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BBA - General Subjects (v.1820, #10)

Editorial Board (pp. i).

A plant peptide: N-glycanase orthologue facilitates glycoprotein ER-associated degradation in yeast by Yuki Masahara-Negishi; Akira Hosomi; Massimiliano Della Mea; Donatella Serafini-Fracassini; Tadashi Suzuki (pp. 1457-1462).

The cytoplasmic peptide: N-glycanase (PNGase) is a deglycosylating enzyme involved in the ER-associated degradation (ERAD) process, while ERAD-independent activities are also reported. Previous biochemical analyses indicated that the cytoplasmic PNGase orthologue in Arabidopsis thaliana (AtPNG1) can function as not only PNGase but also transglutaminase, while its in vivo function remained unclarified.AtPNG1 was expressed in Saccharomyces cerevisiae and its in vivo role on PNGase-dependent ERAD pathway was examined.AtPNG1 could facilitate the ERAD through its deglycosylation activity. Moreover, a catalytic mutant of AtPNG1 (AtPNG1(C251A)) was found to significantly impair the ERAD process. This result was found to be N-glycan-dependent, as the AtPNG(C251A) did not affect the stability of the non-glycosylated RTA∆ (ricin A chain non-toxic mutant). Tight interaction between AtPNG1(C251A) and the RTA∆ was confirmed by co-immunoprecipitation analysis.The plant PNGase facilitates ERAD through its deglycosylation activity, while the catalytic mutant of AtPNG1 impair glycoprotein ERAD by binding to N-glycans on the ERAD substrates.Our studies underscore the functional importance of a plant PNGase orthologue as a deglycosylating enzyme involved in the ERAD.► Plant PNGase orthologue (AtPNG1) acts as a PNGase in yeast, facilitating ERAD. ► A catalytic mutant of AtPNG1 stabilises glycosylated ERAD substrates. ► Non-glycosylated ERAD substrate is degraded in an AtPNG1-independent manner. ► Binding between mutant AtPNG1 and the N-glycans on the ERAD substrate is observed.

Keywords: Abbreviations; PNGase; peptide:; N; -glycanase; ERAD; ER-associated degradation; TG and TGase; transglutaminase; RTA∆; ricin A chain non-toxic mutant; RTL; RTA∆-transmembrane-Leu2; SC; synthetic complete; endo H; endoglycosidase HPeptide:; N; -glycanase; ER-associated degradation; Yeast

Polyunsaturated fatty acids cause apoptosis in C. albicans and C. dubliniensis biofilms by Vuyisile S. Thibane; Ruan Ells; Arno Hugo; Jacobus Albertyn; Walter J. Janse van Rensburg; Pieter W.J. Van Wyk; Johan L.F. Kock; Carolina H. Pohl (pp. 1463-1468).

Polyunsaturated fatty acids (PUFAs) have antifungal properties, but the mode by which they induce their action is not always clear. The aim of the study was to investigate apoptosis as a mode of action of antifungal PUFAs (stearidonic acid, eicosapentaenoic acid and docosapentaenoic acid) which are inhibitory towards biofilm formation of C. albicans and C. dubliniensis. Candida biofilms were grown in the absence or presence of 1mM PUFAs (linoleic acid, stearidonic acid, eicosapentaenoic acid, docosapentaenoic acid) for 48h at 37°C. The effect of these PUFAs on the membrane fatty acid profile and unsaturation index, oxidative stress, mitochondrial transmembrane potential and apoptosis was evaluated.When biofilms of C. albicans and C. dubliniensis were exposed to certain PUFAs there was an increase in unsaturation index of the cellular membranes and accumulation of intracellular reactive oxygen species (ROS). This resulted in apoptosis, evidenced by reduced mitochondrial membrane potential and nuclear condensation and fragmentation. The most effective PUFA was stearidonic acid.The resultant cell death of both C. albicans and C. dubliniensis is due to apoptosis.Due to the increase in drug resistance, alternative antifungal drugs are needed. A group of natural antifungal compounds is PUFAs. However, understanding their mechanisms of action is important for further use and development of these compounds as antifungal drugs. This paper provides insight into a possible mode of action of antifungal PUFAs.► Antifungal PUFAs increase unsaturation index of C. albicans and C. dubliniensis membranes. ► Antifungal PUFAs increase intracellular reactive oxygen species. ► Antifungal PUFAs decrease mitochondrial membrane potential, leading to apoptosis. ► We propose this as a mechanism of action of antifungal PUFAs.

Keywords: Antifungal polyunsaturated fatty acids; Apoptosis; Biofilms; Candida albicans; Candida dubliniensis; Stearidonic acid

Structural basis for catalytic activity of a silkworm Delta-class glutathione transferase by Kohji Yamamoto; Kazuhiro Usuda; Yoshimitsu Kakuta; Makoto Kimura; Akifumi Higashiura; Atsushi Nakagawa; Yoichi Aso; Mamoru Suzuki (pp. 1469-1474).

Glutathione transferase (GST) catalyzes glutathione conjugation, a major detoxification pathway for xenobiotics and endogenous substances. Here, we determined the crystal structure of a Delta-class GST from Bombyx mori (bmGSTD) to examine its catalytic residues.The three-dimensional structure of bmGSTD was resolved by the molecular replacement method and refined to a resolution of 2.0Å.Structural alignment with a Delta-class GST of Anopheles gambiae indicated that bmGSTD contains 2 distinct domains (an N-terminal domain and a C-terminal domain) connected by a linker. The bound glutathione localized at the N-terminal domain. Putative catalytic residues were changed to alanine by site-directed mutagenesis, and the resulting mutants were characterized in terms of catalytic activity using glutathione and 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic analysis of bmGSTD mutants indicated that Ser11, Gln51, His52, Ser67, and Arg68 are important for enzyme function.These results provide structural insights into the catalysis of glutathione conjugation in B. mori by bmGSTD.► We determined the crystal structure of Delta-class GST from Bombyx mori (bmGSTD). ► Secondary and tertiary structures are highly conserved in bmGSTD. ► bmGSTD is stabilized by a hydrophobic lock-and-key motif. ► We determined crucial residues for glutathione binding by site-directed mutagenesis. ► Our study reveals possible catalysis mechanisms in glutathione conjugation of bmGSTD.

Keywords: Abbreviations; CBB; Coomassie Brilliant Blue R250; CDNB; 1-chloro-2,4-dinitrobenzene; ECA; ethacrynic acid; GTX; S; -hexyl glutathione; GSH; glutathione; GST; glutathione transferase; 4-HNE; 4-hydroxynonenal; IPTG; isopropyl 1-thio-β-; d; -galactoside; LB; Luria–Bertani medium; PCR; polymerase chain reaction; RT-PCR; reverse transcriptase PCR; PEG; polyethylene glycol; ROS; reactive oxygen species; SDS‐PAGE; sodium dodecyl sulfate‐polyacrylamide gel electrophoresisCrystal structure; Glutathione; Glutathione transferase; Lepidoptera; Site-directed mutagenesis

Evaluation of sialic acid-analogs for the attenuation of amyloid-beta toxicity by Dhruva Dhavale; James E. Henry (pp. 1475-1480).

Amyloid-beta peptide (Aβ) is the main constituent of senile plaques and is implicated in the pathogenesis of Alzheimer's disease (AD). To that end, agents which either sequester Aβ or interfere with Aβ interaction/binding to cells have been investigated as a means to reduce the pathological effects of Aβ.Different structural analogs of sialic acid (N-acetylneuramic acid) were used to decorate a chitosan backbone using EDC chemistry. FTIR and colorimetric assays were used to characterize the complexes. The ability of these complexes to attenuate Aβ toxicity was investigated in vitro using a model neuroblastoma cell line SH-SY5Y.Oxygen substitution in ring structure is responsible for the increase in toxicity and increase in protective properties of the complexes. Also, the multiOH tail present in sialic acid is critical to attenuate toxicity. Analogs show no protective properties which reinforces the conclusion that clustering of sugars in cellular membranes play a significant role in Aβ binding.Successfully produced compounds that showed varying degree of efficacy in attenuating Aβ toxicity to cells in culture. This work elucidates the impact that certain structures of sialic acid and its analogs can have on Aβ binding. It will allow for more specific and detailed improvements in the therapeutic polysaccharide structures that can be developed and modified to overcome other shortcomings of AD therapeutic development, particularly of penetrating the blood–brain barrier.Oxygen atom plays crucial role on therapeutic effectiveness. This work can help as a general guideline for further therapeutic development.► Successful decoration of chitosan by different sialic acid analogs. ► Results show change in intrinsic toxicity before and after complexation of sugars. ► Demonstrated how unique structures effect Aβ toxicity attenuation. ► Results show importance of oxygen substitution and multi-OH tail of sialic acid. ► Results indicate oxygen containing structures will show therapeutic effectiveness.

Keywords: Abbreviations; Aβ; Amyloid-beta peptide; AD; Alzheimer's disease; KDN; Keto-deoxynonulosonic acid; EDC; 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; Sulfo-NHS; N-hydroxysuccinimide; Pyran; Tetrahydropyran-2-carboxylic acid; GA; D(+) galacturonic acid; CHC; Cyclohexanecarboxylic acid; DMSO; dimethyl sulfoxide; NGF; Nerve growth factor; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MES; 2-(morpholino)ethanesulfonic acidAmyloid-beta toxicity; Therapeutics for Alzheimer's disease; Ganglioside; Sialic acid analog; Subgroups and substructure; Chitosan

Potent low toxicity inhibition of human melanogenesis by novel indole-containing octapeptides by Anan Abu Ubeid; Sylvia Do; Chris Nye; Basil M. Hantash (pp. 1481-1489).

Abnormal production and accumulation of melanin are characteristics of a number of skin disorders, including postinflammatory hyperpigmentation and melasma. Our objective was to develop and validate novel oligopeptides with potent inhibitory activity against mushroom and human tyrosinase with minimal toxicity toward melanocytes, keratinocytes, and fibroblasts.A library of short sequence oligopeptides was docked against the crystal structure of mushroom tyrosinase to screen for favorable binding free energies and direct interaction with the catalytic pocket. The inhibitory activity of the octapeptides and hydroquinone (HQ) was assessed using mushroom and human tyrosinase and melanin content via human primary melanocytes. Effects on cell viability and proliferation were determined using the MTT assay and cytotoxicity via trypan blue exclusion.Octapeptides P16–18 outperformed HQ, the benchmark of hypopigmenting agents, in all tested categories. Prolonged incubation of human keratinocytes, fibroblasts, or melanocytes with 30–3000μM HQ led to 8- to 65-fold greater cell death than with octapeptides. After 6d of incubation with 30μM HQ, we observed 70±3% and 60±2% cell death in melanocytes and fibroblasts, respectively, versus minimal toxicity up to an octapeptide concentration of 3mM.Octapeptides P16–18 are potent competitive tyrosinase inhibitors with minimal toxicity toward the major cell types of human skin.The findings in our study suggest that all three novel octapeptides may serve as safe and efficacious replacements of HQ for the treatment of pigmentary disorders.► P16–18 octapeptides are potent competitive inhibitors of mushroom/human tyrosinase. ► P16–18 are 29% to 58% more potent tyrosinase inhibitors than hydroquinone. ► P16–18 octapeptides were 8- to 65-fold less toxic than hydroquinone. ► P16–18 reduced melanin content by 29–58%. ► Octapeptide inhibition may involve indole moiety binding catalytic site copper ions.

Keywords: Abbreviations; HQ; hydroquinone; l; -DOPA; l; -3-4-dihydroxyphenylalanineCopper chelation; Hydroquinone; Melanogenesis; Molecular docking; Octapeptide; Tyrosinase inhibition

Conjugated linoleic acid in the maternal diet differentially enhances growth and cortical spreading depression in the rat progeny by Juliana K.B. Soares; Ana P. Rocha-de-Melo; Maria C. Medeiros; Rita C.R.E. Queiroga; Marco A.D. Bomfim; Amanda F.O. de Souza; Ana L.V. Nascimento; Rubem C.A. Guedes (pp. 1490-1495).

Conjugated linoleic acids (CLA) are fatty acids that are found in the lipids from goat milk, and appear to protect neurons from excitotoxicity.We investigated in developing rats the effects of a maternal CLA-rich diet (containing 7% lipids from goat milk) on body development and cerebral electrical activity of the progeny from dams receiving the CLA diet during gestation (G), lactation (L) or both periods (G+L).Compared to a control group (C) receiving a diet with 7% soybean oil, body weight increased at 14, 21 and 28days, but not at 35–45days, in L and G+L groups (P<0.05). No intergroup difference was found on body and brain weights, body length, abdominal and thoracic circumferences, body mass index and abdominal to thoracic circumference ratio at 35–45days. In contrast, at this later age the CSD velocities of propagation were significantly higher (P<0.05) in L as compared with the C and G group, and in the L+G, as compared with the C, G and L groups, suggesting a long-lasting brain effect.These data indicate that a maternal CLA-rich diet can differentially influence body weight increment (short-term effect), and CSD propagation (long-term effect) in the progeny, and the lactation is the most critical period for such diet actions.The facilitating effect of the lipids from goat milk on an excitability-related phenomenon in the brain (CSD) can be of clinical relevance, since CSD has been associated to neurological disturbances like migraine and epilepsy.► We explored brain effects of dietary conjugated linoleic acid (CLA), in rats. ► We recorded cortical spreading depression (CSD) and measure its propagation velocity. ► We describe a CSD-facilitation by CLA dietary treatment, compared to CLA-free rats. ► Data are medically relevant as CSD is related to diseases like migraine and epilepsy.

Keywords: Brain development; Brain electrophysiology; Conjugated linoleic acid; Physical parameter; Polyunsaturated fatty acid; Rats

Correlation of serotonin levels in CSF, platelets, plasma, and urine by Tapan Audhya; James B. Adams; Leah Johansen (pp. 1496-1501).

Neurotransmitter levels are best measured in the cerebrospinal fluid (CSF), but that requires an invasive procedure.Samples were collected from humans and rats. Eighteen women age 38–51years with fibromyalgia provided samples of CSF, plasma, platelets, and urine. Samples of CSF, plasma, platelets, and urine were also collected from Sprague–Dawley rats, adult male, 6months old. One group of rats was treated with p-chlorophenylalanine to decrease their levels of serotonin, and another group of rats was treated with amphetamine to increase their levels of serotonin.Methodological improvements include: 1) the use of siliconized glassware, plasticware, and tubing to prevent adsorption of serotonin, 2) the extraction of serotonin from the CSF, plasma, and platelets, 3) repeated washing of the platelets with an improved buffer, and 4) early morning sample collection. HPLC/MS was used to measure serotonin after extraction.For serotonin, the new method of measuring platelet levels resulted in a very high correlation with levels of serotonin in CSF in rats (r=0.97) and humans (r=0.97). There were lower correlations of levels of serotonin in CSF with levels in plasma (r=0.77 for rats and r=0.57 in humans) and urine (r=0.67 in rats and r=0.62 in humans).This method of measuring serotonin levels in platelets results in a very strong correlation with levels in CSF, so in most cases platelet measurements will be preferable since it is much less invasive to collect. Levels of serotonin in plasma and urine are significantly but less strongly correlated with levels in CSF.► An improved method of measuring serotonin levels was developed. ► Levels of serotonin in CSF and platelets were highly correlated in humans (r=0.97). ► First study to show high correlation of serotonin in CSF and blood in humans ► Levels of serotonin in CSF and platelets were highly correlated in rats (r=0.97). ► Lower correlations were found for serotonin in CSF vs. plasma or urine.

Keywords: Serotonin; Platelet; Cerebrospinal fluid; Plasma; Urine; Human

Energetics, conformation, and recognition of DNA duplexes containing a major adduct of an anticancer azolato-bridged dinuclear Pt^II complex by Jarmila Mlcouskova; Jaroslav Malina; Vojtech Novohradsky; Jana Kasparkova; Seiji Komeda; Viktor Brabec (pp. 1502-1511).

The design of anticancer metallodrugs is currently focused on platinum complexes which form on DNA major adducts that cannot readily be removed by DNA repair systems. Hence, antitumor azolato-bridged dinuclear Pt^II complexes, such as [{cis-Pt(NH₃)₂}₂(μ‐OH)(μ-pyrazolate)]²⁺ (AMPZ), have been designed and synthesized. These complexes exhibit markedly higher toxic effects in tumor cell lines than mononuclear conventional cisplatin.Biophysical and biochemical aspects of the alterations induced in short DNA duplexes uniquely and site-specifically modified by the major DNA adduct of AMPZ, namely 1,2-GG intrastrand cross-links, were examined. Attention was also paid to conformational distortions induced in DNA by the adducts of AMPZ and cisplatin, associated alterations in the thermodynamic stability of the duplexes, and recognition of these adducts by high-mobility-group (HMG) domain proteins.Chemical probing of DNA conformation, DNA bending studies and translesion synthesis by DNA polymerase across the platinum adduct revealed that the distortion induced in DNA by the major adduct of AMPZ was significantly less pronounced than that induced by similar cross-links from cisplatin. Concomitantly, the cross-link from AMPZ reduced the thermodynamic stability of the modified duplex considerably less. In addition, HMGB1 protein recognizes major DNA adducts of AMPZ markedly less than those of cisplatin.The experimental evidence demonstrates why the major DNA adducts of the new anticancer azolato-bridged dinuclear Pt^II complexes are poor substrates for DNA repair observed in a previously published report. The relative resistance to DNA repair explains why these platinum complexes show major pharmacological advantages over cisplatin in tumor cells.Display Omitted► Mechanism of anticancer effects of anticancer dinuclear Pt^II complex was examined. ► The new Pt^II complex distorts DNA less extensively than conventional cisplatin. ► The new complex thermodynamically destabilizes DNA much less than cisplatin. ► Rationale behind improved antitumor effects of the new Pt^II complex is outlined.

Keywords: Abbreviations; AMPZ; [{cis-{Pt(NH; ₃; ); ₂; }; ₂; (μ-OH)(μ-pyrazolate)]; ²⁺; cisplatin; [; cis; -diamminedichloridoplatinum(II)]; bp; base pair; CL; cross-link; CT; calf thymus; DSC; differential scanning calorimetry; EMSA; electrophoretic mobility shift assay; FAAS; flameless atomic absorption spectrometry; DMS; dimethyl sulfate; dNTP; deoxyribonucleotide triphosphate; HPLC; high-pressure liquid chromatography; HMG; high mobility group; HMGB1a; domain A of full length HMGB1 protein; HMGB1b; domain B of full length HMGB1 protein; LD; linear dichroism; T; _m; melting temperature; NER; nucleotide excision repair; PAA; polyacrylamide; Polη; DNA polymerase η; r; _b; the number of molecules of the platinum complex bound per nucleotide residue; SDS; sodium dodecyl sulfate; XPA; xeroderma pigmentosum group ADNA conformation; Dinuclear platinum; Thermodynamic stability; Damaged-DNA binding protein; DNA repair; Anticancer

Non-synonymous single nucleotide polymorphisms in genes for immunoregulatory galectins: Association of galectin-8 (F19Y) occurrence with autoimmune diseases in a Caucasian population by Pal Zsuzsanna Pál; Péter Antal; Sanjeev Kumar Srivastava; Hullam Gábor Hullám; Ágnes F. Semsei; Gal János Gál; Svebis Mihály Svébis; Soos Györgyi Soós; Csaba Szalai; Andre Sabine André; Elena Gordeeva; György Nagy; Herbert Kaltner; Nicolai V. Bovin; Molnar Mária Judit Molnár; András Falus; Hans-Joachim Gabius; Buzas Edit Irén Buzás (pp. 1512-1518).

Galectins are potent immune regulators, with galectin-8 acting as a pro-apoptotic effector on synovial fluid cells and thymocytes and stimulator on T-cells. To set a proof-of-principle example for risk assessment in autoimmunity, and for a mutation affecting physiological galectin sensor functions, a polymorphism in the coding region of the galectin-8 gene (rs2737713; F19Y) was studied for its association with two autoimmune disorders, i.e. rheumatoid arthritis and myasthenia gravis.A case–control analysis and a related quantitative trait-association study were performed to investigate the association of this polymorphism in patients (myasthenia gravis 149, rheumatoid arthritis 214 and 134 as primary and repetitive cohorts, respectively) and 365 ethnically matched (Caucasian) healthy controls. Distribution was also investigated in patients grouped according to their antibody status and age at disease onset. Comparative testing for lectin activity was carried out in ELISA/ELLA-based binding tests with both wild-type and F19Y mutant galectin-8 from peripheral blood mononuclear cell lysates of healthy individuals with different genotypes as well as with recombinant wild-type and F19Y mutant galectin-8 proteins.A strong association was found for rheumatoid arthritis, and a mild one with myasthenia gravis. Furthermore, the presence of the sequence deviation also correlated with age at disease onset in the case of rheumatoid arthritis. The F19Y substitution did not appear to affect carbohydrate binding in solid-phase assays markedly.This is the first report of an association between a galectin-based polymorphism leading to a mutant protein and autoimmune diseases, with evidence for antagonistic pleiotropy.► Focuses on a polymorphism in the coding region of the galectin-8 gene. ► Reveals clinical association with rheumatoid arthritis and also myasthenia gravis. ► Directs attention to functional study beyond trisaccharide recognition.

Keywords: Abbreviations; AChR; acetylcholine receptor; ANOVA; analysis of variances; BMLA; Bayesian multilevel analysis; BSA; bovine serum albumin; CCP; cyclic citrullinated peptide; CD; cluster of differentiation; CI; confidence interval; ELISA; enzyme-linked immunosorbent assay; ELLA; enzyme-linked lectin assay; Exp(B); exponentiation of the B value; HLA; human leukocyte antigen; HWE; Hardy–Weinberg equilibrium; LGALS8; human galectin-8; MG; myasthenia gravis; OD; optical density; OR; odds ratio; PBMC; peripheral blood mononuclear cell; PBS; phosphate-buffered saline; RA; rheumatoid arthritis; SD; standard deviation; SE; standard error of mean; SNP; single nucleotide polymorphismAllele; Autoimmunity; Lectin; Neoglycoconjugate; Sialylation

Helicobacter pylori hydrogenase accessory protein HypA and urease accessory protein UreG compete with each other for UreE recognition by Stéphane L. Benoit; Jonathan L. McMurry; Stephanie A. Hill; Robert J. Maier (pp. 1519-1525).

The gastric pathogen Helicobacter pylori relies on nickel-containing urease and hydrogenase enzymes in order to colonize the host. Incorporation of Ni²⁺ into urease is essential for the function of the enzyme and requires the action of several accessory proteins, including the hydrogenase accessory proteins HypA and HypB and the urease accessory proteins UreE, UreF, UreG and UreH.Optical biosensing methods (biolayer interferometry and plasmon surface resonance) were used to screen for interactions between HypA, HypB, UreE and UreG.Using both methods, affinity constants were found to be 5nM and 13nM for HypA–UreE and 8μM and 14μM for UreG-UreE. Neither Zn²⁺ nor Ni²⁺ had an effect on the kinetics or stability of the HypA–UreE complex. By contrast, addition of Zn²⁺, but not Ni²⁺, altered the kinetics and greatly increased the stability of the UreE–UreG complex, likely due in part to Zn²⁺-mediated oligomerization of UreE. Finally our results unambiguously show that HypA, UreE and UreG cannot form a heterotrimeric protein complex in vitro; instead, HypA and UreG compete with each other for UreE recognition.Factors influencing the pathogen's nickel budget are important to understand pathogenesis and for future drug design.► Two different optical biosensing methods were used (biolayer interferometry and surface plasmon resonance). ► Interactions between four hydrogenase and/or urease accessory proteins were analyzed. ► Kinetic constants for HypA–UreE and UreE–UreG were determined. ► HypA–UreE interaction is tighter than UreE–UreG. ► A competition between HypA and UreG for UreE was found.

Keywords: Abbreviations; BLI; biolayer interferometry; SPR; Surface Plasmon ResonanceMetalloenzyme; Metal homeostasis; Hydrogenase; Urease; Nickel; Zinc

Understanding the drug resistance mechanism of hepatitis C virus NS3/4A to ITMN-191 due to R155K, A156V, D168A/E mutations: A computational study by Dabo Pan; Weiwei Xue; Wenqi Zhang; Huanxiang Liu; Xiaojun Yao (pp. 1526-1534).

ITMN-191 (RG7227, Danoprevir), as a potential inhibitor of the NS3/4A protease of hepatitis C virus, has been in phase 2 clinical trial. Unfortunately, several ITMN-191 resistance mutants including R155K, A156V, and D168A/E have been identified.Molecular dynamics simulation, binding free energy calculation and per-residue energy decomposition were employed to explore the binding and resistance mechanism of hepatitis C virus NS3/4A protease to ITMN-191.Based on molecular dynamics simulation and per-residue energy decomposition, the nonpolar energy term was found to be the driving force for ITMN-191 binding. For the studied R155K, A156V, D168A/E mutants, the origin of resistance is mainly from the conformational changes of the S4 and extended S2 binding pocket induced by the studied mutants and further leading to the reduced binding ability to the extended P2 and P4 moieties of ITMN-191.Further structural analysis indicates that the destruction of conservative salt bridges between residues 168 and 155 should be responsible for the large conformation changes of the binding pocket in R155K and D168A/E mutants. For A156V mutation, the occurrence of drug resistance is mainly from the changed binding pocket by a replacement of one bulky residue Val.The obtained drug resistance mechanism of this study will provide useful guidance for the development of new and effective HCV NS3/4A inhibitors with low resistance.► Drug resistance mechanism of hepatitis C virus NS3/4A to ITMN-191 was explored. ► For the mutations at 155 and 168, the main reason is the destroy of key salt bridges. ► For A156V mutation, the origin of drug resistance is the change of binding pocket.

Keywords: Hepatitis C virus; NS3/4A protease; Drug resistance; ITMN-191; Molecular dynamics simulation

Cysteine protease attribute of eukaryotic ribosomal protein S4 by Babu Sudhamalla; Madasu Yadaiah; Dasari Ramakrishna; Abani K. Bhuyan (pp. 1535-1542).

Ribosomal proteins often carry out extraribosomal functions. The protein S4 from the smaller subunit of Escherichia coli, for instance, regulates self synthesis and acts as a transcription factor. In humans, S4 might be involved in Turner syndrome. Recent studies also associate many ribosomal proteins with malignancy, and cell death and survival. The list of extraribosomal functions of ribosomal proteins thus continues to grow.Enzymatic action of recombinant wheat S4 on fluorogenic peptide substrates Ac–XEXD↓–AFC (N-acetyl-residue-Glu-residue-Asp-7-amino-4-trifluoromethylcoumarin) and Z–FR↓–AMC (N-CBZ-Phe-Arg-aminomethylcoumarin) as well as proteins has been examined under a variety of solution conditions.Eukaryotic ribosomal protein S4 is an endoprotease exhibiting all characteristics of cysteine proteases. The K_m value for the cleavage of Z–FR↓–AMC by a cysteine mutant (C41F) is about 70-fold higher relative to that for the wild-type protein under identical conditions, implying that S4 is indeed a cysteine protease. Interestingly, activity responses of the S4 protein and caspases toward environmental parameters, including pH, temperature, ionic strength, and Mg2⁺ and Zn2⁺ concentrations, are quite similar. Respective kinetic constants for their cleavage action on Ac–LEHD↓–AFC are also similar. However, S4 cannot be a caspase, because unlike the latter it also hydrolyzes the cathepsin substrate Z–FR↓–AMC.The eukaryotic S4 is a generic cysteine protease capable of hydrolyzing a broad spectrum of synthetic substrates and proteins. The enzyme attribute of eukaryotic ribosomal protein S4 is a new phenomenon. Its possible involvement in cell growth and proliferations are presented in the light of known extraribosomal roles of ribosomal proteins.► The eukaryotic ribosomal protein S4 is a cysteine protease. ► It cleaves synthetic substrates commonly used for assaying cathepsins and caspases. ► Proteolysis of at least two recombinant proteins, Bcl-x_L and eIF2α, is demonstrated. ► The extraribsosomal activity of S4 could have roles in cell growth and proliferation.

Keywords: Abbreviations; DEVD-CHO; N-acetyl-Asp-Glu-Val-Asp-aldehyde; Ac-XEXD-AFC; N-acetyl-residue-Glu-residue-Asp-7-amino-4-trifluoromethylcoumarin; Z-FR-AMC; N-CBZ-Phe-Arg-aminomethylcoumarinNon-caspase cysteine protease; Ribosomal protein S4; Specificity of caspase substrates; Caspase substrate cleavage

The only exoribonuclease present in Haloferax volcanii has an unique response to temperature changes by Rute G. Matos; Lopez-Vinas Eduardo López-Viñas; Gomez-Puertas Paulino Goméz-Puertas; Cecília M. Arraiano (pp. 1543-1552).

Little is known regarding mRNA degradation mechanisms in archaea. In some of these single-cell organisms the existence of a complex of exoribonucleases called the exosome has been demonstrated. However, in halophilic archaea the RNase R homologue is essential since it is the only enzyme described with exoribonucleolytic activity.In this work we have characterized the mechanism of action of Haloferax volcanii RNase R and its implications for the RNA degradation process. We have determined the salt, pH and divalent ion preference, and set the best conditions for the activity assays. Furthermore, we have determined the activity of the protein at different temperatures using different substrates. The dissociation constants were also calculated by Surface Plasmon Resonance. Finally, we have built a model and compared it with the Escherichia coli counterparts.The results obtained showed that at 37°C, in spite of being named RNase R, this protein behaves like an RNase II protein, halting when it reaches secondary structures, and releasing a 4 nt end-product. However, at 42°C, the optimum temperature of growth, this protein is able to degrade secondary structures, acting like RNase R.This discovery has a great impact for RNA degradation, since this is the first case reported where a single enzyme has two different exoribonucleolytic activities according to the temperature. Furthermore, the results obtained are very important to help to decipher the RNA degradation mechanisms in H. volcanii, since RNase R is the only exoribonuclease involved in this process.► RNase R is the only enzyme described with exoribonucleolytic activity in Haloferax. ► Characterization of H. volcanii RNase R exonucleolytic and RNA binding activities. ► Protein predicted to be like E. coli RNase R but at 37°C behaves like RNase II. ► Unusual protein with two different activities according to the temperature. ► Disclosure of additional levels of regulation by RNases during RNA decay.

Keywords: Abbreviations; RNase; ribonuclease; CSD; Cold Shock Domain; DTT; Dithiothreitol; EDTA; Ethylenediaminetetraacetic acid; SPR; Surface plasmon resonance; PDB; protein data bank; PNPase; Polynucleotide phosphorylase; PAP I; Poly(A) Polymerase; RMSD; Root mean square deviationRNA degradation; RNase R; RNase II; Exosome; Temperature; Archaea

Vitamin D-mediated regulation of CYP21A2 transcription — A novel mechanism for vitamin D action by Johan Lundqvist; Kjell Wikvall; Maria Norlin (pp. 1553-1559).

1α,25-Dihydroxyvitamin D₃ has recently been reported to decrease expression and activity of CYP21A2. In this paper, we have studied the mechanisms for the 1α,25-dihydroxyvitamin D₃-mediated effect on CYP21A2 transcriptional rate.We have studied the effects of 1α,25-dihydroxyvitamin D₃ using luciferase reporter constructs containing different lengths of the CYP21A2 promoter. These constructs were transfected into cell lines derived from human and mouse adrenal cortex. The mechanism for the effects of vitamin D on the CYP21A2 promoter was studied using chromatin immunoprecipitation assay, mutagenesis and gene silencing by siRNA.1α,25-Dihydroxyvitamin D₃ was found to alter the promoter activity via a VDR-mediated mechanism, including the comodulators VDR interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF). The involvement of comodulator VDIR was confirmed by gene silencing. We identified a vitamin D response element in the CYP21A2 promoter. Interaction between this novel response element and VDR, WSTF and VDIR was shown by chromatin immunoprecipitation assay. When this sequence was deleted, the effect of 1α,25-dihydroxyvitamin D₃ was abolished, indicating that this sequence in the CYP21A2 promoter functions as a vitamin D response element. Interestingly, an altered balance between nuclear receptors and comodulators reversed the suppressing effect of vitamin D to a stimulatory effect.This paper reports data important for the understanding of the mechanisms for vitamin D-mediated suppression of gene expression as well as for the vitamin D-mediated effects on CYP21A2. We report a novel mechanism for effects of 1α,25-dihydroxyvitamin D₃.► The transcriptional rate of CYP21A2 is downregulated by 1α,25-dihydroxyvitamin D₃. ► The effect is mediated by nuclear receptor VDR and comodulators VDIR and WSTF. ► The expression level of comodulators may shift the suppression to stimulation. ► This paper reports a functional VDRE in the CYP21A2 promoter. ► Our data indicate a novel mechanism for vitamin D action.

Keywords: Abbreviations; RXR; retinoid X receptor; SF-1; steroidogenic factor-1; VDIR; VDR interacting repressor; VDR; vitamin D receptor; VDRE; vitamin D responsive element; nVDRE; negative vitamin D responsive element; WSTF; Williams syndrome transcription factorSteroidogenesis; Vitamin D; Calcitriol; CYP21A2; Steroid

The mechanism underlying nitroxyl and nitric oxide formation from hydroxamic acids by Yuval Samuni; Uri Samuni; Sara Goldstein (pp. 1560-1566).

The pharmacological effects of hydroxamic acids (RC(O)NHOH, HX) are partially attributed to their ability to serve as HNO and/or NO donors under oxidative stress. Given the development and use of HXs as therapeutic agents, elucidation of the oxidation mechanism is needed for more educated selection of HX-based drugs.Acetohydroxamic and glycine-hydroxamic acids were oxidized at pH 7.0 by a continuous flux of radiolytically generated^·OH or by metmyoglobin and H₂O₂ reactions system. Gas chromatography and spectroscopic methods were used to monitor the accumulation of N₂O, N₂, nitrite and hydroxylamine.Oxidation of HXs by^·OH under anoxia yields N₂O, but not nitrite, N₂ or hydroxylamine. Upon the addition of H₂O₂ to solutions containing HX and metmyoglobin, which is instantaneously and continuously converted into compound II, nitrite and, to a lesser extent, N₂O are accumulated under both anoxia and normoxia.Oxidation of HXs under anoxia by a continuous flux of^·OH, which solely oxidizes the hydroxamate moiety to RC(O)NHO^·, forms HNO. This observation implies that bimolecular decomposition of RC(O)NHO^· competes efficiently with unimolecular decomposition processes such as internal disproportionation, hydrolysis or homolysis. Oxidation by metmyoglobin/H₂O₂ involves relatively mild oxidants (compounds I and II). Compound I reacts with HX forming RC(O)NHO^· and compound II, which oxidizes HX, RC(O)NHO^·, HNO and NO. The latter reaction is the main source of nitrite.HXs under oxidative stress release HNO, but can be considered as NO-donors provided that HNO oxidation is more efficient than its reaction with other biological targets.► Oxidation of hydroxamic acids (HXs) by a continuous generation of^·OH yields HNO. ► HNO is formed via the bimolecular decomposition of the transient nitroxide radical. ► HXs oxidation by metMb/H₂O₂ forms nitrite and HNO under both anoxia and normoxia. ► HXs can be considered as NO-donors under oxidative stress if HNO is oxidized to NO.

Keywords: Abbreviations; aceto-HX; acetohydroxamic acid; MbFe; ^IV; =O; ferryl myoglobin; GC; gas chromatography; gly-HX; glycine-hydroxamic acid; HX; hydroxamic acid; MbFe; ^III; metmyoglobin; PB; phosphate bufferHNO donor; Radiolysis; ^·; OH radical; Compound II; Nitroxide radical; Oxidation

Sulfate uptake in photosynthetic Euglena gracilis. Mechanisms of regulation and contribution to cysteine homeostasis by Garcia-Garcia Jorge Donato García-García; Viridiana Olin-Sandoval; Emma Saavedra; Lourdes Girard; Hernandez Georgina Hernández; Moreno-Sanchez Rafael Moreno-Sánchez (pp. 1567-1575).

Sulfate uptake was analyzed in photosynthetic Euglena gracilis grown in sulfate sufficient or sulfate deficient media, or under Cd²⁺ exposure or Cys overload, to determine its regulatory mechanisms and contribution to Cys homeostasis.In control and sulfate deficient or Cd²⁺-stressed cells, one high affinity and two low affinity sulfate transporters were revealed, which were partially inhibited by photophosphorylation and oxidative phosphorylation inhibitors and ionophores, as well as by chromate and molybdate; H⁺ efflux also diminished in presence of sulfate. In both sulfate deficient and Cd²⁺-exposed cells, the activity of the sulfate transporters was significantly increased. However, the content of thiol-metabolites was lower in sulfate-deficient cells, and higher in Cd²⁺-exposed cells, in comparison to control cells. In cells incubated with external Cys, sulfate uptake was strongly inhibited correlating with 5-times increased intracellular Cys. Re-supply of sulfate to sulfate deficient cells increased the Cys, γ-glutamylcysteine and GSH pools, and to Cys-overloaded cells resulted in the consumption of previously accumulated Cys. In contrast, in Cd²⁺ exposed cells none of the already elevated thiol-metabolites changed.(i) Sulfate transport is an energy-dependent process; (ii) sulfate transporters are over-expressed under sulfate deficiency or Cd²⁺ stress and their activity can be inhibited by high internal Cys; and (iii) sulfate uptake exerts homeostatic control of the Cys pool.► Plasma membrane sulfate uptake was coupled to H⁺ gradient in Euglena gracilis. ► Sulfate transporter activity increased under sulfate-deficiency or Cd²⁺ stress. ► Sulfate uptake was strongly inhibited by Cys accumulation. ► Sulfate uptake exerted control on the Cys concentration under sulfate deficiency.

Keywords: Abbreviations; APS; adenosine 5´-phosphosulfate; EST; expressed sequence tags; γ-EC; γ-glutamylcysteine; γ-ECS; γ-glutamylcysteine synthetase; GSH; glutathione; GS; glutathione synthetase; HAST; high affinity sulfate transporters; LAST; low affinity sulfate transporters; PMSTs; plasma membrane sulfate transporters; PCs; phytochelatinsSulfate uptake kinetics; Sulfate uptake regulation; Sulfate deficiency; Cadmium stress; Cys accumulation

Type III and V collagens modulate the expression and assembly of EDA⁺ fibronectin in the extracellular matrix of defective Ehlers–Danlos syndrome fibroblasts by Nicoletta Zoppi; Marco Ritelli; Marina Colombi (pp. 1576-1587).

Alternative splicing of EDA fibronectin (FN) region is a cell type- and development-regulated mechanism controlled by pathological processes, growth factors and extracellular matrix (ECM). Classic and vascular Ehlers–Danlos syndrome (cEDS and vEDS) are connective tissue disorders caused by COL5A1/ COL5A2 and COL3A1 gene mutations, leading to an in vivo abnormal collagen fibrillogenesis and to an in vitro defective organisation in the ECM of type V (COLLV) and type III collagen (COLLIII). These defects induce the FN-ECM disarray and the decrease of COLLs and FN receptors, the α2β1 and α5β1 integrins. Purified COLLV and COLLIII restore the COLL-FN-ECMs in both EDS cell strains.Real-time PCR, immunofluorescence microscopy, and Western blotting were used to investigate the effects of COLLs on FN1 gene expression, EDA region alternative splicing, EDA⁺-FN-ECM assembly, α5β1 integrin and EDA⁺-FN-specific α9 integrin subunit organisation, α5β1 integrin and FAK co-regulation in EDS fibroblasts.COLLV-treated cEDS and COLLIII-treated vEDS fibroblasts up-regulate the FN1 gene expression, modulate the EDA⁺ mRNA maturation and increase the EDA⁺-FN levels, thus restoring a control-like FN-ECM, which elicits the EDA⁺-FN-specific α9β1 integrin organisation, recruits the α5β1 integrin and switches on the FAK binding and phosphorylation.COLLs regulate the EDA⁺-FN-ECM organisation at transcriptional and post-transcriptional level and activate the α5β1–FAK complexes. COLLs also recruit the α9β1 integrin involved in the assembly of the EDA⁺-FN-ECM in EDS cells.The knowledge of the COLLs-ECM role in FN isotype expression and in EDA⁺-FN-ECM-mediated signal transduction adds insights in the ECM remodelling mechanisms in EDS cells.► Ehlers–Danlos syndrome cells lack collagens and fibronectin extracellular matrix. ► Collagens III and V restore the matrix containing the EDA⁺ fibronectin isotype. ► Collagens III and V induce fibronectin gene expression and maturation of EDA⁺ mRNA. ► Collagens induce recruitment of α5β1 and α9β1 integrin and FAK-mediated signal transduction.

Keywords: Abbreviations; CE; cell extract; CM; culture medium; COLLIII; type III collagen; COLLV; type V collagen; COLLs; collagens; DOC-IS; deoxycholate-insoluble; ECM; extracellular matrix; FAK; focal adhesion kinase; FN; fibronectin; FN1; fibronectin geneFibronectin; EDA region; Alternative splicing; Collagen; Integrins; Ehlers–Danlos syndrome

The selenoproteins GPx2, TrxR2 and TrxR3 are regulated by Wnt signalling in the intestinal epithelium by Anna P. Kipp; Muller Mike F. Müller; Goken Eva M. Göken; Stefanie Deubel; Brigelius-Flohe Regina Brigelius-Flohé (pp. 1588-1596).

The glutathione peroxidase 2 (GPx2) is expressed at crypt bases of the intestinal epithelium and in tumour tissue. The GPx2 promoter is activated by the Wnt pathway, which might be the reason for the specific expression pattern of GPx2. Together with additional selenoproteins, thioredoxin reductases TrxR2 and TrxR3, which are putative Wnt targets based on microarray analysis, Wnt-dependent GPx2 expression was analysed.Two cell culture models for either an activated (3T3 cells with Wnt3a overexpression) or an inhibited Wnt pathway (HT-29 APC cells) were analysed. To provide physiological relevance, crypt base epithelial cells of the jejunum and colon of mice were compared to cells of the villus or crypt table, respectively. In addition, β-catenin was deleted in crypt base cells ex vivo.In cancer cell lines, the endogenous expression of all three selenoproteins was consistently dependent on Wnt pathway activity. Expression was higher in the proliferative crypt compartment, where also the Wnt pathway is active. An inducible knockout of β-catenin in isolated colonic crypt base cells reduced basal GPx2 expression.We, thus, demonstrated the regulation of GPx2 expression by the Wnt pathway in vitro and in vivo. Furthermore, the selenoproteins TrxR2 and TrxR3 have been identified as novel Wnt targets. This may imply a role of GPx2, TrxR2 and TrxR3 in proliferation, apoptosis and, therefore, also during cancer development.Selenium which is essential for the biosynthesis of Wnt-dependent selenoproteins might be important for the renewal of the intestinal epithelium and during carcinogenesis.► TrxR2 and TrxR3 are novel targets of the Wnt pathway. ► GPx2, TrxR2 and TrxR3 expression co-localises with an active Wnt pathway. ► GPx2 expression at crypt bases depends on an active Wnt pathway

Keywords: Abbreviations; GPx; glutathione peroxidase; TrxR; thioredoxin reductase; Gsk3β; glycogen synthase kinase 3β; APC; adenomatous polyposis coli; Dvl; dishevelled; Lgr5; leucine-rich repeat containing G protein-coupled receptor 5; Lrp; low density lipoprotein receptor-related protein; dnTCF4; dominant-negative TCF4; TGR; thioredoxin glutathione reductase; IFN; interferon; PCNA; proliferating cell nuclear antigen; Fabp2; intestinal fatty acid binding protein 2; RAR; retinoic acid receptorGlutathione peroxidase 2; Thioredoxin reductase 2; Thioredoxin reductase 3; Wnt; Selenium; Intestinal epithelium

Trimethylamine N-oxide suppresses the activity of the actomyosin motor by Ryusei Kumemoto; Kento Yusa; Tomohiro Shibayama; Kuniyuki Hatori (pp. 1597-1604).

During actomyosin interactions, the transduction of energy from ATP hydrolysis to motility seems to occur with the modulation of hydration. Trimethylamine N-oxide (TMAO) perturbs the surface of proteins by altering hydrogen bonding in a manner opposite to that of urea. Hence, we focus on the effects of TMAO on the motility and ATPase activation of actomyosin complexes.Actin and heavy meromyosin (HMM) were prepared from rabbit skeletal muscle. Structural changes in HMM were detected using fluorescence and circular dichroism spectroscopy. The sliding velocity of rhodamine-phalloidin-bound actin filaments on HMM was measured using an in vitro motility assay. ATPase activity was measured using a malachite green method.Although TMAO, unlike urea, stabilized the HMM structure, both the sliding velocity and ATPase activity of acto-HMM were considerably decreased with increasing TMAO concentrations from 0–1.0M. Whereas urea-induced decreases in the structural stability of HMM were recovered by TMAO, TMAO further decreased the urea-induced decrease in ATPase activation. Urea and TMAO were found to have counteractive effects on motility at concentrations of 0.6M and 0.2M, respectively.The excessive stabilization of the HMM structure by TMAO may suppress its activities; however, the counteractive effects of urea and TMAO on actomyosin motor activity is distinct from their effects on HMM stability.The present results provide insight into not only the water-related properties of proteins, but also the physiological significance of TMAO and urea osmolytes in the muscular proteins of water-stressed animals.► TMAO stabilized the actomyosin structure. ► TMAO and urea decreased the motility and ATPase activation of actomyosin. ► TMAO counteracts the effects of urea on motility but not ATPase activation.

Keywords: Abbreviations; ATP; adenosine-5′-triphosphate; bis-ANS; 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid; BSA; bovine serum albumin; HMM; heavy meromyosin; PCA; perchloric acid; Pi; inorganic phosphate; TMAO; trimethylamine; N; -oxideMuscular protein; ATP hydrolysis; Motility; Hydration; Urea; Osmolyte

Conformational dynamics of CYP3A4 demonstrate the important role of Arg212 coupled with the opening of ingress, egress and solvent channels to dehydrogenation of 4-hydroxy-tamoxifen by Kiumars Shahrokh; Thomas E. Cheatham III; Garold S. Yost (pp. 1605-1617).

Structure-based methods for P450 substrates are commonly used during drug development to identify sites of metabolism. However, docking studies using available X-ray structures for the major drug-metabolizing P450, CYP3A4, do not always identify binding modes supportive of the production of high-energy toxic metabolites. Minor pathways such as P450-catalyzed dehydrogenation have been experimentally shown to produce reactive products capable of forming biomolecular adducts which can lead to increased risk toxicities. 4-Hydroxy-tamoxifen (4OHT) is metabolized by CYP3A4 via competing hydroxylation and dehydrogenation reactions. Ab initio gas-phase electronic structural characterization of 4OHT was used to develop a docking scoring scheme. Conformational sampling of CYP3A4 with molecular dynamics simulations along multiple trajectories were used to generate representative structures for docking studies using recently published heme parameters. A key predicted binding mode was tested experimentally using site-directed mutagenesis of CYP3A4 and liquid chromatography–mass spectroscopy analysis.Docking with MD-refined CYP3A4 structures incorporating hexa-coordinate heme parameters identifies a unique binding mode involving ARG212 and channel 4, unobserved in the starting PDB ID:1TQN X-ray structure. The models supporting dehydrogenation are consistent with results from in vitro incubations.Our models indicate that coupled structural contributions of the ingress, egress and solvent channels to the CYP3A4 active site geometries play key roles in the observed 4OHT binding modes. Thus adequate sampling of the conformational space of these drug-metabolizing promiscuous enzymes is important for substrates that may bind in malleable regions of the enzyme active-site.► MD-refined P450 structures identify the importance of channels in CYP3A4 docking. ► A unique configuration is identified involving Arg212 in the dehydrogenation of 4OHT. ► CYP3A4 and CYP3A4-R212A show a decreased rate of dehydrogenation versus oxygenation.

Keywords: Abbreviations; P450; cytochrome P450 enzymes; 4OHT; 4-hydroxy-tamoxifen; SOM; sites of metabolism; RALX; raloxifeneP450; 4-Hydroxy‐tamoxifen; Dehydrogenation; Molecular dynamics; Docking; Conformational dynamics

Fibronectin stimulates migration through lipid raft dependent NHE-1 activation in mouse embryonic stem cells: Involvement of RhoA, Ca2⁺/CaM, and ERK by Jae Hong Park; Jung Min Ryu; Seung Pil Yun; Mi Ok Kim; Ho Jae Han (pp. 1618-1627).

Extracellular matrix (ECM) components and intracellular pH (pH_i) may serve as regulators of cell migration in various cell types.The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na⁺/H⁺ exchanger (NHE)-1 activity was evaluated by measuring pH_i and [²²Na⁺] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed.ECM components (FN, laminin, fibrinogen, and collagen type I) increased [²²Na⁺] uptake, pH_i, and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin β1 and subsequently stimulates caveolin-1 phosphorylation and Ca2⁺ influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca2⁺/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [²²Na⁺] uptake and pH_i. Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration.FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca2⁺/CaM signaling pathways.The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways.► This study analyzed relationship between FN and NHE in regulating migration. ► NHE was localized in DRM, suggesting that response to FN is mediated by lipid rafts. ► NHE-1 activity is, in part, regulated by factors such as lipid rafts in ES cells. ► FN-induced cav-1 activated RhoA or Ca2⁺/CaM pathways, which stimulated NHE-1. ► Activated ERK by NHE-1 stimulated cell migration via MMP-2 and F-actin expression.

Keywords: Abbreviations; [Ca; 2; ⁺; ]; _i; intracellular Ca; 2; ⁺; concentration; CaM; calmodulin; ECM; extracellular matrix; ESCs; embryonic stem cells; ERK; extracellular signal-regulated kinase; F-actin; filamentous-actin; FN; fibronectin; MβCD; methyl-β-cyclodextrin; MMP; matrix metalloproteinase; Na; ⁺; sodium; NHE; Na; ⁺; /H; ⁺; exchanger; pH; _i; intracellular pH; RhoA; Ras homolog gene family member A; ROCK; Rho-associated protein kinase; RT-PCR; reverse transcriptase polymerase chain reaction; siRNA; small interfering RNAEmbryonic stem cells; Fibronectin; Caveolin-1; NHE-1; Cell migration

Differential inhibition of α-synuclein oligomeric and fibrillar assembly in parkinson's disease model by cinnamon extract by Ronit Shaltiel-Karyo; Dan Davidi; Moran Frenkel-Pinter; Michael Ovadia; Daniel Segal; Ehud Gazit (pp. 1628-1635).

The oligomeriztion of α-synuclein (α-syn) into ordered assemblies is associated with the symptoms of Parkinson's Disease (PD). Yet, it is still debatable whether oligomers are formed as part of a multistep process towards amyloid fibril formation or alternatively as "off-pathway" aggregates.100μM α-syn was incubated with decreasing amounts of cinnamon extract precipitation (CEppt). The fibril formation was measured using spectroscopy and microscopy analyses and oligomers were detected using western blot analysis. The secondary structure of the protein was analyzed using CD. Drosophila brains were studied using immunostaining and confocal microscopy.Here we probed the inhibition pattern of oligomeric and fibrillar forms of α-syn, using a natural substance, CEppt which was previously shown to effectively inhibit aggregation of β-amyloid polypeptide. We demonstrated that CEppt has a differential inhibitory effect on the formation of soluble and insoluble aggregates of α-synuclein in vitro. This inhibition pattern revokes the possibility of redirection to "off-pathway" oligomers. When administering to Drosophila fly model expressing mutant A53T α-syn in the nervous system, a significant curative effect on the behavioral symptoms of the flies and on α-syn aggregation in their brain was observed.We conclude that CEppt affects the process of aggregation of α-syn without changing its secondary structure and suggest that increasing amounts of CEppt slow this process by stabilizing the soluble oligomeric phase. When administered to Drosophila fly model, CEppt appears to have a curative effect on the defective flies.Our results indicate that CEppt can be a potential therapeutic agent for PD.► We show that CEppt is an effective inhibiter for α-synuclein aggregation. ► CEppt has a differential effect on the formation of soluble and insoluble aggregates. ► High molecular ratio of CEppt stabilizes oligomers. ► Drosophila model shows reduced brain aggregates and defective phenotype correction.

Keywords: α-synuclein; Cinnamon extract; Aggregation inhibitor; Parkinson's disease; Drosophila model for Parkinson

PKA and cAMP stimulate proliferation of mouse embryonic stem cells by elevating GLUT1 expression mediated by the NF-κB and CREB/CBP signaling pathways by Mi Ok Kim; Yu Jin Lee; Jae Hong Park; Jung Min Ryu; Seung Pil Yun; Ho Jae Han (pp. 1636-1646).

Regulation of glucose transporter (GLUT) expression and activity plays a vital role in the supply of glucose to embryonic stem (ES) cells.To observe the effect of 6-phenyl cyclic monophosphate (cAMP) on glucose uptake and cell proliferation, 2-deoxyglucose (2-DG) uptake, immunohistochemistry, Western blotting, and immunoprecipitation were carried out.Among GLUT isoforms in mouse ES cells, GLUT1 was predominantly expressed and 6-phenyl cAMP increased GLUT mRNA levels. Among cAMP agonists, 6-phenyl cAMP increased 2-DG uptake more than that of 8-p-chlorophenylthio-2′-O-methyl-cAMP. 6-Phenyl cAMP increased GLUT1 expression and translocation from the cytosol to the plasma membrane. 6-Phenyl cAMP increased 2-DG uptake in a time- and concentration-dependent manner due to an increase in V_max but not K_m. 6-Phenyl cAMP increased phosphorylation of nuclear factor-κB (NF-κB) and cAMP response element binding (CREB) and expression of the CREB protein (CBP) and transducer of regulated CREB activity 2 (TORC2) in sequence. 6-Phenyl cAMP induced complex formation of NF-κB/CREB/CBP/TORC2, which are involved in the increase of gluconeogenic enzyme expression. 6-Phenyl cAMP also increased cell cycle regulatory protein expression levels, the proportion of S-phase cells, and proto-oncogene expression via protein kinase A (PKA)-dependent NF-κB signaling. Finally, GLUT1 siRNA blocked the 6-phenyl cAMP-induced increase in ES cell proliferation. We conclude that PKA stimulated the complex formation of CREB/CBP/TORC2 via NF-κB, which induced effective coordination of glucose uptake as well as proliferation in ES cells.6-Phenyl cAMP-induced PKA activation modified the proliferation, which may be beneficial for expanding ES cell use to cell therapy.► This study focused on PKA-mediated modulation of glucose uptake and proliferation. ► 6-Phenyl cAMP increased 2-DG uptake and GLUT1 translocation to the plasma membrane. ► Expression of GLUT1 was regulated by CREB/CBP/TORC2 complex formation through NF-κB. ► PKA enhanced the cell proliferation through GLUT1 expression and 2-DG uptake.

Keywords: Abbreviations; PKA; protein kinase A; NF-κB; nuclear factor-κB; CREB; cAMP response element binding; CBP; CREB binding protein; TORC2; transducer of regulated CREB activity 2; GLUT; glucose transporter; 2-DG; 2-deoxyglucoseGlucose transporter; Protein kinase A; Nuclear factor kappa B; Embryonic stem cell; Proliferation

PEP-1–metallothionein-III protein ameliorates the oxidative stress-induced neuronal cell death and brain ischemic insults by Eun Jeong Sohn; Dae Won Kim; Mi Jin Kim; Hoon Jae Jeong; Min Jea Shin; Eun Hee Ahn; Soon Won Kwon; Young Nam Kim; Duk-Soo Kim; Kyu Hyung Han; Jinseu Park; Hyun Sook Hwang; Won Sik Eum; Soo Young Choi (pp. 1647-1655).

Oxidative stress is considered to be involved in a number of human diseases including ischemia. Metallothioneins (MT)-III can protect neuronal cells from the cytotoxicity of reactive oxygen species (ROS). However, MT-III proteins biological function is unclear in ischemia. Thus, we examined the protective effects of MT-III proteins on oxidative stress-induced neuronal cell death and brain ischemic insult.A human MT-III gene was fused with a protein transduction domain, PEP-1 peptide, to construct a cell permeable PEP-1–MT-III protein. PEP-1–MT-III protein was purified using affinity chromatograph. Transduced PEP-1–MT-III proteins were detected by Western blotting and immunoflourescence. Cell viability and DNA fragmentation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay and terminal dexoynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. Brain ischemic injury was detected with immunohistochemistry.Purified PEP-1–MT-III proteins transduced into astrocytes in a time- and dose-dependent manner and protected against oxidative stress-induced cell death. Also, transduced PEP-1–MT-III proteins efficiently protected cells against DNA fragmentation. Furthermore, immunohistochemical analysis revealed that PEP-1–MT-III prevented neuronal cell death in the CA1 region of the hippocampus induced by transient forebrain ischemia. We demonstrated that transduced PEP-1–MT-III protein protects against oxidative stress induced cell death in vitro and in vivo.Transduced PEP-1–MT-III protein has neuroprotective roles as an antioxidant in vitro and in vivo. PEP-1–MT-III protein is a potential therapeutic agent for various human brain diseases such as stroke, Alzheimer's disease, and Parkinson's disease.► The function of Metallothioneins (MT)-III protein in ischemia is still unclear. ► We construct a cell permeable PEP-1-MT-III fusion protein. ► Transduced PEP-1-MT-III protein highly protects against oxidative stress-induced cell death and ischemic insult in animal models. ► PEP-1-MT-III protein could be useful as a therapeutic agent for oxidative stress.

Keywords: Abbreviations; MT; metallothionein; ROS; reactive oxygen species; PTD; protein transduction domain; PBS; phosphate-buffered saline; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide; TUNEL; terminal dexoynucleotidyl transferase-mediated dUTP nick-end labelingAntioxidant; PEP-1–MT-III; Protein transduction; Cell viability; Ischemia; ROS

β-Glucan from Saccharomyces cerevisiae reduces lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages by Xiaojuan Xu; Michiko Yasuda; Masashi Mizuno; Hitoshi Ashida (pp. 1656-1663).

β-Glucans obtained from fungi, such as baker's yeast ( Saccharomyces cerevisiae)-derived β-glucan (BBG), potently activate macrophages through nuclear factor κB (NFκB) translocation and activation of its signaling pathways. The mechanisms by which β-glucans activate these signaling pathways differ from that of lipopolysaccharide (LPS). However, the effects of β-glucans on LPS-induced inflammatory responses are poorly understood. Here, we examined the effects of BBG on LPS-induced inflammatory responses in RAW264.7 mouse macrophages.We explored the actions of BBG in RAW264.7 macrophages.BBG inhibited LPS-stimulated nitric oxide (NO) production in RAW264.7 macrophages by 35–70% at concentrations of 120–200μg/ml. BBG also suppressed mRNA and protein expression of LPS-induced inducible NO synthase (iNOS) and mitogen-activated protein kinase phosphorylation, but not NFκB activation. By contrast, a neutralizing antibody against dectin-1, a β-glucan receptor, did not affect BBG-mediated inhibition of NO production. Meanwhile, BBG suppressed Pam3CSK-induced NO production. Moreover, BBG suppressed LPS-induced production of pro-and anti-inflammatory cytokines, including interleukin (IL)-1α, IL-1ra, and IL-27.Our results indicate that BBG is a powerful inhibitor of LPS-induced NO production by downregulating iNOS expression. The mechanism involves inactivation of mitogen-activated protein kinase and TLR2 pathway, but is independent of dectin-1.BBG might be useful as a novel agent for the chemoprevention of inflammatory diseases.► Baker’s yeast β-glucan inhibited LPS-induced NO production in mouse Mφ. ► β-glucan suppressed both MAPK and TLR2 pathways. ► Dectin-1 was not involved in the inhibitory effects of β-glucan on NO production.

Keywords: Abbreviations; BBG; baker's yeast derived β-glucan; BLC; B-lymphocyte chemoattractant; C; complement; cNOS; constitutive nitric oxide synthase; CCL; CC chemokine ligands; CXCL; chemokine CXC ligand; DMEM; Dulbecco's modified Eagle's medium; ERK; extracellular signal-regulated kinase; FBS; fetal bovine serum; G-CSF; granulocyte colony-stimulating factor; GM-CSF; granulocyte-macrophage colony-stimulating factor; HPRT; hypoxanthineguaninephosphoribosyltransferase; I-TAC; IFN-inducible T-cell α chemoattractant; IL; interleukin; iNOS; inducible nitric oxide synthase; IP; IFNγ-induced protein; JNK; c-Jun N-terminal kinase; KC; keratinocyte chemoattractant; LPS; lipopolysaccharide; M-CSF; macrophage colony-stimulating factor; MAPK; mitogen-activated protein kinase; MCP; monocyte chemoattractant protein; MIG; monokine induced by IFNγ; MIP; macrophage inflammatory protein; NFκB; nuclear factor κB; NO; nitric oxide; RANTES; regulated upon activation normal T-cell expressed and presumably secreted; RT-PCR; reverse transcription-polymerase chain reaction; SDF; stromal cell-derived factor; SDS; sodium dodecyl sulfate; sICAM-1; soluble intercellular adhesion molecule; TARC; thymus and activation regulated chemokine; TIMP; tissue inhibitor of matrix metalloprotease; TNF; tumor necrosis factor; TREM; triggering receptor expressed on myeloid cellsBeta-glucan; Anti-inflammation; RAW264.7 macrophage; Lipopolysaccharide; Inducible nitric oxide synthase

AglR is required for addition of the final mannose residue of the N-linked glycan decorating the Haloferax volcanii S-layer glycoprotein by Lina Kaminski; Ziqiang Guan; Mehtap Abu-Qarn; Zvia Konrad; Jerry Eichler (pp. 1664-1670).

Recent studies of Haloferax volcanii have begun to elucidate the steps of N-glycosylation in Archaea, where this universal post-translational modification remains poorly described. In Hfx. volcanii, a series of Agl proteins catalyzes the assembly and attachment of a N-linked pentasaccharide to the S-layer glycoprotein. Although roles have been assigned to the majority of Agl proteins, others await description. In the following, the contribution of AglR to N-glycosylation was addressed.A combination of bioinformatics, gene deletion, mass spectrometry and metabolic radiolabeling served to show a role for AglR in archaeal N-glycosylation at both the dolichol phosphate and reporter glycoprotein levels.The modified behavior of the S-layer glycoprotein isolated from cells lacking AglR points to an involvement of this protein in N-glycosylation. In cells lacking AglR, glycan-charged dolichol phosphate, including mannose-charged dolichol phosphate, accumulates. At the same time, the S-layer glycoprotein does not incorporate mannose, the final subunit of the N-linked pentasaccharide decorating this protein. AglR is a homologue of Wzx proteins, annotated as flippases responsible for delivering lipid-linked O-antigen precursor oligosaccharides across the bacterial plasma membrane during lipopolysaccharide biogenesis.The effects resulting from aglR deletion are consistent with AglR interacting with dolichol phosphate-mannose, possibly acting as a dolichol phosphate-mannose flippase or contributing to such activity.Little is known of how lipid-linked oligosaccharides are translocated across membrane during N-glycosylation. The possibility of Hfx. volcanii AglR mediating or contributing to flippase activity could help address this situation.► In cells lacking AglR, glycan-charged dolichol phosphates accumulate. ► In Δ aglR cells, the final subunit of the N-linked pentasaccharide is absent. ► AglR is a homologue of Wzx, thought to ‘flip’ lipid-linked glycans in bacteria.

Keywords: Abbreviations; ABC; ATP-binding cassette; CBB; Coomassie brilliant blue; DDW; double-distilled water; DolP; dolichol phosphate; DolPP-Man5; mannose; ₅; -N-acetylglucosamine; ₂; -charged dolichol pyrophosphate; EIC; extracted ion chromatograms; ER; endoplasmic reticulum; LC-ESI/MS; liquid chromatography-electrospray ionization mass spectrometry; LLO; lipid-linked oligosaccharide; Man; mannose; MS/MS; tandem mass spectrometry; RT-PCR; reverse transcriptase-PCRArchaea; Dolichylphosphate-mannose; Haloferax volcanii; N-glycosylation; S-layer glycoprotein

Longevity of elastin in human intervertebral disc as probed by the racemization of aspartic acid by Sarit-Sara Sivan; Benno Van El; Yulia Merkher; Christian E.H. Schmelzer; Anne-Marie Zuurmond; Andrea Heinz; Ellen Wachtel; Peter-Paul Varga; Aron Lazary; Marco Brayda-Bruno; Alice Maroudas (pp. 1671-1677).

Aging and degeneration of human intervertebral disc (IVD) are associated with biochemical changes, including racemization and glycation. These changes can only be counteracted by protein turnover. Little is known about the longevity of IVD elastin in health or disease. Yet, such knowledge is important for a quantitative understanding of tissue synthesis and degradation.We have measured the accumulation ofd-Asp and pentosidine in IVD elastin. Samples representing a broad range of ages (28–82years) and degeneration grades (1–5) were analyzed.d/l-Asp for elastin increased linearly with age from 3.2% (early 30s) to 14.8% (early 80s) for normal tissue (grades 1–2) and from 1.7% (late 20s) to 6.0% (until the mid 50s) for degenerate tissue (grades 3–5) with accumulation rates of 16.2±3.1×10−⁴ and 11.7±3.8×10−⁴year−¹, respectively; no significant difference was found between these values (p<0.05). Above the mid 50s, a decrease ind-Asp accumulation was recorded in the degenerate tissue.d-Asp accumulation correlated well with pentosidine content for elastin from healthy and degenerate tissues combined. We conclude that IVD elastin is metabolically‐stable and long‐lived in both healthy and degenerate human IVDs, with signs of new synthesis in the latter. The correlation ofd‐Asp with pentosidine content suggests that both these agents may be used as markers in the overall aging process of IVD.Accumulation of modified IVD elastin argues for its longevity and may have a negative impact on its role in disc function. Weak signs of newly synthesized molecules may act to counteract this effect in degenerate tissue.►d-Asp content of elastin increased linearly with age for normal and degenerate IVDs. ► Pentosidine content increased linearly for normal and degenerate IVD tissues combined. ► Signs of new elastin synthesis were found for degenerate IVD above 50years. ►d-Asp and pentosidine may be used as markers in the aging process of IVD. ► IVD elastin is metabolically stable and long-lived in human disc tissues.

Keywords: Abbreviations; IVD; intervertebral disc; d; -Asp or; d; _Asp; d; isomer of aspartic acid; l; -Asp or; l; _Asp; l; isomer of aspartic acid; LC; liquid chromatography; MS; mass spectrometry; NP; nucleus pulposus; AF; annulus fibrosus; HPLC; high performance liquid chromatography; AGE; advanced glycation endproductAspartic acid racemization; Aging; Turnover; Elastin; Pentosidine; Intervertebral disc

O-GlcNAc modification affects the ATM-mediated DNA damage response by Yuri Miura; Yoko Sakurai; Tamao Endo (pp. 1678-1685).

O-Linked β- N-acetylglucosamine ( O-GlcNAc) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. The role of O-GlcNAcylation in the ataxia-telangiectasia mutated (ATM)-mediated DNA damage response is unknown. It is unclear whether ATM, which is an early acting and central component of the signal transduction system activated by DNA double strand breaks, is an O-GlcNAc-modified protein.The effect of O-GlcNAc modification on ATM activation was examined using two inhibitors, PUGNAc and DON that increase and decrease, respectively, levels of protein O-GlcNAcylation. To assess O-GlcNAcylation of ATM, immunoprecipitation and immunoblot analyses using anti-ATM or anti- O-GlcNAc antibody were performed in HeLa cells and primary cultured neurons. Interaction of ATM with O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc to target proteins, was examined by immunoprecipitation and immunoblot analyses using anti-ATM.Enhancement of protein O-GlcNAcylation increased levels of X-irradiation-induced ATM activation. However, decreases in protein O-GlcNAcylation did not affect levels of ATM activation, but these decreases did delay ATM activation and ATM recovery processes based on assessment of de-phosphorylation of phospho-ATM. Thus, activation and recovery of ATM were affected by O-GlcNAcylation. ATM was subjected to O-GlcNAcylation, and ATM interacted with OGT. The steady-state O-GlcNAc level of ATM was not significantly responsive to X-irradiation or oxidative stress.ATM is an O-GlcNAc modified protein, and dynamic O-GlcNAc modification affects the ATM-mediated DNA damage response.► O-GlcNAcylation was involved in the DNA damage responses to X-irradiation. ► Activation and recovery of ATM were affected by dynamic O-GlcNAc modifications. ► ATM was subject to O-GlcNAcylation and interacted with O-GlcNAc transferase. ► O-GlcNAcylation may regulate the dissociation of PP2A, a process for ATM activation.

Keywords: Abbreviations; ATM; ataxia telangiectasia mutated; O; -GlcNAc; O; -linked β-; N; -acetylglucosamine; DSBs; double-strand breaks; SMC1; structural maintenance of chromosomes 1; OGT; O; -linked β-; N; -acetylglucosamine transferase; OGA; O; -linked β-; N; -acetylglucosaminidase; PP2A; protein phosphatases of type 2AATM; O; -GlcNAc; Radiation; Phosphorylation

VP2118 has major roles in Vibrio parahaemolyticus response to oxidative stress by Tam Thi Thanh Le; Kazuaki Mawatari; Miki Maetani; Tomomi Yamamoto; Sayaka Hayashida; Hitomi Iba; Mutsumi Aihara; Akiko Hirata; Takaaki Shimohata; Takashi Uebanso; Akira Takahashi (pp. 1686-1692).

Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus, a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus. This study aims to clarify the role of V. parahaemolyticus SODs against ROS. V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured.Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance.VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses.The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.► Vibrio parahaemolyticus has three SOD genes: VP2118, VP2860, and VPA1514 ► The promoter activity of the VP2118 gene was the highest among the SOD genes ► The SOD activity of the VP2118 gene-deleted mutant was significantly decreased ► The absence of the VP2118 gene resulted in significantly lower resistance to ROS ► Complementation of the VP2118 gene recovered SOD activity and resistance to ROS

Keywords: Abbreviations; SOD; superoxide dismutase; ROS; reactive oxygen species; GFP; green fluorescence protein; PCR; polymerase chain reaction; V. parahaemolyticus; Vibrio parahaemolyticus; V. vulnificus; Vibrio vulnificus; E. coli; Escherichia coli; bp; base pair; CFU; colony forming unit; WT; wild typeSuperoxide dismutase; Oxidative stress; Vibrio parahaemolyticus; reactive oxygen species; Hydrogen peroxide

Protective role of quercetin against lead-induced inflammatory response in rat kidney through the ROS-mediated MAPKs and NF-κB pathway by Chan-Min Liu; Yun-Zhi Sun; Jian-Mei Sun; Jie-Qiong Ma; Chao Cheng (pp. 1693-1703).

Lead (Pb) exposure is considered as a risk factor for the development of renal dysfunction. The flavonoid quercetin (QE) in diets exerts the nephroprotective effects. This study investigated the effects of quercetin on renal oxidative stress and inflammation in rats exposed to Pb.Wistar rats were divided into normal, lead exposure groups, lead plus quercetin groups and quercetin groups. Rats were exposed to lead acetate in the drinking water (500mgPb/L) with or without quercetin co-administration (25 and 50mgQU/kg intragastrically once daily). After 75days, serum uric acid, urea, creatinine, renal reactive oxygen species (ROS) production, thiobarbituric acid reactive substances (TBARS) and histopathological analysis were performed. Pb content in kidney was also assayed. The levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), the extracellular-receptor kinases (ERK1/2), the c-Jun N-terminal kinases (JNK1/2), p38 MAPK and nuclear factor-κB (NF-κB) were measured.Quercetin significantly prevented Pb-induced nephrotoxicity in a dose-dependent manner, indicated by both diagnostic indicators and histopathological analysis. Quercetin significantly decreased Pb content in kidney. Pb-induced profound elevations of oxidative stress in kidney were suppressed by quercetin. Furthermore, quercetin significantly inhibited Pb-induced inflammation in rat kidney.These results suggest that quercetin has the nephroprotective actions. The inhibition of Pb-induced kidney inflammation by quercetin is due at least in part to its anti-oxidant activity and its ability to modulate the MAPK and NF-κB signaling pathway.Quercetin might be a potent nephroprotective drug to protect Pb-induced kidney injury.Display Omitted► Quercetin prevented Pb-induced nephrotoxicity. ► Quercetin reduced Pb-induced increase in ROS and TBARS production in kidney of rats. ► Quercetin inhibited the inflammatory cytokine expression in kidney of Pb treated rats. ► Quercetin suppressed the MAPKs and NF-κB activation in rat kidney. ► Quercetin inhibited the Pb-induced oxidative stress and inflammation.

Keywords: Abbreviations; COX-2; cyclooxygenase-2; ERK; the extracellular-receptor kinases; IL-1β; interleukin-1beta; IL-6; interleukin-6; JNK; the c-Jun N-terminal kinases; NF-κB; nuclear factor-κB; MAPKs; mitogen-activated protein kinases; Pb; lead; QU; quercetin; ROS; reactive oxygen species; TBARS; thiobarbituric acid reactive substances; TNF-α; tumor necrosis factor-alphaPb; Quercetin; MAPK; Oxidative stress; NF-κB; Renal inflammation

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BBA - General Subjects (v.1820, #10)