Collagen stimulating effect of peptide amphiphile C16−KTTKS on human fibroblasts

The collagen production of human dermal and corneal fibroblasts in contact with solutions of the peptide amphiphile (PA) C16−KTTKS is investigated and related to its self-assembly into nanotape structures. This PA is used in antiwrinkle cosmeceutical applications (trade name Matrixyl). We prove that C16−KTTKS stimulates collagen production in a concentration-dependent manner close to the critical aggregation concentration determined from pyrene fluorescence spectroscopy. This suggests that self-assembly and the stimulation of collagen production are inter-related.

Aggregation behaviour of hydrophobically modified poly(allylammonium) chloride

The behaviour of hydrophobically modified poly(allylammonium) chloride having octyl, decyl, dodecyl and hexadecyl side chains has been studied in aqueous solution using fluorescence emission techniques. Micropolarity studies using the I-1/I-3 ratio of the vibronic bands of pyrene show that the formation of hydrophobic microdomains depends on both the length of the side chain and the polymer concentration. The I-1/I-3 ratio of the polymers with low hydrophobe content (less than 5% mel) changes substantially when reaching a certain concentration. These changes are assigned to aggregation originating from interchain interactions. This behaviour is also confirmed by the behaviour of the monomer/excimer emission intensities of pyrene- dodecanoic acid used as a probe. For polymers having dodecyl side chains and hydrophobe contents higher than 10%, aggregates are formed independently of the polymer concentration. Anisotropy measurements show that microdomains resulting from the inter- and/or intramolecular interactions are similar to those observed for cationic surfactants. Viscosity measurements show that the coil dimensions are substantially decreased for the polymers having high hydrophobe contents, indicating intramolecular associations.

Comparative studies of AOT and NaDEHP by fluorescence technique and z-potential (ζ) measurements

The aggregation behaviors of two surfactants with the same hydrophobic tail, sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and sodium bis(2-ethylhexyl)phosphate (NaDEHP), have been investigated by the fluorescence technique and z-potential (ζ) measurements. Five fine peaks of the pyrene molecule fluorescence spectroscopy appear in the surfactant solution, and the micropolarity at which pyrene locates is monitored from the intensity ratio of the first (I1) and the third peak (I3). A wide peak around 475 nm, the emission spectra of the excimer of pyrene molecules, is observed in the NaDEHP solution, while this is not found for the AOT system. The value of I1/I3 decreases in a more limited concentration range for the AOT system than for NaDEHP, indicating that small aggregates can be more easily formed by NaDEHP molecules. The z-potential results for the aggregates formed by the two surfactants show that the interaction between AOT and PVP is stronger than that between NaDEHP and PVP.

Pyrene-functionalised, alternating copolyimide for sensing nitroaromatic compounds

A novel, pyrene-functionalised copolymer has been synthesised in a single step via imidisation of poly(maleic anhydride-alt-1-octadecene) with 1-pyrenemethylamine, and its potential for the detection of volatile nitro aromatic compounds (NACs) evaluated. The new copolymer forms complexes in solution with NACs such as 2,5-dinitrobenzonitrile, as shown by H-1 NMR, UV-vis and fluorescence spectroscopy. Moreover, thin films of this copolymer, cast from THF solution, undergo almost instantaneous fluorescence quenching when exposed to the vapour of 2,5-dinitrobenzonitrile (a model for TNT) at ambient temperatures and pressures.

The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A biomimetic model membrane system on oxidic surfaces designed for the investigation of cytochrome c oxidase and other membrane proteins by fluorescence and waveguide spectroscopy

Die transmembrane Potenzialdifferenz Δφm ist direkt mit der katalytischen Aktivität der Cytochrom c Oxidase (CcO) verknüpft. Die CcO ist das terminale Enzym (Komplex IV) in der Atmungskette der Mitochondrien. Das Enzym katalysiert die Reduktion von O2 zu 2 H2O. Dabei werden Elektronen vom natürlichen Substrat Cytochrom c zur CcO übertragen. Der Eleltronentransfer innerhalb der CcO ist an die Protonentranslokation über die Membran gekoppelt. Folglich bildet sich über der inneren Membrane der Mitochondrien eine Differenz in der Protonenkonzentration. Zusätzlich wird eine Potenzialdifferenz Δφm generiert.rnrnDas Transmembranpotenzial Δφm kann mit Hilfe der Fluoreszenzspektroskopie unter Einsatz eines potenzialemfindlichen Farbstoffs gemessen werden. Um quantitative Aussagen aus solchen Untersuchungen ableiten zu können, müssen zuvor Kalibrierungsmessungen am Membransystem durchgeführt werden.rnrnIn dieser Arbeit werden Kalibrierungsmessungen von Δφm in einer Modellmembrane mit inkorporiertem CcO vorgestellt. Dazu wurde ein biomimetisches Membransystem, die Proteinverankerte Doppelschicht (protein-tethered Bilayer Lipid Membrane, ptBLM), auf einem transparenten, leitfähigem Substrat (Indiumzinnoxid, ITO) entwickelt. ITO ermöglicht den simultanen Einsatz von elektrochemischen und Fluoreszenz- oder optischen wellenleiterspektroskopischen Methoden. Das Δφm in der ptBLM wurde durch extern angelegte, definierte elektrische Spannungen induziert. rnrnEine dünne Hydrogelschicht wurde als "soft cushion" für die ptBLM auf ITO eingesetzt. Das Polymernetzwerk enthält die NTA Funktionsgruppen zur orientierten Immobilisierung der CcO auf der Oberfläche der Hydrogels mit Hilfe der Ni-NTA Technik. Die ptBLM wurde nach der Immobilisierung der CcO mittels in-situ Dialyse gebildet. Elektrochemische Impedanzmessungen zeigten einen hohen elektrischen Widerstand (≈ 1 MΩ) der ptBLM. Optische Wellenleiterspektren (SPR / OWS) zeigten eine erhöhte Anisotropie des Systems nach der Bildung der Doppellipidschicht. Cyklovoltammetriemessungen von reduziertem Cytochrom c bestätigten die Aktivität der CcO in der Hydrogel-gestützten ptBLM. Das Membranpotenzial in der Hydrogel-gestützten ptBLM, induziert durch definierte elektrische Spannungen, wurde mit Hilfe der ratiometrischen Fluoreszenzspektroskopie gemessen. Referenzmessungen mit einer einfach verankerten Dopplellipidschicht (tBLM) lieferten einen Umrechnungsfaktor zwischen dem ratiometrischen Parameter Rn und dem Membranpotenzial (0,05 / 100 mV). Die Nachweisgrenze für das Membranpotenzial in einer Hydrogel-gestützten ptBLM lag bei ≈ 80 mV. Diese Daten dienen als gute Grundlage für künftige Untersuchungen des selbstgenerierten Δφm der CcO in einer ptBLM.

Spectroscopic properties of pyrene-containing DNA mimics

DNA mimics containing non-nucleosidic pyrene building blocks are described. The modified oligomers form stable hybrids, although a slight reduction in hybrid stability is observed in comparison to the unmodified DNA duplex. The nature of the interaction between the pyrene residues in single and double stranded oligomers is analyzed spectroscopically. Intra- and inter-strand stacking interactions of pyrenes are monitored by UV-absorbance as well as fluorescence spectroscopy. Excimer formation is observed in both single and double strands. In general, intrastrand excimers show fluorescence emission at shorter wavelengths (approx. 5-10 nm) than excimers formed by interstrand interactions. The existence of two different forms of excimers (intra- vs. interstrand) is also revealed in temperature dependent UV-absorbance spectra. (C) 2007 Elsevier Ltd. All rights reserved.

In Vivo Characterization of Myocardial Tissue Post Myocardial Infarction Using Combined Fluorescence and Diffuse Reflectance Spectroscopy

Accurately assessing the extent of myocardial tissue injury induced by Myocardial infarction (MI) is critical to the planning and optimization of MI patient management. With this in mind, this study investigated the feasibility of using combined fluorescence and diffuse reflectance spectroscopy to characterize a myocardial infarct at the different stages of its development. An animal study was conducted using twenty male Sprague-Dawley rats with MI. In vivo fluorescence spectra at 337 nm excitation and diffuse reflectance between 400 nm and 900 nm were measured from the heart using a portable fiber-optic spectroscopic system. Spectral acquisition was performed on - (1) the normal heart region; (2) the region immediately surrounding the infarct; and (3) the infarcted region - one, two, three and four weeks into MI development. The spectral data were divided into six subgroups according to the histopathological features associated with various degrees / severities of myocardial tissue injury as well as various stages of myocardial tissue remodeling, post infarction. Various data processing and analysis techniques were employed to recognize the representative spectral features corresponding to various histopathological features associated with myocardial infarction. The identified spectral features were utilized in discriminant analysis to further evaluate their effectiveness in classifying tissue injuries induced by MI. In this study, it was observed that MI induced significant alterations (p < 0.05) in the diffuse reflectance spectra, especially between 450 nm and 600 nm, from myocardial tissue within the infarcted and surrounding regions. In addition, MI induced a significant elevation in fluorescence intensities at 400 and 460 nm from the myocardial tissue from the same regions. The extent of these spectral alterations was related to the duration of the infarction. Using the spectral features identified, an effective tissue injury classification algorithm was developed which produced a satisfactory overall classification result (87.8%). The findings of this research support the concept that optical spectroscopy represents a useful tool to non-invasively determine the in vivo pathophysiological features of a myocardial infarct and its surrounding tissue, thereby providing valuable real-time feedback to surgeons during various surgical interventions for MI.

Evaluation of portable Raman spectroscopy and handheld X-ray fluorescence analysis (hXRF) for the direct analysis of glyptics

In archaeometry, the advantages of a combined use of Raman spectroscopy and X-ray fluorescence spectroscopy are extensively discussed for applications such as the analysis of paintings, manuscripts, pottery, etc. Here, we demonstrate for the first time the advantage of using both techniques for analysing glyptics. These engraved gemstones or glass materials were originally used as stamps, to identify the owner, for instance on letters, but also on wine vessels. For this research, a set of 64 glyptics (42 Roman glass specimens and 22 modern ones), belonging to the collection of the museum ‘Quinta das Cruzes’ in Funchal (Madeira, Portugal), was analysed with portable Raman spectroscopy and handheld X-ray fluorescence (hXRF). These techniques were also used to confirm the gemological identification of these precious objects and can give extra information about the glass composition. Raman spectroscopy identifies the molecular composition as well as on the crystalline phases present. On the other hand, hXRF results show that the antique Roman glass samples are characterised with low Pb and Sn levels and that the modern specimens can be discriminated in two groups: lead-based and non-lead-based ones.

Synchronous fluorescence and UV–vis spectroscopic studies of interactions between the tetracycline antibiotic, aluminium ions and DNA with the aid of the Methylene Blue dye probe

Synchronous fluorescence spectroscopy (SFS) was applied for the investigation of interactions of the antibiotic, tetracycline (TC), with DNA in the presence of aluminium ions (Al3+). The study was facilitated by the use of the Methylene Blue (MB) dye probe, and the interpretation of the spectral data with the aid of the chemometrics method, parallel factor analysis (PARAFAC). Three-way synchronous fluorescence analysis extracted the important optimum constant wavelength differences, Δλ, and showed that for the TC–Al3+–DNA, TC–Al3+ and MB dye systems, the associated Δλ values were different (Δλ = 80, 75 and 30 nm, respectively). Subsequent PARAFAC analysis demonstrated the extraction of the equilibrium concentration profiles for the TC–Al3+, TC–Al3+–DNA and MB probe systems. This information is unobtainable by conventional means of data interpretation. The results indicated that the MB dye interacted with the TC–Al3+–DNA surface complex, presumably via a reaction intermediate, TC–Al3+–DNA–MB, leading to the displacement of the TC–Al3+ by the incoming MB dye probe.

Fluorescence spectrometric study on the interactions of isoprocarb and sodium 2-isopropylphenate with bovine serum albumin

The binding interaction of the pesticide Isoprocarb and its degradation product, sodium 2-isopropylphenate, with bovine serum albumin (BSA) was studied by spectrofluorimetry under simulated physiological conditions. Both Isoprocarb and sodium 2-isopropylphenate quenched the intrinsic fluorescence of BSA. This quenching proceeded via a static mechanism. The thermodynamic parameters (ΔH°, ΔS° and ΔG°) obtained from the fluorescence data measured at two different temperatures showed that the binding of Isoprocarb to BSA involved hydrogen bonds and that of sodium 2-isopropylphenate to BSA involved hydrophobic and electrostatic interactions. Synchronous fluorescence spectroscopy of the interaction of BSA with either Isoprocarb or sodium 2-isopropylphenate showed that the molecular structure of the BSA was changed significantly, which is consistent with the known toxicity of the pesticide, i.e., the protein is denatured. The sodium 2-isopropylphenate, was estimated to be about 4–5 times more toxic than its parent, Isoprocarb. Synchronous fluorescence spectroscopy and the resolution of the three-way excitation–emission fluorescence spectra by the PARAFAC method extracted the relative concentration profiles of BSA, Isoprocab and sodium 2-isopropylphenate as a function of the added sodium 2-isopropylphenate. These profiles showed that the degradation product, sodium 2-isopropylphenate, displaced the pesticide in a competitive reaction with the BSA protein.

Electron rich supramolecular polymers as fluorescent sensors for nitroaromatics

Three pi-electron rich fluorescent supramolecular polymers (1-3) have been synthesized incorporating 2-methyl-3-butyn-2-ol groups in reasonable yield by employing Sonagashira coupling. They were characterized by multinuclear NMR (H-1, C-13), ESI-MS and single crystal X-ray diffraction analyses 1 = 1( 2-methyl-3-butyn-2-ol) pyrene; 2 = 9,10-bis(2-methyl-3-butyn-2-ol) anthracene; 3 = 1,3,6,8-tetrakis(2methyl- 3-butyn-2-ol) pyrene]. Single crystal structures of 1-3 indicated that the incorporation of hydroxy (-OH) groups on the peripheral of the fluorophores helps them to self-associate into an infinite supramolecular polymeric network via intermolecular hydrogen bonding interactions between the adjacent discrete fluorophore units. All these compounds showed fluorescence characteristics in chloroform solution due to the extended pi-conjugation and were used as selective fluorescent sensors for the detection of electron deficient nitroaromatics. The changes in photophysical properties of fluorophores (1-3) upon complex formation with electron deficient nitroaromatic explosives were studied in chloroform solution by using fluorescence spectroscopy. All these fluorophores showed the largest quenching response with moderate selectivity for nitroaromatics over various other electron deficient/ rich aromatic compounds tested (Chart 1). Analysis of the fluorescence titration profile of 9,10-bis(2-methyl-3butyn- 2-ol) anthracene fluorophore (2) with 1,3,5-trinitrotoluene/ 2,4-dinitrotoluene provided evidence that this particular fluorophore detects nitroaromatics in the nanomolar range 2.0 ppb for TNT, 13.7 ppb for DNT]. Moreover, sharp visual color change was observed upon mixing nitroaromatic (DNT) with fluorophores (1-3) both in solution as well as in solid phase. Furthermore, the vapor-phase sensing study of thin film of fluorophores (1-3) showed efficient quenching responses for DNT and this sensing process is reproducible. Selective fluorescence quenching response including a sharp visual color change for nitroaromatics make these tested fluorophores (1-3) as potential sensors for nitroaromatic compounds with a detection limit of ppb level.

A Probe for the Selective and Parts-per-Billion-Level Detection of Copper(II) and Mercury(II) using a Micellar Medium and Its Utility in Cell Imaging

A novel colorimetric probe 1 based on the picolyl moiety has been designed and synthesized. Probe 1 is composed of a pyrene and a bispicolyl amine (BPA) unit, in which the BPA moiety acts as a binding unit and the binding phenomenon is sensed from the changes in the signaling subunit. The probe detects Cu2+ specifically in water and both Cu2+ and Hg2+ efficiently in neutral Brij-58 micellar media. The probe shows a color change visible to the naked eye upon addition of metal ions. Notably, in a micellar medium, probe 1 can detect both the Cu2+ and Hg2+ ions even at parts-per-billion levels. Furthermore, the probe shows ratiometric detection of both the metal ions making the sensing quantitative. The two metal ions could be discriminated both visibly under a UV lamp and with the use of fluorescence spectroscopy. The probe could be also used in biological cell lines for the detection of both Hg2+ and Cu2+ ions.

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta(2) subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively. (c) 2006 Elsevier Inc. All rights reserved.