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In vitro and in vivo reactivity of porous, electrosprayed calcium phosphate coatings.

The dissolution and/or precipitation behaviour of porous calcium phosphate (CaP) coatings, deposited using electrostatic spray deposition (ESD), was investigated (a) in vitro after soaking in simulated body fluid (SBF) for several time periods (2, 4, 8, and 12 weeks), and (b) in vivo after subcutaneous implantation of CaP-coated implants in the back of goats for identical time periods. Physical and chemical properties of coatings were characterized before and after in vitro/vivo testing by means of scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and energy dispersive spectroscopy. Moreover, part of the explants was prepared for light microscopical evaluation of the tissue response. In vitro, all apatitic ESD-coatings induced the formation of homogeneous and adherent CaP precipitation layers. Amorphous CaP, however, displayed a delayed precipitation of poorly adherent CaP layers, whereas heterogeneous calcification was observed on top of beta-TCP-coated substrates, indicating that beta-TCP and amorphous CaP coatings exhibit a poor ability of inducing calcification in SBF as compared to crystalline apatitic coatings. In vivo, no adverse tissue reactions (toxic effects/inflammatory cells) were observed using light microscopy, and all coatings became surrounded by a dense, fibrous tissue capsule after implantation. All ESD-coatings degraded gradually at a dissolution rate depending on the chemical phase (order of relative solubility: amorphous CaP approximately carbonate apatite>beta-TCP>carbonated hydroxyapatite), thereby enabling synthesis of CaP coatings with a tailored degradation rate.

Animals↗

Immobilization mechanisms of arsenate in iron hydroxide sludge stabilized with cement.

Leaching tests, Fourier transform infrared spectroscopy (FTIR), extended X-ray absorption fine structure (EXAFS) spectroscopy, and thermodynamic modeling were performed to investigate arsenate [As(V)] immobilization mechanisms in iron hydroxide sludge stabilized with cement. The sludge from a groundwater remediation site in Tacoma, WA was mixed and immobilized with premixed cement to reach cement-to-sludge ratios of 2.5, 3.3, 5, 10, and 20 (wt premixed cement/wt dry sludge). The EXAFS analysis determined that As(V) formed bidentate mononuclear complexes on the iron hydroxide surface in the sludge. The adsorbed As(V) had a characteristic FTIR band at 830 cm(-1). Cement treatment converted the adsorbed As(V) to calcium arsenate precipitate with a FTIR peak at 860 cm(-1). The chemical forms of the As(V) were incorporated in an adsorption triple layer model (TLM) to describe the leaching behavior of As(V) in a pH range between 3 and 12. Cement treatment significantly reduced arsenic mobility because of the formation of the sparingly soluble calcium arsenate.

Arsenates↗

Wild type p53 gene causes reorganization of cytoskeleton and, therefore, the impaired deformability and difficult migration of murine erythroleukemia cells.

We studied the role of p53 gene in the biophysics and biology in murine erythroleukemia cell line (MEL), with the goal of understanding the influence of this tumor suppressor gene on the deformability and metastasis of tumor cells. Experiments were performed on MEL and p53-transfected MEL (MEL-M with mutant p53 gene and MEL-W with wild-type p53 gene). The cell growth curves indicated that the over-expression of wild-type p53 gene significantly suppressed the growth of MEL, with G(0)-G(1) arrest and apoptosis shown by flow cytometric assays. Confocal laser scanning microscopy revealed that the MEL-W had a more compact organization of the F-Actin cytoskeleton than MEL and MEL-M. Fluorescence polarization measurement indicated a higher membrane fluidity of MEL-W than the other two groups. Fourier transform infrared spectroscopy (FT-IR) showed changes in the composition and/or structure of membrane lipids in MEL-W, with decreases in secondary structures of proteins such as alpha-helix, turns and bends and random coil, in comparison to MEL and MEL-M. The osmotic fragility curves indicated that MEL-W was more fragile and micropipette experiments showed that they had increased elasticity and reduced deformability in comparison to MEL and MEL-M. The adhesion assay with the use of the flow chamber revealed a lower adhesion rate of MEL-W to endothelial cells at high shear stress. The present study on the molecular biology with biophysics of MEL cells contributes to our knowledge on the tumor suppressor gene p53.

Actins↗

On the temperature--pressure free-energy landscape of proteins.

We studied the thermodynamic stability of a small monomeric protein, staphylococcal nuclease (Snase), as a function of both temperature and pressure, and expressed it as a 3D free-energy surface on the p,T-plane using a second-order Taylor expansion of the Gibbs free-energy change delta G upon unfolding. We took advantage of a series of different techniques (small-angle X-ray scattering, Fourier-transform infrared spectroscopy, differential thermal analysis, pressure perturbation calorimetry and densitometry) in the evaluation of the conformation of the protein and in evaluating the changes in the thermodynamic parameters upon unfolding, such as the heat capacity, enthalpy, entropy, volume, isothermal compressibility and expansivity. The calculated results of the free-energy landscape of the protein are in good agreement with experimental data of the p,T-stability diagram of the protein over a temperature range from 200 to 400 K and at pressures from ambient pressure to 4000 bar. The results demonstrate that combined temperature--pressure-dependent studies can help delineate the free-energy landscape of proteins and hence help elucidate which features and thermodynamic parameters are essential in determining the stability of the native conformational state of proteins. The approach presented may also be used for studying other systems with so-called re-entrant or Tamman loop-shaped phase diagrams.

Calorimetry, Differential Scanning↗

Fourier transform infrared double-flash experiments resolve bacteriorhodopsin's M1 to M2 transition.

The orientation of the central proton-binding site, the protonated Schiff base, away from the proton release side to the proton uptake side is crucial for the directionality of the proton pump bacteriorhodopsin. It has been proposed that this movement, called the reprotonation switch, takes place in the M1 to M2 transition. To resolve the molecular events in this M1 to M2 transition, we performed double-flash experiments. In these experiments a first pulse initiates the photocycle and a second pulse selectively drives bR molecules in the M intermediate back into the BR ground state. For short delay times between initiating and resetting pulses, most of the M molecules being reset are in the M1 intermediate, and for longer delay times most of the reset M molecules are in the M2 intermediate. The BR-M1 and BR-M2 difference spectra are monitored with nanosecond step-scan Fourier transform infrared spectroscopy. Because the Schiff base reprotonation rate is kM1 = 0.8 x 10(7) s(-1) in the light-induced M1 back-reaction and kM2 = 0.36 x 10(7) s(-1) in the M2 back-reaction, the two different M intermediates represent two different proton accessibility configurations of the Schiff base. The results show only a minute movement of one or two peptide bonds in the M1 to M2 transition that changes the interaction of the Schiff base with Y185. This backbone movement is distinct from the larger one in the subsequent M to N transition. No evidence of a chromophore isomerization is seen in the M1 to M2 transition. Furthermore, the results show time-resolved reprotonation of the Schiff base from D85 in the M photo-back-reaction, instead of from D96, as in the conventional cycle.

Bacteriorhodopsins↗

The prion domain of yeast Ure2p induces autocatalytic formation of amyloid fibers by a recombinant fusion protein.

The Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion-like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the [URE3] phenotype. The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for [URE3] (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD). We expressed both UPD and Ure2 as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble. Upon cleavage of GST-UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid-like characteristics, such as Congo red dye binding and green-gold birefringence. The fibrils exhibited high beta-sheet content by Fourier transform infrared spectroscopy. Fiber formation proceeded in an autocatalytic manner. In contrast, the released, full-length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology. Aggregation of Ure2p could be seeded by UPD fibrils. Our results provide biochemical support for the proposal that the [URE3] state is caused by a self-propagating inactive form of Ure2p. We also found that the uncleaved GST-UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, beta-sheet-rich conformation. The findings on the GST-UPD fusion protein indicate that the ability of the prion domain to mediate a prion-like conversion process is not specific for or limited to the Ure2p.

Amyloid↗

Evolution of wheat gliadins conformation during film formation: a fourier transform infrared study.

The secondary structures of wheat gliadins (a major storage protein fraction from gluten) in film-forming solutions and their evolution during film formation were investigated by Fourier transform infrared spectroscopy. In the film-forming solution, wheat gliadins presented a mixture of different secondary structures, with an important contribution of beta-turns induced by proline residues. The presence of plasticizer did not have any influence on protein secondary structure in the film-forming solution. The evolution of protein conformation was followed during drying; the major feature of this evolution was a clear growing of the infrared band at 1622 cm(-1), characteristic of intermolecular hydrogen-bonded beta-sheets. This revealed the formation of protein aggregates during film drying. The influence of the drying temperature on film properties and gliadin secondary structures was also investigated. Higher drying temperatures induced an increase of both the tensile strength of the films and the amount of beta-sheets aggregates. Although the appearance of heat-induced disulfide bridge cross-links has already been described, there is clear evidence that hydrogen-bonded beta-sheets aggregates are also induced by thermal treatment. It was not possible, however, to determine whether there is a direct relationship between the occurrence of these aggregates and the increase of the tensile strength of the films.

Gliadin↗

In situ transient FTIR and XANES studies of the evolution of surface species in CO oxidation on Au/TiO2.

The adsorption of CO and its reaction with oxygen were investigated using a combination of in situ Fourier transform infrared spectroscopy, step response measurements in a microreactor, (18)O isotopic labeling, and X-ray absorption near edge structure spectroscopy. An as-prepared sample in which Au is present as a surface oxyhydroxy complex does not adsorb CO. On an activated sample in which only metallic Au is detected, 0.18 +/- 0.03 mol CO/(mol Au) are adsorbed on Au at -60 degrees C, which shows an IR band at 2090 cm(-1). When oxygen is present in the gas phase, this species reacts with a turnover rate of 1.4 +/- 0.2 mol CO(mol Au min)(-1), which is close to the steady-state turnover rate. In contrast, there is a very small quantity of adsorbed oxygen on Au. A small IR peak at 1242 cm(-1) appears when an activated sample is exposed to CO. It reacts rapidly with oxygen and is shifted to 1236 cm(-1) if (18)O is used. It is assigned to the possible intermediate hydroxycarbonyl.

Adsorption↗

Release of PEGylated granulocyte-macrophage colony-stimulating factor from chitosan/glycerol films.

We have prepared a new formulation for mucosal delivery of GM-CSF or PEGylated GM-CSF based on a chitosan carrier plus added glycerol to control the rate of release of the protein. Thin dry films comprised of various weight ratios of chitosan to glycerol and containing either granulocyte-macrophage colony-stimulating factor (GM-CSF) or PEGylated GM-CSF, PEG-(GM-CSF), were prepared. The amount of GM-CSF or PEG-(GM-CSF) released from the chitosan/glycerol films was determined using size exclusion high performance liquid chromatography (HPLC-SEC). The amount of PEG-(GM-CSF) released from the films decreased with an increase in the amount of glycerol present in the film. In parallel with this, films with higher glycerol content exhibited a lower degree of equilibrium swelling when immersed in release media. pH measurements of the release media and analysis of the dried films by Fourier-transform infrared spectroscopy (FTIR) suggested that the amount of residual acetic acid in the dry films decreased as the glycerol content increased. This indicates that glycerol may act by displacing and releasing bound acetic acid from the chitosan molecules, resulting in chitosan--glycerol hydrogen bond formation as the film dries. Further, it was found that the release rate and the amount of PEG-(GM-CSF) released decreased with increasing molecular weight of the conjugated PEG. This effect was not observed with films containing physical mixtures of PEG and GM-CSF. The decrease in the fraction of PEG-(GM-CSF) released with increasing PEG molecular weight is believed to be due to the increased steric hindrance of the PEGylated protein molecule during its diffusion out of the swollen chitosan/glycerol film.

Biocompatible Materials↗

Amyloid-like fibril formation in an all beta-barrel protein. Partially structured intermediate state(s) is a precursor for fibril formation.

Acidic fibroblast growth factor from newt (Notopthalmus viridescens) is a approximately 15-kDa, all beta-sheet protein devoid of disulfide bonds. In the present study, we investigate the effects of 2,2,2-trifluoroethanol (TFE) on the structure of newt acidic fibroblast growth factor (nFGF-1). The protein aggregates maximally in 10% (v/v) TFE. Congo red and thioflavin T binding experiments suggest that the aggregates induced by TFE have properties resembling the amyloid fibrils. Transmission electron microscopy and x-ray fiber diffraction data show that the fibrils (induced by TFE) are straight, unbranched, and have a cross-beta structure with an average diameter of 10-15 A. Preformed fibrils (induced by TFE) of nFGF-1 are observed to seed amyloid-like fibril formation in solutions containing the protein (nFGF-1) in the native beta-barrel conformation. Fluorescence, far-UV CD, anilino-8-napthalene sulfonate binding, multidimensional NMR, and Fourier transformed infrared spectroscopy data reveal that formation of a partially structured intermediate state(s) precedes the onset of the fibrillation process. The native beta-barrel structure of nFGF-1 appears to be disrupted in the partially structured intermediate state(s). The protein in the partially structured intermediate state(s) is found to be "sticky" with a solvent-exposed non-polar surface(s). Amyloid fibril formation appears to occur due to coalescence of the protein in the partially structured intermediate state(s) through solvent-exposed non-polar surfaces and intermolecular beta-sheet formation among the extended, linear beta-strands in the protein.

Amyloid beta-Peptides↗

Inhibition of Listeria monocytogenes by food-borne yeasts.

Many bacteria are known to inhibit food pathogens, such as Listeria monocytogenes, by secreting a variety of bactericidal and bacteriostatic substances. In sharp contrast, it is unknown whether yeast has an inhibitory potential for the growth of pathogenic bacteria in food. A total of 404 yeasts were screened for inhibitory activity against five Listeria monocytogenes strains. Three hundred and four of these yeasts were isolated from smear-ripened cheeses. Most of the yeasts were identified by Fourier transform infrared spectroscopy. Using an agar-membrane screening assay, a fraction of approximately 4% of the 304 red smear cheese isolates clearly inhibited growth of L. monocytogenes. Furthermore, 14 out of these 304 cheese yeasts were cocultivated with L. monocytogenes WSLC 1364 on solid medium to test the antilisterial activity of yeast in direct cell contact with Listeria. All yeasts inhibited L. monocytogenes to a low degree, which is most probably due to competition for nutrients. However, one Candida intermedia strain was able to reduce the listerial cell count by 4 log units. Another four yeasts, assigned to C. intermedia (three strains) and Kluyveromyces marxianus (one strain), repressed growth of L. monocytogenes by 3 log units. Inhibition of L. monocytogenes was clearly pronounced in the cocultivation assay, which simulates the conditions and contamination rates present on smear cheese surfaces. We found no evidence that the unknown inhibitory molecule is able to diffuse through soft agar.

Agar↗

Degree of conversion and surface hardness of resin cement cured with different curing units.

OBJECTIVE: The aim of this study was to evaluate the degree of conversion and Vickers surface hardness of resin cement under a simulated ceramic restoration with 3 different curing units: a conventional halogen unit, a high-intensity halogen unit, and a light-emitting diode system. METHODS AND MATERIALS: A conventional halogen curing unit (Hilux 550) (40 s), a high-intensity halogen curing unit used in conventional and ramp mode (Optilux 501) (10 s and 20 s, respectively), and a light-emitting diode system (Elipar FreeLight) (20 s, 40 s) were used in this study. The dual-curing resin cement (Variolink II) was cured under a simulated ceramic restoration (diameter 5 mm, height 2 mm), and the degree of conversion and Vickers surface hardness were measured. For degree of conversion measurement, 10 specimens were prepared for each group. The absorbance peaks were recorded using the diffuse-reflection mode of Fourier transformation infrared spectroscopy. For Vickers surface hardness measurement, 10 specimens were prepared for each group. A load of 200 N was applied for 15 seconds, and 3 evaluations of each of the samples were performed. RESULTS: Degree of conversion achieved with Optilux 501 (20 s) was significantly higher than those of Hilux, Optilux 501 (10 s), Elipar FreeLight (20 s), and Elipar FreeLight (40 s). For Vickers surface hardness measurement, Optilux 501 (20 s) produced the highest surface hardness value. No significant differences were found among the Hilux, Optilux 501 (10 s), Elipar FreeLight (20 s), and Elipar FreeLight (40 s). CONCLUSION: The high-intensity halogen curing unit used in ramp mode (20 s) produced harder resin cement surfaces than did the conventional halogen curing unit, high-intensity halogen curing unit used in conventional mode (10 s) and light-emitting diode system (20 s, 40 s), when cured through a simulated ceramic restoration.

Dental Porcelain↗

Water-soluble inhibitor on microbiologically influenced corrosion in diesel pipeline.

The effect of water-soluble corrosion inhibitor on the growth of bacteria and its corrosion inhibition efficiency were investigated. Corrosion inhibition efficiency was studied by rotating cage test and flow loop techniques. The nature of biodegradation of corrosion inhibitor was also analyzed by using Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (NMR) and Gas chromatography and mass spectrometer (GC-MS). The bacterial isolates (Serratia marcescens ACE2, Bacillus cereus ACE4) have the capacity to degrade the aromatic and aliphatic hydrocarbon present in the corrosion inhibitor. The degraded products of corrosion inhibitor and bacterial activity determine the electrochemical behaviour of API 5LX steel. The influence of bacterial activity on degradation of corrosion inhibitor and its influence on corrosion of API 5LX have been evaluated by employing weight loss techniques and electrochemical studies. The main finding of this paper is that the water-soluble corrosion inhibitor is consumed by the microbial action, which contributes to the decrease in inhibitor efficiency. The present study also emphasis the importance of evaluation of water-soluble corrosion inhibitor in stagnant model (flow loop test) and discusses the demerits of the water-soluble corrosion inhibitors in petroleum product pipeline.

Anti-Bacterial Agents↗

The schiff base of gossypol with 3,6,9,12,15,18,21,24-octaoxa-pentacosylamine complexes and monovalent cations studied by electrospray ionization-mass spectrometry, (1)H nuclear magnetic resonance, Fourier transform infrared, as well as PM5 semiempirical methods.

A Schiff base of gossypol with 3,6,9,12,15,18,21,24-octaoxa-pentacosylamine (GSOB) forms stable complexes with monovalent cations. This process of complex formation was studied by electrospray ionization-mass spectrometry, (1)H-NMR and Fourier transform infrared spectroscopy as well as by the PM5 semiempirical method. It was found that GSOB forms 1:6 complexes with Li(+) and Na(+), and 1:4 complexes with K(+) as well as 1:2 complexes with Rb(+) or Cs(+) cations and exists in all these complexes in the enamine-enamine tautomeric form. Moreover, within these complexes only Li(+) cations can fluctuate between the oxygen atoms of the octaoxaalkyl chains. The interactions of Li(+) cations with hydroxyl groups of the gossypol part is also possible. All other cations are much more localized. In the complex of GSOB with protons, a 1:2 stoichiometry is realized. The two protons are localized on the N atoms of the Schiff base, and the complex exists in the imine-imine tautomeric form. The structures of the complexes are calculated by PM5 semiempirical methods and discussed.

Cations, Monovalent↗

Expression, purification and properties of multidrug efflux proteins.

A general strategy is described for the amplified expression, purification and characterization in Escherichia coli of multidrug efflux proteins from Staphylococcus aureus, Bacillus subtilis, Methanococcus janaschii and E. coli. They all catalyse drug/H(+) antiport of substrates such as quinolones and ethidium and exemplify a family of putatively 12-helix membrane proteins. The gene for each protein was cloned downstream of the tac promoter in plasmid pTTQ18; an oligonucleotide encoding six histidine residues was added, in frame, to the C-terminus to facilitate purification. Growth conditions were optimized in 1-25-litre cultures of E. coli host strains to amplify the expression of each protein; the retention of activity was confirmed by assays of antibiotic resistance in vivo and/or assays of energized transport activity in vitro with synthetic substrates. Proteins were solubilized in dodecylmaltoside and purified to more than 90% homogeneity with Ni(2+)-nitrilotriacetate-affinity column chromography, yielding 5-25 mg per 25 litres of original culture. All the transport proteins migrated anomalously in SDS/PAGE at apparent molecular masses below those predicted from the gene sequence; identity and integrity were therefore confirmed by N-terminal amino acid sequencing and Western blotting for the C-terminal hexahistidine tag. Examination of the secondary structure of detergent-solubilized proteins by CD or Fourier-transform infrared spectroscopy following purification indicated a high content of alpha-helix (more than 75%). Matrix-assisted laser desorption ionization MS confirmed the high degree of purity and the true molecular mass. The formation of three-dimensional crystals is being attempted but crystals have yet to be grown that diffract X-rays. The growth of two-dimensional protein arrays has been more successful, with diffraction of electrons at low resolution. Proteins have been fused to green fluorescent protein or maltose-binding protein to facilitate these structural analyses. In addition, ligands for efflux proteins labelled with (13)C or (15)N have been synthesized to implement solid-state NMR studies of the ligand-binding site.

Bacillus subtilis↗

Soft-tissue response to injectable calcium phosphate cements.

In this study, the soft tissue reaction to two newly developed injectable calcium phosphate bone cements (cement D and W) was evaluated after implantation in the back of goats. For one of the cements (cement D) the tissue reaction was also investigated after varying the concentration of accelerator Na(2)HPO(4) in the cement liquid (resulting in cement D1 and D2). Eight healthy mature female Saanen goats were used. The cement was applied 10min after mixing while it was still moldable and plastic. The material was given a standardized cylindrical shape. Thirty-two implants of each cement formulation were inserted and left in place for 1, 2, 4, and 8weeks. At the end of the study, eight specimens of each material and healing period were available for further analysis. Two specimens were used for X-ray diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FTIR) and six specimens were used for light microscopical evaluation. XRD and FTIR showed that the cements did set as microcrystalline carbonate apatite with the disappearance of monetite from the cements during implantation. Histological analysis showed that after 8weeks of implantation around all materials a thin soft-tissue capsule was formed (thickness ranging from 5 to 15 cell layers) with almost complete absence of inflammatory cells. Only in some specimens a slightly higher inflammatory reaction was observed. This was due to cement surface defects and a zone of dispersed particles near the cement-soft tissue interface. There was almost no resorption of the material after 8 weeks of implantation. In a few 4 and 8weeks samples, small areas of calcification were found in the fibrous capsule surrounding the implants. On the basis of our observations, we conclude that the tested cements were biocompatible and can be used next to soft tissue.

Animals↗

Induction of alpha-helix in the beta-sheet protein tumor necrosis factor-alpha: acid-induced denaturation.

Acid-induced unfolding of proteins often results in an intermediate structure, called the molten globule structure or "A" state, which retains at least partial secondary structure but lacks a rigid tertiary structure. Acid-induced unfolding has been studied extensively for alpha-helical proteins, while few studies have been done on proteins containing only beta-strands. Tumor necrosis factor-alpha (TNF-alpha) is a trimer in which the individual subunits consist of antiparallel beta-sheet, organized into a jellyroll beta-sandwich. We have found previously [Narhi et al. (1996) Biochemistry 35, 11447-11453] that thermal denaturation of TNF-alpha results in an aggregate which contains a substantial amount of alpha-helix and that the addition of trifluoroethanol induces alpha-helix in both murine and human TNF-alpha. Here we show that acid also can induce alpha-helix in these proteins. At acidic pH (below 4), both human and murine TNF-alpha convert to a monomeric form, as determined by sedimentation and diffusion constants obtained from sedimentation velocity experiments. The sedimentation coefficient indicated that this monomer was only slightly expanded relative to the native state. Near-UV circular dichroic (CD) analysis showed a loss of tertiary structure. These structural features coincide with the notion that the acid-induced structure of TNF-alpha is a molten globule. What is unique in this protein is that TNF-alpha acquires alpha-helical structure, which is not present in the native structure as determined by both CD and Fourier transform infrared spectroscopy. Even more surprising is that TNF-alpha at pH 3.3 undergoes a very gradual noncooperative change in secondary structure upon heating, which results in an increase in alpha-helical content. At pH 2.2 in the absence of salt, TNF-alpha shows considerable alpha-helix, although heating does not change the spectrum. At pH 2.2, physiological salt decreases the amount of alpha-helix at ambient temperature, and upon heating, we see the noncooperative increase in alpha-helix as observed at pH 3.3 with low salt. The addition of salt at low pH induces reassociation but to a range of oligomers rather than a unique trimer structure. This acid-induced formation of an alpha-helical monomer of TNF-alpha may be related to its known interaction with lipid bilayers.

Animals↗

Modulation of phospholipase A2 activity by aminoglycosides and daptomycin: a Fourier transform infrared spectroscopic study.

The antibiotics known as aminoglycosides are commonly used to treat severe infections caused by Gram-negative bacteria. Unfortunately, they often lead to acute renal failure after their accumulation in the lysosomes of renal cells, where an inhibition of the phospholipid catabolism is observed. The lipopeptidic antibiotic daptomycin has been shown to reduce the nephrotoxicity of aminoglycosides, but the exact mechanism of this protection is still unknown. In the present study, Fourier transform infrared spectroscopy (FTIR) has been used to monitor the hydrolysis of phosphatidylcholine by phospholipase A2 (PLA2) from Naja mocambique mocambique venom in the presence of various aminoglycosides and/or daptomycin. Gentamicin and amikacin inhibited the reaction in its early stage. Kanamycin A, tobramycin, and especially kanamycin B enhanced the initial enzyme activity by reducing the lag time. After the initiation period, the reaction proceeded at a much slower rate in the presence of gentamicin. On the other hand, daptomycin led to dramatic alterations of the hydrolysis profile: the initial latency period was eliminated, and the maximal extent of hydrolysis was reduced. When both daptomycin and any of the aminoglycosides were present, the latency period also disappeared, and the phospholipase activity was higher than with the lipopeptide alone. The most drastic change occurred with gentamicin, which was the most inhibitory aminoglycoside when used alone but worked synergistically with daptomycin to yield the most dramatic activation of PLA2.

Aminoglycosides↗