Surfactant function and composition after free radical exposure generated by transition metals.
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Surfactant dysfunction in acute lung injury has been postulated as a result of free radical damage to lipid and protein components. This study examines whether transition metals with different redox potentials and different binding affinities for lipids and proteins affect interfacial properties differently. Purified whole calf lung surfactant (CLS) was incubated with 0.125 mM Fe2+, Fe3+, Fe3+-EDTA complex, or Cu2+ either alone or with 0.25 mM H2O2 or H2O2 plus 0.25 mM ascorbate for 4 and 24 h. Lipid peroxidation was assessed by measurement of thiobarbituric acid-reactive substances (TBARS), and free radical-mediated alterations in protein structure were assessed by fluorescamine assay and Western blot analysis. Function was assayed by pulsating bubble surfactometry. Lipid peroxidation was detected in samples incubated with Fe2+, Fe3+, and Fe3+-EDTA but not with Cu2+. All transition metal-based free radical systems affected surfactant protein composition by fluorescamine assay, indicating free radical-mediated modification of protein side chains. Western blot analysis demonstrated surfactant protein A modification, with the generation of higher- and lower-molecular-mass immunoreactive products. Despite biochemical evidence of lipid and protein modification, surfactant dysfunction was minimal and was manifest as an increase in the compression ratio required to achieve surface tension < 1 dyn/cm. This dysfunction was readily reversed by the addition of 5 mM Ca2+ either before or after oxidation. These data indicate that copper- and iron-based free radical-generating systems modify the lipid and protein components of surfactant differently but suggest that these changes have little effect on surfactant function. (+info)
A simple and sensitive fluorometric assay method for taurine using high-voltage paper electrophoresis.
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A simple and sensitive fluorometric assay method for taurine (2-aminoethanesulfonic acid) has been developed. For the separation of taurine, high voltage paper electrophoresis subsequent to column chromatographic procedures was employed. Fluorescent product of taurine was yielded by spraying fluorescamine (4-phenylspiro [furan-2(3H), 1'-phthalan]-3, 3'-dione) and borate buffer on the paper, and the fluorescence was assayed spectro-fluorometrically after eluting with 50% ethanol. The linear relationship between the concentration of taurine and fluorescence developed was achieved over the concentration ranges of 0.5-10 nmoles, and the recoveries obtained were 90-100%. The specificity of this method for taurine was satisfactory and structural analogues involved in the metabolic pathway of taurine did not interfere with the assay. Examples for tissue levels of taurine in various organs of the rat as determined by this new method are also presented. (+info)
Characterisation of the binding interaction between poly(L-lysine) and DNA using the fluorescamine assay in the preparation of non-viral gene delivery vectors.
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A major factor limiting the development of non-viral gene delivery systems is the poor characterisation of polyelectrolyte complexes formed between cationic polymers and DNA. The present study uses the fluorescamine reagent to improve characterisation of poly(L-lysine) (pLL)/DNA complexes post-modified with a multivalent hydrophilic polymer by determining the availability of free amino groups. The results show that the fluorescamine reagent can be used to monitor the self-assembly reaction between pLL and DNA and the degree of surface modification of the resultant complexes with a hydrophilic polymer. This experimental approach should enable the preparation of fully defined complexes whose properties can be better related to their biological activity. (+info)
Modeling the kinetics of acylation of insulin using a recursive method for solving the systems of coupled differential equations.
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This paper describes a theoretical method for solving systems of coupled differential equations that describe the kinetics of complicated reaction networks in which a molecule having multiple reaction sites reacts irreversibly with multiple equivalents of a ligand (reagent). The members of the network differ in the number of equivalents of reagent that have reacted, and in the patterns of sites of reaction. A recursive algorithm generates series, asymptotic, and average solutions describing this kinetic scheme. This method was validated by successfully simulating the experimental data for the kinetics of acylation of insulin. (+info)
Decreased binding to proteins and cells of polymeric gene delivery vectors surface modified with a multivalent hydrophilic polymer and retargeting through attachment of transferrin.
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Binding of serum proteins to polyelectrolyte gene delivery complexes is thought to be an important factor limiting bloodstream circulation and restricting access to target tissues. Protein binding can also inhibit transfection activity in vitro. In this study a multivalent reactive hydrophilic polymer has been used to inhibit protein binding. This polymer is based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA) bearing pendent oligopeptide (Gly-Phe-Leu-Gly) side chains terminated in reactive 4-nitrophenoxy groups (8.6 mol%). The polymer reacts with the primary amino groups of poly(L-lysine) (pLL) and produces a hydrophilic coating on the surface of pLL.DNA complexes (as measured by fluorescamine). The resulting pHPMA-coated complexes show a decreased surface charge (from +14 mV for pLL.DNA complexes to -25 mV for pHPMA-modified complexes) as measured by zeta potential analysis. The pHPMA-coated complexes also show a slightly increased average diameter (approximately 90 nm compared with 60 nm for pLL. DNA complexes) as viewed by atomic force and transmission electron microscopy and around 100 nm as viewed by photon correlation spectroscopy. They are completely resistant to protein interaction, as determined by turbidometry and SDS-polyacrylamide gel electrophoresis analysis of complexes isolated from plasma, and show significantly decreased nonspecific uptake into cells in vitro. Spare reactive ester groups can be used to conjugate targeting ligands (e.g. transferrin) on to the surface of the complex to provide a means of tissue-specific targeting and transfection. The properties of these complexes therefore make them promising candidates for targeted gene delivery, both in vitro and potentially in vivo. (+info)
Molecular cloning, expression, and purification of nuclear inclusion A protease from tobacco vein mottling virus.
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The gene encoding the C-terminal protease domain of the nuclear inclusion protein a (NIa) of tobacco vein mottling virus (TVMV) was cloned from an isolated virus particle and expressed as a fusion protein with glutathione S-transferase in Escherichia coli XL1-blue. The 27-kDa protease was purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography. The purified protease exhibited the specific proteolytic activity towards the nonapeptide substrates, Ac-Glu-Asn-Asn-Val-Arg-Phe-Gln-Ser-Leu-amide and Ac-Arg-Glu-Thr-Val-Arg-Phe-Gln-Ser-Asp-amide, containing the junction sequences between P3 protein and cylindrical inclusion protein and between nuclear inclusion protein b and capsid protein, respectively. The Km and k(cat) values were about 0.2 mM and 0.071 s(-1), respectively, which were approximately five-fold lower than those obtained for the NIa protease of turnip mosaic potyvirus (TuMV), suggesting that the TVMV NIa protease is different in the binding affinity as well as in the catalytic power from the TuMV NIa protease. In contrast to the NIa proteases from TuMV and tobacco etch virus, the TVMV NIa protease was not autocatalytically cleaved into smaller proteins, indicating that the C-terminal truncation is not a common phenomenon occurring in all potyviral NIa proteases. These results suggest that the TVMV NIa protease has a unique biochemical property distinct from those of other potyviral proteases. (+info)
Application of fluorescamine to the study of protein-DNA interactions.
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The reactivity of alpha-amino groups of basic proteins towards fluorescamine is essentially abolished if salt linkages with DNA phosphate groups are formed. This observation prompted the elaboration of a very general assay which allows the determination of binding parameters for the interaction of proteins with DNA and chromatin. Protamines, labeled with fluorescamine prior to their binding by DNA appear to be useful probes to monitor the formation and nature of DNA-protein complexes. (+info)
Lipid antioxidant, etoposide, inhibits phosphatidylserine externalization and macrophage clearance of apoptotic cells by preventing phosphatidylserine oxidation.
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Apoptosis is associated with the externalization of phosphatidylserine (PS) in the plasma membrane and subsequent recognition of PS by specific macrophage receptors. Selective oxidation of PS precedes its externalization/recognition and is essential for the PS-dependent engulfment of apoptotic cells. Because etoposide is a potent and selective lipid antioxidant that does not block thiol oxidation, we hypothesized that it may affect PS externalization/recognition without affecting other features of the apoptotic program. We demonstrate herein that etoposide induced apoptosis in HL-60 cells without the concomitant peroxidation of PS and other phospholipids. HL-60 cells also failed to externalize PS in response to etoposide treatment. In contrast, oxidant (H2O2)-induced apoptosis was accompanied by PS externalization and oxidation of different phospholipids, including PS. Etoposide potentiated H2O2-induced apoptosis but completely blocked H2O2-induced PS oxidation. Etoposide also inhibited PS externalization as well as phagocytosis of apoptotic cells by J774A.1 macrophages. Integration of exogenous PS or a mixture of PS with oxidized PS in etoposide-treated HL-60 cells reconstituted the recognition of these cells by macrophages. The current data demonstrate that lipid antioxidants, capable of preventing PS peroxidation, can block PS externalization and phagocytosis of apoptotic cells by macrophages and hence dissociate PS-dependent signaling from the final common pathway for apoptosis. (+info)