Surfactin production enhances the level of cardiolipin in the cytoplasmic membrane of Bacillus subtilis

Surfactin is a cyclic lipopeptide antibiotic that disturbs the integrity of the cytoplasmic membrane. In this study, the role of membrane lipids in the adaptation and possible surfactin tolerance of the surfactin producer Bacillus subtilis ATCC 21332 was investigated. During a 1-day cultivation, the phospholipids of the cell membrane were analyzed at the selected time points, which covered both the early and late stationary phases of growth, when surfactin concentration in the medium gradually rose from 2 to 84 μmol·l^− 1. During this time period, the phospholipid composition of the surfactin producer's membrane (Sf⁺) was compared to that of its non-producing mutant (Sf⁻). Substantial modifications of the polar head group region in response to the presence of surfactin were found, while the fatty acid content remained unaffected. Simultaneously with surfactin production, a progressive accumulation up to 22% of the stress phospholipid cardiolipin was determined in the Sf⁺ membrane, whereas the proportion of phosphatidylethanolamine remained constant. At 24 h, cardiolipin was found to be the second major phospholipid of the membrane. In parallel, the Laurdan generalized polarization reported an increasing rigidity of the lipid bilayer. We concluded that an enhanced level of cardiolipin is responsible for the membrane rigidification that hinders the fluidizing effect of surfactin. At the same time cardiolipin, due to its negative charge, may also prevent the surfactin-membrane interaction or surfactin pore formation activity.     

Effect of pressure on islet amyloid polypeptide aggregation: revealing the polymorphic nature of the fibrillation process

Type II diabetes mellitus is a disease which is characterized by peripheral insulin resistance coupled with a progressive loss of insulin secretion that is associated with a decrease in pancreatic islet beta-cell mass and the deposition of amyloid in the extracellular matrix of beta-cells, which lead to islet cell death. The principal component of the islet amyloid is a pancreatic hormone called islet amyloid polypeptide (IAPP). High-pressure coupled with FT-IR spectroscopic and AFM studies were carried out to elucidate further information about the aggregation pathway as well as the aggregate structures of IAPP. To this end, a comparative fibrillation study of IAPP fragments was carried out as well. As high hydrostatic pressure (HHP) is acting to weaken or even prevent hydrophobic self-organization and electrostatic interactions, application of HHP has been used as a measure to reveal the importance of these interactions in the fibrillation process of IAPP and its fragments. IAPP preformed fibrils exhibit a strong polymorphism with heterogeneous structures, a large population of which are rather sensitive to high hydrostatic pressure, thus indicating a high percentage of ionic and hydrophobic interactions and loose packing of these species. Conversely, fragments 1-19 and 1-29 are resistant to pressure treatment, suggesting more densely packed aggregate structures with less void volume and strong cooperative hydrogen bonding. Furthermore, the FT-IR data indicate that fragment 1-29 has intermolecular beta-sheet conformational properties different from those of fragment 1-19, the latter exhibiting polymorphic behavior with more disordered structures and less strongly hydrogen bonded fibrillar assemblies. The data also suggest that hydrophobic interactions and/or less efficient packing of amino acids 30-37 region leads to the marked pressure sensitivity observed for full-length IAPP.     

Improvement of four sigma analysis for the investigation of oxygen evolution by Photosystem II

We present here an improvement to the analysis of oxygen evolution with four sigma coefficients (4-S) by computing z, the sum of the S-state probabilities, which was introduced earlier (Delrieu and Rosengard 1987, Biochim Biophys Acta 892: 163–171). We demonstrate that z is equal to the ratio of two consecutive Mean Y (the estimation of the steady state oxygen production based on local properties) found by three sigma analysis. The quantity z is useful for computing double-hits, and for showing the inactivation/activation processes of PS II complexes. Three sigma analysis assumes z=1 exactly; since this is not verified, it is argued that four sigma analysis is closer to the real workings of the water oxidizing complex. Oxygen evolution can then be interpreted in the frame of a modified Kok's model where the sum of the probabilities equals z. We therefore suggest that the closer fitting of four sigma analysis to oxygen production data is not simply due to an extra, unnecessary variable, but to the fact that PS II complexes can be inactivated and reactivated under flashing light. Finally, in order to facilitate the use of four sigma analysis, a computer program is made available upon request.     

Photodynamic antimicrobial therapy

Photodynamic antimicrobial therapy (PACT) involves the utilisation of photosensitizers activated by exposure to visible light in order to eradicate microbes (this method has already been applied in photodynamic therapy of tumours). Photodynamic effect of the particular photosensitive substance (PS) is attributed to its ability to penetrate susceptible microorganisms, to absorb the light of certain wavelength, and to generate reactive cytotoxic oxygen products. The target microorganisms for photoinactivation are bacteria, fungi, viruses and protozoa. Photodynamic antimicrobial therapy is proposed as a potentially topical, non-invasive approach suitable for treatment of locally occurring infection. The fact that bacteria are becoming increasingly resistant to antibiotics and antiseptics has lead to an increased interest in the development of new alternative eradication methods, such as PACT. Research and development of photosensitive substances are aimed at finding effective antimicrobial substances, which would have a broad-spectrum potency.     

Glial Metabolism of Valine

The three essential amino acids, valine, leucine and isoleucine, constitute the group of branched-chain amino acids (BCAAs). BCAAs are rapidly taken up into the brain parenchyma, where they serve several distinct functions including that as fuel material in brain energy metabolism. As one function of astrocytes is considered the production of fuel molecules that support the energy metabolism of adjacent neural cells in brain. Astroglia-rich primary cultures (APC) were shown to rapidly dispose of the BCAAs, including valine, contained in the culture medium. While the metabolisms of leucine and isoleucine by APC have already been studied in detail, some aspects of valine metabolism remained to be determined. Therefore, in the present study an NMR analysis was performed to identify the ¹³C-labelled metabolites that are generated by APC during catabolism of [U-¹³C]valine and that are subsequently released into the incubation medium. The results presented show that APC (1) are potently disposing of the valine contained in the incubation medium; (2) are capable of degrading valine to the tricarboxylic acid (TCA) cycle member succinyl-CoA; and (3) release into the extracellular milieu valine catabolites and compounds generated from them such as [U-¹³C]2-oxoisovalerate, [U-¹³C]3-hydroxyisobutyrate, [U-¹³C]2-methylmalonate, [U-¹³C]isobutyrate, and [U-¹³C]propionate as well as several TCA cycle-dependent metabolites including lactate. This article is dedicated to Dr. George DeVries.     

Interplay of cysteinyl leukotrienes and TGF-β in the activation of hepatic stellate cells from Schistosoma mansoni granulomas

Hepatic stellate cells (HSCs) have a critical role in liver physiology, and in the pathogenesis of liver inflammation and fibrosis. Here, we investigated the interplay between leukotrienes (LT) and TGF-β in the activation mechanisms of HSCs from schistosomal granulomas (GR-HSCs). First, we demonstrated that GR-HSCs express 5-lipoxygenase (5-LO), as detected by immunolocalization in whole cells and confirmed in cell lysates through western blotting and by mRNA expression through RT-PCR. Moreover, mRNA expression of 5-LO activating protein (FLAP) and LTC₄-synthase was also documented, indicating that GR-HSCs have the molecular machinery required for LT synthesis. Morphological analysis of osmium and Oil-Red O-stained HSC revealed large numbers of small lipid droplets (also known as lipid bodies). We observed co-localization of lipid droplet protein marker (ADRP) and 5-LO by immunofluorescence microscopy. We demonstrated that GR-HSCs were able to spontaneously release cysteinyl-LTs (CysLTs), but not LTB_4, into culture supernatants. CysLT production was highly enhanced after TGF-β-stimulation. Moreover, the 5-LO inhibitor zileuton and 5-LO gene deletion were able to inhibit the TGF-β-stimulated proliferation of GR-HSCs, suggesting a role for LTs in HSC activation. Here, we extend the immunoregulatory function of HSC by demonstrating that HSC from liver granulomas of schistosome-infected mouse are able to release Cys-LTs in a TGF-β-regulated manner, potentially impacting pathogenesis and liver fibrosis in schistosomiasis.     

Does developmental acclimatization reduce the susceptibility to predation in newt larvae?

Many organisms respond to the heterogeneity of abiotic environmental conditions by plastic modifications of their phenotypes (acclimation or acclimatization). Despite considerable research efforts in this area, the beneficial (adaptive) effect of acclimation or acclimatization is still debated. We examined whether the development of newt larvae (Ichthyosaura alpestris) under different natural light and thermal conditions subsequently altered their susceptibility to predation in sun‐exposed versus shaded tanks in nature. During predation trials in various light and temperature conditions, newt larvae that developed in sun‐exposed warmer tanks consistently suffered from higher predation by dragonfly nymphs (Aeshna cyanea) compared to larvae from shaded or colder tanks. We conclude that higher sun exposure during embryonic and larval development negatively affects antipredator performance even in sun‐exposed tanks: this result is inconsistent with the beneficial acclimation hypothesis. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ●● , ●●–●●.     

Trophic role of rotifers in the plankton of lake Kozjak (Plitvice Lakes)

The trophic role of rotifers in the zooplankton community of dimictic, oligotrophic lake Kozjak, the largest lake of the Plitvice Lakes, NW Dinarid Mountains, is analyzed. Their spatial and temporal biomass distribution in relation to that of protozoans, cladocerans and copepods shows that they form a significant part of the non-predatory zooplankton of this karstic standing water.     

Cytochrome P450 Monooxygenase CYP53 Family in Fungi: Comparative Structural and Evolutionary Analysis and Its Role as a Common Alternative Anti-Fungal Drug Target

Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family''s role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s'' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative structural (gene and protein structure-level) and evolutionary analysis of a fungal P450 family.     

Plant activation of aromatic amines mediated by cytochromes P450 and flavin-containing monooxygenases

To know the mechanisms involved in the activation of promutagenic aromatic amines mediated by plants, we used Persea americana S117 system (S117) for the activation of 2-aminofluorene (2-AF) and m-phenylenediamine (m-PDA) in Ames assays. In these assays, the effect of the diphenylene iodonium (DPI), an inhibitor of flavin-containing monooxygenases (FMOs), of the 1-aminobenzotriazole (1-ABT), an inhibitor of cytochromes P450 (cyt-P450s) and of the methimazole, a high-affinity substrate for FMOs, was studied. The efficacy of both inhibitors and of the methimazole was verified to find that they did partially inhibit the mutagenesis of both aromatic amines, activated with rat liver S9. Similarly, both inhibitors and methimazole did produce a significant decrease in 2-AF and m-PDA mutagenesis, when the activation system was S117, indicating that, similar to what occurs in mammalian systems, plant FMOs and cyt-P450s can metabolize aromatic amines to mutagenic product(s). However, the affinity of both FMOs and cyt-P450s of plant for 2-AF and m-PDA was different. Data obtained indicate that the activities of plant FMOs must be the main enzymatic system of m-PDA activation while, in 2-AF activation, plant cyt-P450s have the most relevant activities. In addition, peroxidases of the S117 system must contribute to 2-AF activation and some isoforms of FMOs and/or cyt-P450s of the S117 system, uninhibited by the inhibitors used, must be the responsible for a partial activation of m-PDA.