SDS Micelles Research Articles

Entrapment in micellar assemblies switches the excimer population of potential therapeutic luminophore azapodophyllotoxin.

Azapodophyllotoxin is a new class of anti-tumor agent with brilliant therapeutic activity and understanding its physicochemical nature in bio-mimetic microenvironments may provide substantial importance in context of its intercellular localization, efficacy as well as delivery. The present work epitomizes environment-sensitive fluorescence modulation of a prodigy, 4-(2-Hydroxyethyl)-10-phenyl-3,4,6,7,8,10- hexahydro-1H-cyclopenta[g]furo[3,4-b]quinoline-1-one (HPFQ) from the class of anti-cancer agent Azapodophyllotoxin, in differently charged model bio-mimetic micellar microenvironment of cationic CTAB, anionic SDS and neutral Triton X-100 using UV-visible absorption, steady state fluorescence, time-resolved fluorescence and fluorescence anisotropy studies. As a distinct phenomenon, anticancer HPFQ exhibits prolific fluorescence in solvents of varying polarity, originating from a mixed contribution of locally excited, charge transfer and excimer emission. A dramatic modulation in the photophysics of HPFQ has been observed in two types of surfactant consortiums: pre-micellar and post-micellar at physiological and anoxic pH. On photo-excitation, anti-cancer HPFQ exists in monomer-excimer equilibrium with varying ratios in different polarity regions. The marked enhancement in fluorescence intensity of HPFQ in post-micellar region of the surfactant under study probably arises due to regeneration of the monomer from its excimer. This reoccurrence reduces the possibility of Förster resonance energy transfer (FRET) from monomer to excimer, which essentially increases the desired emission intensity. Localization of HPFQ in micellar systems highly depends on polarity gradient inside the micelle, electrostatic, hydrophobic and intermolecular hydrogen bonding interactions. Further corroboration with the polarity sensitive experiments in dioxane-water mixture indicates towards spatial localization of the probe molecule in the stern layer of cationic CTAB, sheer surface of neutral TX100 and outer Gouy-Chapman layer in anionic SDS micelles. A molecular binary logic gate correlates the dominance of micellization over the polarity factor, which enhances the fluorescence response of HPFQ. The enhancement of the emissive potential of anti-cancer HPFQ in biomimetic environments by switching its excimer population may have an immense importance to achieve the status of a dual therapeutic and imaging agent altogether in progressive biomedical research.

Broad Spectrum Tunable Photoluminescent Material Based on Cascade Fluorescence Resonance Energy Transfer between Three Fluorophores Encapsulated within the Self-Assembled Surfactant Systems.

A broad spectrum tunable photoluminescent material with dual encryption based on a two-step fluorescence resonance energy transfer (FRET) between pyrene (Py), coumarin 480 (Cou480), and rhodamine 6G (R6G) in micelles of SDS and bmimDS is presented. The phenomenon is achievable due to the encapsulation of the fluorophores within these micelles. The transfer of energy as FRET between the pair Py and Cou480 showed ON at 336 nm and OFF at 402 nm in contrast to the FRET observed between the pair Cou480 and R6G that showed ON at 402 nm and OFF at 336 nm. However, the transfer of energy as FRET occurs from Py to R6G in the presence of Cou480 when excited at 336 nm, thereby making it a chain of three fluorophores with Cou480 acting as a relay fluorophore receiving energy from Py and transferring it to R6G. The different FRET scenarios between the three fluorophores in micelles provide a window for the generation of a matrix of colors, which occupies a significant 2D area in the chromaticity diagram, having potential applications in security printing. The different fluorophoric ratios generate different colors based on their individual photonic emissions and the FRET processes taking place between them. Writing tests were carried out using varied ratios of the fluorophores in the micellar systems producing different colored outputs under the UV light with insignificant visibility under the white light. We envision that this as-discovered three fluorophoric FRET system could form the basis for the future development of multi-FRET light-harvesting devices and anti-counterfeiting security inks based on much simpler non-covalent interaction aided encapsulation of the fluorophores within the self-assembled soft systems.

Micelle Heterogeneity from the 2D Kinetics of Solute Rotation.

The chemical and physical properties of microstructured materials vary with position. The photophysics of solute molecules can measure these local properties, but they often show multiple rates (rate dispersion), which complicates the interpretation. In the case of micelles, rate dispersion in a solute's anisotropy decay has been assigned to either local anisotropy or heterogeneity in the local viscosity. To resolve this conflict, the rotation of PM597 molecules in SDS micelles has been measured by polarized MUPPETS (multiple population-period transient spectroscopy). This 2D technique shows that heterogeneity is strong and that local anisotropy is minimal. The results suggest that on a subnanosecond time scale, the solute sees only one strong fluctuation of the micelle structure. The anisotropic, average structure emerges on longer time scales.

Characterization and antimicrobial mechanism of CF-14, a new antimicrobial peptide from the epidermal mucus of catfish

In this study, we identified a novel antibacterial peptide, RIVELTLPRVSVRL-NH2 (named CF-14), derived from the epidermal mucus of catfish and characterized its antimicrobial activity. Analysis of antimicrobial activity and hemolytic activity of CF-14 revealed broad spectrum, high levels of antimicrobial activity and low toxicity to eukaryotic cells. CF-14 remained stable at pH values ranging from 4.0 to 12.0 and remained bioactive when exposed to high temperature. CD analysis indicated that CF-14 forms a random coil in PBS buffer and an α-helical conformation in the membrane-mimetic 2.5% SDS micelle. Additionally, the antibacterial mechanism of CF-14 against Shewanella putrefaciens was investigated. Membrane permeability experiments confirmed that CF-14 could increase cell wall membrane permeability and cause nucleotide leakage. Moreover, observations performed using scanning electron and confocal microscopy indicated that CF-14 could penetrate into the cell membranes of S. putrefaciens and accumulate in bacterial cells, but did not break down cell membranes. Further, electrophoresis analysis demonstrated that CF-14 possesses DNA-binding affinity. The results provide a substantial basis for future application of CF-14, a novel cell-penetrating peptide (CPP) derived from catfish.

Laser-Induced Wurtz-Type Syntheses with a Metal-Free Photoredox Catalytic Source of Hydrated Electrons.

Upon irradiation with ns laser pulses at 355 nm, 2-aminoanthracene in SDS micelles readily produces hydrated electrons. These "super-reductants" rapidly attack substrates such as chloro-organics and convert them into carbon-centred radicals through dissociative electron transfer. For a catalytic cycle, the aminoanthracene needs to be restored from its photoionization by-product, the radical cation, by a sacrificial donor. The ascorbate monoanion can only achieve this across the micelle-water interface, but the monoanion of ascorbyl palmitate results in a fully micelle-contained regenerative electron source. The shielding by the micelle in the latter case not only increases the life of the catalyst but also strongly suppresses the interception of the carbon-centred radicals by the hydrogen-donating ascorbate moiety; and in conjunction with the high local concentrations effected by the pulsed laser, termination by radical dimerization thus dominates. We have obtained a complete and consistent picture through monitoring the individual steps and the assembled system by flash photolysis on fast and slow timescales, from microseconds to minutes; and in preparative studies on a variety of substrates, we have achieved up to quantitative dimerization with a turnover on the order of 1 mmol per hour.

Conformational Preferences of Aβ25-35 and Aβ35-25 in Membrane Mimicking Environments

The structural transition of aggregating Abeta peptides is the key event in the progression of Alzheimer's Disease (AD). In the present work, the structural modifications of toxic Aβ25-35 and the scrambled Aβ35-25 were studied in Trifluoroethanol (TFE) and in aqueous SDS micelles. Using CD spectroscopic investigations, the conformational transition of Aβ25-35 and Aβ35-25 peptides were determined in different membrane mimicking environments such as TFE and SDS. An interval scan CD of the peptides on evaporation of TFE was performed. TFE titrations were carried out to investigate the intrinsic ability of the structural conformations of peptides. We show by spectroscopic evidence that Aβ25-35 prefers beta sheet structures upon increasing TFE concentrations. On the other hand, the non-toxic scrambled Aβ35-25 peptide only undergoes a transition from random coil to α-helix conformation with increasing TFE. In the interval scan studies, Aβ25-35 did not show any structural transitions, whereas Aβ35-25 showed transition from α-helix to β-sheet conformation. In membrane simulating aqueous SDS micelles, Aβ25-35 showed a transition from random coil to α-helix while Aβ35-25 underwent transition from random coil to β-sheet conformation. Overall, the current results seek new insights into the structural properties of amyloidogenic and the truncated sequence in membrane mimicking solvents.

Characterization of an OrtT-like toxin of Salmonella enterica serovar Houten.

The Escherichia coli GhoT/GhoS system is a type V toxin-antitoxin system in which the antitoxin GhoS cleaves the GhoT mRNA, controlling its translation. GhoT is a small hydrophobic protein that damages bacterial membranes. OrtT is a GhoT-like toxin, but it apparently lacks a corresponding antitoxin and serves a different physiologic role. Using a profile hidden Markov model approach, a Salmonella enterica serovar Houten genome was screened to obtain homologs of GhoT/OrtT. We only found one protein (referred to here as OrtT-Sal) that shared more sequence identity with OrtT than GhoT. The chromosomal region around the coding sequence of OrtT-Sal suggests that it is an orphan toxin and can be under RpoH activation. To study OrtT-Sal, we chemically synthesized and expressed in E. coli the whole toxin and its N- and C-terminal regions (OrtT-Sal1-29 and OrtT-Sal29-57, respectively). Our findings have shown that the overproduction of the polypeptides resulted in severe growth inhibition and cell lysis. Using circular dichroism, we found that OrtT-Sal, OrtT-Sal1-29, and OrtT-Sal29-57 form an alpha-helical structure in the presence of SDS micelles or TFE. Finally, using carboxyfluorescein-loaded lipid vesicles, we determined that the polypeptides damage lipid membrane directly.

Study on interactions of vitamin B1 with sodium dodecyl sulfate for potential food applications: Conductometric, volumetric, calorimetric and spectroscopic approach

As vitamin B1 (thiamine hydrochloride, THC) is being oxidized by food preservatives and light, therefore the advantages of surfactant micelles as protecting agent are taken into consideration in the present study. Keeping this in mind the impact of THC on the SDS micellar system has been studied by using conductance, volumetric, calorimetric and spectroscopic techniques. From conductance measurements, various parameters like critical micellization concentration (CMC), degree of dissociation (α) and thermodynamics of micellization (ΔGᴼm, ΔHᴼm and ΔSᴼm) have been calculated. Apparent molar volume (VΦ) and apparent molar compressibility (Ks,2,Φ) have been calculated from density and sound velocity measurements which have been discussed in terms of solute-solute and solute-solvent interactions. The binding and partitioning of THC into SDS micelles have been studied by using UV–visible spectroscopy and calorimetric studies. Calorimetric studies have also been used to address the interactions of THC with monomers of SDS. The values of binding/partitioning constant (K), enthalpy, entropy, free energy and stoichiometry of binding have been determined in the pre-micellar and post-micellar regions from isothermal titration calorimetery (ITC). In addition, proton (1H) NMR studies have been performed to determine the locus and orientation of THC within SDS micelles.

Thermodynamic insights and molecular environments into catanionic surfactant systems: Influence of chain length and molar ratio

HypothesisImidazolium-based Ionic liquids as new generation cationic surfactants can provide designable alkyl chain length. In the catanionic surfactant systems, the alkyl chain lengths and molar ratios can greatly influence the interactions such as electrostatic and hydrophobic interaction. The variation in these interactions has a significant effect on the molecular environments of the self-assembly structure, and this process is always accompanied by the transition of aggregates and release or consumption of heat. Hence, it is of interest to study the relationship between intermolecular interactions, molecular environments, self-assembly structure and the change in energy of system in the catanionic surfactant mixed systems. ExperimentsThe enthalpy change ΔH of titrations the imidazolium-based into SDS micelle solution was studied to characterize the heat by using isothermal titration calorimetry (ITC) during the transitions of the aggregate structures. The corresponding self-assembly structure was monitored via cryogenic transmission electron microscopy (cryo-TEM). Employing proton magnetic resonance (1H NMR), we focus on the interactions between imidazolium-based ILs and SDS based on the variations in the molecular environments of aggregates. FindingsOf these imidazolium-based ionic liquids/SDS system, the 1-octyl-3-methylimidazolium ([OMIM]Cl)/SDS system shows several features such as intense energy absorption and releasing processes, which indicate the formation of high entanglement wormlike micelles and vesicles. This is related to the formation of self-adjusting state between the SDS and [OMIM]Cl molecules due to the balance between the electrostatic interaction and hydrophobic interaction. Varying the alkyl chain length appears to cause significant differences to the molecular environments. From the molecular environments, three different models about the polarity of the catanionic surfactant molecules are used to explain the balance of the intermolecular interactions.

Dynamic oligomerization of hRAGE's transmembrane and cytoplasmic domains within SDS micelles.

The human Receptor for Advanced Glycation End Products (hRAGE) is a pattern recognition receptor implicated in inflammation and adhesion. It is involved in both innate and adaptive immunity. Its aberrant signaling is tied to the pathogenesis of diabetic complications, neurodegenerative disorders, and chronic inflammatory responses. Previous structural studies have focused on its extracellular domains with their canonical constant and variable Ig folds, and to a much lesser extent, the intrinsically disorder cytoplasmic domain. No experimental data are reported on the transmembrane domain, which is integral to signaling. We have constructed, expressed and purified the transmembrane domain attached to the cytoplasmic domain of hRAGE in E. coli. Multiple self-associated forms of these domains were observed in vitro. This pattern of mixed oligomers resembled previously reported in vivo forms of the complete receptor. The self-association of these two domains was further characterized using: SDS-PAGE, intrinsic tryptophan fluorescence and heteronuclear NMR spectroscopy. NMR conditions were assessed across time and temperature within micelles. Our data show that the transmembrane and cytoplasmic domains of hRAGE undergo dynamic oligomerizations that can occur in the absence of its extracellular domains or ligand binding. And, such associations are only partially disrupted even with prolonged incubation in strong detergents.

Inhibition of bacterial human pathogens on tomato skin surfaces using eugenol‐loaded surfactant micelles during refrigerated and abuse storage

AbstractThe plant‐derived essential oil component eugenol (1.0% wt/vol), free or loaded into surfactant micelles constructed of sodium dodecyl sulfate (SDS; 1.0% wt/vol), 200 ppm free chlorine (hypochlorous acid [HOCl]; pH 7.0), and sterile distilled water were evaluated for their ability to reduce Salmonella Saintpaul and Escherichia coli O157:H7 on skin surface samples of Roma tomatoes. Samples were treated and then stored aerobically for up to 10 days. All samples were initially stored at 5 °C; one set of samples was shifted to 15 °C after 5 days of storage to simulate temperature abuse during postharvest handling. Encapsulated and free eugenol, HOCl, and empty SDS micelles reduced pathogens to nondetectable counts (detection limit: 0.5 log10 cfu/cm2) during refrigerated storage (p ≥ .05). Conversely, during temperature abuse storage (15 °C), only free and micelle‐loaded eugenol consistently reduced pathogens to nondetection. Eugenol‐loaded micelles can be used to decontaminate harvested tomatoes from enteric bacterial pathogens.Practical applicationsTomatoes may be consumed raw and have been repeatedly implicated in the occurrence of human foodborne disease. Washing of tomatoes with novel antimicrobial interventions that increase the dispersion of antimicrobials over the surface of the tomato should result in greater reduction of microbial foodborne pathogens through enhanced contact of antimicrobial with pathogen. Application of surfactant micelle‐entrapped eugenol onto tomato surfaces reduced Salmonella Saintpaul and Escherichia coli O157:H7 to nondetection during refrigerated (5 °C) and temperature abuse (15 °C) storage. Micelle‐loaded eugenol reduced numbers of aerobic bacteria, Enterobacteriaceae, and fungi to nondetection during temperature abuse storage.

Adevonin, a novel synthetic antimicrobial peptide designed from the Adenanthera pavonina trypsin inhibitor (ApTI) sequence

ABSTRACTThe biological activities and the structural arrangement of adevonin, a novel antimicrobial peptide, were investigated. The trypsin inhibitor ApTI, isolated from Adenanthera pavonina seeds, was used as a template for screening 18-amino acid peptides with predicted antimicrobial activity. Adevonin presented antimicrobial activity and minimum inhibitory concentrations (MIC) ranging from 1.86 to 7.35 µM against both Gram-positive and – negative bacterial strains. Moreover, adevonin exerted time-kill effects within 10 min and both susceptible and drug-resistant bacterial strains were affected by the peptide. In vitro and in vivo assays showed that, at MIC concentration, adevonin did not affect human fibroblasts (MRC-5) viability or Galleria mellonella survival, respectively. Hemolytic activity was observed only at high peptide concentrations. Additionally, nucleic acid efflux assays, gentian violet uptake and time-kill kinetics indicate that the antimicrobial activity of adevonin may be mediated by bacterial membrane damage. Furthermore, molecular dynamic simulation in the presence of SDS micelles and anionic membrane bilayers showed that adevonin acquired a stable α-helix secondary structure. Further studies are encouraged to better understand the mechanism of action of adevonin, as well as to investigate the anti-infective activity of this peptide.

First Micelle-Free Photoredox Catalytic Access to Hydrated Electrons for Syntheses and Remediations with a Visible LED or even Sunlight.

Hydrated electrons are super-reductants, yet can be generated with visible light when two photons are pooled, most efficiently through storing the energy of the first photon in a radical pair formed by the reduction of an excited catalyst by a sacrificial donor. All previous such systems for producing synthetically useable amounts of hydrated electrons with an LED in the visible range had to resort to compartmentalization by SDS micelles to curb the performance-limiting recombination of the pair. To overcome micelle-imposed constraints on sustainability and applications, we have instead attached carboxylate groups to a ruthenium (tris)bipyridyl catalyst such that its pentaanionic radical strongly repels the dianionic radical of the bioavailable donor urate. We have explored the influence of the Coulombic interactions on the electron generation by a time-resolved study from microseconds to hours, including for comparison the unsubstituted complex, which forms a monocationic radical, with and without SDS micelles. The new homogeneous electron source is best with regard to stability and total electron output; it has a broader synthetic scope because it does not entail micellar shielding of less hydrophilic compounds, which particularly facilitates cross-couplings; and it tolerates supramolecular containers as carriers of water-insoluble substrates or products. As an application in the field, we demonstrate the solar remediation of a recalcitrant chloro-organic bulk chemical.

Structural insight into the mechanism of action of antimicrobial peptide BMAP-28(1–18) and its analogue mutBMAP18

Structural characterization of BMAP-28(1–18), a potent bovine myeloid antimicrobial peptide can aid in understanding its mechanism of action at molecular level. We report NMR structure of the BMAP-28(1–18) and its mutated analogue mutBMAP18 in SDS micelles. Structural comparison of the peptides bound to SDS micelles and POPE-POPG vesicles using circular dichroism, suggest that structures in the two lipid preparations are similar. Antimicrobial assays show that even though both these peptides adopt helical conformation, BMAP-28(1–18) is more potent than mutBMAP18 in killing bacterial cells. Our EM images clearly indicate that the peptides target the bacterial cell membrane resulting in leakage of its contents. The structural basis for difference in activity between these peptides was investigated by molecular dynamics simulations. Inability of the mutBMAP18 to retain its helical structure in presence of POPE:POPG membrane as opposed to the BMAP-28(1–18) at identical peptide/lipid ratios could be responsible for its decreased activity. Residues Ser5, Arg8 and Arg12 of the BMAP-28(1–18) are crucial for its initial anchoring to the bilayer. We conclude that along with amphipathicity, a stable secondary structure that can promote/initiate membrane anchoring is key in determining membrane destabilization potential of these AMPs. Our findings are a step towards understanding the role of specific residues in antimicrobial activity of BMAP-28(1–18), which will facilitate design of smaller, cost-effective therapeutics and would also help prediction algorithms to expedite screening out variants of the parent peptide with greater accuracy.

Catecholase activity of mononuclear copper(II) complexes of tridentate 3N ligands in aqueous and aqueous micellar media: Influence of stereoelectronic factors on catalytic activity

A series of mononuclear copper(II) complexes of the type [Cu(L)Cl2] 1–6, where L is a tridentate 3 N ligand such as N-methyl-N′-(pyrid-2-ylmethyl)ethylenediamine (L1), N-ethyl-N′-(pyrid-2-ylmethyl)ethylenediamine (L2), N-phenyl-N′-(pyrid-2-ylmethyl)ethylenediamine (L3), N,N-dimethyl-N′-(pyrid-2-ylmethyl)ethylenediamine (L4), N,N-diethyl-N′-(pyrid-2-ylmethyl)-ethylenediamine (L5) and N-methyl-N′-(pyridin-2-ylmethyl)benzene-1,2-diamine (L6), has been isolated and studied as functional models for catechol oxidase enzymes in different aqueous micellar media. The X-ray crystal structures of 2 and 5 contain a CuN3Cl2 chromophore with a trigonal bipyramidal based coordination geometry. Interestingly, all the complexes form cationic [Cu(L)(DBC)(H2O)]+ adduct species in which DBC2− acts as a monodentate ligand, and the adduct species interacts strongly with anionic SDS micelles. The rates of the oxygenase reaction follow the trend SDS > TX-100 ∼ water > CTAB, revealing that the rate of the reaction in micellar media are higher than those in aqueous solution and that the nature of biomimetic microenvironments of the micellized copper(II) catalyst and also the ligand stereoelectronic factors like donor atom basicity and steric bulk of ligand N-alkyl substituents, determine the catecholase-like activity.

Hydrophobic and Elastic Forces Experienced by a Series of Pyrene End-Labeled Poly(ethylene oxide)s Interacting with Sodium Dodecyl Sulfate Micelles

A series of pyrene end-labeled poly(ethylene oxide)s (Py2-PEO(x) with x equal to 2, 3.4, 5, 6, 7.8, and 10 kg mol–1) were synthesized, and their interactions with SDS micelles were characterized by steady-state and time-resolved fluorescence. Pyrene excimer formation (PEF) indicated that both pyrenyl ends were located in the same SDS micelle whereas the absence of PEF reflected bridging of two SDS micelles by a Py2-PEO molecule having its pyrenyl end groups in separate micelles. PEF efficiency was characterized from the excimer-to-monomer fluorescence intensity ratio (IE/IM) which was determined as a function of PEO chain length and salt concentration. More detailed information about the PEF process was obtained by fitting the monomer and excimer fluorescence decays globally according to the model free analysis (MFA). MFA yielded the average rate constant ⟨k⟩ of PEF and the molar fraction (ffree) of pyrenyl labels that did not form excimer, thus reflecting the extent of bridging undergone by the Py2-PEO c...

Environmental Effects Determine the Structure of Potential β-Amino Acid Based Foldamers.

This work shows that hybrid peptides formed by alternating trans-2-aminocyclopentanecarboxylic acid (trans-ACPC) and trans-2-aminocyclohexanecarboxylic acid (trans-ACHC) do not fold in the solvents typically used in the study of their homo-oligomers. Only when the peptides are assayed in SDS micelles are the predicted helical structures obtained. This indicates that the environment could play an equally important role (as the backbone stereochemistry) in determining their fold, possibly by providing a sequestered environment.

Specific optical rotation for the identification of the locus of solubilization of chiral molecules in achiral micelles

We present the first detailed experimental study of discrete wavelength resolved specific optical rotations (SORs) of chiral molecules embedded in achiral micelles. Eight different chiral compounds embedded in SDS and TX100 micelles have been used to analyze their SORs in comparison to those obtained for the same chiral molecules in achiral solvents. An important observation made is that the SORs of hydrophobic chiral molecules, α-pinene and 2-carene, in hydrophobic achiral solvents (dodecane, heptane and CCl4) are greater than those of same chiral molecules embedded in hydrophobic core of SDS micelles. This difference is envisioned to be due to the differences in microenvironments provided by achiral SDS micelle and organic solvents. The ratio of SOR for a chiral molecule in CCl4 solvent and achiral micelles is found to reflect the locus of solubilization of chiral molecule in achiral micelle. The locus of solubilization of chiral molecules in TX100 micelles has been independently verified using 1H NMR studies. The emerging general observations are: (a) when chiral molecule is located deep inside the hydrophobic core of the micelle, SOR in micelle is smaller than that in CCl4. (b) When chiral molecule is located at the interface of hydrophobic and hydrophilic regions of the micelle, SOR in micelle is slightly greater than, or equal to, that in CCl4. While quantum chemical (QC) calculations to explain these observations in micelles need significantly larger computational time line, the prospect for such calculations has been verified by undertaking QC predictions of SOR for a representative chiral molecule, fenchone, in water (with 4 Å solvation sphere), and comparing the predicted results with the corresponding experimental observations in water. The predictions and observations are found to be in good agreement. The present results are expected to provide impetus for understanding the microenvironment that influences most of the spectroscopic observations.

Fluorescence lifetime imaging of red yeast Cystofilobasidium capitatum during growth

Abstract Red yeast Cystofilobasidium capitatum autofluorescence was studied by means of confocal laser scanning microscopy (CLSM) to reveal distribution of carotenoids inside the cells. Yeasts were cultivated in 2L fermentor on glucose medium at permanent light exposure and aeration. Samples were collected at different times for CLSM, gravimetric determination of biomass and HPLC determination of pigments. To compare FLIM (Fluorescence Lifetime Imaging Microscopy) images and coupled data (obtained by CLSM) with model systems, FLIM analysis was performed on micelles of SDS:ergosterol and SDS:coenzyme Q with different content of ergosterol and coenzyme Q, respectively, and with constant addition of beta-carotene. Liposomes lecithin:ergosterol:beta-carotene were investigated too. Two different intracellular forms of carotenoids were observed during most of cultivations, with third form appeared at the beginning of stationary phase. Observed behavior is probably due to formation of some kind of carotenoid protective system in membranes of different compartments of yeast cell, especially cytoplasmic membrane.

Insights on the structure-activity relationship of peptides derived from Sticholysin II.

Sticholysin II (StII) is a pore-forming actinoporin from the sea anemone Stichodactyla helianthus. A mechanistic model of its action has been proposed: proteins bind to cell membrane, insert their N-termini into the lipid core and assemble into homo-tetramer pores responsible for host-cell death. Because very likely the first 10 residues of StII N-terminus are critical for membrane penetration, to dissect the molecular details of that functionality, we studied two synthetic peptides: StII1-30 and StII16-35 . They show diverse haemolytic and candidacidal activity that correlate with distinct orientations in SDS micelles. NMR shows that StII1-30 partly inserts into the micelle, while StII16-35 lays on the micelle surface. These results justify the diverse concentration dependence of their candidacidal activity supposing a different mechanism of action and providing new hints on StII lytic activity at molecular level. Biotechnological application of these peptides, focused on the development of therapeutic immunocomplexes, may be envisaged.