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BBA - General Subjects (v.1790, #8)

Editorial Board (pp. i).

Verdoheme formation in Proteus mirabilis catalase by Pierre Andreoletti ⁎; Jean-Marie Mouesca; Patrice Gouet; Michel Jaquinod; Chantal Capeillère-Blandin; Hélène Marie Jouve (pp. 741-753).

Heme oxidative degradation has been extensively investigated in peroxidases but not in catalases. The verdoheme formation, a product of heme oxidation which inactivates the enzyme, was studied in Proteus mirabilis catalase.The verdoheme was generated by adding peracetic acid and analyzed by mass spectrometry and spectrophotometry.Kinetics follow-up of different catalase reactional intermediates shows that i) the formation of compound I always precedes that of verdoheme, ii) compound III is never observed, iii) the rate of compound II decomposition is not compatible with that of verdoheme formation, and iv) dithiothreitol prevents the verdoheme formation but not that of compound II, whereas NADPH prevents both of them. The formation of verdoheme is strongly inhibited by EDTA but not increased by Fe³⁺ or Cu²⁺ salts. The generation of verdoheme is facilitated by the presence of protein radicals as observed in the F194Y mutated catalase. The inability of the inactive variant (H54F) to form verdoheme, indicates that the heme oxidation is fully associated to the enzyme catalysis.These data, taken together, strongly suggest that the verdoheme formation pathway originates from compound I rather than from compound II.The autocatalytic verdoheme formation is likely to occur in vivo.

Keywords: Abbreviations; PMC; Proteus mirabilis; catalase; PR; peroxide resistant; Cpd; catalase compound; VSC; Vibrio salmonicida; catalase; BLC; bovine liver catalase; HEC; human erythrocyte catalase; SCA; Saccharomyces cerevisiae; catalase A; HRP; horseradish peroxidase; SOD; superoxide dismutase; DTT; 1,4-dithio-; dl; -threitol; IPTG; isopropyl-1-thio-β-; d; -galactopyranoside; peracetic acid; peroxoacetic acid; P723; pigment of PMC corresponding to an absorbance peak at 723 nm appearing when verdoheme is formed into the protein; Tris–HCl; tris(hydroxymethyl)aminomethane hydrochloride; NADPH; β-nicotinamide adenine dinucleotide phosphate, reduced form; NADP; ⁺; β-nicotinamide adenine dinucleotide phosphate, oxidized form; NADPCl; monochlorinated (chlorine atom at C5 and hydroxy at C6 position) NADPH; β-NMNH; β-nicotinamide mononucleotide, reduced form; EDTA; ethylenediamine-tetraacetic acid; rms; root mean square; Rz; A; _406 nm; /; A; _280 nm; , Reinheitszahl index; SDS-PAGE; sodium dodecyl sufate-polyacrylamide gel electrophoresisCatalase; Proteus mirabilis; Verdoheme; NADPH; Mass spectrometry; X-ray crystal structure

Transfection by particle bombardment: Delivery of plasmid DNA into mammalian cells using gene gun by Masaki Uchida; Xiong Wei Li; Peter Mertens; H. Oya Alpar ⁎ (pp. 754-764).

Recently, particle bombardment has become increasingly popular as a transfection method, because of a reduced dependency on target cell characteristics. In this study, we evaluated in vitro gene transfer by particle bombardment.gWIZ luciferase and gWIZ green fluorescent protein (GFP) plasmids were used as reporter genes. Mammalian cell lines HEK 293, MCF7 and NIH/3T3 were used in the transfection experiments. Transfection was performed by bombardment of the cells with gene-coated gold particles using the Helios Gene Gun. The technology was assessed by analyzing gene expression and cell damage. Cell damage was evaluated by MTT assay.This technology resulted in efficient in vitro transfection, even in the cells which are difficult to transfect. The gene expression was dependent on the gene gun's helium pressure, the sizes of the gold particles, the amount of the particles and DNA loading, while cell viability was mostly dependent on helium pressure and amount of the gold particles.This technology was useful to transfection of cells. Optimal transfection conditions were determined to be between 75 and 100 psi of helium pressure, 1.0 to 1.6 μm gold particle size and 0.5 mg of gold particle amount with a loading ratio of 4 μg DNA/mg gold particles.These findings will be useful in the design of gene gun device, and bring further improvements to the in vitro and in vivo transfection studies including gene therapy and vaccination.

Keywords: Abbreviations; gWIZ-; luc; gWIZ luciferase plasmid; GFP; green fluorescent protein; gWIZ-; GFP; gWIZ-GFP plasmid; HEK 293 cells; human embryonic kidney 293 cells; MCF7 cells; human breast adenocarcinoma cells; NIH/3T3 cells; mouse embryonic fibroblast cells; RLU; relative light units; CLSM; confocal laser scanning microscopyParticle bombardment; Helios Gene Gun; in vitro; transfection; Mammalian cell

In vivo calcium imaging of OFF-responding ASK chemosensory neurons in C. elegans by Tokumitsu Wakabayashi; Yukihiro Kimura; Yusuke Ohba; Ryota Adachi; Yoh-ichi Satoh; Ryuzo Shingai ⁎ (pp. 765-769).

How neurons and neuronal circuits transform sensory input into behavior is not well understood. Because of its well-described, simple nervous system, Caenorhabditis elegans is an ideal model organism to study this issue. Transformation of sensory signals into neural activity is a crucial first step in the sensory–motor transformation pathway in an animal's nervous system. We examined the properties of chemosensory ASK neurons of C. elegans during sensory stimulation.A genetically encoded calcium sensor protein, G-CaMP, was expressed in ASK neurons of C. elegans, and the intracellular calcium dynamics of the neurons were observed.After application of the attractantsl-lysine or food-related stimuli, the level of calcium in ASK neurons decreased. In contrast, responses increased upon stimulus removal. Opposite responses were observed after application and removal of a repellent.The observed changes in response to external stimuli suggest that the activity of ASK neurons may impact stimulus-evoked worm behavior. The stimulus-ON/activity-OFF properties of ASK neurons are similar to those of vertebrate retinal photoreceptors.Analysis of sensory–motor transformation pathways based on the activity and structure of neuronal circuits is an important goal in neurobiology and is practical in C. elegans. Our study provides insights into the mechanism of such transformation in the animal.

Keywords: G-CaMP; calcium imaging; imaging; sensory neuron; OFF-response; L; -lysine; SDS

Plagiochin E, an antifungal bis(bibenzyl), exerts its antifungal activity through mitochondrial dysfunction-induced reactive oxygen species accumulation in Candida albicans by Xiu-Zhen Wu; Ai-Xia Cheng; Ling-Mei Sun; Shu-Juan Sun; Hong-Xiang Lou ⁎ (pp. 770-777).

Plagiochin E (PLE) is an antifungal macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. Its antifungal mechanism is unknown. To elucidate the mechanism of action, its effect on mitochondria function in Candida albicans was studied.We assayed the mitochondrial membrane potential (mtΔ ψ) using rhodamine 123, measured ATP level in mitochondria by HPLC, and detected the activities of mitochondrial F₀F₁-ATPase and dehydrogenases. Besides, the mitochondrial dysfunction-induced reactive oxygen species (ROS) production was determined by a fluorometric assay, and the effects of antioxidant L-cysteine on PLE-induced ROS production and the antifungal effect of PLE on C. albicans were also investigated.Exposure to PLE resulted in an elevation of mtΔ ψ, and a decrease of ATP level in mitochondria. The ATP depletion owed to PLE-induced enhancement of mitochondrial F₀F₁-ATPase and inhibition of the mitochondrial dehydrogenases. These dysfunctions of mitochondria caused ROS accumulation in C. albicans, and this increase in the level of ROS production and PLE-induced decrease in cell viability were prevented by addition of L-cysteine, indicating that ROS was an important mediator of the antifungal action of PLE.PLE exerts its antifungal activity through mitochondrial dysfunction-induced ROS accumulation in C. albicans.The effect of PLE on the mitochondria function in C. albicans was assayed for the first time. These results would conduce to elucidate its underlying antifungal mechanism.

Keywords: ATP depletion; Candida albicans; Mitochondrial hyperpolarization; Plagiochin E; Reactive oxygen species

Modulation of lysozyme function and degradation after nitration with peroxynitrite by Tiana V. Curry-McCoy ⁎; Natalia A. Osna; Terrence M. Donohue Jr. ⁎ (pp. 778-786).

Peroxynitrite (PN) is formed from superoxide and nitric oxide, both of which are increased during hepatic ethanol metabolism. Peroxynitrite forms adducts with proteins, causing structural and functional alterations. Here, we investigated PN-induced alterations in lysozyme structure and function, and whether they altered the protein's susceptibility to proteasome-catalyzed degradation.Hen egg lysozyme was nitrated using varying amounts of either PN or the PN donor, 3-morpholinosydnonimine (SIN-1). The activity, nitration status and the susceptibility of lysozyme to proteasome-catalyzed degradation were assessed.Lysozyme nitration by PN or SIN-1 caused dose-dependent formation of 3-nitrotyrosine-lysozyme adducts, causing decreased catalytic activity, and enhanced susceptibility to degradation by the 20S proteasome. Kinetic analyses revealed an increased affinity by the 20S proteasome toward nitrated lysozyme compared with the native protein.Lysozyme nitration enhances the affinity of the modified enzyme for degradation by the proteasome, thereby increasing its susceptibility to proteolysis.Increased levels of peroxynitrite have been detected in tissues of ethanol-fed animals. The damaging effects from excessive peroxynitrite in the cell increase hepatotoxicity and cellular death by protein modification due to nitration. Cellular defenses against such changes include enhanced proteolysis by the proteasome in order to maintain protein quality control.

Keywords: Peroxynitrite; Lysozyme; 20S proteasome; Kinetics; Ethanol; Oxidative stress

Oxidation of melatonin by AAPH-derived peroxyl radicals: Evidence of a pro-oxidant effect of melatonin by Valdecir F. Ximenes; Adriano S. Pessoa; Camila Z. Padovan; Daniele C. Abrantes; Fabiana Helena F. Gomes; Michele A. Maticoli; Manoel L. de Menezes (pp. 787-792).

Melatonin is well-established as a powerful reducing agent of oxidant generated in the cell medium. We aimed to investigate how readily melatonin is oxidized by peroxyl radicals ROO⋅ generated by the thermolysis of 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and the role of glutathione (GSH) during the reaction course.Chromatographic, mass spectroscopy, and UV–visible spectrometric techniques were used to study the oxidation of melatonin by ROO⋅ or horseradish peroxidase (HRP)/H₂O₂. Our focus was the characterization of products and the study of features of the reaction.We found that N¹-acetyl- N²-formyl-5-methoxykynuramine (AFMK) and a monohydroxylated derivative of melatonin were the main products of the reaction between melatonin and ROO⋅. Higher pH or saturation of the medium with molecular oxygen increased the yield of AFMK but did not affect the reaction rate. Melatonin increased the depletion of intracellular GSH mediated by AAPH. Using the HRP/H₂O₂ as the oxidant system, the addition of melatonin promoted the oxidation of GSH to GSSG.These results show, for the first time, that melatonin radical is able to oxidize GSH.We propose that this new property of melatonin could explain or be related to the recently reported pro-oxidant activities of melatonin.

Keywords: Melatonin; N; ¹; -acetyl-; N; ²; -formyl-5-methoxykynuramine (AFMK); 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH); Glutathione; pro-oxidant activity

Morphine induces DNA damage and P53 activation in CD3⁺ T cells by Hiroshi Tsujikawa ⁎; Takehiro Shoda; Toshiyuki Mizota; Kazuhiko Fukuda (pp. 793-799).

Morphine has been shown to affect the function of immune system, but the precise mechanism remains to be elucidated. The present study was aimed to clarify the mechanism for the morphine-induced immune suppression by analyzing the direct effect of morphine on human CD3⁺ T cells.To identify genes up-regulated by action of morphine on the opioid receptor expressed in CD3⁺ T cells, PCR-select cDNA subtraction was performed by the use of total RNA from human CD3⁺ T cells treated with morphine in the presence and absence of naloxone.We show that p53 and damage-specific DNA binding protein 2 ( ddb2) genes are up-regulated by morphine in a naloxone-sensitive manner. Furthermore, the results indicate that DNA damage, quantified by apurinic–apyrimidinic site counting assay and phosphorylation of Ser-15 in P53 protein, is induced in CD3⁺ T cells by morphine in a naloxone-sensitive manner.Because it was shown that only the κ opioid receptor gene is expressed in CD3⁺ T cells in the opioid receptor family, the present study suggests that morphine induces DNA damage through the action on the κ opioid receptor, which leads to immune suppression by activation of P53-mediated signal transduction.

Keywords: Abbreviations; DDB2; damage-specific DNA binding protein 2; CD3; ⁺; T cells; peripheral human CD3 positive T cells; NKT cells; natural killer T cellsMorphine; DNA damage; P53; DDB2

Tryptophan end-tagging of antimicrobial peptides for increased potency against Pseudomonas aeruginosa by Mukesh Pasupuleti; Anna Chalupka; Matthias Mörgelin; Artur Schmidtchen; Martin Malmsten ⁎ (pp. 800-808).

Due to increasing antibiotics resistance, antimicrobial peptides (AMPs) are receiving increased attention. Pseudomonas aeruginosa is a major pathogen in this context, involved, e.g., in keratitis and wound infections. Novel bactericidal agents against this pathogen are therefore needed.Bactericidal potency was monitored by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was probed by hemolysis. Mechanistic information was obtained from assays on peptide-induced vesicle disruption and lipopolysaccharide binding.End-tagging by hydrophobic amino acids yields increased potency of AMPs against P. aeruginosa, irrespective of bacterial proteinase production. Exemplifying this by two peptides from kininogen, GKHKNKGKKNGKHNGWK and KNKGKKNGKH, potency increased with tag length, correlating to more efficient bacterial wall and vesicle rupture, and to more pronounced P. aeruginosa lipopolysaccharide binding. End-tag effects remained at high electrolyte concentration and in the presence of plasma or anionic macromolecular scavengers. The tagged peptides displayed stability against P. aeruginosa elastase, and were potent ex vivo, both in a contact lens model and in a skin wound model.End-tagging, without need for post-peptide synthesis modification, may be employed to enhance AMP potency against P. aeruginosa at maintained limited toxicity.

Keywords: Abbreviations; AMP; antimicrobial peptide; CFU; colony-forming units; CF; carboxyfluorescein; MIC; minimal inhibitory concentration assay; RDA; radial diffusion assay; TSB; trypticase soy broth; VCA; viable count analysisAntimicrobial peptide; Hydrophobic tagging; P. aeruginosa; Kininogen; Peptide; Minimal inhibitory concentration assay; Potency enhancement

The RH 421 styryl dye induced, pore model-dependent modulation of antimicrobial peptides activity in reconstituted planar membranes by Aurelia Apetrei; Loredana Mereuta; Tudor Luchian ⁎ (pp. 809-816).

Antimicrobial agents, with different pore-formation mechanisms, may be differently influenced by alteration of the dipolar electric field of a lipid membrane.By using electrophysiological measurements on reconstituted lipid membranes, we used alamethicin, melittin and magainin to report on how controlled manipulation of the membrane dipole potential by the styrylpyridinium dye RH 421 affects the kinetic and transport features of peptides within membranes.Our data demonstrate that the increase of the membrane dipole potential caused by RH 421 decreases the activity and single-channel conductance of alamethicin. Surprisingly, we found that RH 421 increases the activity of melittin and magainin, suggesting that RH 421 may contribute via electrostatic repulsions, among others, to an increase in the monolayer spontaneous curvature of the membrane. We propose that RH 421-induced dipole potential and membrane elasticity changes alter the peptide-induced channel dynamics, and the prevalence of one mechanism over the other for particular classes of peptides is dictated by the electrical and mechanical interactions which rule the pore-formation mechanism of such peptides.These results point to a novel paradigm in which electrical and mechanical effects promoted by chemicals which preferentially alter the electrostatics of the membrane, may be employed to help distinguish among various pore-formation mechanisms of membrane-permeabilizing peptides.

Keywords: Single molecule recording; Lipid membrane; Antimicrobial peptide; Dipole potential; Membrane elasticity

The cytosolic sialidase Neu2 is degraded by autophagy during myoblast atrophy by Stefania Rossi; Elena Stoppani; Wim Martinet; Andrea Bonetto; Paola Costelli; Roberta Giuliani; Francesca Colombo; Augusto Preti; Sergio Marchesini; Alessandro Fanzani ⁎ (pp. 817-828).

The sialidase Neu2 is a cytosolic enzyme which is fully expressed in mature muscle myofibers.To investigate Neu2 expression during muscle atrophy, we employed an in vitro model consisting of terminally differentiated C2C12 myotubes exposed to different pro-atrophic stimuli that triggered catabolic pathways involved in proteasome activation or autophagy.Neu2 expression was unchanged in myotubes treated with TNF-alpha, a cytokine known to activate the proteasome. However, Neu2 transcript levels and enzymatic activity were downregulated in starved or dexamethasone-treated myotubes that showed proteosomal activation and several hallmarks of macroautophagy, such as formation of autophagosomes, the accumulation of LC3 dots and bulk degradation of long-lived proteins. Neu2 activity and protein levels were rescued upon cotreatment with the lysosomotropic agent NH₄Cl, the autophagy inhibitor 3-methyladenine or cathepsin inhibitors, as well as by insulin administration, but were unaffected upon pharmacological inhibition of the proteasome. Moreover, HA- or GST-Neu2 recombinant fusion proteins were rapidly degraded in vitro by purified cathepsin L and B. Overall, we may conclude that Neu2 is degraded by lysosomal enzymes in myotubes undergoing autophagy-mediated atrophy.This study demonstrates that Neu2 enzyme degradation occurs in atrophic myotubes via macroautophagy and independently of proteasome activation.

Keywords: Abbreviations; LC3; microtubule-associated protein 1 light chain 3; IGF-1; insulin-like growth factor 1; IL-4; interleukin 4; DMEM; Dulbecco's modified Eagle's Medium; PBS; phosphate buffer solution; MyHC; myosin heavy chain; IPTG; isopropyl-beta-; d; -thiogalactopyranoside; MMLV-RT; Moloney Murine Leukemia Virus Reverse Transcriptase; TNF-alpha; Tumor Necrosis Factor-alphaSialidases; C2C12 myoblasts; Atrophy; Autophagy; Lysosomes; Cathepsin

Small molecule–RNA interaction: Spectroscopic and calorimetric studies on the binding by the cytotoxic protoberberine alkaloid coralyne to single stranded polyribonucleotides by Md. Maidul Islam; Gopinatha Suresh Kumar (pp. 829-839).

RNA has attracted recent attention for its key role in gene expression and hence targeting by small molecules for therapeutic intervention. This study is aimed to elucidate the specificity of the alkaloid coralyne to poly(G), poly(C), poly(I) and poly(U) in the light of its ability in inducing self-structure in poly(A).Multifaceted experimental techniques like competition dialysis, absorption, fluorescence, circular dichroism and calorimetry were employed. Salt dependence and temperature dependence of the binding was also elucidated.Results of competition dialysis, absorption and fluorescence studies revealed that coralyne binds strongly to the polypurines, poly(G) and poly(I) compared to the polypyrimidines, poly(U) and poly(C). Partial intercalative binding due to the stacking of the molecules between the bases was envisaged. The binding was predominantly enthalpy driven with favourable entropy term with a large favourable non-electrostatic contribution revealed from salt dependent data and the dissection of the free energy. The heat capacity change of −125 and −119 cal/mol K−¹ respectively for poly(G) and poly(I) and the partial enthalpy–entropy compensation phenomenon observed confirmed the involvement of multiple weak noncovalent interactions. Circular dichroism studies provided evidence for significant perturbation of the conformation of the RNAs, but no self-structure induction was evident in any of the polymers under the condition of the study.This study presents a complete structural and thermodynamic profile of coralyne interaction to four single stranded RNA polymers.The study for the first time elucidates the base specificity of coralyne–RNA complexation at the single stranded level.

Keywords: ss RNAs; Coralyne interaction; Dialysis; Spectroscopy; Thermodynamics

Induction of apoptosis by Polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma L929 cells by Bo Liu; Bo Zhang; Ming-wei Min; He-jiao Bian; Long-fei Chen; Qian Liu; Jin-ku Bao ⁎ (pp. 840-844).

The Galanthus nivalis agglutinin (GNA)-related lectins have been reported to bear antiproliferative and apoptosis-inducing activities in cancer cells; however, the precise mechanisms by which GNA-related lectins induce cell death are still only rudimentarily understood.In the present study, Polygonatum odoratum lectin (designated POL), a mannose-binding specific GNA-related lectin, possessed a remarkable antiproliferative activity toward murine fibrosarcoma L929 cells. And, this lectin induced L929 cell apoptosis in a caspase-dependent manner. In addition, POL treatment increased the levels of FasL and Fas-Associated protein with Death Domain (FADD) proteins and resulted in caspase-8 activation. Also, POL treatment caused mitochondrial transmembrane potential collapse and cytochrome c release, leading to activations of caspase-9 and caspase-3. Moreover, POL treatment enhanced tumor necrosis factor α (TNFα)-induced L929 cell apoptosis.Our data demonstrate for the first time that this lectin induces apoptosis through both death-receptor and mitochondrial pathways, as well as amplifies TNFα-induced L929 cell apoptosis.These inspiring findings would provide new molecular basis for further understanding cell death mechanisms of the Galanthus nivalis agglutinin (GNA)-related lectins in future cancer investigations.

Keywords: The; Galanthus nivalis; agglutinin (GNA)-related lectins; Polygonatum odoratum; Lectin (POL); Apoptosis; Death-receptor; Mitochondria; Tumor necrosis factor α (TNFα)

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BBA - General Subjects (v.1790, #8)