Structural dynamics of a lytic peptide interacting with a supported lipid bilayer

The interaction of a melittin mutant with a 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)-supported lipid bilayer was studied with the use of time-resolved evanescent wave-induced fluorescence spectroscopy (TREWIFS) and evanescent wave-induced time-resolved fluorescence anisotropy measurements (EW-TRAMs). The mutant peptide was labeled at position K14 with AlexaFluor 430 and retained the lytic activity characteristic of native melittin. The fluorescence decay kinetics of the conjugate was found to be biexponential with a short-lived component, τ₁, due to photoinduced electron transfer between AlexaFluor 430 and proximal side chains within or between the peptides. The longer-lived component, τ₂, was sensitive to the polarity of the microenvironment at or near the K14 position of the peptide. Upon interaction with a DPPC-supported bilayer, the proportional contribution of τ₁ increased, indicating a conformational change of the peptide. The values of τ₁ and τ₂ indicate that the AlexaFluor 430 probe experienced an environment with an equivalent polarity no less than that of methanol. EW-TRAMs data from the melittin mutant revealed hindered rotational motions of the AlexaFluor 430 probe both in the plane and perpendicular to the plane of the supported lipid bilayer. The data indicate a highly ordered and polar environment near the center of the melittin helix consistent with the formation of a toroidal pore.     

Strategies for the identification and purification of ligands for orphan biomolecules

The isolation of related genes with evolutionary conserved motifs by the application ofpolymerase chain reaction-based molecular biology techniques, or from database searchingstrategies, has facilitated the identification of new members of protein families. Many of theseprotein molecules will be involved in protein–protein interactions (e.g. growth factors,receptors, adhesion molecules), since such interactions are intrinsic to virtually every cellularprocess. However, the precise biological function and specific binding partners of these novelproteins are frequently unknown, hence they are known as orphan molecules.Complementary technologies are required for the identification of the specific ligands orreceptors for these and other orphan proteins (e.g., antibodies raised against crude biologicalextracts or whole cells). We describe herein several alternative strategies for the identification,purification and characterisation of orphan peptide and protein molecules, specifically thesynergistic use of micropreparative HPLC and biosensor techniques.     

Adaptive responses to low-dose/low-dose-rate gamma rays in normal human fibroblasts: the role of growth architecture and oxidative metabolism

To investigate low-dose/low-dose-rate effects of low-linear energy transfer (LET) ionizing radiation, we used gamma-irradiated cells adapted to grow in a three-dimensional architecture that mimics cell growth in vivo. We determined the cellular, molecular and biochemical changes in these cells. Quiescent normal human fibroblasts were irradiated with single acute or chronic doses (1-10 cGy) of (137)Cs gamma rays. Whereas exposure to an acute dose of 10 cGy increased micronucleus formation, protraction of the dose over 48 h reduced micronucleus frequency to a level similar to or lower than what occurs spontaneously. The protracted treatment also up-regulated the cellular content of the antioxidant glutathione. These changes correlated with modulation of phospho-TP53 (serine 15), a stress marker that was regulated by doses as low as 1 cGy. The DNA damage that occurred after exposure to an acute dose of 10 cGy was protected against in two ways: (1) up-regulation of cellular antioxidant enzyme activity by ectopic overexpression of MnSOD, catalase or glutathione peroxidase, and (2) inhibition of superoxide anion generation by flavin-containing oxidases. These results support a significant role for oxidative metabolism in mediating low-dose radiation effects and demonstrate that cell culture in three dimensions is ideal to investigate radiation-induced adaptive responses. Expression of connexin 43, a constitutive protein of gap junctions, and the G(1) checkpoint were more sensitive to regulation by gamma rays in cells maintained in a three-dimensional than in a two-dimensional configuration.     

Molecular and morphological divergence in the butterfly genus Lycaeides (Lepidoptera: Lycaenidae) in North America: evidence of recent speciation

Male genital morphology, allozyme allele frequencies and mtDNA sequence variation were surveyed in the butterfly species Lycaeides idas and L. melissa from across much of their range in North America. Despite clear differences in male genital morphology, wing colour patterns and habitat characteristics, genetic variation was not taxonomically or geographically structured and the species were not identifiable by either genetic data set. Genetic distances (Nei's D=0.002–0.078, calculated from allozyme data) between all populations of both species were within the range commonly observed for conspecific populations of other butterflies. The most frequent mtDNA haplotype was present in individuals of both species in populations from southern California to Wisconsin. We conclude that speciation has probably happened recently and the lack of genetic differentiation between the species is the product of either (1) recent or ongoing gene flow at neutral loci, and/or (2) an insufficiency of time for lineage sorting. The evolution of male genital morphology, wing colour patterns and ecological characteristics has proceeded more rapidly than allozyme or mtDNA evolution.     

Physico-chemical characteristics of cell walls from Arabidopsis thaliana microcalli showing different adhesion strengths

Changes in the composition and structure of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Arabidopsis thaliana microcalli. Three growth phases, namely the cell division phase, the cell expansion phase, and the stationary phase, were distinguished and associated with a decreasing cell cluster adhesion strength. Degradation of the homogalacturonan pectic backbone and of linear pectic side chains (1,4)-beta-D-galactan were observed concomitantly with the cell expansion and stationary phases and the decrease in cell adhesion. Also, in the stationary phase, branched (1,5)-alpha-L-arabinans were linearized. The AGP content of the culture medium increased while it decreased in the cell wall during cell growth and as cell adhesion decreased. These data suggest that, in addition to homogalacturonan, pectic side chains and AGP are involved in plant cell development and particularly in cell-cell attachment.     

Anti-microfouling Activity of Lipidic Metabolites from the Invasive Brown Alga Sargassum muticum (Yendo) Fensholt

The purification of the chloroform extract from the brown invasive macroalga Sargassum muticum, through a series of chromatographic separations, yielded 12 fractions that were tested against strains of bacteria, microalgae, and fungi involved in marine biofilm formation. The chemical composition of four (a, c, g, and k) out of the six fractions that exhibited anti-microfouling activity was investigated. Fraction a contained saturated and unsaturated linear hydrocarbons (C₁₂–C₂₇). Arachidonic acid was identified as the major metabolite in fraction c whereas fraction g contained mainly palmitic, linolenic, and palmitoleic acids. Fraction k was submitted to further purification yielding the fraction kAcaF1e that was composed of galactoglycerolipids, active against the growth of two of the four bacterial strains (Shewanella putrefaciens and Polaribacter irgensii) and all tested fungi. These promising results, in particular the isolation and the activity of galactoglycerolipids, attest the potential of the huge biomass of S. muticum as a source of new environmentally friendly antifouling compounds.     

Electrochemistry of cytochrome c immobilized on cardiolipin-modified electrodes: A probe for protein–lipid interactions

Electrochemistry of cytochrome c (cyt c) immobilized on a cardiolipin (CL)/phosphatidylcholine (PC) film supported on a glassy carbon electrode was investigated using variable-frequency AC voltammetry. At low ionic strength, we observed two redox-active subpopulations characterized by distinct values of potential (E_1/2) and electron transfer rate constant (k_ET). At high ionic strength, only one subpopulation was detected, consistent with the existence of very stable cyt c–CL adducts, most probably formed by hydrophobic interactions between the protein and the fatty acid (FA) chains carried by CL. This subpopulation exhibits a comparatively high k_ET value (> 300 s^− 1) apparently changing with the structure of the FA chains of CL, i.e. 18:2(n − 6) or 14:0. Our study suggests that electrochemistry can be a useful technique for probing protein–lipid interactions, and more particularly the role played by the specific structure of the FA chains of CL on cyt c binding.     

Herpes simplex virus type 1 DNA is immunostimulatory in vitro and in vivo

Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically immunostimulatory both in vitro and in vivo, tending to promote Th1-like responses. In contrast, CpG dinucleotides in mammalian DNAs are extensively methylated on cytosines and hence immunologically inert. Since the herpes simplex virus (HSV) genome is unmethylated and G+C rich, we predicted that CpG motifs would be highly prevalent in the HSV genome; hence, we examined the immunostimulatory potential of purified HSV DNA in vitro and in vivo. Mouse splenocyte cultures treated with HSV DNA or HSV-derived oligodeoxyribonucleotides (ODNs) showed strong proliferative responses and production of inflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor [TNF], and interleukin-6 [IL-6]) in vitro, whereas splenocytes treated with mammalian CV-1 DNA or non-CpG ODN did not. After immunization with ovalbumin (OVA), only splenocytes from mice immunized with HSV DNA or HSV-ODN as the adjuvants proliferated strongly and produced typical Th1 responses, including CD8⁺ cytotoxic T-lymphocyte responses, upon restimulation with OVA. Furthermore, HSV-ODN synergized with IFN-γ to induce nitric oxide (NO), IL-6, and TNF production from macrophages. These results demonstrate that HSV DNA and HSV-ODN are immunostimulatory, driving potent Th1 responses both in vitro and in vivo. Considering that HSV DNA has been found to persist in nonneuronal cells, these results fuel speculation that HSV DNA might play a role in pathogenesis, in particular, in diseases like herpes stromal keratitis (HSK) that involve chronic inflammatory responses in the absence of virus or viral antigens.