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BBA - General Subjects (v.1820, #12)

Editorial Board (pp. i).

Antimicrobial peptide pleurocidin synergizes with antibiotics through hydroxyl radical formation and membrane damage, and exerts antibiofilm activity by Hyemin Choi; Dong Gun Lee (pp. 1831-1838).

Pleurocidin, a 25-mer antimicrobial peptide (AMP), is known to exert bactericidal activity. However, the synergistic activity and mechanism(s) of pleurocidin in combination with conventional antibiotics, and the antibiofilm effect of the peptide are poorly understood.The interaction between pleurocidin and antibiotics was evaluated using checkerboard assay. To study the mechanism(s) involved in their synergism, we detected hydroxyl radical formation using 3′-(p-hydroxyphenyl) fluorescein, measured the NAD⁺/NADH ratio by NAD⁺ cycling assay, observed change in bacterial viability with the hydroxyl radical scavenger thiourea, and investigated cytoplasmic membrane damage using propidium iodide. Also, the antibiofilm effect of pleurocidin was examined with the tissue culture plate method.All combinations of pleurocidin and antibiotics showed synergistic interaction against bacterial strains (fractional inhibitory concentration index (FICI)≤0.5) except for Enterococcus faecium treated with a combination of the peptide and ampicillin (FICI=0.75). We identified that pleurocidin alone and in combinations with antibiotics induced formation of hydroxyl radicals. The oxidative stress was caused by a transient NADH depletion and the addition of thiourea prevented bacterial death, especially in the case of the combined treatment of pleurocidin and ampicillin showing synergisms. The combination of pleurocidin and erythromycin increased permeability of bacterial cytoplasmic membrane. Additionally, pleurocidin exhibited a potent inhibitory effect on preformed biofilm of bacterial organisms. In conclusion, pleurocidin synergized with antibiotics through hydroxyl radical formation and membrane-active mechanism, and exerted antibiofilm activity.The synergistic effect between pleurocidin and antibiotics suggests the AMP is a potential therapeutic agent and adjuvant for antimicrobial chemotherapy.► We aimed to evaluate the combination effect of pleurocidin with antibiotics. ► Most combinations between pleurocidin and antibiotics showed synergistic effects. ► We investigated the mechanism(s) involved in the synergistic effects. ► The synergisms were induced by hydroxyl radical production and membrane damage. ► Additionally, we indentified that pleurocidin exerted antibiofilm effect.

Keywords: Abbreviations; AMPs; antimicrobial peptides; EPS; extracellular polymeric substance; CH; ₃; CN; acetonitrile; LB; Luria Bertani; CLSI; Clinical and Laboratory Standards Institute; MIC; minimum inhibitory concentration; NCCLS; National Committee for Clinical Laboratory Standards; FICI; fractional inhibitory concentration index; HPF; 3′-(p-hydroxyphenyl) fluorescein; PES; phenazine ethosulfate; MTT; 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; PBS; phosphate buffered saline; PI; propidium iodide; TCP; tissue culture plate; BHI; brain–heart infusionPleurocidin; Pleuronectes americanus; Synergistic effect; Antibiofilm effect; Hydroxyl radical; Membrane damage

Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition by Ludivine Drougat; Stéphanie Olivier-Van Stichelen; Marlène Mortuaire; François Foulquier; Anne-Sophie Lacoste; Jean-Claude Michalski; Tony Lefebvre; Anne-Sophie Vercoutter-Edouart (pp. 1839-1848).

DNA replication represents a critical step of the cell cycle which requires highly controlled and ordered regulatory mechanisms to ensure the integrity of genome duplication. Among a plethora of elements, post-translational modifications (PTMs) ensure the spatiotemporal regulation of pivotal proteins orchestrating cell division. Despite increasing evidences showing that O-GlcNAcylation regulates mitotic events, the impact of this PTM in the early steps of the cell cycle remains poorly understood.Quiescent MCF7 cells were stimulated by serum mitogens and cell cycle progression was determined by flow cytometry. The levels of O-GlcNAc modified proteins, O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) were examined by Western blotting and OGA activity was measured during the progression of cells towards S phase. A global decrease in O-GlcNAcylation was observed at S phase entry, concomitantly to an increase in the activity of OGA. A combination of two-dimensional electrophoresis, Western blotting and mass spectrometry was then used to detect and identify cell cycle-dependent putative O-GlcNAcylated proteins. 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated through G1/S transition were identified and the O-GlcNAc variations of Cytokeratin 8, hnRNP K, Caprin-1, Minichromosome Maintenance proteins MCM3, MCM6 and MCM7 were validated by immunoprecipitation.The dynamics of O-GlcNAc is regulated during G1/S transition and observed on key proteins involved in the cytoskeleton networks, mRNA processing, translation, protein folding and DNA replication.Our results led us to propose that O-GlcNAcylation joins the PTMs that take part in the regulation of DNA replication initiation.► S phase entry is correlated with a marked decrease of O-GlcNAc modified proteins ► O-GlcNAcase activity increases at the G1/S transition ► Identification of 58 differentially putative O-GlcNAcylated proteins during G1/S transition ► We report cell-cycle dependent O-GlcNAcylation of nucleoplasmic DNA licensing factors MCMs ► O-GlcNAcylation may regulate DNA replication initiation

Keywords: Abbreviations; CDK; Cyclin dependent kinase; CHX; Cycloheximide; CK; Cytokeratin; hnRNP; Heterogeneous nuclear ribonucleoprotein; HU; Hydroxyurea; MCM; Minichromosome maintenance; O; -GlcNAc; O; -linked beta-; N; -acetylglucosaminylation; OGA; O; -; GlcNAcase or beta-; N; -; acetylglucosaminidase; OGT; O; -GlcNAc transferase; PTM; Post-translational modification; pRb; Retinoblastoma protein; UDP-GlcNAc; Uridine diphosphate; N; -acetylglucosamine O; -GlcNAc; Post-translational modifications; G1/S transition; MCM protein; Caprin-1; hnRNP

Photochemical internalization (PCI) of HER2-targeted toxins by Maria Brandal Berstad; Anette Weyergang; Kristian Berg (pp. 1849-1858).

Photochemical internalization (PCI) is a modality for cytosolic release of drugs trapped in endocytic vesicles. The method is based upon photosensitizers localized in the membranes of endocytic vesicles which create membrane rupture upon light exposure by generating reactive oxygen species (ROS), predominantly singlet oxygen (¹O₂).The human epidermal growth factor receptor 2 (HER2)-targeted immunotoxin (IT), trastuzumab–saporin, was evaluated in combination with PCI using TPCS_2a (Amphinex®), a new photosensitizer approved for clinical use.PCI synergistically enhanced the cytotoxicity of trastuzumab–saporin on trastuzumab-resistant HER2⁺ Zr-75-1 cells. The PCI effect was only observed when the IT was administered prior to the photochemical treatment (“light after” strategy), while administration of a non-targeted drug may equally well be performed after light exposure. Mechanistic studies showed reduced ligand-induced HER2 phosphorylation and receptor-mediated endocytosis after TPCS_2a-PDT. Photochemical disruption of the cytoplasmic domain of HER2 was found to be induced by¹O₂ generated both by photosensitizer located in the endocytic vesicles and in the outer leaflet of the plasma membrane.Administration of the HER2-targeted toxin prior to light exposure is a prerequisite for successful PCI-mediated delivery of HER2-targeted toxins.PCI of HER2-targeted toxins is demonstrated as a highly effective treatment modality which may overcome trastuzumab resistance. The mechanistic studies of the lack of PCI effect of the “light first” procedure is of outermost importance when designing a clinical PCI treatment protocol for delivery of HER2-targeted therapies.Display Omitted► This is the first report on photochemical internalization (PCI) of a HER2-targeted immunotoxin. ► Synergistic cytotoxicity of PCI of trastuzumab–saporin in trastuzumab-resistant Zr-75-1 cells ► Photochemical effects on HER2-mediated endocytosis with impact on PCI on trastuzumab–saporin ► PCI effect only observed when the immunotoxin is administered prior to the photochemical treatment ► PCI of trastuzumab–saporin; a method to treat HER2+ and trastuzumab-resistant breast cancer

Keywords: Human epidermal growth factor receptor 2; Trastuzumab; Saporin; Immunotoxin; Photochemical; Photodynamic therapy

Entamoeba histolytica thioredoxin reductase: Molecular and functional characterization of its atypical properties by Diego G. Arias; Erika L. Regner; Alberto A. Iglesias; Sergio A. Guerrero (pp. 1859-1866).

Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. Thioredoxin reductase catalyzes the reversible transfer of reducing equivalents between NADPH and thioredoxin, a small protein that plays key metabolic functions in maintaining the intracellular redox balance.The present work deals with in vitro steady state kinetic studies aimed to reach a better understanding of the kinetic and structural properties of thioredoxin reductase from E. histolytica ( EhTRXR).Our results support that native EhTRXR is a homodimeric covalent protein that is able to catalyze the NAD(P)H-dependent reduction of amoebic thioredoxins and S‐nitrosothiols. In addition, the enzyme exhibited NAD(P)H dependent oxidase activity, which generates hydrogen peroxide from molecular oxygen. The enzyme can reduce compounds like methylene blue, quinones, ferricyanide or nitro-derivatives; all alternative substrates displaying a relative high capacity to inhibit disulfide reductase activity of EhTRXR.Interestingly, EhTRXR exhibited kinetic and structural properties that differ from other low molecular weight TRXR. The TRX system could play an important role in the parasite defense against reactive species. The latter should be critical during the extra intestinal phase of the amoebic infection. So far we know, this is the first in depth characterization of EhTRXR activity and functionality.► The amoebic enzyme can use NADPH or NADH as substrates in μM concentrations. ► EhTRXR catalyzes the thioredoxin-independent reduction of S‐nitrosothiols. ► EhTRXR can use non-physiological substrates with similar catalytic efficiency.

Keywords: Abbreviations; TRXR; thioredoxin reductase; TRX; thioredoxin; GSNO; S; ‐nitrosoglutathione; CySNO; S; -nitrosocysteine; MBQ; 2-methyl-benzoquinone; DTBBQ; 2-5-ditert-buthyl-benzoquinone; DTNB; 5-5´-dithio-nitrobenzoic acid; MB; methylene blue; MV; methyl viologen; CDNB; 1-chloro-2,4-dinitrobenzene Entamoeba; Thioredoxin; S; -nitrosothiol; Antioxidant; Reducing power

Extracellular ATP induces cell death in human intestinal epithelial cells by Carolina O. Souza; Giani F. Santoro; Vanessa R. Figliuolo; Hayandra Ferreira Nanini; Heitor S.P. de Souza; Morgana Teixeira Lima Castelo-Branco; Alessandra Alves Abalo; Mauricio M. Paiva; Claudia M.L.M. Coutinho; Robson Coutinho-Silva (pp. 1867-1878).

Extracellular ATP is an endogenous signaling molecule released by various cell types and under different stimuli. High concentrations of ATP released into the extracellular medium activate the P2X7 receptor in most inflammatory conditions. Here, we seek to characterize the effects of ATP in human intestinal epithelial cells and to evaluate morphological changes in these cells in the presence of ATP.We treated human intestinal epithelial cells with ATP and evaluated the effects of this nucleotide by scanning and transmission electron microscopy analysis and calcium measurements. We used flow cytometry to evaluate apoptosis. We collected human intestinal explants for immunohistochemistry, apoptosis by the TUNEL approach and caspase-3 activity using flow cytometry analyses. We also evaluated the ROS production by flow cytometry and NO secretion by the Griess technique.ATP treatment induced changes characteristic of cell death by apoptosis and autophagy but not necrosis in the HCT8 cell line. ATP induced apoptosis in human intestinal explants that showed TUNEL-positive cells in the epithelium and in the lamina propria. The explants exhibited a significant increase of caspase-3 activity when the colonic epithelial cells were incubated with IFN-gamma followed by ATP as compared to control cells. In addition, it was found that antioxidants were able to inhibit both the ROS production and the apoptosis induced by ATP in epithelial cells.The activation of P2X7 receptors by ATP induces apoptosis and autophagy in human epithelial cells, possibly via ROS production, and this effect might have implications for gut inflammatory conditions.► Extracellular ATP induces apoptosis and autophagy in human intestinal epithelial cells. ► Extracellular ATP induces caspase-3 activation and apoptosis in explants of human gut epithelial tissue from health donors. ► Inflammatory condition can increase the ATP effects in human intestinal epithelial cells.

Keywords: Intestinal epithelial cell; Adenosine triphosphate; Purinergic signaling; Cell death

Calorimetry-based profiling of blood plasma from colorectal cancer patients by Svetla Todinova; Sashka Krumova; Panayot Kurtev; Valentin Dimitrov; Lachezar Djongov; Zlate Dudunkov; Stefka G. Taneva (pp. 1879-1885).

Differential scanning calorimetry (DSC), a highly sensitive technique for resolving thermally-induced protein folding/unfolding transitions, recently was recognized as a novel tool for disease diagnosis and monitoring. To further elaborate this approach we have applied DSC in a study of blood plasma from patients with colorectal cancer (CRC) at different stages of tumor development and localization.Blood plasma from patients diagnosed with CRC was analyzed by DSC. The CRC thermograms were compared to those of healthy individuals and patients with gastric cancer and non-cancerous soft tissue inflammation. The thermodynamic parameters: excess heat capacity and enthalpy of the transitions corresponding to the most abundant plasma proteins, as well as transition and first moment temperatures were determined from the calorimetric profiles.The calorimetric profiles of blood plasma from CRC patients are found to be distinct from those of healthy individuals and those of patients with soft tissue, non-cancerous inflammation. Generally the CRC thermograms exhibit reduced heat capacity of the major albumin/globulin-assigned thermal transitions, lower enthalpy and a featureless high temperature region compared to healthy individuals.A classification of blood plasma proteome from patients with colorectal cancer (CRC1, CRC2 and CRC3 groups, and subgroups within each group CRC1_1–2, CRC2_1–2 and CRC3_1–2) is proposed based on the derived thermodynamic parameters.The presented data demonstrate a proof of principle and confirm that the DSC technique has a potential to monitor changes in the CRC blood plasma proteome. This study is a further step toward the validation of calorimetry as a diagnostic tool.Display Omitted► DSC reveals a strongly modified thermal behavior of blood plasma from CRC patients. ► The heat capacity of the major thermal transitions is altered in CRC thermograms. ► The high temperature region is featureless for majority of the CRC thermograms. ► The CRC blood plasma thermograms are classified in three groups/six subgroups. ► DSC is a sensitive tool for monitoring changes in CRC blood plasma proteome.

Keywords: Abbreviations; DSC; differential scanning calorimetry; CRC; colorectal cancer; GC; gastric cancer; STI; non-cancerous soft tissue inflammation; TNM; (tumor, lymph nodes, metastasis) stage; T; _max; temperature maximum of the transitions; T; _FM; temperature of the first moment; c; _P; ^ex; excess heat capacity; ΔH; _cal; total enthalpyDifferential scanning calorimetry; Colorectal cancer; Blood plasma; Thermal transition; Excess heat capacity; Transition temperature

Different mechanisms of hepatitis C virus RNA polymerase activation by cyclophilin A and B in vitro by Leiyun Weng; Xiao Tian; Yayi Gao; Koichi Watashi; Kunitada Shimotohno; Takaji Wakita; Michinori Kohara; Tetsuya Toyoda (pp. 1886-1892).

Cyclophilins (CyPs) are cellular proteins that are essential to hepatitis C virus (HCV) replication. Since cyclosporine A was discovered to inhibit HCV infection, the CyP pathway contributing to HCV replication is a potential attractive stratagem for controlling HCV infection. Among them, CyPA is accepted to interact with HCV nonstructural protein (NS) 5A, although interaction of CyPB and NS5B, an RNA-dependent RNA polymerase (RdRp), was proposed first.CyPA, CyPB, and HCV RdRp were expressed in bacteria and purified using combination column chromatography. HCV RdRp activity was analyzed in vitro with purified CyPA and CyPB.CyPA at a high concentration (50× higher than that of RdRp) but not at low concentration activated HCV RdRp. CyPB had an allosteric effect on genotype 1b RdRp activation. CyPB showed genotype specificity and activated genotype 1b and J6CF (2a) RdRps but not genotype 1a or JFH1 (2a) RdRps. CyPA activated RdRps of genotypes 1a, 1b, and 2a. CyPB may also support HCV genotype 1b replication within the infected cells, although its knockdown effect on HCV 1b replicon activity was controversial in earlier reports.CyPA activated HCV RdRp at the early stages of transcription, including template RNA binding. CyPB also activated genotype 1b RdRp. However, their activation mechanisms are different.These data suggest that both CyPA and CyPB are excellent targets for the treatment of HCV 1b, which shows the greatest resistance to interferon and ribavirin combination therapy.► Cyclophilin A (CyPA) and B (CyPB) activated HCV RNA polymerase (RdRp). ► CyPA activated all the HCV RdRps so far tested. ► CyPA activation required 50× higher amount than that of RdRp. ► CyPB activated genotype 1b and J6CF RdRps but not genotype 1a or JFH1 RdRps. ► CyPB showed allosteric effect on the HCV RdRp activation.

Keywords: Abbreviations; BSA; bovine serum albumin; CsA; cyclosporine A; CyP; cyclophilin; DTT; dithiothreitol; E; envelope; EDTA; ethylenediaminetetraacetic acid; GST; glutathione S-transferase; HCV; hepatitis C virus; NS; nonstructural protein; PPI; peptidyl prolyl; cis; /; trans; -isomerases; Peg-IFN; pegylated interferon-α; PMSF; phenylmethanesulfonylfluoride; RT-PCR; reverse transcription polymerase chain reaction; RdRp; RNA-dependent RNA polymerase; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis; SVR; sustained virological response; ΔPPI; PPI knockout; wt; wild typeHCV; RNA polymerase; Cyclophilin A; Cyclophilin B

Depletion of SUMO ligase hMMS21 impairs G1 to S transition in MCF-7 breast cancer cells by Huey-Juin Ni; Ying-Nang Chang; Pu-Hong Kao; Shin-Pei Chai; Ya-Hsin Hsieh; Duo-Hsiang Wang; Jim C. Fong (pp. 1893-1900).

hMMS21 is a human SUMO ligase required for DNA damage repair and mitotic progression in HeLa cervical cancer cells. Owing to the diversity of cancer, we further investigated the effect of hMMS21-depletion on MCF-7 breast cancer cells.hMMS21-depletion was achieved by RNA interference. Cellular hMMS21 and E2F1 mRNA levels were estimated by RT-PCR and real-time PCR. Cell cycle profile was assessed by flow cytometry. Western blot and co-immunoprecipitation were used to determine the protein levels of various factors involved in G1–S transition and CDK2- or CDK4-associated p21 and p27. Kinase activity of cyclin E/CDK2 was measured in anti-cyclin E immunoprecipitate.hMMS21-depletion induced slower cell growth and G1–S transition. While it had no effect on cyclin D1 or phospho-Rb (S807/811) levels, hMMS21-depletion provoked lower E2F1 levels and cyclin E/CDK2 activity. The decreased cyclin E/CDK2 activity correlated with increased cellular p21^CIP1 levels and CDK2–p21 association. Moreover, ectopic expression of Flag-hMMS21 but not its ligase-inactive mutant rescued the decreased growth rates of hMMS21-depletd cells. Thus, depletion of hMMS21 seems to impair G1–S transition due to lowered E2F1 protein levels and cyclin E/CDK2 activity. The decreased cyclin E/CDK2 activity is probably attributable to its greater association with p21 as a result of increased p21 levels. In addition, hMMS21-mediated sumoylation appears to be involved.This study demonstrates that hMMS21 is required for G1–S transition in breast cancer cells and implies that manipulation of hMMS21-mediated sumoylation may alter the growth rates of breast cancer cells.► hMMS21-depletion slows MCF-7 breast cancer cell growth. ► hMMS21-depletion impairs G1–S progression. ► hMMS21-depletion lowers E2F1 expression and cyclin E/CDK2 activity. ► hMMS21-depletion increases p21 expression and CDK2-p21 association. ► SUMO ligase activity of hMMS21 is involved.

Keywords: MMS21; G1–S transition; Cyclin E/CDK2; E2F1; MCF-7; p21

Cytosolic ascorbate peroxidase 1 protects organelles against oxidative stress by wounding- and jasmonate-induced H₂O₂ in Arabidopsis plants by Takanori Maruta; Takahiro Inoue; Masahiro Noshi; Masahiro Tamoi; Yukinori Yabuta; Kazuya Yoshimura; Takahiro Ishikawa; Shigeru Shigeoka (pp. 1901-1907).

Reactive oxygen species (ROS) are not only cytotoxic compounds leading to oxidative damage, but also signaling molecules for regulating plant responses to stress and hormones. Arabidopsis cytosolic ascorbate peroxidase 1 (APX1) is thought to be a central regulator for cellular ROS levels. However, it remains unclear whether APX1 is involved in plant tolerance to wounding and methyl jasmonate (MeJA) treatment, which are known to enhance ROS production.We studied the effect of wounding and MeJA treatment on the levels of H₂O₂ and oxidative damage in the Arabidopsis wild-type plants and knockout mutants lacking APX1 (KO-APX1).The KO-APX1 plants showed high sensitivity to wounding and MeJA treatment. In the leaves of wild-type plants, H₂O₂ accumulated only in the vicinity of the wound, while in the leaves of the KO-APX1 plants it accumulated extensively from damaged to undamaged regions. During MeJA treatment, the levels of H₂O₂ were much higher in the leaves of KO-APX1 plants. Oxidative damage in the chloroplasts and nucleus was also enhanced in the leaves of KO-APX1 plants. These findings suggest that APX1 protects organelles against oxidative stress by wounding and MeJA treatment.This is the first report demonstrating that H₂O₂-scavenging in the cytosol is essential for plant tolerance to wounding and MeJA treatment.► Arabidopsis mutants lacking APX1 (KO-APX1) showed high sensitivity to wounding and treatment with methyl jasmonate (MeJA). ► After wounding and MeJA treatment, oxidative damage in the chloroplasts and nucleus was enhanced in the KO-APX1 plants. ► Our findings suggest that APX1 protects organelles against oxidative stress by wounding and MeJA treatment.

Keywords: Abbreviations; APX; ascorbate peroxidase; AsA; ascorbate; DAB; 3,3-diaminobenzidine; MeJA; methyl JA; Rboh; respiratory burst oxidase homolog; ROS; reactive oxygen speciesReactive oxygen species; Ascorbate peroxidase; Wounding; Jasmonate; Oxidative stress; Arabidopsis

Crystal structure of a conformation-dependent rabbit IgG Fab specific for amyloid prefibrillar oligomers by Hiromi Arai; Charles Glabe; Hartmut Luecke (pp. 1908-1914).

Although rabbit antibodies are widely used in research, no structures of rabbit antigen-binding fragments (Fab) have been reported. M204 is a rabbit monoclonal antibody that recognizes a generic epitope that is common to prefibrillar amyloid oligomers formed from many different amyloidogenic sequences. Amyloid oligomers are widely suspected to be a primary causative agent of pathogenesis in several age-related neurodegenerative diseases, such as Alzheimer's disease. The detailed structure of these amyloid oligomers is not known nor is the mechanism for the recognition of the generic epitope by conformation-dependent monoclonal antibodies.As a first approach to understanding the mechanism of conformation-dependent antibody recognition, we have crystallized the Fab of M204.We have determined the structure of the Fab of M204 at 1.54Å resolution. The crystal structure reveals details of the M204 antigen combining site and features unique to rabbit Fabs such as an interdomain disulfide bond on its light chain.Based on the structural features of the antigen-combining site of the M204, we rule out a “steric zipper” formation, as found in numerous amyloid fibril structures, as a mechanism of antibody-antigen recognition. The details of the first rabbit immunoglobulin Fab structure might also be useful for exploiting the potential of rabbit monoclonal antibodies for the development of humanized rabbit antibodies as therapeutic agents.► First rabbit Fab crystal structure ► First structure of a conformation-dependent, amyloid oligomer specific antibody ► Structure shows disulfide bonds unique to rabbit IgG. ► The data rule out steric zipper formation as the Fab antigen-binding mechanism.

Keywords: Abbreviations; Fab; Antigen-binding fragment; PDB; Protein Data Bank; AD; Alzheimer's disease; M204; Monoclonal antibody 204; PFOs; Prefibrillar oligomers; Aβ; Amyloid beta peptide; FOs; Fibrillar oligomers; APFs; Annular protofibrils; IgG; Immunoglobulin G; (HEK293) cells; Human embryonic kidney 293; VH; Variable domain, heavy chain; CH; Constant domain, heavy chain; VL; Variable domain, light chain; CL; Constant domain, light chain; CDRs; Complementarity-determining regions; RMSD; Root-mean-square deviationImmunoglobulin; Rabbit; IgG; Amyloid; Oligomer

Donor substrate binding and enzymatic mechanism of human core α1,6-fucosyltransferase (FUT8) by Kotzler Miriam P. Kötzler; Simon Blank; Frank I. Bantleon; Edzard Spillner; Bernd Meyer (pp. 1915-1925).

Fucosylation is essential for various biological processes including tumorigenesis, inflammation, cell–cell recognition and host–pathogen interactions. Biosynthesis of fucosylated glycans is accomplished by fucosyltransferases. The enzymatic product of core α1,6-fucosyltransferase (FUT8) plays a major role in a plethora of pathological conditions, e.g. in prognosis of hepatocellular carcinoma and in colon cancer. Detailed knowledge of the binding mode of its substrates is required for the design of molecules that can modulate the activity of the enzyme.We provide a detailed description of binding interactions of human FUT8 with its natural donor substrate GDP-fucose and related compounds. GDP-Fuc was placed in FUT8 by structural analogy to the structure of protein -O-fucosyltransferase ( cePOFUT) co-crystallized with GDP-Fuc. The epitope of the donor substrate bound to FUT8 was determined by STD NMR. The in silico model is further supported by experimental data from SPR binding assays. The complex was optimized by molecular dynamics simulations.Guanine is specifically recognized by His363 and Asp453. Furthermore, the pyrophosphate is tightly bound via numerous hydrogen bonds and contributes affinity to a major part. Arg365 was found to bind both the β-phosphate and the fucose moiety at the same time.Discovery of a novel structural analogy between cePOFUT and FUT8 allows the placement of the donor substrate GDP-Fuc. The positioning was confirmed by various experimental and computational techniques.The model illustrates details of the molecular basis of substrate recognition for a human fucosyltransferase for the first time and, thus, provides a basis for structure-based design of inhibitors.► First detailed model of donor substrate binding for human fucosyltransferase FUT8. ► Model is based on X-ray structures combined with ligand-based NMR data and SPR data. ► Refinement with molecular dynamics simulation yields substrate binding mode. ► Major donor binding of FUT8 originates from β-phosphate group. ► The key catalytic residue Arg365 orientates fucose residue and assists GDP release.

Keywords: Abbreviations; ADP; adenosine 5′-diphosphate; BSA; bovine serum albumin; ce; POFUT1; Caenorhabditis elegans; protein O-fucosyltransferase 1; EGF; epidermal growth factor; Fcγ; fragment, crystallizable gamma; FID; free induction decay; FUT8; human α1,6-fucosyltransferase; GDP-Fuc; 6-deoxy-β-; l; -galactopyranosylguanosine-5′-diphosphate; GlcNAc; N-acetyl glucosamine; GT-B; glycosyltransferase B fold; HEK; human embryonic kidney; IDP; inosine 5′-diphosphate; MALDI TOF; matrix-assisted laser desorption/ionization time-of-flight; Mes; 2-(N-morpholino)ethanesulfonic acid; MWCO; molecular weight cut-off; MD; molecular dynamics; MS; mass spectrometry; NMR; nuclear magnetic resonance; NodZ; nodulation factor Z (bacterial α1,6 fucosyltransferase); OPLS; optimized potentials for liquid simulations; PME; particle mesh Ewald; RMSD; root-mean-square deviation; RMSF; root-mean-square fluctuation; SPC; simple point charge; SPR; surface plasmon resonance; STD; saturation transfer difference; T; ₁; longitudinal relaxation time; TGFβ; transforming growth factor beta; VGF; vascular endothelial growth factor; XDP; xanthosine 5′-diphosphateFucosyltransferase; Glycosyltransferase; Saturation transfer difference nuclear magnetic resonance; GDP-fucose; Molecular dynamics simulation; Substrate recognition

Expression of matrix macromolecules and functional properties of breast cancer cells are modulated by the bisphosphonate zoledronic acid by P.G. Dedes; Ch. Gialeli; A.I. Tsonis; I. Kanakis; A.D. Theocharis; D. Kletsas; G.N. Tzanakakis; N.K. Karamanos (pp. 1926-1939).

The extracellular matrix (ECM) components play key roles in the multistep process of cancer growth and progression. Preclinical and clinical data show that bisphosphonates (BPs) may exert direct or indirect antitumoral effects. Despite proven efficiency in cancer treatment, the mechanism by which BPs can interfere with cancer progression remains elusive.We investigated the effects of the third generation BP, zoledronate (zoledronic acid, Zometa®), in the expression of ECM macromolecules as well as the functional properties (proliferation, adhesion, migration and invasion) in two breast cancer cell lines (MDA-MB-231 and MCF-7) with different metastatic potentials.The data highlight that zoledronate effectively inhibits growth of breast cancer cells, functional invasion migration and adhesion to various matrices. At the level of ECM interacting molecules, the expression of specific heparan sulfate proteoglycans implicated in cancer progression, such as syndecan-1, -2 and glypican-1 is downregulated, whereas syndecan-4 expression is upregulated upon treatment with zoledronate. The levels of integrins ανβ3, ανβ5 and α5β1 were significantly reduced following treatment with zoledronate which is in accordance with the reduced cell adhesion on various ECM matrices. The expression of hyaluronan and its receptor CD44 was also significantly suppressed. Moreover, ZOL suppressed the expression of metalloproteinases MMP-2, -9, the membrane type MT1- and MT2-MMP, whereas it increased the expression of their endogenous tissue inhibitors.The obtained results demonstrate that zoledronate is a critical modulator of ECM gene expression and powerful anticancer agent inhibiting growth, migration and the matrix-associated invasion of breast cancer cells.► Zoledronate inhibits invasion, migration and adhesion of breast cancer cells. ► Modulates the expression of matrix molecules implicated in cancer progression. ► Potent pharmaceutical anticancer agent for breast cancer.

Keywords: Abbreviations; BPs; bisphosphonates; ZOL; zoledronate; ECM; extracellular matrix; HA; hyaluronan; MMP; matrix metalloproteinase; PG; proteoglycan; MT-MMP; membrane-type MMP; TIMPs; tissue inhibitors of MMPs; COL I; collagen I; COL II; collagen II; COL-IV; collagen IV; FN; fibronectin; LN; laminin; TN; tenascin; VN; vitronectin; PKCa; protein kinase C-alpha; FAK; focal adhesion kinaseBreast cancer; Extracellular matrix; Migration; Invasion; Adhesion; Zoledronate

Toxicity of depleted uranium on isolated rat kidney mitochondria by Fatemeh Shaki; Mir-Jamal Hosseini; Mahmoud Ghazi-Khansari; Jalal Pourahmad (pp. 1940-1950).

Kidney is known as the most sensitive target organ for depleted uranium (DU) toxicity in comparison to other organs. Although the oxidative stress and mitochondrial damage induced by DU has been well investigated, the precise mechanism of DU-induced nephrotoxicity has not been thoroughly recognized yet.Kidney mitochondria were obtained using differential centrifugation from Wistar rats and mitochondrial toxicity endpoints were then determined in both in vivo and in vitro uranyl acetate (UA) exposure cases.Single injection of UA (0, 0.5, 1 and 2mg/kg, i.p.) caused a significant increase in blood urea nitrogen and creatinine levels. Isolated mitochondria from the UA-treated rat kidney showed a marked elevation in oxidative stress accompanied by mitochondrial membrane potential (MMP) collapse as compared to control group. Incubation of isolated kidney mitochondria with UA (50, 100 and 200μM) manifested that UA can disrupt the electron transfer chain at complex II and III that leads to induction of reactive oxygen species (ROS) formation, lipid peroxidation, and glutathione oxidation. Disturbances in oxidative phosphorylation were also demonstrated through decreased ATP concentration and ATP/ADP ratio in UA-treated mitochondria. In addition, UA induced a significant damage in mitochondrial outer membrane. Moreover, MMP collapse, mitochondrial swelling and cytochrome c release were observed following the UA treatment in isolated mitochondria.Both our in vivo and in vitro results showed that UA-induced nephrotoxicity is linked to the impairment of electron transfer chain especially at complex II and III which leads to subsequent oxidative stress.► Depleted uranium caused mitochondrial dysfunction after vivo and in vitro treatment. ► Depleted uranium inhibited complex II and III of mitochondrial respiratory chain. ► Respiratory chain disruption increased oxidative stress markers in mitochondria. ► Depleted uranium caused mitochondrial permeability transition and cytochrome c release. ► Depleted uranium induced cytochrome c release from mitochondria.

Keywords: Abbreviations; U; Uranium; DU; Depleted uranium; UA; uranyl acetate; ROS; Reactive oxygen species; GSH; reduced glutathione; DCF-DA; 2′,7′-dichlorofluorescein diacetate; TBARs; thiobarbituric acid reactive substances; Cs A; Cyclosporin A; MDA; Malondialdehyde; Rh123; Rhodamine 123; BSA; bovine serum albumin; MPT; mitochondrial permeability transition; MMP; mitochondrial membrane potential; BHT; butylatedhydroxytoluene; BUN; blood urea nitrogenDepleted uranium; Mitochondria; Nephrotoxicity; Respiratory chain; Mitochondrial permeability transition; Cytochrome c

Synergistic effect of conjugated linolenic acid isomers against induced oxidative stress, inflammation and erythrocyte membrane disintegrity in rat model by Siddhartha S. Saha; Pritha Dasgupta; Sumita Sengupta (Bandyopadhyay); Mahua Ghosh (pp. 1951-1970).

α-Eleostearic acid and punicic acid, two typical conjugated linolenic acid (CLnA) isomers present in bitter gourd and snake gourd oil respectively, exhibit contrasting cis- trans configuration which made them biologically important.Rats were divided into six groups. Group 1 was control and group 2 was treated control. Rats in the groups 3 and 4 were treated with mixture of α-eleostearic acid and punicic acid (1:1) (0.5% and 1.0% respectively) while rats in the groups 5 and 6 were treated with 0.5% of α-eleostearic acid and 0.5% of punicic acid respectively along with sodium arsenite by oral gavage once per day.Results showed that increase in nitric oxide synthase (NOS) activity, inflammatory markers expression, platelet aggregation, lipid peroxidation, protein oxidation, DNA damage and altered expression of liver X receptor-α (LXR-α) after arsenite treatment were restored with the supplementation of oils containing CLnA isomers. Altered activities of different antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and ferric reducing ability of plasma (FRAP) also restored after oil supplementation. Altered morphology and fluidity of erythrocyte membrane studied by atomic force and scanning electron microscopy, after stress induction were significantly improved due to amelioration in cholesterol/phospholipid ratio and fatty acid profile of membrane. Oils treatment also improved morphology of liver and fatty acid composition of hepatic lipid.Overall two isomers showed synergistic antioxidant and anti-inflammatory effect against induced perturbations and membrane disintegrity.Synergistic antioxidant and anti-inflammatory role of these CLnA isomers were established by this study.► Vegetable oils containing CLnA isomers showed protection against induced genotoxicity. ► CLnA isomers efficiently restored the biochemical perturbations and lipid profile ► Antioxidant parameters and fluidity of erythrocyte membrane were also restored. ► CLnA isomers caused increase in expression of hepatic LXR-α and decrease in NF-κB. ► Mixture of oils showed synergistic effect due to contrasting cis-trans configuration.

Keywords: Abbreviations; AFM; atomic force microscope; ALA; alfa-linoleic acid; ARA; arachidonic acid; CAT; Catalase; CLnA; conjugated linolenic acid; CLA; conjugated linoleic acid; DHA; docosahexaenoic acid; EM; erythrocyte membrane; EPA; eicosapentaenoic acid; FRAP; ferric reducing ability of plasma; GLA; gamma linolenic acid; GSH; reduced glutathione; GSSG; oxidized glutathione; GPx; glutathione peroxidase; GR; glutathione reductase; LMPA; low-melting point agarose; LXR; liver X receptor; MDA; malondialdehyde; NF-Κb; nuclear factor κB; NOS; nitric oxide synthase; PGE2; prostaglandin E2; PPARγ; peroxisome proliferator activated receptor gamma; ROS; reactive oxygen species; Sa; sodium arsenite; SEM; scanning electron microscope; SH-groups; sulfhydryl groups; SOD; superoxide dismutase; TBA; 2-thiobarbituric acid; TCA; trichloro acetic acid; TNF-α; tumor necrosis factor-αConjugated linolenic acid; Anti-inflammatory; Antioxidant; Cytokines; Membrane fluidity; Synergistic

Fucanomics and galactanomics: Current status in drug discovery, mechanisms of action and role of the well-defined structures by Vitor H. Pomin (pp. 1971-1979).

With the recent advent of glycomics, many medically relevant glycans have been discovered. Sulfated fucans (SFs) and sulfated galactans (SGs) are one of these classes of glycans with increasing interest to both glycomics and medicine. Besides having very unique structures, some of these molecules exhibit a broad range of pharmacological actions. In certain cases, high levels of effectiveness may be reached when the proper structural requirements are found.Here, we cover the fundamental biochemical mechanisms of some of these medicinal properties. We particularly focus on the beneficial activities of SFs and SGs in inflammation, hemostasis, vascular biology, and cancer.In these clinical systems, intermolecular complexes directly driven by electrostatic interactions of SFs and SGs with P- and L-selectins, chemokines, antithrombin, heparin cofactor II, thrombin, factor Xa, bFGF, and VEGF, overall govern the resultant therapeutic effects. In spite of that, the structural features of SFs and SGs have shown to be essential determinants for formation and stability of those molecular complexes, which consequently account to the differential levels of the biomedical responses.Accurate structure–function relationships have mostly been achieved when SFs and SGs of well-defined structures are used for study. Therefore, these types of glycans have become of great usefulness to identify the chemical requirements needed to achieve satisfactory clinical responses.Display Omitted► Sulfated fucans and galactans are classes of medicinal glycans. ► The main molecular mechanisms of their biomedical actions are explained herein. ► The structurally well-defined glycans offer good perspective for clinical assays. ► Fucanomics and galactanomics are promising glycomics projects in drug discovery.

Keywords: Abbreviations; aptt; activated partial thromboplastin time; AT; antithrombin; bFGF; basic fibroblast growth factor; DSPG; dermatan sulfate proteoglycan; FGFR; fibroblast growth factor receptor; GAG; glycosaminoglycans; HCII; heparin cofactor II; HSPG; heparan sulfate proteoglycan; HUVEC; human umbilical vein endothelial cells; ICAM; intercellular adhesion molecule; MSP; marine sulfated polysaccharide; PSGL-1; P-selectin glycoprotein ligand-1; SG; sulfated galactan; SF; sulfated fucan; SP; sulfated polysaccharide; TGF-β; tumor growth factor-β; TNF; tumor necrosis factor; UFH; unfractionated heparin; VEGF; vascular endothelial growth factorAngiogenesis; Cancer; Cardiovascular biology; Inflammation; Sulfated fucan; Sulfated galactan

The exclusive use of flow cytometry to evaluate the antibiotic-susceptibility by Takashi Soejima; Jun-ichi Minami; Keiji Iwatsuki (pp. 1980-1986).

Live and injured bacteria cannot be successfully discriminated using flow cytometric methods (FCM) with commercial live/dead staining agents because injured cells have intact cell membranes and are counted as live cells. We previously reported that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro (Microbiol. Immunol. 51:763-775, 2007).We report that EMA cleaves the chromosomal DNA of antibiotic-injured, but not live, Listeria monocytogenes. The combination of FCM and EMA treatment was evaluated as a rapid method to discriminate between live and antibiotic-injured L. monocytogenes. Additionally, we evaluated our methodology using blood from pediatric patients infected with other gram-negative and gram-positive bacteria.For antibiotic-injured, but not live, L. monocytogenes in blood, photoactivated EMA suppressed SYTO9 staining, as the SYTO9 staining of the antibiotic-injured L. monocytogenes was weak compared with that of live cells. Similarly, the rapid and clear discrimination between live and injured bacteria (gram-negative and gram-positive) was performed using the blood of pediatric patients administered antibiotics.The combination of FCM with EMA treatment is a rapid method for evaluating the susceptibility of live pathogens in infants with bacteremia without the need for bacterial culture.This assay is more rapid than other currently available techniques due to the elimination of the time-consuming culture step and could be used in clinical settings to rapidly determine the success of antibiotic treatment in pediatric bacteremia through the discrimination of injured (i.e., susceptible to the administered antibiotics) and live pathogens.► Live and injured bacteria cannot be evaluated by a routine flow cytometry alone. ► EMA cleaves the DNA of antibiotic-injured, but not live, Listeria monocytogenes. ► EMA-FCM distinguishes live and injured pathogen in pediatric bacteremia patient. ► EMA-FCM allows physicians to rapidly evaluate antibiotic-susceptibility. ► Physicians can rapidly determine if antibiotic treatment is successful.

Keywords: Live; Injured; Bacteremia; Diagnosis; FCM; EMA

Porphyrin binding mechanism is altered by protonation at the loops in G-quadruplex DNA formed near the transcriptional activation site of the human c-kit gene by Sintayehu Manaye; Ramon Eritja; Avino Anna Aviñó; Joaquim Jaumot; Raimundo Gargallo (pp. 1987-1996).

G-quadruplex DNA structures are hypothesized to be involved in the regulation of gene expression and telomere homeostasis. The development of small molecules that modulate the stability of G-quadruplex structures has a potential therapeutic interest in cancer treatment and prevention of aging.Molecular absorption and circular dichroism spectra were used to monitor thermal denaturation, acid base titration and mole ratio experiments. The resulting data were analyzed by multivariate data analysis methods. Surface plasmon resonance was also used to probe the kinetics and affinity of the DNA–drug interactions.We investigated the interaction between a G-quadruplex-forming sequence in the human c-kit proto-oncogene and the water soluble porphyrin TMPyP4. The role of cytosine and adenine residues at the loops of G-quadruplex was studied by substitution of these residues by thymidines.Here, we show the existence of two binding modes between TMPyP4 and the considered G-quadruplex. The stronger binding mode (formation constant around 107) involves end-stacking, while the weaker binding mode (formation constant around 106) is probably due to external loop binding. Evidence for the release of TMPyP4 upon protonation of bases at the loops has been observed.The results may be used for the design of porphyrin-based anti-cancer molecules with a higher affinity to G-quadruplex structures which may have anticancer properties.Protonation pushes away TMPyP4 molecules from the loops in G-quadruplex structures. The interaction of TMPyP4 porphyrin with the G-quadruplex structure formed by a guanine-rich sequence in the promoter region of c-kit gene was studied. Up to three ligand molecules may be bound to the G-quadruplex structure. Protonation at the loops induces the release of one TMPyP4 molecule.Display Omitted► Multivariate analysis and SPR were used to study the porphyrin–quadruplex interaction. ► The role of cytosine and adenine bases at the loops of G-quadruplex was studied. ► Two binding modes are shown with pH strongly influencing one of them.

Keywords: G-quadruplex; c-kit; Ligand; Conformational analysis; Multivariate analysis; Stacking interactions

Sleep deprivation impairs calcium signaling in mouse splenocytes and leads to a decreased immune response by Lisandro Lungato; Marcos L. Gazarini; Edgar J. Paredes-Gamero; Ivarne l.S. Tersariol; Sergio Tufik; Vânia D'Almeida (pp. 1997-2006).

Sleep is a physiological event that directly influences health by affecting the immune system, in which calcium (Ca2⁺) plays a critical signaling role. We performed live cell measurements of cytosolic Ca2⁺ mobilization to understand the changes in Ca2⁺ signaling that occur in splenic immune cells after various periods of sleep deprivation (SD).Adult male mice were subjected to sleep deprivation by platform technique for different periods (from 12 to 72h) and Ca2⁺ intracellular fluctuations were evaluated in splenocytes by confocal microscopy. We also performed spleen cell evaluation by flow cytometry and analyzed intracellular Ca2⁺ mobilization in endoplasmic reticulum and mitochondria. Additionally, Ca2⁺ channel gene expression was evaluatedSplenocytes showed a progressive loss of intracellular Ca2⁺ maintenance from endoplasmic reticulum (ER) stores. Transient Ca2⁺ buffering by the mitochondria was further compromised. These findings were confirmed by changes in mitochondrial integrity and in the performance of the store operated calcium entry (SOCE) and stromal interaction molecule 1 (STIM1) Ca2⁺ channels.These novel data suggest that SD impairs Ca2⁺ signaling, most likely as a result of ER stress, leading to an insufficient Ca2⁺ supply for signaling events. Our results support the previously described immunosuppressive effects of sleep loss and provide additional information on the cellular and molecular mechanisms involved in sleep function.► Sleep deprivation impairs calcium signaling in mice splenocytes. ► Sleep deprivation results in decrease in calcium stores in endoplasmic reticulum. ► Mitochondrial membrane potential in splenocytes is lost after sleep deprivation. ► Sleep deprivation results in splenocytes lysosomal integrity leakage. ► STIM expression is augmented after sleep deprivation in mice spleen.

Keywords: Ca; 2; ⁺; signaling; Splenocytes; Sleep deprivation; Mitochondrial dysfunction; Mice

Expression of glycogenes in differentiating human NT2N neurons. Downregulation of fucosyltransferase 9 leads to decreased Lewis^x levels and impaired neurite outgrowth by Ricardo Gouveia; Lana Schaffer; Suzanne Papp; Nicolas Grammel; Sebastian Kandzia; Steven R. Head; Ralf Kleene; Melitta Schachner; Harald S. Conradt; Júlia Costa (pp. 2007-2019).

Several glycan structures are functionally relevant in biological events associated with differentiation and regeneration which occur in the central nervous system. Here we have analysed the glycogene expression and glycosylation patterns during human NT2N neuron differentiation. We have further studied the impact of downregulating fucosyltransferase 9 (FUT9) on neurite outgrowth.The expression of glycogenes in human NT2N neurons differentiating from teratocarcinoma NTERA-2/cl.D1 cells has been analysed using the GlycoV4 GeneChip expression microarray. Changes in glycosylation have been monitored by immunoblot, immunofluorescence microscopy, HPLC and MALDI-TOF MS. Peptide mass fingerprinting and immunoprecipitation have been used for protein identification. FUT9 was downregulated using silencing RNA.One hundred twelve mRNA transcripts showed statistically significant up-regulation, including the genes coding for proteins involved in the synthesis of the Lewis^x motif ( FUT9), polysialic acid ( ST8SIA2 and ST8SIA4) and HNK-1 ( B3GAT2). Accordingly, increased levels of the corresponding carbohydrate epitopes have been observed. The Lewis^x structure was found in a carrier glycoprotein that was identified as the CRA-a isoform of human neural cell adhesion molecule 1. Downregulation of FUT9 caused significant decreases in the levels of Lewis^x, as well as GAP-43, a marker of neurite outgrowth. Concomitantly, a reduction in neurite formation and outgrowth has been observed that was reversed by FUT9 overexpression.These results provided information about the regulation of glycogenes during neuron differentiation and they showed that the Lewis^x motif plays a functional role in neurite outgrowth from human neurons.► Changes in glycogene expression were found during NT2N neuron differentiation. ► Increase in FUT9 expression that encodes the enzyme synthesizing Lewis^x was observed. ► NCAM is the predominant glycoprotein carrier of Lewis^x in NT2N neurons. ► Downregulation of FUT9 caused decreased Lewis^x levels and impaired neurite outgrowth.

Keywords: Abbreviations; BSA; bovine serum albumin; dHex; deoxyhexose; EDTA; etylenediaminetetraacetic acid; EGTA; ethylene glycol tetraacetic acid; Fuc; fucose; Gal; galactose; GlcNAc; N; -acetylglucosamine; Hex; hexose; HexNAc; N; -acetylhexosamine; HNK-1; human natural killer-1; HPAEC-PAD; high-performance anion-exchange chromatography with pulsed-amperometric detection; HRP; horseradish peroxidase; Le; ^x; Lewis; ^x; MALDI-TOF MS; matrix-assisted laser desorption ionization with time-of-flight mass spectrometry; Man; mannose; PBS; phosphate-buffered saline; PNGase F; peptide; N; -glycosidase F; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBS; Tris-buffered salineNeuron glycosylation; Neurite outgrowth; Fucosyltransferase 9; Lewis; ^x; Neuronal differentiation

Heme binding to human alpha-1 proteinase inhibitor by Elena Karnaukhova; Sonia Silinsky Krupnikova; Mohsen Rajabi; Abdu I. Alayash (pp. 2020-2029).

Heme is a unique prosthetic group of various hemoproteins that perform diverse biological functions; however, in its free form heme is intrinsically toxic in vivo. Due to its potential toxicity, heme binding to plasma proteins is an important safety issue in regard to protein therapeutics derived from human blood. While heme binding by hemopexin, albumin and α₁-microglobulin has been extensively studied, the role of other plasma proteins remains largely unknown.We examined two acute-phase plasma proteins, haptoglobin (Hp) and alpha-1 proteinase inhibitor (α₁-PI) for possible interactions with heme and bilirubin (BR), the final product of heme degradation, using various techniques: UV/Vis spectroscopy, fluorescence, circular dichroism (CD), and surface plasmon resonance (SPR).According to our data, Hp exhibits a very weak association with both heme and BR; α₁-PI's affinity to BR is also very low. However, α₁-PI's affinity to heme (K_D 2.0×10−⁸M) is of the same order of magnitude as that of albumin (1.26×10−⁸M). The data for α₁-PI binding with protoporphyrin IX (PPIX) suggest that the elimination of the iron atom from the porphyrin structure results in almost 350-fold lower affinity (K_D 6.93×10−⁶M), thus indicating that iron is essential for the heme coordination with the α₁-PI.This work demonstrates for the first time that human α₁-PI is a heme binding protein with an affinity to heme comparable to that of albumin.Our data may have important implications for safety and efficacy of plasma protein therapeutics.► A role of non-traditional plasma heme scavengers is evaluated. ► Human α₁-PI and Hp are examined for binding with heme, PPIX and BR. ► Methods include UV/Vis, fluorescence, SPR, CD and functional assays. ► This study for the first time defines human α₁-PI as a heme binding protein. ► Affinity of α₁-PI to heme is of the same order of magnitude as that of albumin.

Keywords: Abbreviations; α; ₁; -PI; alpha-1 proteinase inhibitor; BR; bilirubin; CD; circular dichroism; HA; human albumin; Hp; haptoglobin; L/P; ligand-to-protein (molar) ratio; PBS; phosphate buffer saline; PPIX; protoporphyrin IX; SPR; surface plasmon resonanceAlpha-1 proteinase inhibitor; Heme; Protoporphyrin; Bilirubin; Circular dichroism; Surface plasmon resonance

Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions by M.C. Ribeiro; M.S. Costa-Alves; M. Wengert; J.R. Meyer-Fernandes; P. Zancan; C. Caruso-Neves; A.A.S. Pinheiro (pp. 2030-2036).

The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-³²P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.This activity was linear with time up to 20min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5mM MgCl₂ was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN₃) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN₃. The dose–response of ATP revealed a hyperbolic profile with maximal velocity of 25.2±1.2nmol Pixmg−¹xmin−¹ and K_0.5 of 0.07±0.01mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60min of ischemia.Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.► The ecto-ATPase found in the surface of LLC-PK1 cells resembles a type 3 E-NTPDase. ► Luminal proximal tubule E-NTPDase activity is inhibited by ischemic insult. ► These results could explain the high extracellular levels of ATP during ischemia.

Keywords: Abbreviations; BSA; bovine serum albumin; DMEM; Dulbecco's modified Eagle's medium; E-NTPDase; ecto-nucleoside triphosphate diphosphohydrolase; E-NPP; ecto-nucleotide pyrophosphatase phosphodiesterase; HEPES; N; -2-hydroxyethylpiperazine; N; -2-ethanesulfonic acid; LDH; lactate dehydrogenase; PI; propidium iodide; Tris; (tris350 hydroxymethyl)-aminomethaneE-NTPDase; Pathology; Proximal tubule cell; Purine receptor

The abasic site lesions in the human telomeric sequence d[TA(G₃T₂A)₃G₃]: A thermodynamic point of view by Antonella Virgilio; Luigi Petraccone; Veronica Esposito; Giuseppe Citarella; Concetta Giancola; Aldo Galeone (pp. 2037-2043).

The abasic sites represent one of the most frequent lesions of DNA and most of the events able to generate such modifications involve guanine bases. G-rich sequences are able to form quadruplex structures that have been proved to be involved in several important biological processes.In this paper, we report investigations, based on calorimetric, UV, CD and electrophoretic techniques, on 12 oligodeoxynucleotides analogues of the quadruplex forming human telomere sequence d[TA(G₃T₂A)₃G₃], in which each guanine has been replaced, one at a time, by an abasic site mimic.Although all data show that the modified sequences preserve their ability to form quadruplex structures, the thermodynamic parameters clearly indicate that the presence of an abasic site decreases their thermal stability compared to the parent unmodified sequence, particularly if the replacement concerns one of the guanosines involved in the formation of the central G-tetrad.The collected data indicate that the effects of the presence of abasic site lesions in telomeric quadruplex structures are site-specific. The most dramatic consequences come out when this lesion involves a guanosine in the centre of a G-run.Abasic sites, by facilitating the G-quadruplex disruption, could favour the formation of the telomerase primer. Furthermore they could have implications in the pharmacological approach targeting telomere.Display Omitted► The effects of abasic sites in telomeric quadruplex are sequence-dependent. ► Abasic site lesions decrease the stability of the telomeric quadruplex structure. ► Thermal stability decrease is more evident if the lesion concerns the G-run centre.

Keywords: Quadruplex structure; Abasic site; Telomere; Differential scanning calorimetry (DSC); Thermal difference spectrum (TDS)

Propylbenzmethylation at Val-1(α) markedly increases the tetramer stability of the PEGylated hemoglobin: A comparison with propylation at Val-1(α) by Tao Hu; Dongxia Li; Jun Wang; Qingqing Wang; Yangui Liang; Yanlei Su; Guanghui Ma; Zhiguo Su; Shihua Wang (pp. 2044-2051).

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are potential pharmaceutical agents that can be used in surgery or emergency medicine. PEGylation can modulate the vasoactivity of Hb and is a widely used approach to develop HBOCs. However, PEGylation can significantly enhance the tetramer–dimer dissociation of Hb, which may perturb the structure of Hb and increase its observed adverse effect. Thus, it is necessary to increase the tetramer stability of the PEGylated Hb.Propylbenzmethylation at Val-1(α) of HbA was carried out to stabilize the Hb tetramer. The propylbenzmethylated Hb at Val-1(α) (PrB-Hb) was used as the starting material for site-specific PEGylation at Cys-93(β) of Hb using maleimide PEG. Structural and functional properties, autoxidation rate and thermal stability of the resultant product (PEG-PrB-Hb) were measured.Propylbenzmethylation at Val-1(α) led to 25-fold and 24-fold decreases in the tetramer–dimer dissociation constant of HbA and PEG-Hb, respectively. The increased tetramer stability is due to the enhanced hydrophobicity of the area around Val-1(α) and the increased polar interaction of Hb upon propylbenzmethylation. Thus, the structural and functional properties of PEG-Hb were improved, and its autoxidation rate and thermal denaturation were decreased.Propylbenzmethylation at Val-1(α) showed higher ability than propylation at Val-1(α) to improve the structural and functional properties and decrease the side effect of PEG-Hb.Our study can facilitate the biotechnological development of stable PEGylated Hb as more advanced HBOC. Our study is also expected to improve the stability of the tetrameric or dimeric proteins (e.g., uric oxidase) by propylbenzmethylation at their N-terminus.► Propylbenzmethylation at Val-1(α) enhances the tetramer stability of PEG-Hb. ► Propylbenzmethylation at Val-1(α) stabilizes the structure of PEG-Hb. ► Propylbenzmethylation at Val-1(α) decreases the side effect of PEG-Hb.

Keywords: PEGylation; Hemoglobin-based oxygen carrier; Propylbenzmethylation; Hemoglobin; Tetramer stability

Stimulation of cytosolic and mitochondrial calcium mobilization by indomethacin in Caco-2 cells: Modulation by the polyphenols quercetin, resveratrol and rutin by Catalina Carrasco-Pozo; Edgar Pastene; Carola Vergara; Moises Zapata; Cristian Sandoval; Martin Gotteland (pp. 2052-2061).

The effect of indomethacin (INDO) on Ca2⁺ mobilization, cytotoxicity, apoptosis and caspase activation and the potential protective effect of quercetin (QUE), resveratrol (RES) and rutin (RUT) were determined in Caco-2 cells.Caco-2 cells were incubated with INDO in the presence or absence of QUE, RES or RUT. The concentrations of Ca2⁺ in the cytosol (Fluo-3 AM) and mitochondria (Rhod-2 AM) were determined as well as the cytotoxicity (MTT reduction and LDH leakage), apoptosis (TUNEL) and caspase-3 and 9 activities.INDO promoted Ca2⁺ efflux from the endoplasmic reticulum (ER), resulting in an early, but transient, increment of cytosolic Ca2⁺ at 3.5min, followed by a subsequent increment of intra-mitochondrial Ca2⁺ at 24min. INDO also induced cytotoxicity, apoptosis, and increased caspase activities and cytochrome c release. All these alterations were prevented by the inhibitors of the IP3R and RyR receptors, 2-Aminoethoxydiphenyl borate (2-APB) and dantrolene. QUE was the most efficient polyphenol in preventing Ca2⁺ mobilization induced by INDO and all of its consequences including cytotoxicity and apoptosis.In Caco-2 cells, INDO stimulates ER Ca2⁺ mobilization, probably through the activation of IP3R and RyR receptors, and the subsequent entry of Ca2⁺ into the mitochondria. Polyphenols protected the cells against the Ca2⁺ mobilization induced by INDO and its consequences on cytotoxicity and apoptosis.These results confirm the possibility of using polyphenols and particularly QUE for the protection of the gastroduodenal mucosa in subjects consuming NSAIDs.► INDO increases Ca2⁺ concentrations first in cytosol and then in mitochondria. ► Inhibitors of IP3R and RyR receptors prevent the INDO induced caspases activation. ► Polyphenols prevent the Ca2⁺ mobilization induced by INDO. ► Quercetin prevents cytotoxicity, apoptosis and caspases activation induced by INDO.

Keywords: Abbreviations; 2-APB; 2-Aminoethoxydiphenyl borate; QUE; quercetin; RES; resveratrol; RUT; rutin; PPs; polyphenols; IP3R; inositol trisphosphate receptors; RyR; ryanodine receptors; EGTA; ethylene glycol tetraacetic acid; GI; gastrointestinal; INDO; indomethacin; MMP; mitochondrial membrane potential; NSAID; nonsteroidal anti-inflammatory drug; RFU; relative fluorescence unit; mPTP; mitochondrial permeability transition pore; ER; endoplasmic reticulum; DMSO; dimethylsulphoxide; SOCI; store-operated Ca; 2; ⁺; influx; SOCC; Store-Operated Ca; 2; ⁺; Channel; SERCA; Sarcoplasmic reticulum Ca; 2; ⁺; ATPaseQuercetin; Rutin; Resveratrol; Indomethacin; Ca; 2; ⁺; mobilization; Apoptosis

A pentapeptide signature motif plays a pivotal role in Leishmania DNA topoisomerase IB activity and camptothecin sensitivity by Christopher F. Prada; Alvarez-Velilla Raquel Álvarez-Velilla; Diaz-Gonzalez Rosario Diaz-González; Carlos Prieto; Perez-Pertejo Yolanda Pérez-Pertejo; Balana-Fouce Rafael Balaña-Fouce; Rosa M. Reguera (pp. 2062-2071).

Leishmania donovani – the causative agent of visceral leishmaniasis – has several evolutionary characteristics that make the disease difficult to combat. Among these differences, a rare heterodimeric DNA topoisomerase IB has been reported thus opening a new promising field in the therapy of leishmaniasis. Several studies of the human enzyme have pointed to the importance of the linker domain in respect to camptothecin sensitivity. At present, it has been impossible to pinpoint the regions that make up the linker domain in Leishmania.Several site-directed mutations as well as internal and linear truncations involving both subunits were assayed on both, relaxation activity and sensitivity to camptothecin.Truncations performed on the trypanosomatids conserved motif (RPPVVRS) of the small subunit of leishmanial DNA topoisomerase IB demonstrated that elimination of pentapeptide RPPVV produced a nonfunctional enzyme. However, the removal of the dipeptide RS led to an enzyme with reduced relaxation activity and less sensitivity to camptothecin. The basic structure, both sensitive to camptothecin and able to fully relax DNA, composed of amino acids 1–592 and 175–262 in the large and small subunits, respectively.It has been established that the region between amino acids 175 and 180 (RPPVV) of the small subunit plays a pivotal role in both interaction with the large subunit and sensitivity to camptothecin in Leishmania.The present report describes a functional analysis of the leishmanial DNA topoisomerase IB regions directly involved both in sensitivity to poisons and in the conformation of the linker domain.Display Omitted► We have performed a functional analysis of leishmanial DNA topoisomerase IB. ► Constructs were assayed on relaxation activity and sensitivity to camptothecin. ► Amino acids 175 to 180 of the small subunit play a pivotal role in enzyme activity. ► RPPVV pentapeptide motif is responsible for CPT sensitivity in the small subunit.

Keywords: Abbreviations; Top; DNA topoisomerases; LdTopIB; Leishmania TopIB; hTopIB; human TopIB; LdTopIL; gene encoding the large subunit of LdTopIB; LdTopIS; gene encoding the small subunit of LdTopIB; CPT; camptothecin; CL; cleavage band Leishmania donovani; DNA topoisomerase IB; Camptothecin sensitivity; Relaxation activity; Linker domain; Protein interactions

Entrapment of human flavin-containing monooxygenase 3 in the presence of gold nanoparticles: TEM, FTIR and electrocatalysis by Castrignano Silvia Castrignanò; Sheila J. Sadeghi; Gianfranco Gilardi (pp. 2072-2078).

Nanosized particles of gold are widely used as advanced materials for enzyme catalysis investigations. In some bioanalytical methods these nanoparticles can be exploited to increase the sensitivity by enhancing electron transfer to the biological component i.e. redox enzymes such as drug metabolizing enzymes.In this work, we describe the characterization of human flavin-containing monooxygenase 3 (hFMO3) in a nanoelectrode system based on AuNPs stabilized with didodecyldimethylammonium bromide (DDAB) on glassy carbon electrodes. Once confirmed by FTIR spectroscopy that in the presence of DDAB-AuNPs the structural integrity of hFMO3 is preserved, the influence of AuNPs on the electrochemistry of the enzyme was studied by cyclic voltammetry and square wave voltammetry.Our results show that AuNPs improve the electrochemical performance of hFMO3 on glassy carbon electrodes by enhancing the electron transfer rate and the current signal-to-noise ratio. Moreover, the electrocatalytic activity of hFMO3-DDAB-AuNP electrodes which was investigated in the presence of two well known substrates, benzydamine and sulindac sulfide, resulted in K_M values of 52μM and 27μM, with V_max of 8nmolmin−¹mg−¹ and 4nmolmin−¹mg−¹, respectively, which are in agreement with data obtained with the microsomal enzyme.The immobilization of hFMO3 protein in DDAB stabilized AuNP electrodes improves the bioelectrochemical performance of this important phase I drug metabolizing enzyme.This bio-analytical method can be considered as a promising advance in the development of new techniques suitable for the screening of novel hFMO3 metabolized pharmaceuticals.Display Omitted► First report of the entrapment of human FMO3 in the presence of gold nanoparticles ► Confirmation of the structural integrity of the enzyme upon entrapment by FTIR ► Enhanced electrochemical response of the enzyme ► Metabolism of drugs by the entrapped human FMO3

Keywords: Abbreviations; AuNPs; gold nanoparticles; hFMO3; human flavin-containing monooxygenase isoform 3; DDAB; didodecyldimethylammonium bromide; TEM; transmission electron microscopy; FTIR; Fourier transform infrared spectroscopy; HPLC; high performance liquid chromatographyHuman flavin-containing monooxygenase isoform 3; Stabilized gold nanoparticle; Glassy carbon electrode; FTIR; Enzyme catalysis

Site-specific protein O-glycosylation modulates proprotein processing — Deciphering specific functions of the large polypeptide GalNAc-transferase gene family by Katrine T.-B.G. Schjoldager; Henrik Clausen (pp. 2079-2094).

Posttranslational modifications (PTMs) greatly expand the function and regulation of proteins, and glycosylation is the most abundant and diverse PTM. Of the many different types of protein glycosylation, one is quite unique; GalNAc-type (or mucin-type) O-glycosylation, where biosynthesis is initiated in the Golgi by up to twenty distinct UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). These GalNAc-Ts are differentially expressed in cells and have different (although partly overlapping) substrate specificities, which provide for both unique functions and considerable redundancy. Recently we have begun to uncover human diseases associated with deficiencies in GalNAc-T genes (GALNTs). Thus deficiencies in individual GALNTs produce cell and protein specific effects and subtle distinct phenotypes such as hyperphosphatemia with hyperostosis (GALNT3) and dysregulated lipid metabolism (GALNT2). These phenotypes appear to be caused by deficient site-specific O-glycosylation that co-regulates proprotein convertase (PC) processing of FGF23 and ANGPTL3, respectively.Here we summarize recent progress in uncovering the interplay between human O-glycosylation and protease regulated processing and describes other important functions of site-specific O-glycosylation in health and disease.Site-specific O-glycosylation modifies pro-protein processing and other proteolytic events such as ADAM processing and thus emerges as an important co-regulator of limited proteolytic processing events.Our appreciation of this function may have been hampered by our sparse knowledge of the O-glycoproteome and in particular sites of O-glycosylation. New strategies for identification of O-glycoproteins have emerged and recently the concept of SimpleCells, i.e. human cell lines made deficient in O-glycan extension by zinc finger nuclease gene targeting, was introduced for broad O-glycoproteome analysis.► Up to 20 GalNAc-T isoforms catalyze the initiation of GalNAc-type O-glycosylation. ► O-glycosylation modulates proteolytic processing by PC convertases and ADAMs. ► Deficiencies in GalNAc-Ts cause protein specific effects and distinct phenotypes. ► New glyco-proteomic strategy for discovery of new targets of O-glycosylation/processing

Keywords: O-glycosylation; GalNAc-transferase (UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase); Zinc finger nuclease gene targeting; Pro-protein convertase (PC) processing; Congenital disorder of glycosylation (CDG)

Novel aminobenzyl-acetamidine derivative modulate the differential regulation of NOSs in LPS induced inflammatory response: Role of PI3K/Akt pathway by Antonia Patruno; Sara Franceschelli; Mirko Pesce; Cristina Maccallini; Marialuigia Fantacuzzi; Lorenza Speranza; Alessio Ferrone; Maria Anna De Lutiis; Emanuela Ricciotti; Rosa Amoroso; Mario Felaco (pp. 2095-2104).

Previous reports suggest that NO may contribute to the pathophysiology of septic shock. Recently, we have synthesized and characterized a series of benzyl- and dibenzyl derivative of N-(3-aminobenzyl)acetamidine, a potent and selective inhibitor of iNOS, in vitro assay. We evaluated the molecular mechanisms by which these compounds are involved in the regulation of NOSs expression.H9c2 cells were stimulated with lipopolysaccharide (LPS) in the presence or absence of acetamidine-derivative. The NOSs mRNA and protein, and activation of signaling pathways (Akt and NF-κB) were assayed.The induction of endotoxic shock in H9c2 with LPS caused an increase of inducible NOS and a down-regulation of constitutive NOS. The molecular mechanism involved in the modulation of NOSs expression in H9c2 cells upon LPS stimulation resulted in the modification of the redox state responsible for NF-kB nuclear translocation via NIK -IKKα/β-IkBα, simultaneously to the inactivation of the PI3K/Akt pathway. The compounds acted as an anti-inflammatory modulator.These results suggest that LPS regulates the opposite NOS expression in H9c2 cells by modifying the redox state of these cells responsible for the NF-kB nuclear translocation via NIK–IKKα/β‐IkBα, simultaneous to the inactivation of the PI3K/Akt pathway. The new molecule acts as an anti-inflammatory modulator in LPS-induced inflammation in H9c2 cells by the restoration of eNOS and nNOS expressions, mechanistically involving the PI3K/Akt pathway.This study delineates the underlying mechanisms of opposite NOSs expression in H9c2 cells stimulated with LPS.► LPS stimulation of H9c2 cells resulted in an opposite modulation of NOS isoforms. ► LPS induces iNOS through the activation of NF-kB nuclear translocation via NIK–IKKα/β‐IkBα. ► Effects of a new molecule as an anti-inflammatory modulator involving the PI3K/Akt pathway.

Keywords: Abbreviations; NOS; Nitric Oxide Synthase; LPS; lipopolysaccharide; ROS; reactive oxygen speciesNO synthases; Inflammation; NF-κB; H9c2; Aminobenzyl acetamidine-derivative

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