Passive coupling of membrane tension and cell volume during active response of cells to osmosis.

During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. Using a fluorescent membrane tension probe (fluorescent lipid tension reporter [Flipper-TR]), we investigated the coupling between tension and volume during these asymmetric recoveries. Caveolae depletion and pharmacological inhibition of ion transporters and channels, mTORCs, and the cytoskeleton all affected tension and volume responses. Treatments targeting mTORC2 and specific downstream effectors caused identical changes to both tension and volume responses, their coupling remaining the same. This supports that the coupling of tension and volume responses to osmotic shocks is primarily regulated by mTORC2.

Molecular Basis of the Anticancer and Antibacterial Properties of CecropinXJ Peptide: An In Silico Study.

Esophageal cancer is an aggressive lethal malignancy causing thousands of deaths every year. While current treatments have poor outcomes, cecropinXJ (CXJ) is one of the very few peptides with demonstrated in vivo activity. The great interest in CXJ stems from its low toxicity and additional activity against most ESKAPE bacteria and fungi. Here, we present the first study of its mechanism of action based on molecular dynamics (MD) simulations and sequence-property alignment. Although unstructured in solution, predictions highlight the presence of two helices separated by a flexible hinge containing P24 and stabilized by the interaction of W2 with target biomembranes: an amphipathic helix-I and a poorly structured helix-II. Both MD and sequence-property alignment point to the important role of helix I in both the activity and the interaction with biomembranes. MD reveals that CXJ interacts mainly with phosphatidylserine (PS) but also with phosphatidylethanolamine (PE) headgroups, both found in the outer leaflet of cancer cells, while salt bridges with phosphate moieties are prevalent in bacterial biomimetic membranes composed of PE, phosphatidylglycerol (PG) and cardiolipin (CL). The antibacterial activity of CXJ might also explain its interaction with mitochondria, whose phospholipid composition recalls that of bacteria and its capability to induce apoptosis in cancer cells.

Insertion of Calcium-Permeable AMPA Receptors during Epileptiform Activity In Vitro Modulates Excitability of Principal Neurons in the Rat Entorhinal Cortex.

Epileptic activity leads to rapid insertion of calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) into the synapses of cortical and hippocampal glutamatergic neurons, which generally do not express them. The physiological significance of this process is not yet fully understood; however, it is usually assumed to be a pathological process that augments epileptic activity. Using whole-cell patch-clamp recordings in rat entorhinal cortex slices, we demonstrate that the timing of epileptiform discharges, induced by 4-aminopyridine and gabazine, is determined by the shunting effect of Ca2+-dependent slow conductance, mediated predominantly by K+-channels. The blockade of CP-AMPARs by IEM-1460 eliminates this extra conductance and consequently increases the rate of discharge generation. The blockade of NMDARs reduced the additional conductance to a lesser extent than the blockade of CP-AMPARs, indicating that CP-AMPARs are a more significant source of intracellular Ca2+. The study's main findings were implemented in a mathematical model, which reproduces the shunting effect of activity-dependent conductance on the generation of discharges. The obtained results suggest that the expression of CP-AMPARs in principal neurons reduces the discharge generation rate and may be considered as a protective mechanism.

Functional Modification of Cellulose Acetate Microfiltration Membranes by Supercritical Solvent Impregnation.

This study investigates the modification of commercial cellulose acetate microfiltration membranes by supercritical solvent impregnation with thymol to provide them with antibacterial properties. The impregnation process was conducted in a batch mode, and the effect of pressure and processing time on thymol loading was followed. The impact of the modification on the membrane's microstructure was analyzed using scanning electron and ion-beam microscopy, and membranes' functionality was tested in a cross-flow filtration system. The antibiofilm properties of the obtained materials were studied against Staphyloccocus aureus and Pseudomonas aeruginosa, while membranes' blocking in contact with bacteria was examined for S. aureus and Escherichia coli. The results revealed a fast impregnation process with high thymol loadings achievable after just 0.5 h at 15 MPa and 20 MPa. The presence of 20% of thymol provided strong antibiofilm properties against the tested strains without affecting the membrane's functionality. The study showed that these strong antibacterial properties could be implemented to the commercial membranes' defined polymeric structure in a short and environmentally friendly process.

Synergy between plant phenols and carotenoids in stabilizing lipid-bilayer membranes of giant unilamellar vesicles against oxidative destruction.

We have investigated the synergism between plant phenols and carotenoids in protecting the phosphatidylcholine (PC) membranes of giant unilamellar vesicles (GUVs) from oxidative destruction, for which chlorophyll-a (Chl-a) was used as a lipophilic photosensitizer. The effect was examined for seven different combinations of ß-carotene (ß-CAR) and plant phenols. The light-induced change in GUV morphology was monitored via conventional optical microscopy, and quantified by a dimensionless image-entropy parameter, ΔE. The ΔE-t time evolution profiles exhibiting successive lag phase, budding phase and ending phase could be accounted for by a Boltzmann model function. The length of the lag phase (LP in s) for the combination of syringic acid and ß-CAR was more than seven fold longer than for ß-CAR alone, and those for other different combinations followed the order: salicylic acid < vanillic acid < syringic acid > rutin > caffeic acid > quercetin > catechin, indicating that moderately reducing phenols appeared to be the most efficient membrane co-stabilizers. The same order held for the residual contents of ß-CAR in membranes after light-induced oxidative degradation as determined by resonance Raman spectroscopy. The dependence of LP on the reducing power of phenols coincided with the Marcus theory plot for the rate of electron transfer from phenols to the radical cation ß-CAR˙+ as a primary oxidative product, suggesting that the plant phenol regeneration of ß-CAR plays an important role in stabilizing the GUV membranes, as further supported by the involvement of CAR˙+ and the distinct shortening of its lifetime as shown by transient absorption spectroscopy.

Lysosome Membrane Permeabilization and Disruption of the Molecular Target of Rapamycin (mTOR)-Lysosome Interaction Are Associated with the Inhibition of Lung Cancer Cell Proliferation by a Chloroquinoline Analog.

Lysosomes degrade cellular proteins and organelles and regulate cell signaling by providing a surface for the formation of critical protein complexes, notably molecular target of rapamycin (mTOR) complex 1 (mTORC1). Striking differences in the lysosomes of cancer versus normal cells suggest that they could be targets for drug development. Although the lysomotropic drugs chloroquine (CQ) and hydroxychloroquine (HCQ) have been widely investigated, studies have focused on their ability to inhibit autophagy. We synthesized a novel compound, called EAD1, which is structurally related to CQ but is a 14-fold more potent inhibitor of cell proliferation. Here we find that EAD1 causes rapid relocation, membrane permeabilization (LMP), and deacidification of lysosomes, and it induces apoptosis and irreversibly blocks proliferation of human lung cancer H460, H520, H1299, HCC827, and H1703 cells. EAD1 causes dissociation of mTOR from lysosomes and increases mTOR's perinuclear versus cytoplasmic localization, changes previously shown to inactivate mTORC1. The effect on mTOR was not seen with HCQ, even at >10-fold greater concentrations. Phosphorylation of a downstream target of mTORC1, ribosomal protein S6, was inhibited by EAD1. Although EAD1 also inhibited autophagy, it retained full antiproliferative activity in autophagy-deficient H1650 lung cancer cells, which have a biallelic deletion of Atg7, and in H460 Atg7-knockout cells. As Atg7 is critical for the canonical autophagy pathway, it is likely that inhibition of autophagy is not how EAD1 inhibits cell proliferation. Further studies are needed to determine the relationship of LMP to mTORC1 disruption and their relative contributions to drug-induced cell death. These studies support the lysosome as an underexplored target for new drug development.

Sterilizing Processes and Mechanisms for Treatment of Escherichia coli with Dielectric-Barrier Discharge Plasma.

With increasing attention toward novel sterilization methods, plasma sterilization has gained more and more interest. However, the underlying mechanisms are still unknown. In this paper, we investigated the inactivation of Escherichia coli using dielectric-barrier discharge (DBD) plasma in saline water. There were three processes shown in the survival curve, namely, during the preparation period, the reaction period, and the saturation period. Observations under a transmission electron microscope (TEM) and detection by Fourier transform infrared spectroscopy (FT-IR) supplied adequate details regarding these processes. Based on these results, we infer that during the preparation period, the main process is the accumulation of chemical substances. During the reaction period, adequate amounts of chemicals decompose and denature cell membranes and macromolecules to kill bacteria in large quantities. During the saturation period, the killing effect decreases because of the protection by clustered cells and the saturation of pH. This study of sterilizing processes systematically reveals the mechanisms of plasma sterilization.IMPORTANCE Compared with traditional methods, plasma sterilization has advantages of high efficiency, easy operation, and environmental protection. This may be more suitable for air and sewage sterilization in specific spaces, such as hospitals, laboratories, and pharmaceutical factories. However, the mechanisms of sterilization are still relatively unknown, especially for bactericidal activities. Knowledge of sterilization processes provides guidance for practical applications. For example, the bactericidal action mainly occurs during the reaction period, and the treatment time can be set based on the reaction period, which could save a lot of energy. The results of this study will help to improve the efficiency of plasma sterilization devices.

Understanding the Coacervate-to-Vesicle Transition of Globular Fusion Proteins to Engineer Protein Vesicle Size and Membrane Heterogeneity.

Protein-rich coacervates are liquid phases separate from the aqueous bulk phase that are used by nature for compartmentalization and more recently have been exploited by engineers for delivery and formulation applications. They also serve as an intermediate phase in an assembly path to more complex structures, such as vesicles. Recombinant fusion protein complexes made from a globular protein fused with a glutamic acid-rich leucine zipper (globule-ZE) and an arginine-rich leucine zipper fused with an elastin-like polypeptide (ZR-ELP) show different phases from soluble, through an intermediate coacervate phase, and finally to vesicles with increasing temperature of the aqueous solution. We investigated the phase transition kinetics of the fusion protein complexes at different temperatures using dynamic light scattering and microscopy, along with mathematical modeling. We controlled coacervate growth by aging the solution at an intermediate temperature that supports coacervation and confirmed that the size of the coacervate droplets dictates the size of vesicles formed upon further heating. With this understanding of the phase transition, we developed strategies to induce heterogeneity in the organization of globular proteins in the vesicle membrane through simple mixing of coacervates containing two different globular fusion proteins prior to the vesicle transition. This study gives fundamental insights and practical strategies for development of globular protein-rich coacervates and vesicles for drug delivery, microreactors, and protocell applications.

Rhodomyrtone (Rom) is a membrane-active compound.

Particularly in Asia medicinal plants with antimicrobial activity are used for therapeutic purpose. One such plant-derived antibiotic is rhodomyrtone (Rom) isolated from Rhodomyrtus tomentosa leaves. Rom shows high antibacterial activity against a wide range of Gram-positive bacteria, however, its mode of action is still unclear. Reporter gene assays and proteomic profiling experiments in Bacillus subtilis indicate that Rom does not address classical antibiotic targets like translation, transcription or DNA replication, but acts at the cytoplasmic membrane. In Staphylococcus aureus, Rom decreases the membrane potential within seconds and at low doses, causes release of ATP and even the excretion of cytoplasmic proteins (ECP), but does not induce pore-formation as for example nisin. Lipid staining revealed that Rom induces local membrane damage. Rom's antimicrobial activity can be antagonized in the presence of a very narrow spectrum of saturated fatty acids (C15:0, C16:0, or C18:0) that most likely contribute to counteract the membrane damage. Gram-negative bacteria are resistant to Rom, presumably due to reduced penetration through the outer membrane and its neutralization by LPS. Rom is cytotoxic for many eukaryotic cells and studies with human erythrocytes showed that Rom induces eryptosis accompanied by erythrocyte shrinkage, cell membrane blebbing, and membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Rom's distinctive interaction with the cytoplasmic membrane reminds on the amphipathic, alpha-helical peptides, the phenol-soluble modulins (PSMs), and renders Rom an important tool for the investigation of membrane physiology.

Cytochrome b561, copper, ß-cleaved amyloid precursor protein and niemann-pick C1 protein are involved in ascorbate-induced release and membrane penetration of heparan sulfate from endosomal S-nitrosylated glypican-1.

Ascorbate-induced release of heparan sulfate from S-nitrosylated heparan sulfate proteoglycan glypican-1 takes place in endosomes. Heparan sulfate penetrates the membrane and is transported to the nucleus. This process is dependent on copper and on expression and processing of the amyloid precursor protein. It remains unclear how exogenously supplied ascorbate can generate HS-anMan in endosomes and how passage through the membrane is facilitated. Here we have examined wild-type, Alzheimer Tg2576 and amyloid precursor protein (-/-) mouse fibroblasts and human fetal and Niemann-Pick C1 fibroblasts by using deconvolution immunofluorescence microscopy, siRNA technology and [S35]sulfate-labeling, vesicle isolation and gel chromatography. We found that ascorbate-induced release of heparan sulfate was dependent on expression of endosomal cytochrome b561. Formation and nuclear transport of heparan sulfate was suppressed by inhibition of ß-processing of the amyloid precursor protein and formation was restored by copper (I) ions. Membrane penetration was not dependent on amyloid beta channel formation. Inhibition of endosomal exit resulted in accumulation of heparan sulfate in vesicles that exposed the C-terminal of the amyloid precursor protein externally. Endosome-to-nucleus transport was also dependent on expression of the Niemann-Pick C1 protein. We propose that ascorbate is taken up from the medium and is oxidized by cytochrome b561 which, in turn, reduces copper (II) to copper (I) present in the N-terminal, ß-cleaved domain of the amyloid precursor protein. Re-oxidation of copper (I) is coupled to reductive, deaminative release of heparan sulfate from glypican-1. Passage through the membrane may be facilitated by the C-terminal, ß-cleaved fragment of the amyloid precursor protein and the Niemann-Pick C1 protein.

Virion-associated phosphatidylethanolamine promotes TIM1-mediated infection by Ebola, dengue, and West Nile viruses.

Phosphatidylserine (PS) receptors contribute to two crucial biological processes: apoptotic clearance and entry of many enveloped viruses. In both cases, they recognize PS exposed on the plasma membrane. Here we demonstrate that phosphatidylethanolamine (PE) is also a ligand for PS receptors and that this phospholipid mediates phagocytosis and viral entry. We show that a subset of PS receptors, including T-cell immunoglobulin (Ig) mucin domain protein 1 (TIM1), efficiently bind PE. We further show that PE is present in the virions of flaviviruses and filoviruses, and that the PE-specific cyclic peptide lantibiotic agent Duramycin efficiently inhibits the entry of West Nile, dengue, and Ebola viruses. The inhibitory effect of Duramycin is specific: it inhibits TIM1-mediated, but not L-SIGN-mediated, virus infection, and it does so by blocking virus attachment to TIM1. We further demonstrate that PE is exposed on the surface of apoptotic cells, and promotes their phagocytic uptake by TIM1-expressing cells. Together, our data show that PE plays a key role in TIM1-mediated virus entry, suggest that disrupting PE association with PS receptors is a promising broad-spectrum antiviral strategy, and deepen our understanding of the process by which apoptotic cells are cleared.

Membrane-binding properties of gating modifier and pore-blocking toxins: Membrane interaction is not a prerequisite for modification of channel gating.

Many venom peptides are potent and selective inhibitors of voltage-gated ion channels, including channels that are validated therapeutic targets for treatment of a wide range of human diseases. However, the development of novel venom-peptide-based therapeutics requires an understanding of their mechanism of action. In the case of voltage-gated ion channels, venom peptides act either as pore blockers that bind to the extracellular side of the channel pore or gating modifiers that bind to one or more of the membrane-embedded voltage sensor domains. In the case of gating modifiers, it has been debated whether the peptide must partition into the membrane to reach its binding site. In this study, we used surface plasmon resonance, fluorescence spectroscopy and molecular dynamics to directly compare the lipid-binding properties of two gating modifiers (µ-TRTX-Hd1a and ProTx-I) and two pore blockers (ShK and KIIIA). Only ProTx-I was found to bind to model membranes. Our results provide further evidence that the ability to insert into the lipid bilayer is not a requirement to be a gating modifier. In addition, we characterised the surface of ProTx-I that mediates its interaction with neutral and anionic phospholipid membranes and show that it preferentially interacts with anionic lipids.

Changes in membrane biophysical properties induced by the Budesonide/Hydroxypropyl-ß-cyclodextrin complex.

Budesonide (BUD), a poorly soluble anti-inflammatory drug, is used to treat patients suffering from asthma and COPD (Chronic Obstructive Pulmonary Disease). Hydroxypropyl-ß-cyclodextrin (HPßCD), a biocompatible cyclodextrin known to interact with cholesterol, is used as a drug-solubilizing agent in pharmaceutical formulations. Budesonide administered as an inclusion complex within HPßCD (BUD:HPßCD) required a quarter of the nominal dose of the suspension formulation and significantly reduced neutrophil-induced inflammation in a COPD mouse model exceeding the effect of each molecule administered individually. This suggests the role of lipid domains enriched in cholesterol for inflammatory signaling activation. In this context, we investigated the effect of BUD:HPßCD on the biophysical properties of membrane lipids. On cellular models (A549, lung epithelial cells), BUD:HPßCD extracted cholesterol similarly to HPßCD. On large unilamellar vesicles (LUVs), by using the fluorescent probes diphenylhexatriene (DPH) and calcein, we demonstrated an increase in membrane fluidity and permeability induced by BUD:HPßCD in vesicles containing cholesterol. On giant unilamellar vesicles (GUVs) and lipid monolayers, BUD:HPßCD induced the disruption of cholesterol-enriched raft-like liquid ordered domains as well as changes in lipid packing and lipid desorption from the cholesterol monolayers, respectively. Except for membrane fluidity, all these effects were enhanced when HPßCD was complexed with budesonide as compared with HPßCD. Since cholesterol-enriched domains have been linked to membrane signaling including pathways involved in inflammation processes, we hypothesized the effects of BUD:HPßCD could be partly mediated by changes in the biophysical properties of cholesterol-enriched domains.

Neglected antibacterial activity of ethylene glycol as a common solvent.

For the first time, the antibacterial activity of ethylene glycol (EG), a routine frequently used solvent against Escherichia coli (E. coli) bacterium, was assessed. The antibacterial activity of EG against E. coli was measured using colony counting and broth turbidity assays. The influence of EG concentration (1.5-25.0%v/v) and exposure time on the growth of E. coli was investigated. By increasing EG concentration, its antibacterial activity against E. coli increased so that for 24.0% of EG, the bacteria growth was totally inhibited within 4 h. The MIC and MBC values of EG are 18.0 and 24.0%v/v, respectively. Since the ratio of MBC to MIC is less than four, EG acts as a bactericidal agent. Also, a model for the slopes of the linear part of the growth curves was proposed. The SEM images of bacteria cells before and after exposure to EG show that most E. coli were seriously distorted.

Functional activation of Gαq coupled to 5-HT2A receptor and M1 muscarinic acetylcholine receptor in postmortem human cortical membranes.

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) play a pivotal role in a wide range of signal transduction pathways, and receptor/G-protein coupling has been implicated in the pathophysiology of mental disorders. In this study, guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation assay for Gαq was applied to postmortem human brains. After its optimization for human prefrontal cortical membranes, we selected 5-hydroxytryptamine (5-HT) and carbachol as efficient agonists for subsequent experiments. The concentration-response curve of 5-HT shifted towards the right by the addition of increasing concentrations of ketanserin (with a pA 2 value of 9.18), indicating the involvement of the 5-HT2A receptor. Besides, the carbachol-stimulated [35S]GTPγS binding to Gαq was competitively antagonized by telenzepine (with a pA 2 value of 8.81), indicating the involvement of the M1 muscarinic acetylcholine receptor (mAChR). Concentration-response curves of 5-HT2A receptor- and M1 mAChR-mediated Gαq activation were determined in 40 subjects. The mean maximum percentage increase (%E max) was 155 and 470%, respectively, and the mean half-maximal effect concentration (EC50) was 131 nM and 15.2 µM, respectively. When the pharmacological parameters were correlated with age, postmortem delay, freezing storage period, and tissue pH, no statistically significant correlation was observed except for the negative correlation between age and %E max value of carbachol-stimulated [35S]GTPγS binding to Gαq. The %E max values for 5-HT2A receptor- and M1 mAChR-mediated Gαq activation also tended to correlate with each other. These results provide fundamental information of Gαq-coupled 5-HT2A receptor and M1 mAChR in native human brains, and lay the foundation for future studies in mental disorder patients.

Association of Permeability and Lipid Content of Membrane.

BACKGROUND: Rat skin and goat cul de sac are mostly used in optimization of formulations as the model of human skin and cul de sac. AIM: To explore the correlation between lipid content of rat skin and goat cul de sac and permeability. MATERIALS AND METHODS: Find out wavelength maximum for Sapat plus malam®, Ciplox eye ointment® and chloramphenicol eye caps and the standard curve was also derived. In vitro studies using Cellophane® membrane and ex vivo studies using rat skin or goat cul de sac of the formulations. Permeability coefficient, % dislodgeable dose, lag time, diffusion parameter, and partition coefficient were found for both studies after six and a half hours of penetration studies. Student's unpaired t-test with equal variance was used to find any statistically significant difference in the ex vivo and in vitro diffusion transport studies at 95% level of confidence. RESULTS: Permeability coefficient of Sapat plus malam®, Ciplox eye ointment® and chloramphenicol eye caps were 0.000316 ± 0.0000625, 0.00416 ± 0.0001, 0.0034 ± 0.00004 for Cellophane® membrane and 0.0001 ± 0.000001, 0.002254 ± 0.0002, 0.00303 ± 0.0001 for ex vivo membrane in cm2/min, respectively. For all three formulations, there were calculated t values which were higher than tabulated t values at 95% of confidence level (P<0.05). CONCLUSION: Cellophane® membrane shows a better diffusion than rat skin or goat cul de sac. In the optimization of formulation, only Cellophane® membrane is advisable to use.

Salinity induces membrane structure and lipid changes in maize mesophyll and bundle sheath chloroplasts.

The membranes of Zea mays (maize) mesophyll cell (MC) chloroplasts are more vulnerable to salinity stress than are those of bundle sheath cell (BSC) chloroplasts. To clarify the mechanism underlying this difference in salt sensitivity, we monitored changes in the glycerolipid and fatty acid compositions of both types of chloroplast upon exposure to salinity stress. The monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) contents were higher in MC chloroplasts than in BSC chloroplasts, in both the presence and absence of salt treatment. Under salt conditions, the MGDG level in MC chloroplasts was significantly lower than under normal conditions, while it was unchanged in BSC chloroplasts. In both types of chloroplast, the contents of DGDG, phosphatidylglycerol and phosphatidylinositol remained at the same levels in control and salt-treated plants, whereas sulfoquinovosyldiacylglycerol and phosphatidylcholine were significantly lower and higher, respectively, upon salt treatment. In addition, the fatty acid composition and double bond index of individual lipid classes were changed by salt treatment in both BSC and MC chloroplasts, although these factors had no effect on glycerolipid content. These findings suggest that the difference in salt sensitivity of MC and BSC chloroplast membranes is related to differences in MGDG responses to salinity. Thus, we propose that the low MGDG content and the low sensitivity of MGDG to salinity in BSC chloroplasts render them more tolerant than MC chloroplasts to salinity stress.

Transmittance and Autofluorescence of Neonatal Rat Stratum Corneum: Nerolidol Increases the Dynamics and Partitioning of Protoporphyrin IX into Intercellular Membranes.

In this work, we developed an experimental apparatus to directly measure transmittance and fluorescence in the stratum corneum (SC) ex vivo. The SC transmittance varied from ~6 to ~52 % in the wavelength range of 280-850 nm. For 260-300 nm excitation, the SC autofluorescence showed a strong emission band between 290 and 425 nm, which is associated with tryptophan, and another in the 600-670 nm range, which we attributed to a process involving resonance energy transfer to very hydrophobic keratin filaments. Weaker emission associated with less hydrophobic keratin filaments was also observed in the wavelength range of 350-480 nm. Protoporphyrin IX (PpIX) was incorporated into SC membranes, and its penetration was further increased by the addition of nerolidol to the treatment suspension. Both PpIX and the endogenous porphyrins showed fluorescence anisotropy consistent with their localization in SC membranes, and their molecular dynamics increased significantly in the presence of 1 % nerolidol. The emission and excitation spectra of PpIX and the endogenous SC porphyrins showed similar alterations during the photobleaching induced by 405-nm irradiation. This work also highlights the SC contribution to skin autofluorescence, which could be useful for fluorescence spectroscopy applications in the early diagnosis of skin diseases.

Trichoplaxin - a new membrane-active antimicrobial peptide from placozoan cDNA.

A method based on the use of signal peptide sequences from antimicrobial peptide (AMP) precursors was used to mine a placozoa expressed sequence tag database and identified a potential antimicrobial peptide from Trichoplax adhaerens. This peptide, with predicted sequence FFGRLKSVWSAVKHGWKAAKSR is the first AMP from a placozoan species, and was named trichoplaxin. It was chemically synthesized and its structural properties, biological activities and membrane selectivity were investigated. It adopts an α-helical structure in contact with membrane-like environments and is active against both Gram-negative and Gram-positive bacterial species (including MRSA), as well as yeasts from the Candida genus. The cytotoxic activity, as assessed by the haemolytic activity against rat erythrocytes, U937 cell permeabilization to propidium iodide and MCF7 cell mitochondrial activity, is significantly lower than the antimicrobial activity. In tests with membrane models, trichoplaxin shows high affinity for anionic prokaryote-like membranes with good fit in kinetic studies. Conversely, there is a low affinity for neutral eukaryote-like membranes and absence of a dose dependent response. With high selectivity for bacterial cells and no homologous sequence in the UniProt, trichoplaxin is a new potential lead compound for development of broad-spectrum antibacterial drugs.

Resistance to and killing by the sporicidal microbicide peracetic acid.

OBJECTIVES: To elucidate the mechanisms of spore resistance to and killing by the oxidizing microbicide peracetic acid (PAA). METHODS: Mutants of Bacillus subtilis lacking specific spore structures were used to identify resistance properties in spores and to understand the mechanism of action of PAA. We also assessed the effect of PAA treatment on a number of spore properties including heat tolerance, membrane integrity and germination. RESULTS: The spore coat is essential for spore PAA resistance as spores with defective coats were greatly sensitized to PAA treatment. Small acid-soluble spore proteins apparently provide no protection against PAA. Defects in spore germination, specifically in germination via the GerB and GerK but not the GerA germination receptors, as well as leakage of internal components suggest that PAA is active at the spore inner membrane. It is therefore likely that the inner membrane is the major site of PAA's sporicidal activity. CONCLUSIONS: PAA treatment targets the spore membrane, with some of its activity directed specifically against the GerB and GerK germination receptors.