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BBA - General Subjects (v.1790, #4)
How to make siRNA lipoplexes efficient? Add a DNA cargo by Hervé Rhinn; Céline Largeau; Pascal Bigey; René Lai Kuen; Magali Richard; Daniel Scherman; Virginie Escriou ⁎ (pp. 219-230).
We recently reported an efficient formulation of siRNA targeting TNF-α, that was able to restore immunological balance in a mouse arthritis model following intravenous injection.Since this efficient formulation included the pre association of siRNA with a DNA cargo, we decided to extensively characterise siRNA lipoplexes with or without DNA cargo, in order to better understand the DNA cargo enhancing effect.We showed that addition of DNA cargo to siRNA lipoplexes led to specific gene extinction in vitro, using reduced siRNA concentration. This procedure is also applicable to other lipid vectors, like Lipofectamine or DMRIE-C. No structural modification could be observed in siRNA lipoplexes upon addition of DNA cargo using dynamic light scattering or transmission electronic microscopy. Nevertheless, we observed some slight differences, in the amount of lipid required to obtain neutrality of the complex and in stability of the complex towards incubation with heparan sulfate.These results suggest that the addition of DNA cargo to siRNA complexes is an easy procedure that leads to more efficient complexes to transfer siRNA at low concentration and in the presence of serum.
Keywords: RNA interference; siRNA; Lipoplex; Cargo; Cationic lipid; Gene silencing
Structural and catalytic roles of residues located in β13 strand and the following β-turn loop in Fibrobacter succinogenes 1,3-1,4-β-d-glucanase by Yu-Shiun Lin; Li-Chu Tsai; Shu-Hua Lee; Hanna S. Yuan; Lie-Fen Shyur (pp. 231-239).
Fibrobacter succinogenes 1,3-1,4-β-d-glucanase (Fsβ-glucanase) is the only naturally occurring circularly permuted β-glucanase among bacterial glucanases with reverse protein domains. We characterized the functional and structural significance of residues 200–209 located in the domain B of Fsβ-glucanase, corresponding to the major surface loop in the domain A region of Bacillus licheniformis glucanase.Rational design approaches including site-directed mutagenesis, initial-rate kinetics, and structural modeling analysis were used in this study.Our kinetic data showed that D202N and D206N exhibited a 1.8- and 1.5-fold increase but G207N, G207−, F205L, N208G and T204F showed a 7.0- to 2.2-fold decrease, in catalytic efficiency ( kcat/ KM) compared to the wild-type enzyme. The comparative energy ΔΔGb value in individual mutant enzymes was well correlated to their catalytic efficiency. D206R mutant enzyme exhibited the highest relative activity at 50 °C over 10 min, whereas K200F was the most heat-sensitive enzyme.This study demonstrates that Phe205, Gly207, and Asn208 in the Type II turn of the connecting loop may play a role in the catalytic function of Fsβ-glucanase.Residues 200–209 in Fsβ-glucanase resided at the similar structural topology to that of Bacillus enzyme were found to play some similar catalytic function in glucanase.
Keywords: 1,3-1,4-β-; d; -glucanase; Fibrobacter succinogenes; Catalytic efficiency; Comparative energy ΔΔG; b; Structural modeling
Olive phenols efficiently inhibit the oxidation of serum albumin-bound linoleic acid and butyrylcholine esterase by Marjolaine Roche; Claire Dufour ⁎; Michèle Loonis; Marianne Reist; Pierre-Alain Carrupt; Olivier Dangles (pp. 240-248).
Olive phenols are widely consumed in the Mediterranean diet and can be detected in human plasma. Here, the capacity of olive phenols and plasma metabolites to inhibit lipid and protein oxidations is investigated in two plasma models.The accumulation of lipid oxidation products issued from the oxidation of linoleic acid bound to human serum albumin (HSA) by AAPH-derived peroxyl radicals is evaluated in the presence and absence of phenolic antioxidants. Phenol binding to HSA is addressed by quenching of the Trp214 fluorescence and displacement of probes (quercetin, dansylsarcosine and dansylamide). Next, the esterase activity of HSA-bound butyrylcholine esterase (BChE) is used as a marker of protein oxidative degradation.Hydroxytyrosol, oleuropein, caffeic and chlorogenic acids inhibit lipid peroxidation as well as HSA-bound BChE as efficiently as the potent flavonol quercetin. Hydroxycinnamic derivatives bind noncompetitively HSA subdomain IIA whereas no clear site could be identified for hydroxytyrosol derivatives.In both models, olive phenols and their metabolites are much more efficient inhibitors of lipid and protein oxidations compared to vitamins C and E. Low postprandial concentrations of olive phenols may help to preserve the integrity of functional proteins and delay the appearance of toxic lipid oxidation products.
Keywords: Abbreviations; HSA; human serum albumin; BChE; butyrylcholine esterase; PUFA; polyunsatured fatty acid; HODE; hydroxyoctadecadienoic acid; HPODE; hydroperoxyoctadecadienoic acid; KODE; oxooctadecadienoic acid; AAPH; 2,2′-azo-bis(2-methylpropionamidine dihydrochloride)Antioxidant; Olive phenols; Serum albumin; Butyrylcholine esterase; Lipid peroxidation; Binding site
Multivalent human blood group ABH and Lewis glycotopes are key recognition factors for alFuc>Man binding lectin from phytopathogenic Ralstonia solanacearum by Albert M. Wu ⁎; June H. Wu; Tanuja Singh; Biswajit Singha; Dvora Sudakevitz; Nechama Gilboa-Garber (pp. 249-259).
Ralstonia solanacearum lectin (RSL), that might be involved in phytopathogenicity, has been defined aslFuc≫Man specific. However, the effects of polyvalency of glycotopes and mammalian structural units on binding have not been established. In this study, recognition factors of RSL were comprehensively examined with natural multivalent glycotopes and monomeric ligands using enzyme linked lectin-sorbent and inhibition assays. Among the glycans tested, RSL reacted strongly with multivalent blood groupAh (GalNAcα1–3[Fucα1–2]Gal) andH (Fucα1–2Gal) active glycotopes, followed byBh (Galα1–3[Fucα1–2]Gal),Lea (Galβ1–3[Fucα1–4]GlcNAc) andLeb (Fucα1–2Galβ1–3[Fucα1–4]GlcNAc) active glycotopes. But weak or negligible binding was observed for blood group precursors having Galβ1–3/4GlcNAcβ1- (Iβ/IIβ) residues or Galβ1–3GalNAcα1- (Tα), GalNAcα1-Ser/Thr (Tn) bearing glycoproteins. These results indicate that the density and degree of exposure of multivalent ligands of α1–2 linkedlFuc to Gal at the non-reducing end is the most critical factor for binding. An inhibition study with monomeric ligands revealed that the combining site of RSL should be of a groove type to fit trisaccharide binding with highest complementarity to blood groupH trisaccharide (HL; Fucα1–2Galβ1–4Glc). The outstandingly broad RSL saccharide-binding profile might be related to the unusually wide spectrum of plants that suffer from R. solanacearum pathogenicity and provide ideas for protective antiadhesion strategies.
Keywords: Binding property; Multivalent glycotope; Human blood group; Bacterial lectin; Ralstonia solanacearum; RSL
Implication of mitogen-activated protein kinase in the induction of G1 cell cycle arrest and gadd45 expression by the carotenoid fucoxanthin in human cancer cells by Yoshiko Satomi; Hoyoku Nishino (pp. 260-266).
The precise mechanism of the anti-tumor action of fucoxanthin has yet to be elucidated. We previously reported that gadd45a and gadd45b might play a role in the G1 arrest induced by fucoxanthin. In the present study, we show that several MAPKs modulate the induction of gadd45 and G1 arrest.HepG2 and DU145 cells were used. The cell cycle was analyzed using flow cytometry. Expression of gadd45 was assayed by Northern blot and/or quantitative RT-PCR analyses. Activation of MAPK was assayed by Western blot analysis.Inhibition of p38 MAPK enhanced the induction of gadd45a expression and G1 arrest by fucoxanthin in HepG2 cells. Inhibition of ERK enhanced gadd45b expression but had no effect on the induction of G1 arrest by fucoxanthin in HepG2 cells. Inhibition of SAPK/JNK suppressed the induction of gadd45a expression and G1 arrest by fucoxanthin in DU145 cells.These data suggest that gadd45a is closely related with the G1 arrest induced by fucoxanthin, and that the pattern of MAPK involvement in the induction of gadd45a and G1 arrest by fucoxanthin differs depending on the cell type.The implication of GADD45 and MAPK involvement in the anti-tumor action of carotenoids is first described.
Keywords: Carotenoid; Fucoxanthin; GADD45; MAPK; G1 arrest
Polymorphism distribution and structural conservation in RNA-sensing Toll-like receptors 3, 7, and 8 in pigs by Takeya Morozumi; Hirohide Uenishi ⁎ (pp. 267-274).
Viral genomic RNA—both single-stranded (ss) and double-stranded (ds)—is recognized by RNA-sensing Toll-like receptors (TLRs), notably TLR3 (dsRNA), TLR7 (ssRNA), and TLR8 (ssRNA). However, our knowledge of the roles of porcine TLR3, 7, and 8 in antiviral immunity is inadequate.From information on exon–intron boundaries obtained through comparisons of the genomic and cDNA sequences, polymorphisms in the coding sequences of each gene were detected in 84 male pigs of 11 breeds.Genomic structures are conserved between pigs and humans. The RNA-sensing TLR genes had fewer polymorphisms causing amino acid alterations than did the cell-surface TLR genes, but the alterations were distributed with a similar bias toward ectodomains.The low level of diversity of substitutive polymorphisms in RNA-sensing TLRs than cell-surface ones implies that polymorphisms severely affecting function have been eliminated by selection pressure during longstanding pig breeding.Recognition of virus-derived RNA is critical in host defense against infection. These results should provide a useful clue to analysis of the association between polymorphisms in RNA-sensing TLRs and disease resistance.
Keywords: Swine; Toll-like receptor; Genomic structure; Single nucleotide polymorphism
Protective protein/cathepsin A rescues N-glycosylation defects in neuraminidase-1 by Dongning Wang; Slava Zaitsev; Garry Taylor; Alessandra d'Azzo; Erik Bonten ⁎ (pp. 275-282).
Neuraminidase-1 (NEU1) catabolizes the hydrolysis of sialic acids from sialo-glycoconjugates. NEU1 depends on its interaction with the protective protein/cathepsin A (PPCA) for lysosomal compartmentalization and catalytic activation. Murine NEU1 contains 4 N-glycosylation sites, 3 of which are conserved in the human enzyme. The expression of NEU1 gives rise to differentially glycosylated proteins.We generated single-point mutations in mouse NEU1 at each of the 4 N-glycosylation sites. Mutant enzymes were expressed in NEU1-deficient cells in the presence and absence of PPCA.All 4 N-glycosylation variants were targeted to the lysosomal/endosomal compartment. All N-glycans, with the exception of the most C-terminal glycan, were important for maintaining stability or catalytic activity. The loss of catalytic activity caused by the deletion of the second N-glycan was rescued by increasing PPCA expression. Similar results were obtained with a human NEU1 N-glycosylation mutant identified in a sialidosis patient. The N-terminal N-glycan of NEU1 is indispensable for its function, whereas the C-terminal N-glycan appears to be non-essential. The omission of the second N-glycan can be compensated for by upregulating the expression of PPCA.These findings could be relevant for the design of target therapies for patients carrying specific NEU1 mutations.
Keywords: Abbreviations; D; aspartic acid; DMEM; Dulbecco's modified Eagle's medium; DTT; dl; -dithio-threitol; FACS; fluorescence activated cell sorting; FBS; fetal bovine serum; GFP; green fluorescent protein; LSD; lysosomal storage disease; N, asparagine NEU1; lysosomal neuraminidase-1; PCR; polymerase chain reaction; PPCA; protective protein/cathepsin A; YFP; yellow fluorescent proteinGlycosylation; Sialidosis; Chaperone therapy; Neuraminidase; Sialidase; NEU1; Protective protein/cathepsin A