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BBA - General Subjects (v.1810, #7)
The Galβ-(syn)-gauche configuration is required for galectin-recognition disaccharides by Jun Iwaki; Hiroaki Tateno; Nozomu Nishi; Toshikazu Minamisawa; Sachiko Nakamura-Tsuruta; Yoko Itakura; Junko Kominami; Tadasu Urashima; Takanori Nakamura; Jun Hirabayashi (pp. 643-651).
Galectins form a large family of animal lectins, individual members having variously divergent carbohydrate-recognition domains (CRDs) responsible for extensive physiological phenomena. Sugar-binding affinities of galectins were previously investigated by us using frontal affinity chromatography (FAC) with a relatively small set (i.e., 41) of oligosaccharides. However, total understanding of a consensus rule for galectin-recognition saccharides is still hampered by the lack of fundamental knowledge about their sugar-binding specificity toward a much larger panel of oligosaccharides in terms of dissociation constant ( Kd).In the present study, we extended a FAC analysis from a more systematic viewpoint by using 142 fluorescent-labeled oligosaccharides, initially with focus on functional human galectins-1–9. Binding characteristics were further validated with 11 non-human galectins and 13 non-galectin Gal/GalNAc-binding lectins belonging to different families.An empirical [Galβ-equatorial] rule for galectin-recognition disaccharides was first derived by our present research and previous works by others. However, this rule was not valid for a recently reported nematode disaccharide, “Galβ1-4-L-Fuc” [Butschi et al. PLoS Pathog, 2010; 6(1):e1000717], because this glycosidic linkage was directed to ‘axial’ 4-OH of L-Fuc. After careful reconsideration of the structural data, we reached an ultimate rule of galectin-recognition disaccharides, which all of the galectins so far identified fulfilled, i.e., under the re-defined configuration “Galβ-(syn)-gauche”. The rule also worked perfectly for differentiation of galectins from other types of lectins.The present attempt should provide a basis to solve the riddle of the glyco-code as well as to develop therapeutic inhibitors mimicking galectin ligands.► Galectins require “Galβ-(syn)-gauche” configuration of their ligands. ► Galectins are differentiated from other Gal/GalNAc-binding lectins by FAC. ► FAC reveals consensus for galectin-recognition disaccharides.
Keywords: Abbreviations; CRD; carbohydrate-recognition domain; DTT; dithiothreitol; EDTA; ethylenediaminetetraacetic acid; FAC; frontal affinity chromatography; GAG; glycosaminoglycan; IPTG; isopropyl-β-D-thiogalactopyranoside; LNB; lacto-; N; -biose; LDN; LacdiNAc; PA-; pyridylaminated; p; NP-; p; -nitrophenyl; p; MP-; p; -methoxyphenylCarbohydrate-binding specificity; Comparative glycomics; Dissociation constant; Frontal affinity chromatography; Galectin; Pyridylamination
Role of endothelial dysfunction in modulating the plasma redox homeostasis in visceral leishmaniasis by Kaustav Dutta Chowdhury; Gargi Sen; Avik Sarkar; Tuli Biswas (pp. 652-665).
Evidence in the literature suggests that down-regulation of nitric oxide (NO) is associated with the pathophysiological conditions during visceral leishmaniasis (VL). Here we have investigated the mechanism that leads to the down regulation of systemic NO in the infected condition. Moreover, we have determined whether down regulation of NO is associated with increased generation of reactive oxygen species (ROS) during this disease. Therapeutic strategy targeting signaling molecules of these events was evaluated.Plasma protein-nitrotyrosine was examined by ELISA kit. Generation of superoxides and peroxynitrites was investigated by flow cytometry. NO bioavailability in endothelial cells was evaluated using DAF-2DA fluorescence. Ceramide contents were evaluated using FACS analysis, HPTLC and HPLC. L. donovani infected reticulo-endothelial cells regulated the activity of eNOS and NAD(P)H oxidase in the endothelial cells through the generation of intercellular messenger, ceramide. Activation of SMases played an important role in the generation of ceramide in animals during chronic infection. These events led to generation of ROS within endothelial cells. Modulation of redox status of plasma and accumulation of ROS in endothelial cells were critically involved in the regulation of NO bioavailability in plasma of the infected animal. Endothelial dysfunction and decline of NO were resulted from an increased production of superoxide where upregulation of eNOS expression appeared as an ineffective compensatory event. Inhibition of ceramide generation increased NO bioavailability, prevented endothelial dysfunction and concomitant oxidative stress.Decreased NO bioavailability and endothelial dysfunction were the downstream of ceramide signaling cascade. ROS accumulation promoted peroxynitrite generation and reduced NO bioavailability. Inhibition of ceramide generation may be a potential therapeutic option in preventing the co-morbidity associated with VL.► Increase in plasma nitrotyrosine was observed during visceral leishmaniasis (VL). ► Peroxynitrite generation led to decline in bioavailability of NO. ► Enhanced NADPH oxidase activity and eNOS expression occurred in endothelial cells. ► Ceramide signaled endothelial dysfunction during VL.
Keywords: Abbreviations; NO; nitric oxide; VL; visceral leishmaniasis; eNOS; endothelial nitric oxide synthase; FITC; fluorescein isothiocyanate; FCS; fetal calf serum; DAF-2DA; 4,5-diaminofluorescein diacetate; VCl; 3; vanadium tri-chloride; NEDD; N-(1-naphthyl) ethylenediamine dihydrochloride; NADPH; nicotinamide adenine dinucleotide phosphate reduced form; DHE; dihydroethidium, HPF, 3’-(p-hydroxyphenyl) fluorescein; DMSO; dimethyl sulfoxide; HPTLC; high performance thin layer chromatography; HPLC; high performance liquid chromatography; NEM; N-ethylmaleimide; DTNB; 5,5’-dithio-bis(2-nitrobenzoic acid); ELISA; enzyme linked immunosorbent assay; ABAP; azobis (2-amidinopropane) hydrochloride; FACS; fluorescence–activated cell sortingNitric oxide (NO); Endothelial cell; Superoxide; Plasma; Nitrotyrosine; Visceral leishmaniasis (VL)
Inhibition of Rac activity alleviates lipopolysaccharide-induced acute pulmonary injury in mice by Hong-yi Yao; Lihua Chen; Chengyun Xu; Jirong Wang; Jiqiang Chen; Qiang-min Xie; Ximei Wu; Xiao-feng Yan (pp. 666-674).
Rac small GTPases play important roles in cytoskeleton and many cell functions including cell cycle, cell growth, cell adhesion and gene transcription. Here, we investigated the roles of Rac including Rac1 and Rac2 in lipopolysaccharide (LPS)-induced pulmonary injury.After LPS was intratracheally instilled to lungs in mice, Rac, CDC42 and RhoA activation assay by pull-down and West blot, inflammatory cell infiltration assay by counting cell numbers and lung histological examination, pro-inflammatory mediator mRNA expression assay by quantitative RT-PCR, measurement of myeloperoxidase (MPO) activity, Evans Blue and albumin accumulation by spectrophotometry were performed to evaluate the roles of Rac in pulmonary injury by using its specific inhibitor, NSC23766.LPS challenge led to increases of both Rac1 and Rac2, but not CDC42 or RhoA activities in lungs, and intraperitoneal administration with NSC23766 inhibited both Rac1 and Rac2, but not CDC42 or RhoA activities. Treatment with NSC23766 at 1 or 3mg/kg not only reduced the inflammatory cells infiltration and MPO activities, but also inhibited pro-inflammatory mediators, tumor necrosis factor-α and interleukin-1β, mRNA expression. Moreover, in vitro neutrophil migration assay and in vivo microvascular permeability assay indicated that NSC23766 not only inhibited neutrophil transwell migration toward a chemoattractant, fMLP, but also reduced Evans Blue and albumin accumulation in LPS-challenged lungs. LPS activated both Rac1 and Rac2, but not CDC42 or RhoA activities in lungs, and specific inhibition of Rac activities by NSC23766 effectively alleviated LPS-induced injury.Rac could be a potential target for therapeutic intervention of pulmonary inflammation.► Both Rac1 and Rac2 but not CDC42 or RhoA are activated in LPS-challenged lung injury. ► Activation of Rac is important in lung injury, as evidenced by using Rac inhibitor. ► Inhibition of Rac attenuates injury through decreasing migration and permeability. ► Rac could be a potential target for therapeutic intervention of pulmonary inflammation.
Keywords: Rac GTPase; NSC23766; Neutrophil; Chemotaxis; Endothelial permeability; Pulmonary inflammation
A conserved isoleucine in the LOV1 domain of a novel phototropin from the marine alga Ostreococcus tauri modulates the dark state recovery of the domain by Sindhu Kandoth Veetil; Chitvan Mittal; Peeyush Ranjan; Suneel Kateriya (pp. 675-682).
Phototropins are UV-A/blue light receptor proteins with two LOV (Light-Oxygen-Voltage) sensor domains at their N terminus and a kinase domain at the C-terminus in photoautotrophic organisms. This is the first research report of a canonical phototropin from marine algae Ostreococcus tauri.We synthesized core LOV1 (OtLOV1) domain-encoding portion of the phototropin gene of O. tauri, the domain was heterologously expressed, purified and assessed for its spectral properties and dark recovery kinetics by UV–Visible, fluorescence spectroscopy and mutational studies. Quaternary structure characteristics were studied by SEC and glutaraldehyde crosslinking.The absorption spectrum of OtLOV1 lacks the characteristic 361nm peak shown by other LOV1 domains. It undergoes a photocycle with a dark state recovery time of approximately 30min (τ=300.35s). Native OtLOV1 stayed as dimer in aqueous solution and the dimer formation was light and concentration independent. Mutating isoleucine at 43rd position to valine accelerated the dark recovery time by more than 10-fold. Mutating it to serine reduced sensitivity to blue light, but the dark recovery time remained unaltered. I43S mutation also destabilized the FMN binding to a great extent.The OtLOV1 domain of the newly identified OtPhot is functional and the isoleucine at position 43 of OtLOV1 is the key residue responsible for fine-tuning the domain properties.This is the first characterized LOV1 domain of a canonical phototropin from a marine alga and spectral properties of the domain are similar to that of the LOV1 domain of higher plants.► This is the first identified canonical phototropin from a marine alga. ► I43V mutation accelerates dark state recovery by more than 10 folds. ► I43S mutant reduces photosensitivity and chromophore binding. ► Salt bridge between E60 and K101 is dispensable. ► Dimerization of OtLOV1 is manifested by hydrophobic interactions.
Keywords: Abbreviations; IPTG; isopropyl-β-d-thiogalactopyranoside; SDS; sodium dodecyl sulfate; FMN; flavin mononucleotide; TCA; Trichloro Acetic Acid; SEC; size exclusion chromatography; FPLC; Fast Performance Liquid Chromatography; UPLC; Ultra Performance Liquid Chromatography; LOV1; Light Oxygen Voltage 1; Co++-IMAC; Cobalt Immobilized Metal Affinity Chromatography; BLAST; Basic Local Alignment Search Tool; CDART; Conserved Domain Architecture Retrieval Tool; PCR; Polymerase Chain Reaction; cps; counts per second Ostreococcus tauri; Marine algae; Phototropin; LOV domain; Dimerization; Dark recovery kinetics
Cytotoxicity and inhibition of platelet aggregation caused by anl-amino acid oxidase from Bothrops leucurus venom by Gustavo B. Naumann; Liliana F. Silva; Luciana Silva; Gilson Faria; Michael Richardson; Karla Evangelista; Markus Kohlhoff; Celia M.F. Gontijo; Alexei Navdaev; Flavia F. de Rezende; Johannes A. Eble; Eladio F. Sanchez (pp. 683-694).
Multifunctionall-amino acid oxidases (LAAOs) occur widely in snake venoms.Thel-AAO from Bothrops leucurus ( Bl-LAAO) venom was purified using a combination of molecular exclusion and ion-exchange chromatographies. We report some biochemical features of Bl-LAAO associated with its effect on platelet function and its cytotoxicity. Bl-LAAO is a 60kDa monomeric glycoprotein. Its N-terminal sequence shows high homology to other members of the snake-venom LAAO family. Bl-LAAO catalyzes oxidative deamination ofl-amino acids with the generation of H2O2. The best substrates were:l-Met,l-Norleu,l-Leu,l-Phe andl-Trp. The effects of snake venom LAAOs in hemostasis, especially their action on platelet function remain largely unknown. Bl-LAAO dose-dependently inhibited platelet aggregation of both human PRP and washed platelets. Moreover, the purified enzyme exhibited a killing effect in vitro against Leishmania sp., promastigotes, with a very low EC50 of 0.07μM. Furthermore, the cytotoxicity of Bl-LAAO was observed in the stomach cancer MKN-45, adeno carcinoma HUTU, colorectal RKO and human fibroblast LL-24 cell lines. The enzyme released enough H2O2 in culture medium to induce apoptosis in cells in a dose- and time-dependent manner. The biological effects were inhibited by catalase. Bl-LAAO, a major component of B. leucurus venom, is a cytotoxin acting primarily via the generation of high amounts of H2O2 which kill the cells.These results allow us to consider the use of LAAOs as anticancer agents, as tools in biochemical studies to investigate cellular processes, and to obtain a better understanding of the envenomation mechanism.► Chromatographic purification of anl-amino acid oxidase of Bothrops leucurus. ► Purity confirmed by MS, 2-DE, N-terminal sequencing. ►l-Met,l-Norleu,l-Leu,l-Phe andl-Trp were best substrates. ► Bl-LAAO inhibited ADP- and collagen-induced platelet aggregation. ► Strong killing effect on various cancer cell lines.
Keywords: Abbreviations; MTT; [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium]; IC; 50; inhibitory concentration that causes 50% inhibition; OPD; o-phenylenediamine; DMC; dimethylcasein; Bl; -LAAO; l; -amino acid oxidase from; Bothrops leucurus; venom; i.p; intraperitoneal; ACN; acetonitrile; TFA; trifluoroacetic acid; 2-DE; two-dimensional gel electrophoresisAnimal toxin; Bl; -LAAO; l; -amino acid oxidase; Platelet function; Apoptosis
Brazilian propolis-derived components inhibit TNF-α-mediated downregulation of adiponectin expression via different mechanisms in 3T3-L1 adipocytes by Rie Ikeda; Masayoshi Yanagisawa; Nobuyuki Takahashi; Teruo Kawada; Shigenori Kumazawa; Noriyuki Yamaotsu; Izumi Nakagome; Shuichi Hirono; Takanori Tsuda (pp. 695-703).
Previous reports suggest that Brazilian propolis has multiple biological functions and may help to restore adiponectin expression and insulin sensitivity. However, little is known about the molecular mechanisms by which these compounds inhibit the downregulation of adiponectin.The effect of various Brazilian propolis-derived components on inhibition of tumor necrosis factor-α (TNF-α)-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes and molecular mechanism was investigated.Pretreatment with either artepillin C (C3) or its derivative (C4) significantly inhibited TNF-α-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes. Interestingly, C3 strongly activated peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity. Treatment of adipocytes with C3 resulted in the upregulation of adiponectin and fatty acid-binding protein 4 expression, but C4 did not significantly induce PPARγ transactivation. C4 did, however, inhibit the TNF-α-induced c-Jun-NH2-terminal kinase (JNK) signaling that is involved in adiponectin expression. Molecular docking studies based on hPPARγ with C3 and JNK1 with C4 clearly supported our experimental results. These data demonstrate that 1) both C3 and C4 significantly inhibit the TNF-α-mediated downregulation of adiponectin in adipocytes, 2) C3 functions as a PPARγ agonist, and its inhibition of the effect of TNF-α is due to this PPARγ transactivation, and 3) C4 is an effective inhibitor of JNK activation, thus inhibiting the TNF-α-mediated downregulation of adiponectin.Brazilian propolis-derived components (C3 and C4) can significantly inhibit TNF-α-mediated downregulation of adiponectin in adipocytes, although they do so via different mechanisms.Display Omitted► Two types of propolis components inhibit downregulation of adiponectin via different mechanisms. ► Artepillin C (C3) functions as a PPARγ agonist. ► C3 can form a stable complex with the hPPARγ LBD based on molecular docking studies. ► C4 is an effective inhibitor of JNK activation. The predicted binding mode of C4 was similar to the binding modes of known JNK inhibitors.
Keywords: Abbreviations; aP2; adipocyte fatty acid-binding protein 4; E; MM; molecular mechanics energy; FBS; fetal bovine serum; JNK; c-Jun-NH; 2; -terminal kinase; MAPK; mitogen-activated protein kinase; MKK; mitogen-activated protein kinase kinase; MM-PBSA; molecular mechanics Poisson-Boltzmann surface area; PPAR; peroxisome proliferator-activated receptor; SEK1/MKK4; stress-activated protein kinase/extracellular signal-regulated kinase kinase 1/ mitogen-activated protein kinase kinase 4; TNF-α; tumor necrosis factor-α; TTBS; Tris–HCl-buffered saline containing 0.05% Tween 20; TZD; troglitazone; vdW; van der WaalsPropolis; Adipocyte; Adiponectin; c-Jun-NH; 2; -terminal kinase; Peroxisome proliferator-activated receptor
Hydogen peroxide-dependent photocytotoxicity by phloxine B, a xanthene-type food colorant by Hang Qi; Hiroshi Takano; Yoji Kato; Qian Wu; Chiharu Ogata; Beiwei Zhu; Yoshiyuki Murata; Yoshimasa Nakamura (pp. 704-712).
Phloxine B (PhB; 2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachloro-fluorescein), an artificial xanthene colorant, has been used as a red coloring agent in drugs and cosmetics as well as foods in some countries. However, little effort has been devoted to the study of this colorant as a potentially useful medicinal agent.We investigated the daily light-induced photocytotoxicity of PhB in two human leukemia cells, HL-60 and Jurkat, and its underlying mechanisms by in vitro experiments using antioxidants.PhB inhibited cell proliferation more preferentially to HL-60 cells than to Jurkat cells. Co-treatment of catalase completely blocked the photocytotoxicity by PhB in HL-60 cells, whereas the effect of histidine was only partial, suggesting that hydrogen peroxide (H2O2), rather than singlet oxygen, might be a prerequisite for the PhB-induced HL-60 cell death. Actually, PhB produced a significant amount of H2O2 in the media as well as in the cells in concentration- and light-dependent manners. Furthermore, methionine, a hypochlorous acid (HOCl) scavenger, also significantly attenuated the cytotoxicity in HL-60 cells, but not in Jurkat cells, indicating the involvement of myeloperoxidase (MPO)-dependent hypohalous acid formation during the photocytotoxicity. In vitro experiments revealed that halogenated tyrosine was generated from the reaction of bovine serum albumin with PhB and HL-60 cell lysate. The present findings suggested that PhB induced a differential photodynamic action in the MPO-containing leukemia cells through an H2O2-dependent mechanism.Our findings provide new insights into the molecular mechanisms underlying the PhB-induced apoptosis and also evaluated PhB as a promising PDT agent.► This is the first investigation of the significant photosensitizing effects of phloxine B (PhB) on human leukemia cell lines by the weak light. ► Our findings provide new insights into the molecular mechanisms underlying the PhB-induced apoptosis and also evaluated PhB as a promising PDT agent. ► We also revealed how the PhB-induced effects can be modulated by the H2O2-MPO-HOCl system to potentially compromise medicinal and therapeutic outcomes.
Keywords: Abbreviations; FDA; Food and Drug Administration; PhB; phloxine B; H; 2; O; 2; hydrogen peroxide; MPO; myeloperoxidase; H; 2; DCF-DA; 2′,7′-dichlorodihydrofluorescein diacetate; FOX; ferrous ion oxidation–xylenol orange; JNK; cJun-; N; -terminal kinase; HOCl; hypochlorous acid; HOX; hypohalous acid; diBr-Y; dibromated tyrosine; PDT; Photodynamic therapy; MAPK; mitogen activated protein kinase; ASK1; apoptosis signal regulating kinase 1Phloxine B; Hydrogen peroxide; Hypohalous acid; Myeloperoxidase; HL-60 cells
Hardening of bio-silica in sponge spicules involves an aging process after its enzymatic polycondensation: Evidence for an aquaporin-mediated water absorption by Muller Werner E.G. Müller; Xiaohong Wang; Matthias Wiens; Schlossmacher Ute Schloßmacher; Klaus Peter Jochum; Schroder Heinz C. Schröder (pp. 713-726).
Spicules, the siliceous skeletal elements of the siliceous sponges, are synthesized enzymatically via silicatein. The product formed, bio-silica, constitutes their inorganic matrix. It remained unexplored which reactions are involved in molding of the amorphous bio-silica and formation of a solid and rigid biomaterial.Cell and molecular biological techniques have been applied to analyze processes resulting in the hardening of the enzymatically synthesized bio-silica. The demosponge Suberites domuncula has been used for the studies.Cell aggregates (primmorphs) from the sponge S. domuncula, grown in the presence of Mn-sulfate, form spicules that comprise, instead of a smooth, a rough and porous surface which is decorated with irregular bio-silica deposits. During this process, the expression of the aquaporin-8 gene becomes down-regulated. Further in vitro studies showed that aquaporin is required for dehydration, and hardening of bio-silica following its enzymatic formation. The data show that in cell aggregates grown in the presence of Mn-sulfate, aquaporin-8 is down-regulated. We conclude that in cell aggregates grown in the presence of Mn-sulfate, the removal of reaction water, produced during the bio-silica polycondensation reaction, is inhibited.This study highlights that besides the silicatein-driven polycondensation reaction, the spicule formation also requires a phase of syneresis that results in a hardening of the material.► Sponges as a model system for evolution of metazoan body plans. ► Sponges as a model system for basic studies of skeleton formation in animals. ► Enzymatic polycondensation reaction for the formation of bio-silica. ► Expression of aquaporin gene for the first time shown during bio-silica formation. ► First description that bio-silica formation in animals requires a de-hydration step.
Keywords: Abbreviations; AQP; aquaporin; Cu; copper; DAPI; 4′,6-diamidino-2-phenylindole; EDX; energy-dispersive X-ray; Fe; ferrum (iron); HF; hydrofluoric acid; IEF; isoelectric focusing; Mn; manganese; PCR; polymerase chain reaction; PEG; poly(ethylene glycol); qRT-PCR; quantitative reverse transcription PCR; SEM; scanning electron microscopy; TEOS; tetraethyl orthosilicate; Zn; zincSponges; Spicules; Bio-silica; Hardening; Suberites domuncula; Primmorphs