BBA - General Subjects (v.1830, #4)

A novel compound derived from danshensu inhibits apoptosis via upregulation of heme oxygenase-1 expression in SH-SY5Y cells by Li-Long Pan; Xin-Hua Liu; Yao-Ling Jia; Dan Wu; Qing-Hui Xiong; Qi-Hai Gong; Yang Wang; Yi-Zhun Zhu (2861-2871).
Heme oxygenase-1 (HO-1) has potential anti-apoptotic properties. A novel compound [4-(2-acetoxy-3-((R)-3-(benzylthio)-1-methoxy-1-oxopropan-2- ylamino)-3-oxopropyl)-1,2-phenylene diacetate (DSC)] was synthesized by joining danshensu and cysteine through an appropriate linker. This study investigated if the cytoprotective properties of DSC involved the induction of HO-1.We evaluated the cytoprotective effects of DSC on H2O2-induced cell damage, apoptosis, intracellular and mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) loss, and apoptosis-related proteins expression and its underlying mechanisms.DSC concentration-dependently attenuated cell death, lactate dehydrogenase release, intracellular and mitochondrial ROS production, and ΔΨm collapse, modulated apoptosis-related proteins (Bcl-2, Bax, caspase-3, p53, and cleaved PARP) expression, and inhibited phosphorylation of extracellular signal-regulated kinase 1/2 in SH-SY5Y cells induced by H2O2. In addition, DSC concentration-dependently induced HO-1 expression associated with nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf-2), while the effect of DSC was inhibited by a phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Furthermore, the protective effect of DSC on H2O2-induced cell death was abolished by HO-1 inhibitor ZnPP, but was mimicked by carbon monoxide-releasing moiety CORM-3 or HO-1 by-product bilirubin. Finally, DSC inhibited H2O2-induced changes of Bcl-2, Bax, and caspase-3 expression, and all of these effects were reversed by HO-1 silencing.Induction of HO-1 may be, at least in part, responsible for the anti-apoptotic property of DSC, an effect that involved the activation of PI3K/Akt/Nrf-2 axis.DSC might have the potential for beneficial therapeutic interventions for neurodegenerative diseases.Display Omitted► DSC induces expression of HO-1 protein. ► H2O2-induced apoptosis is inhibited by DSC pretreatment. ► DSC scavenges reactive oxygen species and protects mitochondrial function which protects H2O2-induced apoptosis. ► Silencing of HO-1 abolishes the anti-apoptotic effects of DSC.
Keywords: Danshensu-cysteine derivative; Apoptosis; Heme oxygenase-1; Neurodegenerative disorder;

Oxidized quercetin inhibits α-synuclein fibrillization by Min Zhu; Shubo Han; Anthony L. Fink (2872-2881).
α-Synucein is a small (14 kDa), abundant, intrinsically disordered presynaptic protein, whose aggregation is believed to be a critical step in Parkinson's disease (PD). Oxidative stress is reported to be a risk factor for dopamine cell degeneration in PD. Flavonoids are suggested to be important antioxidant against oxidative stress. Flavonoids were reported to inhibit fibrillization and disaggregate the preformed fibrils of α-synucein, but the molecular mechanism was still not clear.Quercetin, a well-recognized flavonoid antioxidant, was tested for its inhibition of α-synucein aggregation by thioflavin T assay, light scattering measurement, size-exclusion high performance liquid chromatography, atomic force microscopy, etc.The pre-incubated quercetin exhibited a noticeably stronger inhibition behavior to the fibril formation than that of the freshly prepared. The inhibition is significant in the presence of ortho- and para-benzenediol isomers and inconsiderable in the presence of meta-isomer. The oxidized quercetin species (i.e., chalcantrione, benzyfuranone, quercetinchinone, and other derivatives) cause stronger inhibition than quercetin does because of the elevated polarity and hydrophilicity. Presence of quercetin disaggregates α-synucein fibrils, rather than oligomers and amorphous aggregations.Instead of the antioxidant activity, the 1:1 covalent binding of quercetin with α-synucein, and the increased hydophilicity of the covalently modified α-synucein oligomers or monomers, account for the inhibition of α-synucein fibrillation.Clarification of the molecular mechanism of the inhibition and disaggregation may help to screen safer and more effective flavonoid therapeutic in combating PD.► Oxidized quercetin inhibits fibrillization of α-synucein more strongly than quercetin does. ► Quercetin disaggregates α-synucein fibrils. ► Quercetin inhibits fibrillization and stabilizes oligomers. ► Quercetin binds with α-synucein in 1:1 ratio. ► Increased hydrophilicity of quercetin and α-synucein adducts accounts for inhibition.
Keywords: Quercetin; α-Synuclein; Parkinson's disease; Hydrophilicity; Inhibition; Aggregation;

Adenosine, adenosine receptors and glaucoma: An updated overview by Yisheng Zhong; Zijian Yang; Wei-Chieh Huang; Xunda Luo (2882-2890).
Glaucoma, a leading cause of blindness worldwide, is an optic neuropathy commonly associated with elevated intraocular pressure (IOP). The major goals of glaucoma treatments are to lower IOP and protect retinal ganglion cells. It has been revealed recently that adenosine and adenosine receptors (ARs) have important roles in IOP modulation and neuroprotection.This article reviews recent studies on the important roles of adenosine and ARs in aqueous humor formation and outflow facility, IOP and retinal neuroprotection.Adenosine and several adenosine derivatives increase and/or decrease IOP via A2A AR. Activation of A1 AR can reduce outflow resistance and thereby lower IOP, A3 receptor antagonists prevent adenosine-induced activation of Cl channels of the ciliary non-pigmented epithelial cells and thereby lower IOP. A1 and A2A agonists can reduce vascular resistance and increase retina and optic nerve head blood flow. A1 agonist and A2A antagonist can enhance the recovery of retinal function after ischemia attack. Adenosine acting at A3 receptors can attenuate the rise in calcium and retinal ganglion cells death accompanying P2X(7) receptor activation.Evidence suggested that the adenosine system is one of the potential target systems for therapeutic approaches in glaucoma.Display Omitted► Effects of adenosine/adenosine receptor on glaucoma are reviewed. ► Adenosine system can modulate aqueous humor formation and outflow facility. ► Adenosine system has the neuroprotective properties. ► Adenosine system is a target system for therapeutic approaches in glaucoma.
Keywords: Adenosine; Adenosine receptor; Intraocular pressure; Neuroprotection; Glaucoma;

Glycolysis–respiration relationships in a neuroblastoma cell line by Russell H. Swerdlow; Lezi E.; Daniel Aires; Jianghua Lu (2891-2898).
Although some reciprocal glycolysis–respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized.We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions.Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased.These data document a novel intracellular glycolysis–respiration effect in which restricting glycolysis flux increases mitochondrial respiration.Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis–respiration effect can practically inform the development of new mitochondrial medicine approaches.► Inhibiting glycolysis flux increases mitochondrial respiration ► Reducing net glycolytic ATP production increases mitochondrial respiration ► These studies define a specific, reciprocal glycolysis–respiration relationship ► This reciprocal relationship can be used to manipulate bioenergetics infrastructures
Keywords: Crabtree effect; Glycolysis; Mitochondria; Oxidative phosphorylation; Pasteur effect; Respiration;

Insights into eukaryotic Rubisco assembly — Crystal structures of RbcX chaperones from Arabidopsis thaliana by Piotr Kolesinski; Przemyslaw Golik; Przemyslaw Grudnik; Janusz Piechota; Michał Markiewicz; Miroslaw Tarnawski; Grzegorz Dubin; Andrzej Szczepaniak (2899-2906).
Chloroplasts were formed by uptake of cyanobacteria into eukaryotic cells ca. 1.6 billion years ago. During evolution most of the cyanobacterial genes were transferred from the chloroplast to the nuclear genome. The rbcX gene, encoding an assembly chaperone required for Rubisco biosynthesis in cyanobacteria, was duplicated. Here we demonstrate that homologous eukaryotic chaperones (AtRbcX1 and AtRbcX2) demonstrate different affinities for the C-terminus of Rubisco large subunit and determine their crystal structures.Three-dimensional structures of AtRbcX1 and AtRbcX2 were resolved by the molecular replacement method. Equilibrium binding constants of the C-terminal RbcL peptide by AtRbcX proteins were determined by spectrofluorimetric titration. The binding mode of RbcX–RbcL was predicted using molecular dynamic simulation.We provide crystal structures of both chaperones from Arabidopsis thaliana providing the first structural insight into Rubisco assembly chaperones form higher plants. Despite the low sequence homology of eukaryotic and cyanobacterial Rubisco chaperones the eukaryotic counterparts exhibit surprisingly high similarity of the overall fold to previously determined prokaryotic structures. Modeling studies demonstrate that the overall mode of the binding of RbcL peptide is conserved among these proteins. As such, the evolution of RbcX chaperones is another example of maintaining conserved structural features despite significant drift in the primary amino acid sequence.The presented results are the approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants.► Structural details of eucaryotic Rubisco chaperones are shown for the first time. ► Crystal structures of two RbcX homologs from Arabidopsis thaliana are provided. ► Both AtRbcX share the same overall topology with their cyanobacterial equivalents. ► Structural details differ significantly in the case of AtRbcX1. ► High conservation of the binding pockets of distant RbcX chaperones is revealed.
Keywords: RbcX; Rubisco assembly; Arabidopsis thaliana;

The recent morphological studies on chaperonins have revealed that nearly equivalent amount of symmetric GroEL–(GroES)2 (football-shaped) and asymmetric GroEL–GroES (bullet-shaped) complexes coexist during the chaperonin reaction cycle, which prompted us to reexamine the equatorial split observed for chaperonin from Thermus thermophilus by implementing semi-empirical molecular orbital (MO) calculations, since it is now believed that the symmetric formation is a precursor to the equatorial split.Semi-empirical MO calculations were employed to investigate the intersubunit interactions within the bullet-shaped T. thermophilus chaperonin capturing the substrate of folding intermediates. Interaction energies between each cis-GroEL subunit and closely related remaining subunits in cis-GroEL ring, or in trans-GroEL ring across the equatorial plane, and further, interaction energies between each GroES subunit and adjacent subunits in the same GroES ring and in cis-GroEL ring were simulated.Anisotropic intensities and energy distribution of the subunits were revealed by the calculations, which are consistent with two conformations of the subunits forming cis-GroEL ring as revealed by X-ray crystal structure, and with an anisotropic critical binding site on cis-GroEL ring for chaperonin functioning.This is the first application of semi-empirical MO calculations to the macromolecular complex of the native bullet-shaped chaperonin (GroEL–GroES–ADP homolog) from T. thermophilus.The results also appear to support the occurrence of the equatorial split for T. thermophilus chaperonin observed via electron microscopy, but has not yet been fully observed for Escherichia coli GroEL–GroES system.► Anisotropic intensities of intersubunit interactions in the bullet-shaped GroEL/ES. ► Anisotropic perturbation of inter-ring interactions in the bullet-shaped GroEL/ES. ► Subunit G of the cis-GroEL ring is the initial substrate binding site. ► Then, GroES binds to the complex through the remaining six binding sites. ► The equatorial split of Thermus chaperonin via EM is supported by the MO calculation.
Keywords: GroEL–GroES; Molecular orbital calculation; MOPAC; Local SCF; Thermus thermophilus; Electron microscopy;

Albumin domain II mutant with high bilirubin binding affinity has a great potential as serum bilirubin excretion enhancer for hyperbilirubinemia treatment by Ai Minomo; Yu Ishima; Victor T.G. Chuang; Yoshiaki Suwa; Ulrich Kragh-Hansen; Toru Narisoko; Hiroshi Morioka; Toru Maruyama; Masaki Otagiri (2917-2923).
4Z,15Z-bilirubin-IXα (BR), an endogenous toxic compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. Our previous findings suggest that both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of BR, and especially Lys199 in stand-alone domain II plays a prominent role in the renal elimination of BR. Our hypothesis is that HSA-domain II with high BR binding would be a useful therapeutic agent to treat hyperbilirubinemia in patients with impaired liver function.Unbound BR concentrations were determined using a modified HRP assay. To evaluate the effect of pan3_3-13 domain II mutant in promoting urinary BR excretion, the serum concentration and urinary excretion amount of BR were determined using bile duct ligation mice.After three or six rounds of panning, pan3_3-13 and pan6_4 were found to have a significantly higher affinity for BR than wild-type domain II. Administration of pan3_3-13 significantly reduced serum BR level and increased its urinary excretion in the disease model mice as compared to wild-type domain II treatment.These results suggest that pan3_3-13 has great potential as a therapeutic agent that promotes urinary BR excretion in hyperbilirubinemia.This is the first study to be applied to other HSA bound toxic compounds that are responsible for the progression of disease, thereby paving the way for the development of non-invasive and cost effective blood purification treatment methods.Display Omitted► We investigated the effect of HSA-domain II with high BR binding on hyperbilirubinemia treatment. ► To evaluate the effect of HSA-domain II mutant in promoting BR excretion, bile duct ligation mice were used. ► We examined the serum concentration and urinary excretion amount of BR in bile duct ligation mice. ► HSA-domain II mutant has great potential as a therapeutic agent that promotes urinary BR excretion in hyperbilirubinemia.
Keywords: Human serum albumin; Bilirubin; Liver failure; Hyperbilirubinemia; Phage display;

The effects of ferulic acid on β-amyloid fibrillar structures investigated through experimental and computational techniques by Antonella Sgarbossa; Susanna Monti; Francesco Lenci; Emilia Bramanti; Ranieri Bizzarri; Vincenzo Barone (2924-2937).
Current research has indicated that small natural compounds could interfere with β-amyloid fibril growth and have the ability to disassemble preformed folded structures. Ferulic acid (FA), which possesses both hydrophilic and hydrophobic moieties and binds to peptides/proteins, is a potential candidate against amyloidogenesis. The molecular mechanisms connected to this action have not been elucidated in detail yet.Here the effects of FA on preformed fibrils are investigated by means of a concerted experimental–computational approach. Spectroscopic techniques, such as FTIR, fluorescence, size exclusion chromatography and confocal microscopy in combination with molecular dynamics simulations are used to identify those features which play a key role in the destabilization of the aggregates.Experimental findings highlight that FA has disruptive effects on the fibrils. The computational analysis suggests that dissociation of peptides from the amyloid superstructures could take place along the fibril axis and be primarily determined by the cooperative rupture of the backbone hydrogen bonds and of the Asp-Lys salt bridges.FA clusters could induce a sort of stabilization and tightening of the fibril structure in the short term and its disruption in the long term, inhibiting further fibril re-assembly through FA screening effects.The combination of experimental and computational techniques could be successfully used to identify the disrupting action of FA on preformed Aβ fibrils in water solution.Display Omitted► Ferulic acid (FA) covers the Aβ fibrillar structures and inserts between the oligopeptide chains. ► FA could interact with Asp23-Lys28 salt bridges disrupting the Asp-Lys arrangement. ► FA causes fibril solvation, unpacking of the chains, and Aβ fibril disruption. ► FA–fibril interaction could inhibit Aβ peptide re-association.
Keywords: Fibrillogenesis inhibition; Hydroxycinnamic acid; Protein aggregation;

CK2 phosphorylation of human Sec63 regulates its interaction with Sec62 by Emmanuel Ampofo; Sabrina Welker; Martin Jung; Linda Müller; Markus Greiner; Richard Zimmermann; Mathias Montenarh (2938-2945).
Protein kinase CK2 is a pleiotropic enzyme which is ubiquitously expressed in eukaryotic cells. Several years ago CK2 was found to be associated with the mammalian endoplasmic reticulum. So far nothing is known about the function of CK2 at the ER.CK2 phosphorylation sites in the polypeptide chain of Sec63 were mapped using deletion mutants and a peptide library. Binding of Sec63 to CK2 and to Sec62 was analyzed by pull-down assays and by co-immunoprecipitationSec63 was identified as a novel substrate and binding partner of protein kinase CK2.We identified serine 574, serine 576 and serine 748 as CK2 phosphorylation sites. Phosphorylation of Sec63 by CK2 enhanced its binding to Sec62.Protein kinase CK2 phosphorylation of Sec63 leads to an enhanced binding of Sec63 to Sec62. This complex formation is a prerequisite for a functional ER protein translocon.Thus, our present data indicate a regulatory role of CK2 in the ER protein translocation.Display Omitted► The ER-membrane resident protein Sec63 is phosphorylated by protein kinase CK2. ► The CK2 phosphorylation sites were mapped within the cytoplasmic domain of Sec63. ► Sec63 bound to CK2. ► CK2 phosphorylation of Sec63 enhanced its binding to Sec62.
Keywords: Protein kinase; Phosphorylation; Protein–protein interaction; Protein translocation;

All reported plant ferritins are heteropolymers comprising two different H-type subunits. Whether or not homopolymeric plant ferritin occurs in nature is an open question.A homopolymeric phytoferritin from adzuki bean seeds (ASF) was obtained by various protein purification techniques for the first time, which shares the highest identity (89.6%) with soybean seed H-1 ferritin (rH-1). Therefore, we compared iron oxidation activity and protein stability of ASF with those of rH-1 by stopped-flow combined with light scattering or UV/Vis spectrophotography, SDS- and native- PAGE analyses. Additionally, a new rH-1 variant (S68E) was prepared by site-directed mutagenesis approach to elucidate their difference in protein stability.At high iron loading of protein, the extension peptide (EP) of plant ferritin was involved in iron oxidation, and the EP of ASF exhibited a much stronger iron oxidative activity than that of rH-1. Besides, ASF is more stable than rH-1 during storage, which is ascribed to one amino acid residue, Ser68.ASF exhibits a different mechanism in iron oxidation from rH-1 at high iron loading of protein, and a higher stability than rH-1. These differences are mainly stemmed from their different EP sequences.This work demonstrates that plant cells have evolved the EP of phytoferritin to control iron chemistry and protein stability by exerting a fine tuning of its amino acid sequence.► A novel homopolymeric plant ferritin is firstly purified from adzuki bean seed. ► The EP of ASF exhibits a much stronger iron oxidative activity than that of rH-1. ► Both ASF and rH-1 variant are much more stable than rH-1. ► The degradation of rH-1 is ascribed to one amino acid residue, Ser68.
Keywords: Plant ferritin; Extension peptide (EP); Adzuki bean seed; Homopolymer; Iron oxidation;

Macrophage elastase (MMP-12) in expanding murine adipose tissue by D. Bauters; M. Van Hul; H.R. Lijnen (2954-2959).
Matrix metalloproteinases (MMPs) are known to play a role in adipose tissue development, but little information is available on the role of individual proteinases. Expansion of adipose tissue is associated with an increased macrophage content. Macrophage elastase (MMP-12) has an important role in macrophage infiltration, which induces pro-inflammatory effects in adipose tissue.The role of MMP-12 was investigated in adipose tissues of MMP-12 deficient and wild-type control mice kept on normal chow or on high fat diet for 15 weeks.MMP-12 deficiency had no significant effect on total body weight or on subcutaneous (SC) or gonadal (GON) adipose tissue mass. Adipocyte and blood vessel size and density in SC and GON adipose tissues of obese mice were also comparable in MMP-12 deficient and control mice. Macrophage infiltration in SC and GON adipose tissues was not affected by MMP-12 deficiency, but the amount of crown-like structures (CLS) was significantly lower. MMP-12 deficiency did not affect elastin content in the extracellular matrix of SC or GON adipose tissue.Adipose tissue mass and composition in mice with nutritionally induced obesity was not markedly affected by MMP-12 deficiency, except for an apparently lower degree of CLS.MMP-12 does not seem to be essential for macrophage infiltration in adipose tissue, but contributes to the formation of CLS surrounding moribund adipocytes.►MMP-12 is not essential in adipose tissue development and associated angiogenesis. ► Macrophage infiltration in adipose tissue is not dependent on MMP-12 activity. ► MMP-12 deficiency attenuates formation of CLS surrounding moribund adipocytes.
Keywords: Obesity; Adipose tissue; Macrophage; MMP-12; Elastase;

In silico and in vitro characterization of anti-amyloidogenic activity of vitamin K3 analogues for Alzheimer's disease by Pham Dinh Quoc Huy; Yao-Chung Yu; Son Tung Ngo; Tran Van Thao; Chin-piao Chen; Mai Suan Li; Yi-Cheng Chen (2960-2969).
Aggregation of amyloid-beta (Aβ) has been proposed as the main cause of Alzheimer's disease (AD). Vitamin K deficiency has been linked to the pathogenesis of AD. Therefore, 15 synthesized vitamin K3 (VK3) analogues were studied for their anti-amyloidogenic activity.Biological and spectroscopic assays were used to characterize the effect of VK3 analogues on amyloidogenic properties of Aβ, such as aggregation, free radical formation, and cell viability. Molecular dynamics simulation was used to calculate the binding affinity and mode of VK3 analogue binding to Aβ.Both numerical and experimental results showed that several VK3 analogues, including VK3-6, VK3-8, VK3-9, VK3-10, and VK3-224 could effectively inhibit Aβ aggregation and conformational conversion. The calculated inhibition constants were in the μM range for VK3-10, VK3-6, and VK3-9 which was similar to the IC50 of curcumin. Cell viability assays indicated that VK3-9 could effectively reduce free radicals and had a protective effect on cytotoxicity induced by Aβ.The results clearly demonstrated that VK3 analogues could effectively inhibit Aβ aggregation and protect cells against Aβ induced toxicity. Modified VK3 analogues can possibly be developed as effective anti-amyloidogenic drugs for the treatment of AD.VK3 analogues effectively inhibit Aβ aggregation and are highly potent as anti-amyloidogenic drugs for therapeutic treatment of AD.► VK3 analogues effectively inhibit Aβ aggregation. ► The binding affinity of VK3 analogues to Aβ is in the μM range. ► VK3-9 can reduce the free radical generation of Aβ. ► VK3-9 has a protective effect on cytotoxicity induced by Aβ. ► VK3 analogues have potential as anti-amyloidogenic drugs for the treatment of AD.
Keywords: Amyloid-beta; Degenerative disease; Alzheimer disease; Vitamin K3 analog; Anti-amyloidogenic activity; Molecular dynamics simulation;

Protein dynamics influence protein function and stability and modulate conformational changes. Such motions depend on the underlying networks of intramolecular interactions and communicating residues within the protein structure. Here, we provide the first characterization of the dynamic fingerprint of the dimeric alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 (VAP), which is among the APs with the highest known k cat at low temperatures.Multiple all-atom explicit solvent molecular dynamics simulations were employed in conjunction with different metrics to analyze the dynamical patterns and the paths of intra- and intermolecular communication.Interactions and coupled motions at the interface between the two VAP subunits have been characterized, along with the networks of intramolecular interactions. It turns out a low number of intermolecular interactions and coupled motions, which result differently distributed in the two monomers. The paths of long-range communication mediated from the catalytic residues to distal sites were also characterized, pointing out a different information flow in the two subunits.A pattern of asymmetric flexibility is evident in the two identical subunits of the VAP dimer that is intimately linked to a different distribution of intra- and intermolecular interactions. The asymmetry was also evident in pairs of cross-correlated residues during the dynamics.The results here discussed provide a structural rationale to the half-of-site mechanism previously proposed for VAP and other APs, as well as a general framework to characterize asymmetric dynamics in homomeric enzymes.► Dimeric enzyme dynamics and underlying intra- and intermolecular interaction networks. ► Asymmetric flexibility. ► Different paths of long-range communication in the individual subunits.
Keywords: Cold-adapted; Psychrophilic; Alkaline phosphatase; Molecular dynamics; Crown domain; Asymmetric flexibility;

Edible blue-green algae reduce the production of pro-inflammatory cytokines by inhibiting NF-κB pathway in macrophages and splenocytes by Chai Siah Ku; Tho X. Pham; Youngki Park; Bohkyung Kim; Min Sun Shin; Insoo Kang; Jiyoung Lee (2981-2988).
Chronic inflammation contributes to the development of pathological disorders including insulin resistance and atherosclerosis. Identification of anti-inflammatory natural products can prevent the inflammatory diseases.Anti-inflammatory effects of blue-green algae (BGA), i.e., Nostoc commune var. sphaeroides Kützing (NO) and Spirulina platensis (SP), were compared in RAW 264.7 and mouse bone marrow-derived macrophages (BMM) as well as splenocytes from apolipoprotein E knockout (apoE−/− ) mice fed BGA.When macrophages pretreated with 100 μg/ml NO lipid extract (NOE) or SP lipid extract (SPE) were activated by lipopolysaccharide (LPS), expression and secretion of pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), and IL-6, were significantly repressed. NOE and SPE also significantly repressed the expression of TNFα and IL-1β in BMM. LPS-induced secretion of IL-6 was lower in splenocytes from apoE−/− fed an atherogenic diet containing 5% NO or SP for 12 weeks. In RAW 264.7 macrophages, NOE and SPE markedly decreased nuclear translocation of NF-κB. The degree of repression of pro-inflammatory gene expression by algal extracts was much stronger than that of SN50, an inhibitor of NF-κB nuclear translocation. Trichostatin A, a pan histone deacetylase inhibitor, increased basal expression of IL-1β and attenuated the repression of the gene expression by SPE. SPE significantly down-regulated mRNA abundance of 11 HDAC isoforms, consequently increasing acetylated histone 3 levels.NOE and SPE repress pro-inflammatory cytokine expression and secretion in macrophages and splenocytes via inhibition of NF-κB pathway. Histone acetylation state is likely involved in the inhibition.This study underscores natural products can exert anti-inflammatory effects by epigenetic modifications such as histone acetylation.► Anti-inflammatory effects of edible blue-green algae in macrophages ► Inhibition of NF-κB pathway for the anti-inflammatory effects in macrophages ► Repression of IL-6 secretion from splenocytes of mice fed blue-green algae ► Role of histone deacetylases in the anti-inflammatory role of blue-green algae
Keywords: Blue-green algae; Anti-inflammation; RAW 264.7 macrophage; NF-κB; Cytokine array; Histone deacetylation;

The pH of a biological system is a crucial determinant of the structures and reactivity of its components and cellular homeostasis of H+ is critical for cell viability. Control and monitoring of cellular acidity are highly desirable for the purpose of studying biochemical processes in vivo.The effect of photolysis of a caged strong acid, the ester 1-(2-nitrophenyl)-ethylhexadecyl sulfonate (HDNS) is used to cause a controlled drop in pH in single cells. An isolated cell is selected under the IR microscope, irradiated with near-UV light and monitored by FTIR.We demonstrate the use of FTIR spectromicroscopy to monitor light-induced acidification of the cellular medium by measuring the increased concentration of CO2 and corresponding decrease of HCO3 in the cell and in the surrounding medium.We have demonstrated a method to control and accurately monitor the changes in pH of a cellular system by coupling a caged proton-releasing agent with FTIR spectromicroscopy detection. The overall implementation of photolysis and spectroscopic detection in a microscope optical configuration ensures single cell selectivity in both acidification and monitoring. We show the viability of monitoring of pH changes by FTIR spectromicroscopy with sensitivity comparable to that of glass electrodes, better than the existing methods for determining cell pH.Reporting the effect of small variations of cellular acidity provides a major improvement in the understanding of the interplay between molecular properties as assessed in vitro and cell physiology.Display Omitted► Proton caged HDNS is incubated in 3T3 cells. ► Proton release is induced by near-UV irradiation, inducing pH change in the cellular medium and cytoplasm. ► pH variation is monitored by FTIR measurements of bicarbonate and carbon dioxide.
Keywords: Cell; IR microscopy; Acidity; Caged molecule;

In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials by Andrea Heinz; Christoph K.H. Ruttkies; Günther Jahreis; Christoph U. Schräder; Kanin Wichapong; Wolfgang Sippl; Fred W. Keeley; Reinhard H.H. Neubert; Christian E.H. Schmelzer (2994-3004).
Elastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, however, the cross-linking pattern is still unknown.Small peptides containing reactive allysine residues based on sequences of cross-linking domains of human elastin were incubated in vitro to form cross-links characteristic of mature elastin. The resultant insoluble polymeric biomaterials were studied by scanning electron microscopy. Both, the supernatants of the samples and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry.MS2 data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally cross-linked peptides, but for the first time also tri- and tetrafunctionally cross-linked species. Thus, it was possible to identify intra- and intermolecular cross-links including allysine aldols, dehydrolysinonorleucines and dehydromerodesmosines. The formation of the tetrafunctional cross-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations.The study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin peptides. MALDI-TOF/TOF mass spectrometry and the newly developed software PolyLinX proved suitable for sequencing of native cross-links in proteolytic digests of elastin-like biomaterials.The study provides important insight into the formation of native elastin cross-links and represents a considerable step towards the characterization of the complex cross-linking pattern of mature elastin.► In vitro cross-linking of short elastin peptides yields elastin-like biomaterials. ► Software was developed to allow identification of native polyfunctional cross-links. ► Intra- and intermolecular cross-links characteristic of elastin were identified. ► In vitro formation of desmosine was proven by means of mass spectrometry.
Keywords: Desmosine; Native cross-links; Dehydrolysinonorleucine; Allysine aldol; Dehydromerodesmosine; Mass spectrometry;

Iron nanoparticles from animal blood for cellular imaging and targeted delivery for cancer treatment by M. Chamundeeswari; T.P. Sastry; B.S. Lakhsmi; V. Senthil; Enzo Agostinelli (3005-3010).
Iron nanoparticles (INPs) are usually prepared from inorganic sources, but we have prepared it from goat blood using incineration method. These INPs are then coated with chitosan (C) and coupled with folic acid (F) to form bionanocomposite for folate receptors.The bionanocomposite was characterized for its physicochemical properties and cancer cell targeting studies using Fourier transform infrared spectroscopy, transmission electron microscopy, Zeta potential analysis, scanning electron microscopy–energy dispersive X-ray spectroscopy and magnetic resonance imaging analyses.The results have shown that the particle size of the INP-CF was found to be 80–300 nm and confirmed the presence of chitosan and folic acid in the bionanocomposite. Cancer and normal mouse embryonic cell line study confirmed the internalization of INP-CF and this phenomenon was also supported by physicochemical studies.Thus, nanobiocomposite prepared using natural sources as a raw material will be beneficial compared to commercially available synthetic sources and can be used as receptor targeting agent for cancer treatment. This nanobiocomposite when coupled with substances such as monoclonal antibodies might act as a theranostic nanoagent for cancer therapy in the years to come.The prepared novel nanobiocomposite containing INPs isolated from natural source may be used as multifunctional agent due its paramagnetic property apart from its drug delivery effect.Display Omitted► Magnetic nanoparticles were prepared from natural source. ► The prepared composite is purely composed of natural substances. ► The bionanocomposite is biocompatible and biodegradable. ► It finds dual applications, as MRI contrast agent and as targeting delivery agent. ► The preparation of iron nanoparticles from blood is highlighted in “Nature India”.
Keywords: Breast cancer cell; Chitosan; Folic acid; MRI scan; Iron nanoparticle;

CD53, a suppressor of inflammatory cytokine production, is associated with population asthma risk via the functional promoter polymorphism − 1560 C>T by Haeyong Lee; Sungmin Bae; Jaewoong Jang; Byoung Whui Choi; Choon-Sik Park; Jong Sook Park; Seung-Hyo Lee; Yoosik Yoon (3011-3018).
In this study, the association of asthma with CD53, a member of the tetraspanin family, was assessed for the first time in a mechanism-based study.Genetic polymorphisms of CD53 were analyzed in 591 subjects and confirmed in a replication study of 1001 subjects. CD53 mRNA and protein levels were measured in peripheral blood leukocytes, and the effects of the promoter polymorphisms on nuclear factor binding were examined by electrophoretic mobility shift assay. Cellular functional studies were conducted by siRNA transfections.Among tagging SNPs of CD53, the − 1560 C>T in the promoter region was significantly associated with asthma risk. Compared with the CC genotype, the CT and TT genotypes were associated with a higher asthma risk, with odd ratios of 1.74 (P  = 0.009) and 2.03 (P  = 0.004), respectively. These findings were confirmed in the replication study with odd ratios of 1.355 (P  = 0.047) and 1.495 (P  = 0.039), respectively. The − 1560 C>T promoter SNP had functional effects on nuclear protein binding as well as mRNA and protein expression levels in peripheral blood leukocytes. When CD53 was knocked down by siRNA in THP-1 human monocytic cells stimulated with house dust mite, the production of inflammatory cytokines as well as NFκB activity was significantly over-activated, suggesting that CD53 suppresses over-activation of inflammatory responses.The − 1560 C>T SNP is a functional promoter polymorphism that is significantly associated with population asthma risk, and is thought to act by directly modulating nuclear protein binding, thereby altering the expression of CD53, a suppressor of inflammatory cytokine production.Display Omitted► − 1560 C>T in the promoter of CD53 was associated with asthma risk with replication. ► − 1560 C>T affects nuclear protein binding in electrophoretic mobility shift assay. ► − 1560 C>T affects mRNA/protein levels of CD53 in peripheral blood leukocytes ► CD53 suppressed inflammatory cytokine production and NFκB activity.
Keywords: CD53; Tetraspanin; SNP; Polymorphism; Asthma; Cytokine;

Mapping of the binding sites involved in PSP94–CRISP-3 interaction by molecular dissection of the complex by Ananya A. Breed; Amanda Gomes; Binita Sur Roy; Smita D. Mahale; Bhakti R. Pathak (3019-3029).
Human Prostate Secretory Protein of 94 amino acids (PSP94) has been shown to bind human CRISP-3 (cysteine-rich secretory protein 3) with very high affinity. CRISP-3 belongs to the CRISP family of proteins having a PR-1 (pathogenesis related protein 1) domain at its N-terminal and ion channel regulatory (ICR) domain at its C-terminal connected by a hinge region. Functional significance of this complex is not yet known.In order to identify the residues and/or regions involved in PSP94–CRISP-3 interaction, site-directed mutagenesis was employed. Effect of the mutations on the interaction was studied by co-immunoprecipitation (Co-IP).For PSP94, amino acids Y3, F4, P56 and the C-terminal β-strand were found to be crucial for interacting with CRISP-3. A disulfide bond between the two domains of PSP94 (C37A–C73A) was also important for this interaction. In case of CRISP-3, the N-terminal domain alone could not maintain a strong interaction with PSP94 but it required presence of the hinge region and not the C-terminal domain. Apart from CRISP-3, CRISP-2 was also found to interact with human PSP94. Based on our findings the most likely model of PSP94–CRISP-3 complex has been proposed.The terminal β-strands of PSP94 contact the first α-helix and the hinge region of CRISP-3.Involvement of the hinge region of CRISPs in interaction with PSP94 may affect the domain movement of CRISPs essential for the ion-channel regulatory activity resulting in inhibition of this activity.► Generation of different mutants of PSP94 and CRISP-3 and study of their interaction by Co-IP ► Molecular evidence for involvement of terminal β-strands of PSP94 in binding with CRISP-3 ► Demonstration of the importance of hinge region of CRISP-3 in binding with PSP94 ► Identification of CRISP-2 as an interacting partner of PSP94 ► Proposing the most likely model of PSP94–CRISP-3 complex
Keywords: PSP94; β-MSP; CRISP-3; CRISP-2;

Proteomic characterization of EPCs and CECs “in vivo” from acute coronary syndrome patients and control subjects by L. Mourino-Alvarez; E. Calvo; J. Moreu; L.R. Padial; J.A. Lopez; M.G. Barderas; F. Gil-Dones (3030-3053).
Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) represent two scarce blood populations that are thought to play important roles in tissue vascularization. They have also been proposed as potential markers for more than a dozen pathologies. Moreover, EPCs have arisen as a new therapeutic option for cardiovascular disease. However nowadays there is certain controversy about their roles and a better understanding of EPC biology is required to develop new strategies for forthcoming therapies.Flow cytometry analysis was performed on freshly isolated mononuclear cells from control subjects and Acute Coronary Syndrome (ACS) patients. EPCs and CECs for both groups were isolated and quantified. Statistical analyses were performed to test the potential biomarker usefulness of both populations in ACS together with the first “in vivo” proteomic characterizations of these populations.Our results do not show statistical differences in the quantification of CECs and EPCs in control subjects and ACS patients. The proteomic characterization allowed us to identify 673 proteins associated to CECs (389 in controls and 462 in ACS patients), and another 502 proteins in EPCs (350 in controls and 274 in ACS patients).Our data show the necessity to obtain a more accurate and specific phenotype of CECs and EPCs cells as well as a flow cytometry “golden standard” protocol, before they can be considered useful clinical markers.The proteomic data suggest a potential effect of ACS in the protein profiles of these cells.► CECs and EPCs might be potential biomarkers of different diseases. ► EPCs have arisen as a novel therapeutic option for cardiovascular disorders. ► Still there is controversy about their roles, potentials and capabilities. ► Proteomics is a powerful tool to decrease this controversy. ► This technology could help to improve our knowledge in EPC biology.
Keywords: EPCs; CECs; Biomarker; Proteomics; Acute coronary syndrome;

Sulfation pattern of the fucose branch is important for the anticoagulant and antithrombotic activities of fucosylated chondroitin sulfates by Shiguo Chen; Guoyun Li; Nian Wu; Xin Guo; Ningbo Liao; Xingqian Ye; Donghong Liu; Changhu Xue; Wengang Chai (3054-3066).
The aim is to compare the structures, anticoagulant and antithrombotic activities of two fucosylated chondroitin sulfates isolated from sea cucumbers Isostichopus badionotus (fCS-Ib) and Pearsonothuria graeffei (fCS-Pg), which were known to have different sulfation patterns on the fucose branches.The structures of fCSs were identified using 2D NMR. Anticoagulant activities were measured by activated partial thromboplastin time (APTT) and thrombin time (TT), and inhibition of factors IIa, Xa and XIIa was assessed in vitro. Antithrombotic activity was determined ex vivo by measuring the length and weight of the thrombus generated.The two fCSs had identical chondroitin sulfate E backbones and similar fucose branches, but different sulfation patterns of the fucose branches. The fucose branch in fCS-Ib was mainly 2,4-O-sulfated whereas that in fCS-Pg was mainly 3,4-O-sulfated. In vitro assay indicated that fCS-Pg possessed much lower potency than fCS-Ib in prolonging APTT/TT and in inhibition of thrombin. However, they both exhibited similar inhibitory effects on factor X activation by intrinsic tenase complex, and on thrombus generation. Furthermore, both fCSs significantly activated factor XII, which has been proved to be associated with adverse clinical events associated with heparin contaminated by oversulfated chondroitin sulfate.The 2,4-O-sulfated fucose branch is the key structural factor of fCSs for prolonged APTT/TT and inhibition of thrombin, whereas the inhibitory effect of fCSs on factor X, XII activation and thrombus generation was attributed to the overall structure of fCS polysaccharide.Both fCSs have well defined structures and can be readily quality-controlled, and therefore may be potential alternatives for heparin as anticoagulant and antithrombotic drugs.► Fuc2,4S branches are the key for prolonged APTT/TT and inhibition of thrombin. ► FXa, FXIIa and thrombus generation are affected by stereotype structures of fCSs. ► fCS-Pg has high antithrombotic but low anticoagulant activity. ► fCS-Ib has both high anticoagulant activity and antithrombotic activity. ► Both two fCSs have well repeated unit and can be readily quality controlled.
Keywords: Sea cucumber; Fucosylated chondroitin sulfate; Anticoagulant; Antithrombotic; Sulfation pattern;

MiR-182 is a member of the miR-183 cluster located at human chromosome 7q32 region and is up-regulated in human cancers. We study the regulation of miR-182 expression and its oncogenic role.MiR-182 level was investigated by real-time reverse transcription-PCR. Chromatin immunoprecipitation assay was used to confirm promoter binding of transcription factors. The correlation between miR-182 and RECK was analyzed by Western blotting, real-time RT-PCR and 3-untranslated region reporter assay. Zymography, matrix metalloproteinase activity, invasion and colony formation were used to study the tumorigenic activity.MiR-182 is over-expressed in human breast tumor tissues and cell lines. Inhibition or knockdown of β-catenin reduced miR-182 level in MDA-MB-231 cells. ChIP assay confirmed the binding of β-catenin on miR-182 promoter. Anti-miR-182 increased the MMP inhibitor RECK protein in MDA-MB-231 cells while pre-miR-182 reduced RECK protein but not mRNA in normal mammary epithelial H184B5F5/M10 cells. Restoration of RECK protein by anti-miR-182 attenuated MMP-9 activity, cell invasion and colony formation. Ectopic expression of miR-182 inhibited restoration of RECK protein by β-catenin inhibitor indicating miR-182 is important for β-catenin-induced down-regulation of RECK. An inverse association between miR-182 and RECK was demonstrated in breast tumor tissues.We provide evidence that miR-182 is up-regulated by β-catenin signaling pathway in breast cancer and its up-regulation increases tumorigenicity and invasiveness by repressing RECK.Our data demonstrate for the first time that miR-182 expression is controlled by β-catenin. In addition, we identify a new miR-182 target RECK which is important for miR-182-induced tumorigenesis.► MiR-182 is over-expressed in different stages of breast tumor. ► Wnt/β-catenin pathway is involved in the regulation of miR-182 in breast cancer. ► MiR-182 inhibits a new target RECK to increase MMP activity and invasion ability.
Keywords: Reversion-inducing cysteine-rich protein with Kazal motif (RECK); Matrix metalloproteinase; MicroRNA; β-catenin; miR-182;

DNA-maleimide: An improved maleimide compound for electrophoresis-based titration of reactive thiols in a specific protein by Satoshi Hara; Tatsuya Nojima; Kohji Seio; Masasuke Yoshida; Toru Hisabori (3077-3081).
Thiol-mediated redox regulation of proteins plays a key role in many cellular processes.To understand the redox status of cysteinyl thiol groups of the desired proteins, we developed a new maleimide reagent: a maleimide-conjugated single strand DNA, DNA-maleimide (DNA-Mal).DNA-Mal labelled proteins run as a distinct band on SDS-PAGE, with a discrete 9.32 kDa mobility shift per label regardless of the protein species or electrophoretic conditions.DNA-Mal labels free thiols like standard maleimide reagents, but possesses practical advantages in titration of the number and relative content of free thiols in a protein.The versatility of DNA molecule enhances the application of DNA-Mal in a broader range of cysteine containing proteins.Display Omitted
Keywords: Thiol reagent; Thiol redox status; Maleimide conjugated DNA; Cysteine; Sulfhydryl;

Stimulation of σ1-receptor restores abnormal mitochondrial Ca2 + mobilization and ATP production following cardiac hypertrophy by Hideaki Tagashira; Chen Zhang; Ying-mei Lu; Hideyuki Hasegawa; Hiroshi Kanai; Feng Han; Kohji Fukunaga (3082-3094).
We previously reported that the σ1-receptor (σ1R) is down-regulated following cardiac hypertrophy and dysfunction in transverse aortic constriction (TAC) mice. Here we address how σ1R stimulation with the selective σ1R agonist SA4503 restores hypertrophy-induced cardiac dysfunction through σ1R localized in the sarcoplasmic reticulum (SR).We first confirmed anti-hypertrophic effects of SA4503 (0.1–1 μM) in cultured cardiomyocytes exposed to angiotensin II (Ang II). Then, to confirm the ameliorative effects of σ1R stimulation in vivo, we administered SA4503 (1.0 mg/kg) and the σ1R antagonist NE-100 (1.0 mg/kg) orally to TAC mice for 4 weeks (once daily).σ1R stimulation with SA4503 significantly inhibited Ang II-induced cardiomyocyte hypertrophy. Ang II exposure for 72 h impaired phenylephrine (PE)-induced Ca2 + mobilization from the SR into both the cytosol and mitochondria. Treatment of cardiomyocytes with SA4503 largely restored PE-induced Ca2 + mobilization into mitochondria. Exposure of cardiomyocytes to Ang II for 72 h decreased basal ATP content and PE-induced ATP production concomitant with reduced mitochondrial size, while SA4503 treatment completely restored ATP production and mitochondrial size. Pretreatment with NE-100 or siRNA abolished these effects. Chronic SA4503 administration also significantly attenuated myocardial hypertrophy and restored ATP production in TAC mice. SA4503 administration also decreased hypertrophy-induced impairments in LV contractile function.σ1R stimulation with the specific agonist SA4503 ameliorates cardiac hypertrophy and dysfunction by restoring both mitochondrial Ca2 + mobilization and ATP production via σ1R stimulation.Our observations suggest that σ1R stimulation represents a new therapeutic strategy to rescue the heart from hypertrophic dysfunction.► The σ1R agonist, SA4503 inhibits cardiac hypertrophy and contractile dysfunction. ► SΑ4503 ameliorates mitochondrial membrane potential and downregulation of mitodfusin-2 and GRP75. ► SA4503 rescues IP3R-mediated SR-mitochondrial Ca2 + mobilization following hypertrophy. ► SA4503 rescues mitochondrial ATP production following hypertrophy.
Keywords: ATP; Calcium signaling; ATP production; Mitochondria; Sigma receptor;

Starch is a main source of carbohydrate in human diets, but differences are observed in postprandial glycaemia following ingestion of different foods containing identical starch contents. Such differences reflect variations in rates at which different starches are digested in the intestine. In seeking explanations for these differences, we have studied the interaction of α-amylase with starch granules. Understanding this key step in digestion should help with a molecular understanding for observed differences in starch digestion rates.For enzymes acting upon solid substrates, a Freundlich equation relates reaction rate to enzyme adsorption at the surface. The Freundlich exponent (n) equals 2/3 for a liquid-smooth surface interface, 1/3 for adsorption to exposed edges of ordered structures and 1.0 for solution–solution interfaces. The topography of a number of different starch granules, revealed by Freundlich exponents, was compared with structural data obtained by differential scanning calorimetry and Fourier transform infrared spectroscopy with attenuated total internal reflectance (FTIR-ATR).Enzyme binding rate and FTIR-ATR peak ratio were directly proportional to n and Δgel H was inversely related to n. Amylase binds fastest to solubilised starch and to granules possessing smooth surfaces at the solid–liquid interface and slowest to granules possessing ordered crystalline surfaces.Freundlich exponents provide information about surface blocklet structures of starch that supplements knowledge obtained from physical methods.Nanoscale structures at the surface of starch granules influence hydrolysis by α-amylase. This can be important in understanding how dietary starch is digested with relevance to diabetes, cardiovascular health and cancer.Display Omitted► α-Amylase action on starch was studied by Freundlich kinetics and physical methods. ► A Freundlich exponent of 2/3 was obtained for wheat, maize and lam pea starches. ► For adsorption to potato and waxy rice starches, the exponents approached 1/3. ► A 2/3 value indicates adsorption to a smooth surface, 1/3 adsorption to cracks/edges. ► This novel analysis provided evidence for the existence of starch blocklets.
Keywords: Starch; α-Amylase; Binding kinetics; DSC; FTIR-ATR; Freundlich model;

The insulin-mimetic effect of Morin: A promising molecule in diabetes treatment by Paolo Paoli; Paolo Cirri; Anna Caselli; Francesco Ranaldi; Giulia Bruschi; Alice Santi; Guido Camici (3102-3111).
Type-2 diabetes is a worldwidely diffuse disease characterized by insulin resistance that arises from alterations of receptor and/or post-receptor events of insulin signalling. Studies performed with PTP1B-deficent mice demonstrated that PTP1B is the main negative regulator of insulin signalling. Inhibition or down regulation of this enzyme causes enhanced insulin sensitivity. Hence this enzyme represents the most attractive target for development of innovative anti-diabetic drugs.Selection of new PTP1B inhibitors among an in house library of polyphenolic compounds was carried out screening their activity. The inhibition mechanism of Morin was determined by kinetic analyses. The cellular action of Morin was assayed on HepG2 cells. Analyses of the insulin signalling pathways was carried out by Western blot methods, glycogen synthesis was estimated by measuring the incorporation of [3H]-glucose, gluconeogenesis rate was assayed by measuring the glucose release in the cell medium. Cell growth was estimated by cell count. Docking analysis was conducted with SwissDock program.We demonstrated that Morin: i) is a non-competitive inhibitor of PTP1B displaying a Ki in the μM range; ii) increases the phosphorylation of the insulin receptor and Akt; iii) inhibits gluconeogenesis and enhances glycogen synthesis. Morin does not enhance cell growth.We have identified Morin as a new small molecular non-competitive inhibitor of PTP1B, which behaves as an activator and sensitizer of the insulin receptor stimulating the metabolic pathways only.Our study suggests that Morin is a useful lead for development of new low Mr compounds potentially active as antidiabetic drugs.► Morin acts as a competitive inhibitor of PTP1B, displaying Ki in the low micromolar range. ► Morin activates the insulin receptor in HepG2 cells. ► Morin enhances glycogen synthesis and inhibits gluconeogenesis. ► Morin increases insulin sensitivity. ► Morin possesses potential antidiabetic activity.
Keywords: Morin; Flavonoid; PTP1B inhibitor; PTP; Antidiabetic drugs;

Selenoprotein W serves as an antioxidant in chicken myoblasts by Hai-Dong Yao; Qiong Wu; Zi-Wei Zhang; Shu Li; Xiao-Long Wang; Xin-Gen Lei; Shi-Wen Xu (3112-3120).
Selenoprotein W (SelW) was thought to play an antioxidant role in mammals. Because chicken SelW has no cysteine (Cys) at the residue 37 (Cys37) that is required for the presumed antioxidant function in mammals, this study was conducted to determine whether chicken SelW possessed the same function.Small interfering RNAs (siRNAs) technology was applied to suppress the SelW expression in chicken embryonic myoblasts. Thereafter, these myoblasts were treated with different concentrations of H2O2 and assayed for cell viability, apoptosis rate, reactive oxygen species (ROS) status, and expression levels of apoptosis-related genes and proteins (Bax, Bcl-2, and caspase-3).Silencing of the myoblast SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H2O2. While the knockout down of SelW up-regulated Bax and caspase-3 and down-regulated Bcl-2, the induced oxidative injuries were alleviated by treatment with a ROS scavenger, N-acetyl-l-cysteine (NAC).Chicken SelW protected embryonic myoblasts against cell apoptosis mediated by endogenous and exogenous H2O2.Chicken SelW possesses antioxidant function similar to the mammalian homologues despite the lack of Cys37 in the peptide.Display Omitted► The siRNA suppressed the SelW expression by > 77% in chicken myoblasts ► SelW gene silencing enhanced ROS formation in chicken myoblasts ► SelW gene silencing activated cell apoptosis and related signaling ► SelW gene silencing sensitized cells to the H2O2-mediated apoptosis ► Oxidative damages induced by the SelW knockdown was alleviated by NAC
Keywords: SelW; Chicken embryonic myoblasts; Antioxidation; ROS; Apoptosis;

Salinomycin induces apoptosis and senescence in breast cancer: Upregulation of p21, downregulation of survivin and histone H3 and H4 hyperacetylation by Yusra Al Dhaheri; Samir Attoub; Kholoud Arafat; Synan AbuQamar; Ali Eid; Nesreen Al Faresi; Rabah Iratni (3121-3135).
In the present study, we investigated the effect of Salinomycin on the survival of three human breast cancer cell lines MCF-7, T47D and MDA-MB-231 grown in adherent culture conditions.Cell viability was measured by CellTiter-Glo and Trypan blue exclusion assay. Apoptosis was determined by caspase 3/7 activation, PARP cleavage and Annexin V staining. Cell cycle distribution was assessed by propidium iodide flow cytometry. Senescence was confirmed by measuring the senescence-associated β-galactosidase activity. Changes in protein expression and histone hyperacetylation was determined by western blot and confirmed by immunofluorescence assay.Salinomycinwas able to inhibit the growth of the three cell lines in time- and concentration-dependent manners. We showed that depending on the concentrations used, Salinomycin elicits different effects on theMDA-MB-231 cells. High concentrations of Salinomycin induced a G2 arrest, downregulation of survivin and triggered apoptosis. Interestingly, treatment with low concentrations of Salinomycin induced a transient G1 arrest at earlier time point and G2 arrest at later point and senescence associatedwith enlarged cellmorphology, upregulation of p21 protein, increase in histone H3 and H4 hyperacetylation and expression of SA-β-Gal activity. Furthermore, we found that Salinomycin was able to potentiate the killing of the MCF-7 and MDA-MB-231 cells, by the chemotherapeutic agents, 4-Hydroxytamoxifen and frondoside A, respectively.Our data are the first to link senescence and histone modifications to Salinomycin.This study provides a new insight to better understand the mechanism of action of Salinomycin, at least in breast cancer cells.► Salinomycin elicits different effects on the MDA-MB 231 cells. ► The magnitude of DNA damage determines the response of the cells to these damages. ► Salinomycin-treated MDA-MB 231cells exhibited markers of senescence. ► Histone H3 and H4 hyperacetylation and elevated expression of the CDK inhibitor, p21 ► Salinomycin potentiates the anticancer activity of frondoside A and 4-hydroxytamoxifen.
Keywords: Apoptosis; DNA damage; Histone hyperacetylation; p21; Salinomycin; Senescence-associated beta galactosidase (SA-β-Gal);