABC proteins have been found to play important physiological roles by transporting hydrophobic compounds. Defects in their functions cause various diseases, indicating that endogenous hydrophobic compounds, as well as water-soluble compounds, are properly transported by transmembrane proteins. MDR1 transports a large number of structurally unrelated drugs and is involved in their pharmacokinetics, and thus is a key factor in drug interaction. ABCA1, an ABC protein, eliminates excess cholesterol in peripheral cells by generating HDL. Because ABCA1 is a key molecule in cholesterol homeostasis, its function and expression are highly regulated. Eukaryote ABC proteins function on the body surface facing the outside and in organ pathways to adapt to the extracellular environment and protect the body to maintain optimal health.

  4. Pathogen receptor discovery with a microfluidic human membrane protein array.

    PubMed

    Glick, Yair; Ben-Ari, Ya'ara; Drayman, Nir; Pellach, Michal; Neveu, Gregory; Boonyaratanakornkit, Jim; Avrahami, Dorit; Einav, Shirit; Oppenheim, Ariella; Gerber, Doron

    2016-04-19

    The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein-pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism.

  5. Chemoproteomic profiling and discovery of protein electrophiles in human cells

    NASA Astrophysics Data System (ADS)

    Matthews, Megan L.; He, Lin; Horning, Benjamin D.; Olson, Erika J.; Correia, Bruno E.; Yates, John R.; Dawson, Philip E.; Cravatt, Benjamin F.

    2016-10-01

    Activity-based protein profiling (ABPP) serves as a chemical proteomic platform to discover and characterize functional amino acids in proteins on the basis of their enhanced reactivity towards small-molecule probes. This approach, to date, has mainly targeted nucleophilic functional groups, such as the side chains of serine and cysteine, using electrophilic probes. Here we show that 'reverse-polarity' (RP)-ABPP using clickable, nucleophilic hydrazine probes can capture and identify protein-bound electrophiles in cells. Using this approach, we demonstrate that the pyruvoyl cofactor of S-adenosyl-L-methionine decarboxylase (AMD1) is dynamically controlled by intracellular methionine concentrations. We also identify a heretofore unknown modification—an N-terminally bound glyoxylyl group—in the poorly characterized protein secernin-3. RP-ABPP thus provides a versatile method to monitor the metabolic regulation of electrophilic cofactors and discover novel types of electrophilic modifications on proteins in human cells.

  6. Structural studies of human glioma pathogenesis-related protein 1.

    PubMed

    Asojo, Oluwatoyin A; Koski, Raymond A; Bonafé, Nathalie

    2011-10-01

    Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn2+ complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn2+ similarly to snake-venom CRISPs, which are involved in Zn2+-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.

  7. Characterization of the human GARP (Golgi associated retrograde protein) complex

    SciTech Connect

    Liewen, Heike; Meinhold-Heerlein, Ivo; Oliveira, Vasco; Schwarzenbacher, Robert; Luo Guorong; Wadle, Andreas; Jung, Martin; Pfreundschuh, Michael; Stenner-Liewen, Frank . E-mail: stenlie@t-online.de

    2005-05-15

    The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the 'orphan' SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells.

  8. Mass spectrometry of peptides and proteins from human blood.

    PubMed

    Zhu, Peihong; Bowden, Peter; Zhang, Du; Marshall, John G

    2011-01-01

    It is difficult to convey the accelerating rate and growing importance of mass spectrometry applications to human blood proteins and peptides. Mass spectrometry can rapidly detect and identify the ionizable peptides from the proteins in a simple mixture and reveal many of their post-translational modifications. However, blood is a complex mixture that may contain many proteins first expressed in cells and tissues. The complete analysis of blood proteins is a daunting task that will rely on a wide range of disciplines from physics, chemistry, biochemistry, genetics, electromagnetic instrumentation, mathematics and computation. Therefore the comprehensive discovery and analysis of blood proteins will rank among the great technical challenges and require the cumulative sum of many of mankind's scientific achievements together. A variety of methods have been used to fractionate, analyze and identify proteins from blood, each yielding a small piece of the whole and throwing the great size of the task into sharp relief. The approaches attempted to date clearly indicate that enumerating the proteins and peptides of blood can be accomplished. There is no doubt that the mass spectrometry of blood will be crucial to the discovery and analysis of proteins, enzyme activities, and post-translational processes that underlay the mechanisms of disease. At present both discovery and quantification of proteins from blood are commonly reaching sensitivities of ∼1 ng/mL.

  9. Human growth hormone prevents the protein catabolic side effects of prednisone in humans.

    PubMed Central

    Horber, F F; Haymond, M W

    1990-01-01

    Prednisone treatment causes protein wasting and adds additional risks to a patient, whereas human growth hormone (hGH) treatment causes positive nitrogen balance. To determine whether concomitant administration of hGH prevents the protein catabolic effects of prednisone, four groups of eight healthy volunteers each were studied using isotope dilution and nitrogen balance techniques after 7 d of placebo, hGH alone (0.1 mg.kg-1.d-1), prednisone alone (0.8 mg.kg-1.d-1), or prednisone plus hGH (n = 8 in each group). Whether protein balance was calculated from the leucine kinetic data or nitrogen balance values, prednisone alone induced protein wasting (P less than 0.001), whereas hGH alone resulted in positive (P less than 0.001) protein balance, when compared to the placebo-treated subjects. When hGH was added to prednisone therapy, the glucocorticoid-induced protein catabolism was prevented. Using leucine kinetic data, negative protein balance during prednisone was due to increased (P less than 0.05) proteolysis, whereas hGH had no effect on proteolysis and increased (P less than 0.01) whole body protein synthesis. During combined treatment, estimates of proteolysis and protein synthesis were similar to those observed in the placebo treated control group. In conclusion, human growth hormone may have a distinct role in preventing the protein losses associated with the administration of pharmacologic doses of glucocorticosteroids in humans. PMID:2195062

  10. Evaluation of silica nanoparticle binding to major human blood proteins

    NASA Astrophysics Data System (ADS)

    Hata, Katsutomo; Higashisaka, Kazuma; Nagano, Kazuya; Mukai, Yohei; Kamada, Haruhiko; Tsunoda, Shin-ichi; Yoshioka, Yasuo; Tsutsumi, Yasuo

    2014-12-01

    Nanomaterials are used for various biomedical applications because they are often more effective than conventional materials. Recently, however, it has become clear that the protein corona that forms on the surface of nanomaterials when they make contact with biological fluids, such as blood, influences the pharmacokinetics and biological responses induced by the nanomaterials. Therefore, when evaluating nanomaterial safety and efficacy, it is important to analyze the interaction between nanomaterials and proteins in biological fluids and to evaluate the effects of the protein corona. Here, we evaluated the interaction of silica nanoparticles, a commonly used nanomaterial, with the human blood proteins albumin, transferrin, fibrinogen, and IgG. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the amount of albumin, transferrin, and IgG binding to the silica particles increased as the particle size decreased under conditions where the silica particle mass remained the same. However, under conditions in which the specific surface area remained constant, there were no differences in the binding of human plasma proteins to the silica particles tested, suggesting that the binding of silica particles with human plasma proteins is dependent on the specific surface area of the silica particles. Furthermore, the amount of albumin, transferrin, and IgG binding to silica nanoparticles with a diameter of 70 nm (nSP70) and a functional amino group was lower than that with unmodified nSP70, although there was no difference in the binding between nSP70 with the surface modification of a carboxyl functional group and nSP70. These results suggest that the characteristics of nanomaterials are important for binding with human blood proteins; this information may contribute to the development of safe and effective nanomaterials.

  11. Ionic liquids as surfactants

    NASA Astrophysics Data System (ADS)

    Smirnova, N. A.; Safonova, E. A.

    2010-10-01

    Problems of self-assembling in systems containing ionic liquids (ILs) are discussed. Main attention is paid to micellization in aqueous solutions of dialkylimidazolium ILs and their mixtures with classical surfactants. Literature data are reviewed, the results obtained by the authors and co-workers are presented. Thermodynamic aspects of the studies and problems of molecular-thermodynamic modeling receive special emphasis. It is shown that the aggregation behavior of dialkylimidazolium ILs is close to that of alkyltrimethylammonium salts (cationic surfactants) though ILs have a higher ability to self-organize, especially as it concerns long-range ordering. Some aspects of ILs applications are outlined where their common features with classical surfactants and definite specificity are of value.

  12. Proteome-wide protein concentrations in the human heart.

    PubMed

    Aye, Thin Thin; Scholten, Arjen; Taouatas, Nadia; Varro, Andras; Van Veen, Toon A B; Vos, Marc A; Heck, Albert J R

    2010-10-01

    The largest component of the human heart, the left ventricle (LV), plays a major role in delivering blood throughout the body. Therefore, an in-depth detailed quantitative proteome analysis of the human LV is a valuable resource. For this purpose, a multifaceted proteomics approach combining differential sample fractionations (gel, strong cation exchange (SCX)), enzymatic digestions (trypsin, chymotrypsin, LysN), and peptide fragmentation techniques (CID and ETcaD) was used to enhance protein sequence coverage, identification confidence and quantitative abundance determination. Using stringent criteria, 3584 distinct proteins could be identified from the latest well-annotated Swissprot database (23,000 entries). Commutatively, the over 130,000 identified MS/MS spectra were used to assess concentrations of each identified LV protein through a combination of spectral counting methods. Among the most concentrated proteins, many currently used biomarkers for detection of myocardial infarction reside. These cardiac leakage markers have a good diagnostic power, but their prognostic potential seems limited. Discovery of markers that represent etiological determinants of cardiac disease require a shift of focus towards the signaling proteome. Therefore, a protein-class centered quantitative analysis of kinases, phosphatases and GTPases was adopted. These comparative analyses revealed many cardiac involved kinases (PKA, CaMKII, ERK) to reside among the most abundant signaling proteins, and also to mediate many observed in vivo phosphorylation sites. The abundance chart of signaling proteins may assist in identifying novel functional pathways, for instance through the abundant, but relatively little known, kinases STK38L and OXSR1. The obtained quantitative protein library of the human left ventricle is a valuable resource to isolate signaling based, putative biomarkers with concentrations likely to be detectable in plasma.

  13. Phospholipid transfer protein in human plasma associates with proteins linked to immunity and inflammation.

    PubMed

    Cheung, Marian C; Vaisar, Tomás; Han, Xianlin; Heinecke, Jay W; Albers, John J

    2010-08-31

    Phospholipid transfer protein (PLTP), which associates with apolipoprotein A-I (the major HDL protein), plays a key role in lipoprotein remodeling. Because its level in plasma increases during acute inflammation, it may also play previously unsuspected roles in the innate immune system. To gain further insight into its potential physiological functions, we isolated complexes containing PLTP from plasma by immunoaffinity chromatography and determined their composition. Shotgun proteomics revealed that only 6 of the 24 proteins detected in the complexes were apolipoproteins. The most abundant proteins were clusterin (apoJ), PLTP itself, coagulation factors, complement factors, and apoA-I. Remarkably, 20 of the 24 proteins had known protein-protein interactions. Biochemical studies confirmed two previously established interactions and identified five new ones between PLTP and proteins. Moreover, clusterin, apoA-I, and apoE preserved the lipid-transfer activity of recombinant PLTP in the absence of lipid, indicating that these interactions may have functional significance. Unexpectedly, lipids accounted for only 3% of the mass of the PLTP complexes. Collectively, our observations indicate that PLTP in human plasma resides on lipid-poor complexes dominated by clusterin and proteins implicated in host defense and inflammation. They further suggest that protein-protein interactions drive the formation of PLTP complexes in plasma.

  14. Comparative interactomics for virus-human protein-protein interactions: DNA viruses versus RNA viruses.

    PubMed

    Durmuş, Saliha; Ülgen, Kutlu Ö

    2017-01-01

    Viruses are obligatory intracellular pathogens and completely depend on their hosts for survival and reproduction. The strategies adopted by viruses to exploit host cell processes and to evade host immune systems during infections may differ largely with the type of the viral genetic material. An improved understanding of these viral infection mechanisms is only possible through a better understanding of the pathogen-host interactions (PHIs) that enable viruses to enter into the host cells and manipulate the cellular mechanisms to their own advantage. Experimentally-verified protein-protein interaction (PPI) data of pathogen-host systems only became available at large scale within the last decade. In this study, we comparatively analyzed the current PHI networks belonging to DNA and RNA viruses and their human host, to get insights into the infection strategies used by these viral groups. We investigated the functional properties of human proteins in the PHI networks, to observe and compare the attack strategies of DNA and RNA viruses. We observed that DNA viruses are able to attack both human cellular and metabolic processes simultaneously during infections. On the other hand, RNA viruses preferentially interact with human proteins functioning in specific cellular processes as well as in intracellular transport and localization within the cell. Observing virus-targeted human proteins, we propose heterogeneous nuclear ribonucleoproteins and transporter proteins as potential antiviral therapeutic targets. The observed common and specific infection mechanisms in terms of viral strategies to attack human proteins may provide crucial information for further design of broad and specific next-generation antiviral therapeutics.

  15. Antibody humanization by structure-based computational protein design.

    PubMed

    Choi, Yoonjoo; Hua, Casey; Sentman, Charles L; Ackerman, Margaret E; Bailey-Kellogg, Chris

    2015-01-01

    Antibodies derived from non-human sources must be modified for therapeutic use so as to mitigate undesirable immune responses. While complementarity-determining region (CDR) grafting-based humanization techniques have been successfully applied in many cases, it remains challenging to maintain the desired stability and antigen binding affinity upon grafting. We developed an alternative humanization approach called CoDAH ("Computationally-Driven Antibody Humanization") in which computational protein design methods directly select sets of amino acids to incorporate from human germline sequences to increase humanness while maintaining structural stability. Retrospective studies show that CoDAH is able to identify variants deemed beneficial according to both humanness and structural stability criteria, even for targets lacking crystal structures. Prospective application to TZ47, a murine anti-human B7H6 antibody, demonstrates the approach. Four diverse humanized variants were designed, and all possible unique VH/VL combinations were produced as full-length IgG1 antibodies. Soluble and cell surface expressed antigen binding assays showed that 75% (6 of 8) of the computationally designed VH/VL variants were successfully expressed and competed with the murine TZ47 for binding to B7H6 antigen. Furthermore, 4 of the 6 bound with an estimated KD within an order of magnitude of the original TZ47 antibody. In contrast, a traditional CDR-grafted variant could not be expressed. These results suggest that the computational protein design approach described here can be used to efficiently generate functional humanized antibodies and provide humanized templates for further affinity maturation.

  16. A family of human cdc2-related protein kinases.

    PubMed Central

    Meyerson, M; Enders, G H; Wu, C L; Su, L K; Gorka, C; Nelson, C; Harlow, E; Tsai, L H

    1992-01-01

    The p34cdc2 protein kinase is known to regulate important transitions in the eukaryotic cell cycle. We have identified 10 human protein kinases based on their structural relation to p34cdc2. Seven of these kinases are novel and the products of five share greater than 50% amino acid sequence identity with p34cdc2. The seven novel genes are broadly expressed in human cell lines and tissues with each displaying some cell type or tissue specificity. The cdk3 gene, like cdc2 and cdk2, can complement cdc28 mutants of Saccharomyces cerevisiae, suggesting that all three of these protein kinases can play roles in the regulation of the mammalian cell cycle. The identification of a large family of cdc2-related kinases opens the possibility of combinatorial regulation of the cell cycle together with the emerging large family of cyclins. Images PMID:1639063

  17. Effects of early surfactant treatment persisting for one week after lung transplantation in rats.

    PubMed

    Erasmus, M E; Hofstede, G J; Petersen, A H; Haagsman, H P; Oetomo, S B; Prop, J

    1997-08-01

    We investigated whether pulmonary surfactant in rat lung transplants recovered during the first week post-transplantation, along with symptoms of the reimplantation response, and whether this recovery was affected by early surfactant treatment. The severity of pulmonary injury was varied by transplanting left lungs with 6-h and 20-h ischemia (n = 12 and 19, respectively). Half of the transplants were treated by instillation of surfactant before reperfusion. Lungs from sham operated, and normal rats (n = 4 and 5, respectively) served as controls. The pulmonary injury severely impaired lung transplant function; 10 of the worst affected animals died. After 1 wk, symptoms of reimplantation response and properties of pulmonary surfactant were assessed. If untreated, the reimplantation response had almost resolved in the 6-h but not in the 20-h ischemia group; pulmonary surfactant, however, continued to be deficient in both ischemia groups (low amounts of surfactant phospholipids and surfactant protein A [SP-A]). Surfactant treatment improved the recovery from injury in the 20-h ischemia group resulting in normal lung function and amounts of surfactant phospholipids. Amounts of SP-A were not improved by surfactant treatment. In conclusion, early surfactant treatment enhances recovery from transplantation injury and is persistently beneficial for pulmonary surfactant in lung transplants.

  18. Tight binding of proteins to membranes from older human cells.

    PubMed

    Truscott, Roger J W; Comte-Walters, Susana; Ablonczy, Zsolt; Schwacke, John H; Berry, Yoke; Korlimbinis, Anastasia; Friedrich, Michael G; Schey, Kevin L

    2011-12-01

    The lens is an ideal model system for the study of macromolecular aging and its consequences for cellular function, since there is no turnover of lens fibre cells. To examine biochemical processes that take place in the lens and that may also occur in other long-lived cells, membranes were isolated from defined regions of human lenses that are synthesised at different times during life, and assayed for the presence of tightly bound cytosolic proteins using quantitative iTRAQ proteomics technology. A majority of lens beta crystallins and all gamma crystallins became increasingly membrane bound with age, however, the chaperone proteins alpha A and alpha B crystallin, as well as the thermally-stable protein, βB2 crystallin, did not. Other proteins such as brain-associated signal protein 1 and paralemmin 1 became less tightly bound in the older regions of the lens. It is evident that protein-membrane interactions change significantly with age. Selected proteins that were formerly cytosolic become increasingly tightly bound to cell membranes with age and are not removed even by treatment with 7 M urea. It is likely that such processes reflect polypeptide denaturation over time and the untoward binding of proteins to membranes may alter membrane properties and contribute to impairment of communication between older cells.

  19. Surfactant mixing rules applied to surfactant enhanced alkaline flooding

    SciTech Connect

    Taylor, K.C. )

    1992-01-01

    This paper discusses surfactant mixing rules which have been used to describe crude oil/alkali/surfactant phase behavior, using David Lloydminster crude oil and the surfactant Neodol 25-3S. It was found that at a fixed salinity and alkali concentration, a specific mole fraction of synthetic surfactant to petroleum soap was required to produce optimal phase behavior as the water-to-oil ratio varied. This methodology is useful in understanding the relationship between the variables of water-to-oil ratio and synthetic surfactant concentration in phase behavior systems that produce a petroleum soap.

  20. Identification of Cellular Proteins that Interact with Human Cytomegalovirus Immediate-Early Protein 1 by Protein Array Assay

    PubMed Central

    Puerta Martínez, Francisco; Tang, Qiyi

    2013-01-01

    Human cytomegalovirus (HCMV) gene expression during infection is characterized as a sequential process including immediate-early (IE), early (E), and late (L)-stage gene expression. The most abundantly expressed gene at the IE stage of infection is the major IE (MIE) gene that produces IE1 and IE2. IE1 has been the focus of study because it is an important protein, not only for viral gene expression but also for viral replication. It is believed that IE1 plays important roles in viral gene regulation by interacting with cellular proteins. In the current study, we performed protein array assays and identified 83 cellular proteins that interact with IE1. Among them, seven are RNA-binding proteins that are important in RNA processing; more than half are nuclear proteins that are involved in gene regulations. Tumorigenesis-related proteins are also found to interact with IE1, implying that the role of IE1 in tumorigenesis might need to be reevaluated. Unexpectedly, cytoplasmic proteins, such as Golgi autoantigen and GGA1 (both related to the Golgi trafficking protein), are also found to be associated with IE1. We also employed a coimmunoprecipitation assay to test the interactions of IE1 and some of the proteins identified in the protein array assays and confirmed that the results from the protein array assays are reliable. Many of the proteins identified by the protein array assay have not been previously reported. Therefore, the functions of the IE1-protein interactions need to be further explored in the future. PMID:24385082

  1. Singlet CH domain containing human multidomain proteins: an inventory.

    PubMed

    Friedberg, Felix

    2010-03-01

    The actin cytoskeleton presents the basic force in processes such as cytokinesis, endocytosis, vesicular trafficking and cell migration. Here, we list 30 human singlet CH (calpononin homology/actin binding) containing multidomain molecules, each encoded by one gene. We show the domain distributions as given by the SMART program. These mosaic proteins organize geographically the placement of selected proteins in proximity within the cell. In most instances, their precise location, their actin binding capacity by way of the singlet CH (or by other domains?) and their physiological functions need further elucidation. A dendrogram based solely on the relationship for the human singlet CH domains (in terms of AA sequences) for the various molecules that possess the domain, implies that the singlet descended from a common ancestor which in turn sprouted three main branches of protein products. Each branch bifurcated multiple times thus accounting for a cornucopia of products. Wherever, additional (unassigned), highly homologous regions exist in related proteins (e.g., in LIM and LMO7 or in Tangerin and EH/BP1), these unrecognized domain regions await assignment as specific functional domains. Frequently genes coding multidomain proteins duplicated. The varying modular nature within multidomain proteins should have accelerated evolutionary changes to a degree not feasible to achieve by means of mere post-duplication mutational changes.

  2. Beer consumption and changes in stability of human serum proteins.

    PubMed

    Gorinstein, S; Caspi, A; Goshev, I; Moncheva, S; Zemser, M; Weisz, M; Libman, I; Lerner, H T; Trakhtenberg, S; Martín-Belloso, O

    2001-03-01

    The aim of this study was to evaluate the influence of beer consumption (BC) on the functional and structural properties of human serum proteins (HSP). Thirty-eight volunteers (after coronary bypass) were divided into two groups: experimental (EG) and control (CG). Nineteen volunteers of the EG consumed 330 mL per day of beer (about 20 g of alcohol) for 30 consecutive days. The CG volunteers consumed mineral water instead of beer. Blood samples were collected from EG and CG patients before and after the experiment. Albumin (Alb), globulin (Glo), and methanol-precipitable proteins (MPP) from human serum were denatured with 8 M urea. Fluorescence and electrophoresis were employed in order to elucidate urea-induced conformational changes and structural behavior of proteins. The measured fluorescence emission spectra were used to estimate the stability of native and denatured protein fractions before and after BC. It was found that before BC the fractions most stable to urea denaturation were Glo, Alb, and MPP fractions. After BC in most of the beer-consuming patients (EG) some changes in native and denatured protein fractions were detected: a tendency to lower stability and minor structural deviations. These qualitative changes were more profound in MPP than in Alb and Glo. Thus, Glo is more resistible to alcohol influence than Alb, which in turn is more resistible than MPP. No serum protein changes were detected in patients of CG.

  3. Human milk proteins: an interactomics and updated functional overview.

    PubMed

    D'Alessandro, Angelo; Scaloni, Andrea; Zolla, Lello

    2010-07-02

    Milk and milk fractions are characterized by a wide array of proteins, whose concentration spans across several orders of magnitude. By exploiting a combined approach based on functional gene ontology enrichment (FatiGO/Babelomics), hierarchical clustering, and pathway and network analyses, we merged data from literature dealing with protein-oriented studies on human milk. A total of 285 entries defined a nonredundant list upon comparison with the Ingenuity Knowledge Base from the Ingenuity Pathway Analysis software. Results were compared with an inventory of bovine milk proteins gathered from dedicated proteomic studies. A protein core of 106 proteins was found, with most of the entries associated to three main biological functions, namely nutrient transport/lipid metabolism, concretization of the immune system response and cellular proliferation processes. Our analyses confirm and emphasize that the biological role of the human milk proteins is not only limited to the provision of external nutrients and defense molecules against pathogens to the suckling but also to the direct stimulation of the growth of neonate tissues/organs and to the development of a proper independent immune system, both through the induction of a number of molecular cascades associated with cell proliferation/differentiation. The latter aspects were previously investigated by single-molecule dedicated studies, missing the holistic view that results from our analysis.

  4. Nuclear and nucleolar targeting of human ribosomal protein S6.

    PubMed Central

    Schmidt, C; Lipsius, E; Kruppa, J

    1995-01-01

    Chimeric proteins were constructed to define the nuclear localization signals (NLSs) of human ribosomal protein S6. The complete cDNA sequence, different cDNA fragments and oligonucleotides of the human ribosomal proteins S6, respectively, were joined to the 5' end of the entire LacZ gene of Escherichia coli by using recombinant techniques. The hybrid genes were transfected into L cells, transiently expressed, and the intracellular location of the fusion proteins was determined by their beta-galactosidase activity. Three NLSs were identified in the C-terminal half of the S6 protein. Deletion mutagenesis demonstrated that a single NLS is sufficient for targeting the corresponding S6-beta-galactosidase chimera into the nucleus. Removal of all three putative NLSs completely blocked the nuclear import of the resulting S6-beta-galactosidase fusion protein, which instead became evenly distributed in the cytoplasm. Chimeras containing deletion mutants of S6 with at least one single NLS or unmodified S6 accumulated in the nucleolus. Analysis of several constructs reveals the existence of a specific domain that is essential but not sufficient for nucleolar accumulation of S6. Images PMID:8590812

  5. Fibrinogen stability under surfactant interaction.

    PubMed

    Hassan, Natalia; Barbosa, Leandro R S; Itri, Rosangela; Ruso, Juan M

    2011-10-01

    Differential scanning calorimetry (DSC), circular dichroism (CD), difference spectroscopy (UV-vis), Raman spectroscopy, and small-angle X-ray scattering (SAXS) measurements have been performed in the present work to provide a quantitatively comprehensive physicochemical description of the complexation between bovine fibrinogen and the sodium perfluorooctanoate, sodium octanoate, and sodium dodecanoate in glycine buffer (pH 8.5). It has been found that sodium octanoate and dodecanoate act as fibrinogen destabilizer. Meanwhile, sodium perfluorooctanoate acts as a structure stabilizer at low molar concentration and as a destabilizer at high molar concentration. Fibrinogen's secondary structure is affected by all three studied surfactants (decrease in α-helix and an increase in β-sheet content) to a different extent. DSC and UV-vis revealed the existence of intermediate states in the thermal unfolding process of fibrinogen. In addition, SAXS data analysis showed that pure fibrinogen adopts a paired-dimer structure in solution. Such a structure is unaltered by sodium octanoate and perfluoroctanoate. However, interaction of sodium dodecanoate with the fibrinogen affects the protein conformation leading to a complex formation. Taken together, all results evidence that both surfactant hydrophobicity and tail length mediate the fibrinogen stability upon interaction.

  6. Localization of luteinizing hormone receptor protein in the human ovary.

    PubMed

    Yung, Y; Aviel-Ronen, S; Maman, E; Rubinstein, N; Avivi, C; Orvieto, R; Hourvitz, A

    2014-09-01

    The luteinizing hormone receptor (LHR) plays a pivotal role during follicular development. Consequently, its expression pattern is of major importance for research and has clinical implications. Despite the accumulated information regarding LHR expression patterns, our understanding of its expression in the human ovary, specifically at the protein level, is incomplete. Therefore, our aim was to determine the LHR protein localization and expression pattern in the human ovary. We examined the presence of LHR by immunohistochemical staining of human ovaries and western blots of mural granulosa and cumulus cells aspirated during IVF treatments. We were not able to detect LHR protein staining in primordial or primary follicles. We observed equivocal positive staining in granulosa cells and theca cells of secondary follicles. The first appearance of a clear signal of LHR protein was observed in granulosa cells and theca cells of small antral follicles, and there was evidence of increasing LHR production as the follicles mature to the pre-ovulatory stage. After ovulation, LHR protein was ubiquitously produced in the corpus luteum. To confirm the expression pattern in granulosa cells and cumulus cells, we performed western blots and found that LHR expression was stronger in granulosa cells than in cumulus cells, with the later demonstrating low, but still significant, amounts of LHR protein. In summary, we conclude that LHR protein starts to appear on granulosa cells and theca cells of early antral follicles, and low but significant expression of LHR exists also in the cumulus cells. These results may have implications for the future design of clinical protocols and culture mediums for in vitro fertilization and especially in vitro maturation of oocytes.

  7. Presence of hypochlorite-modified proteins in human atherosclerotic lesions.

    PubMed Central

    Hazell, L J; Arnold, L; Flowers, D; Waeg, G; Malle, E; Stocker, R

    1996-01-01

    Oxidation of LDL may contribute to atherogenesis, though the nature of the in vivo oxidant(s) remains obscure. Myeloperoxidase, the enzyme responsible for hypochlorous acid/hypochlorite (HOCl) production in vivo, is present in active form in human atherosclerotic lesions, and HOCl aggregates and transforms LDL into a high-uptake form for macrophages in vitro. Here we demonstrate HOCl-modified proteins in human lesions using an mAb raised against HOCl-modified LDL that recognizes HOCl-oxidized proteins but does not cross-react with Cu2+-, malondialdehyde-, or 4-hydroxynonenal-modified LDL. This antibody detected significantly more material in advanced atherosclerotic lesions than normal arteries, even though azide and methionine were included during sample work-up to inhibit myeloperoxidase and to scavenge HOCl. The epitope(s) recognized was predominantly cell associated and present in monocyte/macrophages, smooth muscle, and endothelial cells. The intima and cholesterol clefts stained more heavily than the center of the thickened vessels; adventitial staining was apparent in some cases. Immunostaining was also detected in a very early lesion from an accident victim, beside healthy areas that were unreactive. LDL oxidized by HOCl in vitro, but not native LDL, effectively competed with the epitopes in lesions for antibody binding. Density centrifugation of plaque homogenates and Western blot analysis showed that, in the apo B-containing lipoprotein fraction, the mAb recognized protein(s) of molecular mass greater than apo B, similar to those produced during oxidation of LDL with HOCl in vitro. Three major proteins were recognized by the anti-HOCl-modified protein antibody but not by an anti-apo B antibody in the apo B-free fraction. Together, these results demonstrate HOCl-oxidized proteins in human atherosclerotic lesions, implicating this oxidant in LDL modification in vivo. PMID:8617887

  8. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    SciTech Connect

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-06-15

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates.

  9. A Drosophila gene encoding a protein resembling the human. beta. -amyloid protein precursor

    SciTech Connect

    Rosen, D.R.; Martin-Morris, L.; Luo, L.; White, K. )

    1989-04-01

    The authors have isolated genomic and cDNA clones for a Drosophila gene resembling the human {beta}-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human {beta}-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development.

  10. A Drosophila gene encoding a protein resembling the human beta-amyloid protein precursor.

    PubMed Central

    Rosen, D R; Martin-Morris, L; Luo, L Q; White, K

    1989-01-01

    We have isolated genomic and cDNA clones for a Drosophila gene resembling the human beta-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human beta-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development. Images PMID:2494667

  11. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    PubMed Central

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-01-01

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. PMID:23566956

  12. Peptide-protein interactions within human hair keratins.

    PubMed

    Cruz, Célia F; Matamá, Teresa; Cavaco-Paulo, Artur

    2017-03-15

    We selected 1235 decapeptides from human hair proteins encoded by human genes of keratins and keratin associated proteins. The peptides were linked to glass arrays and screened for their affinity towards a solution of human hair extracted keratin fraction. Based on the physicochemical properties of the peptides, ten variables were studied: content of different types of amino acid side chains (cysteine, hydrophobic, polar, basic, acidic, aromatic rings, amide, alcohol side chains), isoelectric point, and net charge. We found differences statistically significant on the binding affinity of peptides based on their content of cysteine, hydrophobic and polar amino acids, mainly containing alcohols. These results point to the formation of hydrophobic interactions and disulfide bonds between small peptides and human hair keratins as the main driving forces for the interaction of possible cosmetic peptides, namely designed to strength human hair. As so, our results enlighten the nature of the interaction of keratin based materials with human hair, which are claimed to enhance hair fiber strength, and enable a more directed and sustained hair care peptide design.

  13. Effect of curcumin on the binding of cationic, anionic and nonionic surfactants with myoglobin

    NASA Astrophysics Data System (ADS)

    Mondal, Satyajit; Ghosh, Soumen

    2017-04-01

    Interaction of a globular protein, myoglobin and different surfactants has been studied in the absence and presence of curcumin in phosphate buffer at pH = 7.4 by UV-VIS spectrophotometry, fluorimetry and fluorescence polarization anisotropy methods. Results show that heme environment of myoglobin is changed by cationic cetyltrimethylammonium bromide (CTAB) and sodium N-dodecanoyl sarcosinate (SDDS). In the presence of curcumin, CTAB cannot change the heme; but SDDS can make change. Nonionic surfactant N-decanoyl-N-methylglucamine (Mega 10) cannot change the heme environment. Protein is unfolded by the surfactant. Curcumin can prevent the unfolding of protein in the low concentration region of ionic surfactants such as CTAB and SDDS. In nonionic surfactant media, curcumin accelerates the denaturation process. Due to myoglobin-curcumin complex formation, rotational motion of curcumin decreases in surfactant media and so anisotropy increases.

  14. The effects of non-ionic polymeric surfactants on the cleaning of biofouled hydrogel materials.

    PubMed

    Guan, Allan; Li, Zhenyu; Phillips, K Scott

    2015-01-01

    Block co-polymer surfactants have been used for cleaning hydrogel medical devices that contact the body (e.g., contact lenses) because of their biocompatibility. This work examined the relationship between concentration and detergency of two non-ionic polymeric surfactants (Pluronic F127 and Triton X-100) for cleaning protein soil, with anionic surfactants (sodium dodecyl sulfate and sodium laureth sulfate) as positive controls. Surface plasmon resonance was used to quantify removal of simulated tear soil from self-assembled monolayer surfaces, and a microplate format was used to study the removal of fluorescently labeled soil proteins from contact lenses. While detergency increased as a function of concentration for anionic surfactants, it decreased with concentration for the two polymeric surfactants. The fact that the protein detergency of some non-ionic polymeric surfactants did not increase with concentration above the critical micelle concentration could have implications for optimizing the tradeoff between detergency and biocompatibility.

  15. The inhibitory effect of some amphoteric surfactants on the irritation potential of alkylsulphates.

    PubMed

    Domínguez, J G; Balaguer, F; Parra, J L; Pelejero, C M

    1981-04-01

    Synopsis The physico-chemical and biological properties of an amphoteric/anionic system and its behaviour against a proteinic support have been thoroughly investigated. A considerable inhibition of adsorption of SLS (sodium lauryl sulphate) on human callus caused by the presence of definite amounts of AABet (alkyl-amido-betaines) in the treatment bath is observed. These physico-chemical results are in agreement with those obtained by some in vivo biological tests. A mechanism of the process via the formation of mixed micelles is postulated emphasizing the stability of such systems as a function of pH, the influence of the chain length of the amphoteric surfactant and the molar relative ratio SLS/AABet. Consequently, our work offers the possibility of a wide applicability of the synergic mixtures of both types of surfactants to inhibit considerably skin irritation of cosmetic finished products.

  16. Surfactant-enhanced bioremediation

    SciTech Connect

    Churchill, P.F.; Dudley, R.J.; Churchill, S.A.

    1995-12-31

    This study was undertaken to examine the effect of three structurally related, non-ionic surfactants, Triton X-45, Triton X-100 and Triton X-165, as well as the oleophilic fertilizer, Inipol EAP 22, on the rate of biodegradation of phenanthrene by pure bacterial cultures. Each surfactant dramatically increased the apparent aqueous solubility of phenanthrene. Model studies were conducted to investigate the ability of these surfactants to enhance the rate of transport and uptake of polycyclic aromatic hydrocarbons into bacterial cells, and to assess the impact that increasing the aqueous solubility of hydrocarbons has on their rate of biodegradation. The results indicate that increasing the apparent aqueous solubility of hydrocarbons can lead to enhanced biodegradation rates by two Pseudomonas saccharophila strains. However, the experiments also suggest that some surfactants can inhibit aromatic hydrocarbon biodegradation by certain bacteria. The data also support the hypothesis that surface-active components present in the oleophilic fertilizer formulation, Inipol EAP 22, may have significantly contributed to the positive results reported in tests of remedial agent impact on bioremediation, which was used as a supplemental clean-up technology on Exxon Valdez crude oil-contaminated Alaskan beaches.

  17. Surfactant and adhesive formulations from alkaline biomass extracts

    NASA Astrophysics Data System (ADS)

    Baxter, Matthew

    This work studies the ability to produce effective surfactant and adhesive formulations using surface active biological material extracted from different biomass sources using alkaline extraction methods. Two urban waste biomass sources were used to produce surfactants, Return Activated Sludge (RAS), and solid Urban Refuse (UR). The third biomass source investigated was isolated mustard protein (MP). RAS and MP extracts were investigated for adhesive production. The results indicate that extracts from the waste biomass sources, RAS and UR, can be combined with a commercial surfactant, sodium dioctyl sulfosuccinate (AOT), to produce surfactants with low interfacial tensions against various oils. These highly surface-active formulations were shown to be useful in the removal of bitumen from contaminated sand. RAS and MP showed potential as protein-based wood adhesives. These sources were used in adhesive formulations to produce a strong bond strength under low-pressure, ambient pressing conditions.

  18. Expression and biochemical characterization of recombinant human epididymis protein 4.

    PubMed

    Hua, Ling; Liu, Yunhui; Zhen, Shuai; Wan, Deyou; Cao, Jiyue; Gao, Xin

    2014-10-01

    Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion gene encoding the HE4 protein fused to an IgG1 Fc domain was constructed. The recombinant HE4 protein was expressed as a secretory protein in Pichia pastoris and mammalian HEK293-F cells and was subsequently purified. Our data suggested that the HE4 protein produced by these two expression systems bound to both gram-negative and gram-positive bacteria, but demonstrated slightly inhibitory activity towards the growth of Staphylococcus aureus. Moreover, HE4 exhibited proteinase inhibitory activity towards trypsin, elastase, matrix metallopeptidase 9, and the secretory proteinases from Bacillus subtilis. The effects of glycosylation on the biochemical characterization of HE4 were also investigated. LC-ESI-MS glycosylation analysis showed that the high-mannose glycosylated form of HE4 expressed by P. pastoris has lower biological activity when compared to its complex-glycosylated form produced from HEK293-F cells. The implications of this are discussed, which may be provide theoretical basis for its important role in the development of cancer and innate immune system.

  19. Semiconductor Microcavity Flow Spectroscopy of Intracellular Protein in Human Cells

    NASA Astrophysics Data System (ADS)

    Gourley, Paul; Cox, Jim; Hendricks, Judy; McDonald, Anthony; Copeland, Guild; Sasaki, Darryl; Skirboll, Steve; Curry, Mark

    2001-03-01

    The speed of light through a biofluid or biological cell is inversely related to the biomolecular concentration of proteins and other complex molecules that modify the refractive index at wavelengths accessible to semiconductor lasers. By placing a fluid or cell into a semiconductor microcavity laser, these decreases in light speed can be sensitively recorded in picoseconds as frequency red-shifts in the laser output spectrum. This biocavity laser equipped with microfluidics for transporting cells at high speed through the laser microcavity has shown potential for rapid analysis of biomolecular mass of normal and malignant human cells in their physiologic condition without time-consuming fixing, staining, or tagging. We have used biocavity laser spectroscopy to measure the optical properties of solutions of standard biomolecules (sugars, proteins, DNA, and ions) and human cells. The technique determines the frequency shift, relative to that of water, of spontaneous or stimulated emission from cavity filled with a biomolecular solution. The shift was also measured in human glioblastoma cells that had been sorted by conventional fluorescence-activated cell sorting according to protein content. The results show a direct correlation between protein measured by fluorescence and the frequency shift observed in the microcavity laser.

  20. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins

    PubMed Central

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-01-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  1. The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase

    PubMed Central

    Francis, Kevin; Sapienza, Paul J.; Lee, Andrew L.; Kohen, Amnon

    2016-01-01

    Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H→C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs) that are sensitive to the physical nature of the chemical step, and protein mass-modulation that slows down fast dynamics (femto- to picosecond timescale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5–45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction’s transition state (or tunneling ready state – TRS). Mass modulation of these enzymes through isotopic labeling with 13C, 15N, and 2H at nonexchangeable hydrogens yield an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass-modulation of the human DHFR affects neither DAD distribution nor the DAD’s conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H→C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically-modulated heavy enzymes in general. PMID:26813442

  2. Neutron scattering studies on protein dynamics using the human myelin peripheral membrane protein P2

    NASA Astrophysics Data System (ADS)

    Laulumaa, Saara; Kursula, Petri; Natali, Francesca

    2015-01-01

    Myelin is a multilayered proteolipid membrane structure surrounding selected axons in the vertebrate nervous system, which allows the rapid saltatory conduction of nerve impulses. Deficits in myelin formation and maintenance may lead to chronic neurological disease. P2 is an abundant myelin protein from peripheral nerves, binding between two apposing lipid bilayers. We studied the dynamics of the human myelin protein P2 and its mutated P38G variant in hydrated powders using elastic incoherent neutron scattering. The local harmonic vibrations at low temperatures were very similar for both samples, but the mutant protein had increased flexibility and softness close to physiological temperatures. The results indicate that a drastic mutation of proline to glycine at a functional site can affect protein dynamics, and in the case of P2, they may explain functional differences between the two proteins.

  3. Characterization of Tight Junction Proteins in Cultured Human Urothelial Cells

    PubMed Central

    Rickard, Alice; Dorokhov, Nikolay; Ryerse, Jan; Klumpp, David J.; McHowat, Jane

    2010-01-01

    Tight junctions (TJs) are essential for normal function of epithelia, restricting paracellular diffusion and contributing to the maintainance of cell surface polarity. Superficial cells of the urothelium develop TJs, the basis for the paracellular permeability barrier of the bladder against diffusion of urinary solutes. Focusing on the superficial cell layer of stratified cell cultures of an immortalized human ureteral cell line, TEU-2 cells, we have examined the presence of TJ and TJ-associated proteins. TEU-2 cells were treated with calcium chloride and fetal bovine serum culture conditions used to induce stratification that resembles the normal transitional epithelial phenotype. Cultures were examined for TJ and TJ-associated proteins by confocal immuno-fluorescence microscopy and evaluated for TJ mRNA by reverse transcriptase-polymerase chain reaction (RT- PCR). TEU-2 cultures exhibited immunoreactivity at intercellular margins for claudins 1, 4, 5, 7, 14 and 16 whereas claudins 2, 8 and 12 were intracellular. RT-PCR corroborated the presence of these claudins at the mRNA level. The TJ-associated proteins occludin, JAM-1, and zonula occludens (ZO-1, ZO-2 and ZO-3) were localized at cell margins. We have found that numerous TJs and TJ-associated proteins are expressed in stratified TEU-2 cultures. Further, we propose TEU-2s provide a useful ureteral model for future studies on the involvement of TJs proteins in the normal and pathological physiology of the human urinary system. PMID:18553212

  4. Determination of in vivo protein synthesis in human palatine tonsil.

    PubMed

    Januszkiewicz, Anna; Klaude, Maria; Loré, Karin; Andersson, Jan; Ringdén, Olle; Rooyackers, Olav; Wernerman, Jan

    2005-02-01

    The palatine tonsils are constantly exposed to ingested or inhaled antigens which, in turn, lead to a permanent activation of tonsillar immune cells, even in a basic physiological state. The aim of the present study was to investigate if the immunological activation of the human palatine tonsil is reflected by a high metabolic activity, as determined by in vivo measurement of protein synthesis. The protein synthesis rate of the tonsil was also compared with that of the circulating T-lymphocytes, the total blood mononuclear cells and the whole population of blood leucocytes. Phenotypic characterization of immune-competent cells in tonsil tissue and blood was performed by flow cytometry. Pinch tonsil biopsies were taken after induction of anaesthesia in healthy adult patients (n=12) scheduled for ear surgery, uvulopalatopharyngoplasty or nose surgery. Protein synthesis was quantitatively determined during a 90-min period by a flooding-dose technique. The in vivo protein synthesis rate in the palatine tonsils was 22.8+/-5.7%/24 h (mean+/-S.D.), whereas protein synthesis in the circulating T-lymphocytes was 10.7+/-3.4%/24 h, in mononuclear cells was 10.8+/-2.8%/24 h and in leucocytes was 3.2+/-1.2%/24 h. CD3+ lymphocytes were the most abundant cell population in the tonsil. The in vivo protein synthesis rate in human tonsils was higher compared with the circulating immune cells. This high metabolic rate may reflect the permanent immunological activity present in human tonsils, although cell phenotypes and activity markers do not explain the differences.

  5. Prognostic implications of Kindlin proteins in human osteosarcoma

    PubMed Central

    Ning, Kai; Zhang, Haoshaqiang; Wang, Zhigang; Li, Kun

    2017-01-01

    The Kindlin protein family, comprising Kindlin-1, Kindlin-2 and Kindlin-3, play important roles in various human cancers. Here, to explore the clinical significance of Kindlins in human osteosarcomas, quantitative real-time PCR and Western blot analyses were performed to detect the expression of Kindlin-1, Kindlin-2 and Kindlin-3 mRNAs and proteins in 20 self-pairs of osteosarcoma and adjacent noncancerous tissues. Then, immunohistochemistry was performed to examine subcellular localizations and expression patterns of Kindlin proteins in 100 osteosarcoma and matched adjacent noncancerous tissues. Kindlin-1, Kindlin-2 and Kindlin-3 protein immunostainings were localized in the cytoplasm, nucleus and cytoplasm, respectively, of tumor cells in primary osteosarcoma tissues. Statistically, the expression levels of Kindlin-1 and Kindlin-2 mRNAs and proteins in osteosarcoma tissues were all significantly higher (both P<0.01), but those of Kindlin-3 mRNA and protein were both dramatically lower (both P<0.05), than in matched adjacent noncancerous tissues. In addition, the overexpressions of Kindlin-1 and Kindlin-2 proteins were both significantly associated with high tumor grade (both P=0.01), presence of metastasis (both P=0.006), recurrence (both P=0.006) and poor response to chemotherapy (both P=0.02). Moreover, Kindlin-1 and Kindlin-2 expressions were both identified as independent prognostic factors for overall (both P=0.01) and disease-free (P=0.02 and 0.01, respectively) survivals of osteosarcoma patients. However, no associations were observed between Kindlin-3 expression and various clinicopathologic features and patients’ prognosis. In conclusion, aberrant expression of Kindlin-1 and Kindlin-2 may function as reliable markers for progression and prognosis in osteosarcoma patients, especially for tumor recurrence. PMID:28223823

  6. Human erythrocyte dematin and protein 4.2 (pallidin) are ATP binding proteins.

    PubMed

    Azim, A C; Marfatia, S M; Korsgren, C; Dotimas, E; Cohen, C M; Chishti, A H

    1996-03-05

    Dematin and protein 4.2 are peripheral membrane proteins associated with the cytoplasmic surface of the human erythrocyte plasma membrane. Isoforms of dematin and protein 4.2 exist in many nonerythroid cells. In solution, dematin is a trimeric protein containing two subunits of 48 kDa and one subunit of 52 kDa. Recent determination of the primary structure of the 52 kDa subunit of dematin showed that it contains an additional 22-amino acid sequence in the headpiece domain. An alignment of the 22-amino acid insertion sequence revealed that the 52 kDa subunit of dematin shares a novel 11-amino acid motif with protein 4.2. In this communication, we report that the conserved 11-amino acid motif in dematin52 and protein 4.2 contains a nucleotide binding P-loop. Direct binding of ATP is demonstrated to the glutathione S-transferase fusion proteins containing corresponding segments of dematin52 and protein 4.2 as well as to purified protein 4.2. The binding of ATP to the recombinant domains of dematin52 and protein 4.2 is specific, saturable, and of high affinity. The nucleotide specificity of the P-loop is restricted to ATP since no detectable binding was observed with GTP. These results show that the 11-amino acid motif provides an ATP binding site in dematin52 and protein 4.2. Although the functional significance of ATP binding is not yet clear, our findings open new perspectives for the function of dematin and protein 4.2 in vivo.

  7. Surfactant treatments alter endogenous surfactant metabolism in rabbit lungs

    SciTech Connect

    Oetomo, S.B.; Lewis, J.; Ikegami, M.; Jobe, A.H. )

    1990-04-01

    The effect of exogenous surfactant on endogenous surfactant metabolism was evaluated using a single-lobe treatment strategy to compare effects of treated with untreated lung within the same rabbit. Natural rabbit surfactant, Survanta, or 0.45% NaCl was injected into the left main stem bronchus by use of a Swan-Ganz catheter. Radiolabeled palmitic acid was then given by intravascular injection at two times after surfactant treatment, and the ratios of label incorporation and secretion in the left lower lobe to label incorporation and secretion in the right lung were compared. The treatment procedure resulted in a reasonably uniform surfactant distribution and did not disrupt lobar pulmonary blood flow. Natural rabbit surfactant increased incorporation of palmitate into saturated phosphatidylcholine (Sat PC) approximately 2-fold (P less than 0.01), and secretion of labeled Sat PC increased approximately 2.5-fold in the surfactant-treated left lower lobe relative to the right lung (P less than 0.01). Although Survanta did not alter incorporation, it did increase secretion but not to the same extent as rabbit surfactant (P less than 0.01). Alteration of endogenous surfactant Sat PC metabolism in vivo by surfactant treatments was different from that which would have been predicted by previous in vitro studies.

  8. Nanoparticle interaction with model lung surfactant monolayers

    PubMed Central

    Harishchandra, Rakesh Kumar; Saleem, Mohammed; Galla, Hans-Joachim

    2010-01-01

    One of the most important functions of the lung surfactant monolayer is to form the first line of defence against inhaled aerosols such as nanoparticles (NPs), which remains largely unexplored. We report here, for the first time, the interaction of polyorganosiloxane NPs (AmorSil20: 22 nm in diameter) with lipid monolayers characteristic of alveolar surfactant. To enable a better understanding, the current knowledge about an established model surface film that mimics the surface properties of the lung is reviewed and major results originating from our group are summarized. The pure lipid components dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol have been used to study the biophysical behaviour of their monolayer films spread at the air–water interface in the presence of NPs. Film balance measurements combined with video-enhanced fluorescence microscopy have been used to investigate the formation of domain structures and the changes in the surface pattern induced by NPs. We are able to show that NPs are incorporated into lipid monolayers with a clear preference for defect structures at the fluid–crystalline interface leading to a considerable monolayer expansion and fluidization. NPs remain at the air–water interface probably by coating themselves with lipids in a self-assembly process, thereby exhibiting hydrophobic surface properties. We also show that the domain structure in lipid layers containing surfactant protein C, which is potentially responsible for the proper functioning of surfactant material, is considerably affected by NPs. PMID:19846443

  9. Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

    PubMed

    He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

    2000-08-01

    Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

  10. Protein carbonylation and heat shock proteins in human skeletal muscle: relationships to age and sarcopenia.

    PubMed

    Beltran Valls, Maria R; Wilkinson, Daniel J; Narici, Marco V; Smith, Kenneth; Phillips, Bethan E; Caporossi, Daniela; Atherton, Philip J

    2015-02-01

    Aging is associated with a gradual loss of muscle mass termed sarcopenia, which has significant impact on quality-of-life. Because oxidative stress is proposed to negatively impact upon musculoskeletal aging, we investigated links between human aging and markers of oxidative stress, and relationships to muscle mass and strength in young and old nonsarcopenic and sarcopenic adults. Sixteen young and 16 old males (further subdivided into "old" and "old sarcopenic") were studied. The abundance of protein carbonyl adducts within skeletal muscle sarcoplasmic, myofibrillar, and mitochondrial protein subfractions from musculus vastus lateralis biopsies were determined using Oxyblot immunoblotting techniques. In addition, concentrations of recognized cytoprotective proteins (eg, heat shock proteins [HSP], αβ-crystallin) were also assayed. Aging was associated with increased mitochondrial (but not myofibrillar or sarcoplasmic) protein carbonyl adducts, independently of (stage-I) sarcopenia. Correlation analyses of all subjects revealed that mitochondrial protein carbonyl abundance negatively correlated with muscle strength ([1-repetition maximum], p = .02, r (2) = -.16), but not muscle mass (p = .13, r (2) = -.08). Abundance of cytoprotective proteins, including various HSPs (HSP 27 and 70), were unaffected by aging/sarcopenia. To conclude, these data reveal that mitochondrial protein carbonylation increases moderately with age, and that this increase may impact upon skeletal muscle function, but is not a hallmark of (stage-I) sarcopenia, per se.

  11. Redox proteomics identification of oxidatively modified myocardial proteins in human heart failure: implications for protein function.

    PubMed

    Brioschi, Maura; Polvani, Gianluca; Fratto, Pasquale; Parolari, Alessandro; Agostoni, Piergiuseppe; Tremoli, Elena; Banfi, Cristina

    2012-01-01

    Increased oxidative stress in a failing heart may contribute to the pathogenesis of heart failure (HF). The aim of this study was to identify the oxidised proteins in the myocardium of HF patients and analyse the consequences of oxidation on protein function. The carbonylated proteins in left ventricular tissue from failing (n = 14) and non-failing human hearts (n = 13) were measured by immunoassay and identified by proteomics. HL-1 cardiomyocytes were incubated in the presence of stimuli relevant for HF in order to assess the generation of reactive oxygen species (ROS), the induction of protein carbonylation, and its consequences on protein function. The levels of carbonylated proteins were significantly higher in the HF patients than in the controls (p<0.01). We identified two proteins that mainly underwent carbonylation: M-type creatine kinase (M-CK), whose activity is impaired, and, to a lesser extent, α-cardiac actin. Exposure of cardiomyocytes to angiotensin II and norepinephrine led to ROS generation and M-CK carbonylation with loss of its enzymatic activity. Our findings indicate that protein carbonylation is increased in the myocardium during HF and that these oxidative changes may help to explain the decreased CK activity and consequent defects in energy metabolism observed in HF.

  12. Redox Proteomics Identification of Oxidatively Modified Myocardial Proteins in Human Heart Failure: Implications for Protein Function

    PubMed Central

    Brioschi, Maura; Polvani, Gianluca; Fratto, Pasquale; Parolari, Alessandro; Agostoni, Piergiuseppe; Tremoli, Elena; Banfi, Cristina

    2012-01-01

    Increased oxidative stress in a failing heart may contribute to the pathogenesis of heart failure (HF). The aim of this study was to identify the oxidised proteins in the myocardium of HF patients and analyse the consequences of oxidation on protein function. The carbonylated proteins in left ventricular tissue from failing (n = 14) and non-failing human hearts (n = 13) were measured by immunoassay and identified by proteomics. HL-1 cardiomyocytes were incubated in the presence of stimuli relevant for HF in order to assess the generation of reactive oxygen species (ROS), the induction of protein carbonylation, and its consequences on protein function. The levels of carbonylated proteins were significantly higher in the HF patients than in the controls (p<0.01). We identified two proteins that mainly underwent carbonylation: M-type creatine kinase (M-CK), whose activity is impaired, and, to a lesser extent, α-cardiac actin. Exposure of cardiomyocytes to angiotensin II and norepinephrine led to ROS generation and M-CK carbonylation with loss of its enzymatic activity. Our findings indicate that protein carbonylation is increased in the myocardium during HF and that these oxidative changes may help to explain the decreased CK activity and consequent defects in energy metabolism observed in HF. PMID:22606238

  13. Mining topological structures of protein-protein interaction networks for human brain-specific genes.

    PubMed

    Cui, W J; Gong, X J; Yu, H; Zhang, X C

    2015-10-16

    Compared to other placental mammals, humans have unique thinking and cognitive abilities because of their developed cerebral cortex composed of billions of neurons and synaptic connections. As the primary effectors of the mechanisms of life, proteins and their interactions form the basis of cellular and molecular functions in the living body. In this paper, we developed a pipeline for mining topological structures, identifying functional modules, and analyzing their functions from publically available datasets. A human brain-specific protein-protein interaction network with 1482 nodes and 3105 edges was built using a MapReduce based shortest path algorithm. Within this, 7 functional cliques were identified using a network clustering method, 98 hub proteins were obtained by the calculation of betweenness and connectivity, and 5 closest relationship to clique connector proteins were recognized by the combination scores of topological distance and gene ontology similarity. Furthermore, we discovered functional modules interacting with TP53 protein, which involves several fragmented research study conclusions and might be an important clue for further in vivo or in silico experiments to confirm these associations.

  14. Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.

    PubMed Central

    Provost, P; Doucet, J; Stock, A; Gerisch, G; Samuelsson, B; Rådmark, O

    2001-01-01

    Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction. PMID:11583571

  15. Preparation and application of vegetable proteins, especially proteins from sunflower seed, for human consumption. An approach.

    PubMed

    Gassmann, B

    1983-01-01

    About 80% of the world protein production are of vegetable origin. More than half the vegetable protein is fed to animals, whereas merely 10 kg protein per capita are obtained from meat, milk and eggs per year. Therefore, and because of rising prices for raw materials and energy the production and the firsthand utilisation of proteinacous plants for foodstuffs are a worldwide problem. As future source of protein for human nutrition sunflower seed and oil extraction residues from sunflower seed, respectively, are of great significance. Sunflower seed does not contain anti-nutritive and toxical compounds. After crossing of species having a high oil content, the today cultivated sunflower hybrids bring seeds containing 17-22% crude protein and 30-52% oil. The cultivation also has led to a considerable reduction of the hull content. In processing of sunflower proteins colour problems occur resulting from finely ground particles of dark hulls and from polyphenolic acids which are easily oxidized and converted into brown polymerics. Essential components of the sunflower protein production are, therefore, the at least 98% dehulling before processing as well as the separation of polyphenolic acids and/or the prevention of their oxidation. In principle, the variation and combination of technological steps in pre-treating and defatting of sunflower seed, in extracting, precipitating, washing and drying of proteins, the chemical modification of proteins obtained, the interaction with neutral salts or complexing agents, and the admixture of lysine or proteins of high lysine content allow to adapt sunflower proteins to each type of application.

  16. The p53 gene and protein in human brain tumors

    SciTech Connect

    Louis, D.N. )

    1994-01-01

    Because p53 gene alterations are commonplace in human tumors and because p53 protein is involved in a number of important cellular pathways, p53 has become a topic of intensive investigation, both by basic scientists and clinicians. p53 was initially identified by two independent laboratories in 1979 as a 53 kilodalton (kD) protein that complexes with the large T antigen of SV40 virus. Shortly thereafter, it was shown that the E1B oncoprotein of adenovirus also binds p53. The binding of two different oncogenic viral tumor proteins to the same cellular protein suggested that p53 might be integral to tumorigenesis. The human p53 cDNA and gene were subsequently cloned in the mid-1980s, and analysis of p53 gene alterations in human tumors followed a few year later. During these 10 years, researchers grappling with the vagaries of p53 first characterized the gene as an oncogene, then as a tumor suppressor gene, and most recently as both a tumor suppressor gene and a so-called [open quotes]dominant negative[close quotes] oncogene. The last few years have seen an explosion in work on this single gene and its protein product. A review of a computerized medical database revealed approximately 650 articles on p53 in 1992 alone. p53 has assumed importance in neuro-oncology because p53 mutations and protein alterations are frequent in the common diffuse, fibrillary astrocytic tumors of adults. p53 mutations in astrocytomas were first described in 1989 and were followed by more extensive analyses of gene mutations and protein alterations in adult astrocytomas. The gene has also been studied in less common brain tumors. Elucidating the role of p53 in brain tumorigenesis will not only enhance understanding of brain tumor biology but may also contribute to improved diagnosis and therapy. This discussion reviews key aspects of the p53 gene and protein, and describe their emerging roles in central nervous system neoplasia. 102 refs., 6 figs., 1 tab.

  17. Interaction of p53 with the human Rad51 protein.

    PubMed Central

    Buchhop, S; Gibson, M K; Wang, X W; Wagner, P; Stürzbecher, H W; Harris, C C

    1997-01-01

    p53 is thought to function in the maintenance of genomic stability by modulating transcription and interacting with cellular proteins to influence the cell cycle, DNA repair and apoptosis. p53 mutations occur in >50% of human cancers, and cells which lack wild type p53 accumulate karyotypic abnormalities such as amplifications, deletions, inversions and translocations. We propose that p53 hinders these promiscuous recombinational events by interacting with cellular recombination and repair machinery. We recently reported that p53 can directly bind in vivo to human Rad51 (hRad51) protein and in vitro to its bacterial homologue RecA. We used GST-fusion and his-tagged protein systems to further investigate the physical interaction between p53 and hRad51, homologue of the yeast Rad51 protein that is involved in recombination and DNA double strand repair. The hRad51 binds to wild-type p53 and to a lesser extent, point mutants 135Y, 249S and 273H. This binding is not mediated by a DNA or RNA intermediate. Mapping studies using a panel of p53 deletion mutants indicate that hRad51 could bind to two regions of p53; one between amino acids 94 and 160 and a second between 264 and 315. Addition of anti-p53 antibody PAb421 (epitope 372-381 amino acids) inhibited the interaction with hRad51. In contrast, p53 interacts with the region between aa 125 and 220 of hRad51, which is highly conserved among Rad51 related proteins from bacteria to human. In Escherichia coli ecA protein, this region is required for homo-oligomerization, suggesting that p53 might disrupt the interaction between RecA and Rad51 subunits, thus inhibiting biochemical functions of Rad51 like proteins. These data are consistent with the hypothesis that p53 interaction with hRAD51 may influence DNA recombination and repair and that additional modifications of p53 by mutation and protein binding may affect this interaction. PMID:9380510

  18. Treatment of the Thylakoid Membrane with Surfactants 1

    PubMed Central

    Markwell, John P.; Thornber, J. Philip

    1982-01-01

    Treatment of higher plant (Nicotiana tabacum L. var. Samsun) chloroplast thylakoid membranes with surfactants results in a shift of the chlorophyll a absorption maximum in the red spectral region from its in vivo value of 678.5 nanometers to shorter wavelengths. The magnitude of this shift is correlated with membrane disruption, and is not necessarily due to the release of pigment from pigment-protein complexes present in the membrane. Membrane disruption has been measured by the amount of pigment in the supernatant fraction after centrifugation of surfactant treated membranes. For an equivalent amount of disruption, the extent of the blue-shift is influenced by the ionic nature of the surfactant: anionic surfactants cause small shifts, cationic surfactants cause the largest (∼10 nanometers) shifts, and nonionic surfactants produce intermediate shifts. The wavelength of maximum absorbance of chlorophyll a in the red region is a convenient criterion for assessing the potential utility of different surfactants for studies on the structure, composition and function of higher plant thylakoid membranes. PMID:16662547

  19. [Study of molecular function of proteins in human immunodeficiency virus].

    PubMed

    Fujita, Mikako

    2013-01-01

    Human immunodeficiency virus (HIV) has no more than nine genes expressing approximately twenty proteins. When T lymphocytes and macrophages in a body are infected with HIV, these proteins work in turn at specific time and location, causing acquired immunodeficiency syndrome (AIDS), a disease yet to be overcome. Since the elucidation of molecular mechanism of HIV proteins should lead to remedy of AIDS, the author has been engaged in the study of HIV protein in the past decade. Described herein are viral protein X (Vpx), uniquely found in HIV-2, and its homologous protein Vpr found both in HIV-1 and -2. We found that Vpx enhances genome nuclear import in T lymphocytes, and is critical for reverse transcription of viral RNA in macrophages. This finding on the function in macrophages corrected long-term misleading belief. Furthermore, functional region mapping of Vpx was performed. In 2011, the protein SAMHD1 was identified as the host restriction factor counteracted by Vpx, by foreign researchers. After that, our independent study demonstrated the presence of SAMHD1-independent functions of Vpx in T cells, in addition to its SAMHD1-dependent functions in macrophages. Another topic of this review is Gag protein. Recently, it has reported by overseas researchers that PI(4,5)P2 (one of phosphoinositide) regulates Pr55(Gag) localization and assembly. In this study, we determined the binding affinity between N-terminal MA domain of Pr55(Gag) and various phosphoinositide derivatives using surface plasmon resonance. The results suggested that both negatively charged inositol phosphates and hydrophobic acyl chain are required for the MA binding.

  20. Insights into bacterial protein glycosylation in human microbiota

    PubMed Central

    Zhu, Fan; Wu, Hui

    2017-01-01

    The study of human microbiota is an emerging research topic. The past efforts have mainly centered on studying the composition and genomic landscape of bacterial species within the targeted communities. The interaction between bacteria and hosts is the pivotal event in the initiation and progression of infectious diseases. There is a great need to identify and characterize the molecules that mediate the bacteria-host interaction. Bacterial surface exposed proteins play an important role in the bacteria-host interaction. Numerous surface proteins are glycosylated, and the glycosylation is crucial for their function in mediating the bacterial interaction with hosts. Here we present an overview of surface glycoproteins from bacteria that inhabit three major mucosal environments across human body: oral, gut and skin. We describe the important enzymes involved in the process of protein glycosylation, and discuss how the process impacts the bacteria-host interaction. Emerging molecular details underlying glycosylation of bacterial surface proteins may lead to new opportunities for designing anti-infective small molecules, and developing novel vaccines in order to treat or prevent bacterial infection. PMID:26712033

  1. Expression of heat stress proteins by human periodontal ligament cells.

    PubMed

    Sauk, J J; Norris, K; Foster, R; Moehring, J; Somerman, M J

    1988-11-01

    The purpose of the present report was to document the stress response produced by physical and chemical abuses to human periodontal ligament cells, and to review some of the known functions of stress response proteins produced as a result of such treatments. For these studies human PDL cells were exposed to sublethal challenges of 43 degrees C heat, sodium arsenite and the amino acid analog L-azetidine-2-carboxylic acid (AZC). The cells were labelled with [35S]-methionine and the proteins produced were examined by autofluorography of SDS-PAGE gels. Heat challenges were shown to induce hsps with an apparent mol. wts. of 90K, 68-72K, 41-47K, and 36 K. Arsenite-treated cells produced similar hsps including a 30k protein not produced by other forms of stress. AZC treatment resulted in the production of apparent functionless hsps with apparent molecular weights of 90,000, 72,000, 68,000 and 36,000. The function of these proteins and their possible role in periodontal disease is discussed.

  2. Differential DNA binding properties of three human homeodomain proteins.

    PubMed Central

    Corsetti, M T; Briata, P; Sanseverino, L; Daga, A; Airoldi, I; Simeone, A; Palmisano, G; Angelini, C; Boncinelli, E; Corte, G

    1992-01-01

    The products of three human homeobox containing (HOX) genes, 2C, 3C and 4B, were produced in insect cells using the Baculovirus expression system and purified to near homogeneity. In this system we observed that the DNA binding forms of the three proteins are not glycosylated. HOX 3C and 4B are phosphorylated in insect cells, while HOX 2C is not. The three HOX proteins bind to a DNA sequence known to be a target site for Antennapedia protein with a very similar affinity (Kd = 1-2 x 10(-9) M). We then measured their binding properties to four human sequences present in the HOX 3D, 4C, 1C and 4B promoters. Two of these sequences have been reported to be binding sites for HOX proteins. HOX 2C, 3C and 4B behaved quite differently, showing low affinity for promoters of genes located upstream from their own gene in the HOX clusters and a higher affinity for regulatory sequences of their own gene and downstream HOX genes. Images PMID:1357628

  3. Characterization of Disease-Associated Mutations in Human Transmembrane Proteins

    PubMed Central

    Molnár, János; Szakács, Gergely; Tusnády, Gábor E.

    2016-01-01

    Transmembrane protein coding genes are commonly associated with human diseases. We characterized disease causing mutations and natural polymorphisms in transmembrane proteins by mapping missense genetic variations from the UniProt database on the transmembrane protein topology listed in the Human Transmembrane Proteome database. We found characteristic differences in the spectrum of amino acid changes within transmembrane regions: in the case of disease associated mutations the non-polar to non-polar and non-polar to charged amino acid changes are equally frequent. In contrast, in the case of natural polymorphisms non-polar to charged amino acid changes are rare while non-polar to non-polar changes are common. The majority of disease associated mutations result in glycine to arginine and leucine to proline substitutions. Mutations to positively charged amino acids are more common in the center of the lipid bilayer, where they cause more severe structural and functional anomalies. Our analysis contributes to the better understanding of the effect of disease associated mutations in transmembrane proteins, which can help prioritize genetic variations in personal genomic investigations. PMID:26986070

  4. Insights into bacterial protein glycosylation in human microbiota.

    PubMed

    Zhu, Fan; Wu, Hui

    2016-01-01

    The study of human microbiota is an emerging research topic. The past efforts have mainly centered on studying the composition and genomic landscape of bacterial species within the targeted communities. The interaction between bacteria and hosts is the pivotal event in the initiation and progression of infectious diseases. There is a great need to identify and characterize the molecules that mediate the bacteria-host interaction. Bacterial surface exposed proteins play an important role in the bacteria- host interaction. Numerous surface proteins are glycosylated, and the glycosylation is crucial for their function in mediating the bacterial interaction with hosts. Here we present an overview of surface glycoproteins from bacteria that inhabit three major mucosal environments across human body: oral, gut and skin. We describe the important enzymes involved in the process of protein glycosylation, and discuss how the process impacts the bacteria-host interaction. Emerging molecular details underlying glycosylation of bacterial surface proteins may lead to new opportunities for designing anti-infective small molecules, and developing novel vaccines in order to treat or prevent bacterial infection.

  5. Tunable protein synthesis by transcript isoforms in human cells

    PubMed Central

    Floor, Stephen N; Doudna, Jennifer A

    2016-01-01

    Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. DOI: http://dx.doi.org/10.7554/eLife.10921.001 PMID:26735365

  6. The closer we look the more we see? Quantitative microscopic analysis of the pulmonary surfactant system.

    PubMed

    Ochs, Matthias

    2010-01-01

    The surfactant system of the lung has essential biophysical and immunomodulatory functions. Only at the electron microscopic level does surfactant reveal its morphological complexity--and beauty. Therefore, morphological tools are indispensible to characterize the surfactant system in health and disease. Stereology provides the gold standard for obtaining quantitative (morphometric) data in microscopy. The combination of microscopy and stereology allows for qualitative and quantitative analysis of the intraalveolar as well as the intracellular surfactant pool, both in its preserved microorganization and localization within the lung. Surfactant-producing alveolar epithelial type II cells can be counted and sampled for size estimation with physical disectors at a high magnification light microscopic level. The number of their surfactant storing lamellar bodies can be estimated using physical disectors at the electron microscopic level. Electron tomography allows for high resolution 3D visualization of lamellar body fusion pores. Intraalveolar surfactant subtypes can be quantitated in situ, thus reflecting the functional state of the intraalveolar surfactant pool. By immunoelectron microscopy, surfactant protein distribution can be analyzed. These methods allow for a comprehensive quantitative analysis of surfactant (ultra-)structure. Here, we give an overview on the analysis of the normal and disordered surfactant system by electron microscopy and stereology.

  7. Serologic Cross-Reactions between Nucleocapsid Proteins of Human Respiratory Syncytial Virus and Human Metapneumovirus

    PubMed Central

    Zhang, Yange; Pohl, Jan; Brooks, W. Abdullah

    2015-01-01

    Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) share virologic and epidemiologic features and cause clinically similar respiratory illness predominantly in young children. In a previous study of acute febrile respiratory illness in Bangladesh, we tested paired serum specimens from 852 children presenting fever and cough for diagnostic increases in titers of antibody to hRSV and hMPV by enzyme immunoassay (EIA). Unexpectedly, of 93 serum pairs that showed a ≥4-fold increase in titers of antibody to hRSV, 24 (25.8%) showed a concurrent increase in titers of antibody to hMPV; of 91 pairs showing an increase to hMPV, 13 (14.3%) showed a concurrent increase to hRSV. We speculated that common antigens shared by these viruses explain this finding. Since the nucleocapsid (N) proteins of these viruses show the greatest sequence homology, we tested hyperimmune antisera prepared for each virus against baculovirus-expressed recombinant N (recN) proteins for potential cross-reactivity. The antisera were reciprocally reactive with both proteins. To localize common antigenic regions, we first expressed the carboxy domain of the hMPV N protein that was the most highly conserved region within the hRSV N protein. Although reciprocally reactive with antisera by Western blotting, this truncated protein did not react with hMPV IgG-positive human sera by EIA. Using 5 synthetic peptides that spanned the amino-terminal portion of the hMPV N protein, we identified a single peptide that was cross-reactive with human sera positive for either virus. Antiserum prepared for this peptide was reactive with recN proteins of both viruses, indicating that a common immunoreactive site exists in this region. PMID:25740767

  8. Mixed surfactant systems for enhanced oil recovery

    SciTech Connect

    Llave, F.M.; Gall, B.L.; Noll, L.A.

    1990-12-01

    The results of an evaluation of mixed surfactant systems for enhanced oil recovery are described. Several surfactant combinations have been studied. These include alkyl aryl sulfonates as primary surfactants and carboxymethylated ethoxylated (CME) surfactants and ethoxylated sulfonates (ES) as secondary surfactants. The ethoxylated surfactants increase the salinity tolerance of the primary surfactants and, in theory, allow tailoring of the surfactant system to match selected reservoir conditions. The experiments conducted included interfacial tension (IFT) measurements, phase behavior measurements, adsorption and/or chromatographic separation of mixed surfactant systems, measurements of solution properties such as the critical micelle concentration (CMC) of surfactant mixtures, and crude oil displacement experiments. The effects of temperature, surfactant concentration, salinity, presence of divalent ions, hydrocarbon type, and component proportions in the mixed surfactant combinations, and injection strategies on the performance potential of the targeted surfactant/hydrocarbon systems were studied. 40 refs., 37 figs., 8 tabs.

  9. The Cerebral Surfactant System and Its Alteration in Hydrocephalic Conditions

    PubMed Central

    Friedrich, Benjamin; Bernhard, Matthias K.; Gebauer, Corinna; Dieckow, Julia; Gawlitza, Matthias; Pirlich, Mandy; Saur, Dorothee; Bräuer, Lars; Bechmann, Ingo; Hoffmann, Karl-Titus; Mahr, Cynthia V.; Nestler, Ulf; Preuß, Matthias

    2016-01-01

    Introduction Pulmonary Surfactant reduces surface tension in the terminal airways thus facilitating breathing and contributes to host’s innate immunity. Surfactant Proteins (SP) A, B, C and D were recently identified as inherent proteins of the CNS. Aim of the study was to investigate cerebrospinal fluid (CSF) SP levels in hydrocephalus patients compared to normal subjects. Patients and Methods CSF SP A-D levels were quantified using commercially available ELISA kits in 126 patients (0–84 years, mean 39 years). 60 patients without CNS pathologies served as a control group. Hydrocephalus patients were separated in aqueductal stenosis (AQS, n = 24), acute hydrocephalus without aqueductal stenosis (acute HC w/o AQS, n = 16) and idiopathic normal pressure hydrocephalus (NPH, n = 20). Furthermore, six patients with pseudotumor cerebri were investigated. Results SP A—D are present under physiological conditions in human CSF. SP-A is elevated in diseases accompanied by ventricular enlargement (AQS, acute HC w/o AQS) in a significant manner (0.67, 1.21 vs 0.38 ng/ml in control, p<0.001). SP-C is also elevated in hydrocephalic conditions (AQS, acute HC w/o AQS; 0.87, 1.71 vs. 0.48 ng/ml in controls, p<0.001) and in Pseudotumor cerebri (1.26 vs. 0.48 ng/ml in controls, p<0.01). SP-B and SP-D did not show significant alterations. Conclusion The present study confirms the presence of SPs in human CSF. There are significant changes of SP-A and SP-C levels in diseases affecting brain water circulation and elevation of intracranial pressure. Cause of the alterations, underlying regulatory mechanisms, as well as diagnostic and therapeutic consequences of cerebral SP’s requires further thorough investigations. PMID:27656877

  10. Evolution, Development, and Function of the Pulmonary Surfactant System in Normal and Perturbed Environments.

    PubMed

    Orgeig, Sandra; Morrison, Janna L; Daniels, Christopher B

    2015-12-15

    Surfactant lipids and proteins form a surface active film at the air-liquid interface of internal gas exchange organs, including swim bladders and lungs. The system is uniquely positioned to meet both the physical challenges associated with a dynamically changing internal air-liquid interface, and the environmental challenges associated with the foreign pathogens and particles to which the internal surface is exposed. Lungs range from simple, transparent, bag-like units to complex, multilobed, compartmentalized structures. Despite this anatomical variability, the surfactant system is remarkably conserved. Here, we discuss the evolutionary origin of the surfactant system, which likely predates lungs. We describe the evolution of surfactant structure and function in invertebrates and vertebrates. We focus on changes in lipid and protein composition and surfactant function from its antiadhesive and innate immune to its alveolar stability and structural integrity functions. We discuss the biochemical, hormonal, autonomic, and mechanical factors that regulate normal surfactant secretion in mature animals. We present an analysis of the ontogeny of surfactant development among the vertebrates and the contribution of different regulatory mechanisms that control this development. We also discuss environmental (oxygen), hormonal and biochemical (glucocorticoids and glucose) and pollutant (maternal smoking, alcohol, and common "recreational" drugs) effects that impact surfactant development. On the adult surfactant system, we focus on environmental variables including temperature, pressure, and hypoxia that have shaped its evolution and we discuss the resultant biochemical, biophysical, and cellular adaptations. Finally, we discuss the effect of major modern gaseous and particulate pollutants on the lung and surfactant system.

  11. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

    PubMed

    Yu, Xiaobo; LaBaer, Joshua

    2015-05-01

    AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

  12. Biosynthesis of von Willebrand protein by human megakaryocytes.

    PubMed

    Sporn, L A; Chavin, S I; Marder, V J; Wagner, D D

    1985-09-01

    Immunofluorescence staining of buffy coat smears from a patient with chronic myelogenous leukemia in accelerated phase showed that approximately 13% of all nucleated cells contained von Willebrand protein and, therefore, appeared to be of megakaryocytic origin. This was confirmed by positive staining with antisera against platelet factor 4 and platelet glycoproteins. Short-term cultures of the buffy coat, which lacked endothelial cells, were metabolically labeled with [35S]methionine, and von Willebrand protein was immunopurified from cell lysates and culture medium. Cultures from this patient synthesized and secreted von Willebrand protein, in contrast with cultures from other patients with leukemia, who lacked circulating megakaryocytes, and from normal volunteers. The subunit composition of the megakaryocytic von Willebrand protein was very similar to that of human umbilical vein endothelial cells. The size of the processed subunit (220 kD) and of the cellular (260 kD) and secreted (275 kD) precursors from the two cell types were indistinguishable by gel electrophoresis. Furthermore, the ratio of precursor to processed subunit and the pattern of cellular and secreted nonreduced multimers were very similar. It appears, therefore, that the processing steps in biosynthesis of von Willebrand protein used by the megakaryocytes are very similar to those of umbilical vein endothelial cells.

  13. Pre-fusion structure of a human coronavirus spike protein.

    PubMed

    Kirchdoerfer, Robert N; Cottrell, Christopher A; Wang, Nianshuang; Pallesen, Jesper; Yassine, Hadi M; Turner, Hannah L; Corbett, Kizzmekia S; Graham, Barney S; McLellan, Jason S; Ward, Andrew B

    2016-03-03

    HKU1 is a human betacoronavirus that causes mild yet prevalent respiratory disease, and is related to the zoonotic SARS and MERS betacoronaviruses, which have high fatality rates and pandemic potential. Cell tropism and host range is determined in part by the coronavirus spike (S) protein, which binds cellular receptors and mediates membrane fusion. As the largest known class I fusion protein, its size and extensive glycosylation have hindered structural studies of the full ectodomain, thus preventing a molecular understanding of its function and limiting development of effective interventions. Here we present the 4.0 Å resolution structure of the trimeric HKU1 S protein determined using single-particle cryo-electron microscopy. In the pre-fusion conformation, the receptor-binding subunits, S1, rest above the fusion-mediating subunits, S2, preventing their conformational rearrangement. Surprisingly, the S1 C-terminal domains are interdigitated and form extensive quaternary interactions that occlude surfaces known in other coronaviruses to bind protein receptors. These features, along with the location of the two protease sites known to be important for coronavirus entry, provide a structural basis to support a model of membrane fusion mediated by progressive S protein destabilization through receptor binding and proteolytic cleavage. These studies should also serve as a foundation for the structure-based design of betacoronavirus vaccine immunogens.

  14. Determining protein concentrations of the human ventricular proteome.

    PubMed

    Scholten, Arjen; Heck, Albert J R

    2013-01-01

    Proteomics is mostly used for measurements of relative differences in protein concentrations. Although such analyses are meaningful for comparing differences between two and more conditions, they do not directly provide details on the absolute protein concentrations within a system. Now, proteomics is heading more towards absolute quantitative strategies with results being expressed in copies/cell or ng/mg tissue. In the cardiac context, such quantitative information is crucial for (1) evaluating the feasibility of selecting a certain protein as potential novel drug target, (2) the expected concentration excreted into the circulation when selecting a biomarker, and (3) to build a model of cardiac function at the molecular level. At the same time, by mass spectrometry-based proteomics, a wealth of spectral information is gathered that can be used to evaluate protein levels of a select set of novel disease-altered proteins using, for instance, single reaction monitoring. Here we describe how to build a quantitative map of the human left ventricular proteome using a simple yet effective mass spectrometry-based spectral count method.

  15. Biological and immunological characterization of a human liver immunoregulatory protein.

    PubMed

    Schrempf-Decker, G E; Baron, D P; Brattig, N W; Bockhorn, H; Berg, P A

    1983-01-01

    The liver immunoregulatory protein (LIP) was originally characterized as human liver-derived soluble factor which inhibited the alloantigen and phytohemagglutinin-induced proliferation of human lymphocytes (1). Soluble extracts prepared under the same experimental conditions from kidney, spleen, heart, lymph nodes, and erythrocytes did not exert any inhibitory activity (2). The purpose of this study was to characterize the immunobiological properties of LIP. In the primary one-way mixed lymphocyte culture, LIP depressed the generation of suppressor T cells which inhibited the lymphocyte proliferation induced by phytohemagglutinin or alloantigens. In addition, LIP suppressed in primary mixed lymphocyte culture the induction of cytotoxic T cells and memory cells as determined by cell-mediated lympholysis and secondary mixed lymphocyte culture, respectively. In the presence of LIP, the concanavalin A-mediated induction of suppressor T cells, the pokeweed mitogen-induced IgG synthesis in vitro and the cytolytic activity of K cells reacting in the antibody-dependent cell-mediated cytotoxicity were also inhibited. Cytotoxic effects could be excluded since the viability of human lymphoblastoid cells, hepatocytes, and allogeneically stimulated lymphocytes was not affected by LIP. LIP was shown to be different from other liver-derived substances like acute phase proteins, immunoregulatory alpha-globulins, C-reactive protein, lipoproteins, and F antigen. Furthermore, LIP is not identical to other serum components like the immunoregulatory rosette inhibition factor and the serum inhibitory factor (3). However, the characteristics described herein strongly indicate that LIP is very similar to the liver extract described by Chisari (4) and the liver-derived inhibitory protein (LIP) described by Grol and Schumacher (5).(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Distribution of SR protein exonic splicing enhancer motifs in human protein-coding genes.

    PubMed

    Wang, Jinhua; Smith, Philip J; Krainer, Adrian R; Zhang, Michael Q

    2005-01-01

    Exonic splicing enhancers (ESEs) are pre-mRNA cis-acting elements required for splice-site recognition. We previously developed a web-based program called ESEfinder that scores any sequence for the presence of ESE motifs recognized by the human SR proteins SF2/ASF, SRp40, SRp55 and SC35 (http://rulai.cshl.edu/tools/ESE/). Using ESEfinder, we have undertaken a large-scale analysis of ESE motif distribution in human protein-coding genes. Significantly higher frequencies of ESE motifs were observed in constitutive internal protein-coding exons, compared with both their flanking intronic regions and with pseudo exons. Statistical analysis of ESE motif frequency distributions revealed a complex relationship between splice-site strength and increased or decreased frequencies of particular SR protein motifs. Comparison of constitutively and alternatively spliced exons demonstrated slightly weaker splice-site scores, as well as significantly fewer ESE motifs, in the alternatively spliced group. Our results underline the importance of ESE-mediated SR protein function in the process of exon definition, in the context of both constitutive splicing and regulated alternative splicing.

  17. Surfactants at the Design Limit.

    PubMed

    Czajka, Adam; Hazell, Gavin; Eastoe, Julian

    2015-08-04

    This article analyzes how the individual structural elements of surfactant molecules affect surface properties, in particular, the point of reference defined by the limiting surface tension at the aqueous cmc, γcmc. Particular emphasis is given to how the chemical nature and structure of the hydrophobic tails influence γcmc. By comparing the three different classes of surfactants, fluorocarbon, silicone, and hydrocarbon, a generalized surface packing index is introduced which is independent of the chemical nature of the surfactants. This parameter ϕcmc represents the volume fraction of surfactant chain fragments in a surface film at the aqueous cmc. It is shown that ϕcmc is a useful index for understanding the limiting surface tension of surfactants and can be useful for designing new superefficient surfactants.

  18. The Pulmonary Surfactant: Impact of Tobacco Smoke and Related Compounds on Surfactant and Lung Development

    PubMed Central

    Scott, J Elliott

    2004-01-01

    Cigarette smoking, one of the most pervasive habits in society, presents many well established health risks. While lung cancer is probably the most common and well documented disease associated with tobacco exposure, it is becoming clear from recent research that many other diseases are causally related to smoking. Whether from direct smoking or inhaling environmental tobacco smoke (ETS), termed secondhand smoke, the cells of the respiratory tissues and the lining pulmonary surfactant are the first body tissues to be directly exposed to the many thousands of toxic chemicals in tobacco. Considering the vast surface area of the lung and the extreme attenuation of the blood-air barrier, it is not surprising that this organ is the primary route for exposure, not just to smoke but to most environmental contaminants. Recent research has shown that the pulmonary surfactant, a complex mixture of phospholipids and proteins, is the first site of defense against particulates or gas components of smoke. However, it is not clear what effect smoke has on the surfactant. Most studies have demonstrated that smoking reduces bronchoalveolar lavage phospholipid levels. Some components of smoke also appear to have a direct detergent-like effect on the surfactant while others appear to alter cycling or secretion. Ultimately these effects are reflected in changes in the dynamics of the surfactant system and, clinically in changes in lung mechanics. Similarly, exposure of the developing fetal lung through maternal smoking results in postnatal alterations in lung mechanics and higher incidents of wheezing and coughing. Direct exposure of developing lung to nicotine induces changes suggestive of fetal stress. Furthermore, identification of nicotinic receptors in fetal lung airways and corresponding increases in airway connective tissue support a possible involvement of nicotine in postnatal asthma development. Finally, at the level of the alveoli of the lung, colocalization of nicotinic

  19. The Pulmonary Surfactant: Impact of Tobacco Smoke and Related Compounds on Surfactant and Lung Development

    PubMed Central

    Scott, J Elliott

    2004-01-01

    Cigarette smoking, one of the most pervasive habits in society, presents many well established health risks. While lung cancer is probably the most common and well documented disease associated with tobacco exposure, it is becoming clear from recent research that many other diseases are causally related to smoking. Whether from direct smoking or inhaling environmental tobacco smoke (ETS), termed secondhand smoke, the cells of the respiratory tissues and the lining pulmonary surfactant are the first body tissues to be directly exposed to the many thousands of toxic chemicals in tobacco. Considering the vast surface area of the lung and the extreme attenuation of the blood-air barrier, it is not surprising that this organ is the primary route for exposure, not just to smoke but to most environmental contaminants. Recent research has shown that the pulmonary surfactant, a complex mixture of phospholipids and proteins, is the first site of defense against particulates or gas components of smoke. However, it is not clear what effect smoke has on the surfactant. Most studies have demonstrated that smoking reduces bronchoalveolar lavage phospholipid levels. Some components of smoke also appear to have a direct detergent-like effect on the surfactant while others appear to alter cycling or secretion. Ultimately these effects are reflected in changes in the dynamics of the surfactant system and, clinically in changes in lung mechanics. Similarly, exposure of the developing fetal lung through maternal smoking results in postnatal alterations in lung mechanics and higher incidents of wheezing and coughing. Direct exposure of developing lung to nicotine induces changes suggestive of fetal stress. Furthermore, identification of nicotinic receptors in fetal lung airways and corresponding increases in airway connective tissue support a possible involvement of nicotine in postnatal asthma development. Finally, at the level of the alveoli of the lung, colocalization of nicotinic

  20. EML proteins in microtubule regulation and human disease.

    PubMed

    Fry, Andrew M; O'Regan, Laura; Montgomery, Jessica; Adib, Rozita; Bayliss, Richard

    2016-10-15

    The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104: , 445-450; J. Cell Sci. (1987) 87: (Pt 1), 71-84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8: , 110; Dev. Genes Evol. (2000) 210: , 2-10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy.

  1. [Study of novel artificial lung surfactants incorporating partially fluorinated amphiphiles].

    PubMed

    Nakahara, Hiromichi

    2012-01-01

    Lung surfactants (LS), a complex of ∼90 wt% lipids (mainly dipalmitoylphosphatidylcholine or DPPC) and ∼10 wt% surfactant proteins (SP-A, -B, -C, and -D), adsorb to an air-alveolar fluid interface and then lower its surface tension down to near zero during expiration. Intratracheal instillation of exogenous LS preparations can effectively