BBA - General Subjects (v.1840, #12)

Deregulation of Bmi-1 is associated with enhanced migration, invasion and poor prognosis in salivary adenoid cystic carcinoma by Boyang Chang; Su Li; Qianting He; Zhonghua Liu; Luodan Zhao; Tingting Zhao; Anxun Wang (3285-3291).
Bmi-1 had been found to involve in self renewal of stem cells and tumorigenesis in various malignancies. In this study, we investigated the role of Bmi-1 in the development of salivary adenoid cystic carcinoma (SACC).At first, we confirmed that the deregulation of Bmi-1 was a frequent event in SACC; up-regulation of Bmi-1 was correlated with clinical stages, vital status and distant metastasis and associated with reduced overall survival and disease free survival. SACC-LM cells, higher migration and invasion abilities, elevated the expression of Bmi-1 protein, epithelial-mesenchymal transition (EMT) related proteins (Snail, Slug and Vimentin) and cancer stem cells (CSCs) related proteins (ABCG2, Notch, ALDH-1, Oct-4, Nanog and Epcam) compared to the SACC-83 cells (lower migration and invasion abilities). The migration and invasion abilities were inhibited in SACC-LM cells upon Bmi-1 knockdown. Meanwhile, Bmi-1 knockdown resulted in simultaneous loss of stem cell markers and EMT markers in SACC-LM cells.Our studies confirm that Bmi-1 deregulation plays an important role in the development of SACC and contributes to the migration and the invasion abilities of SACC, which is involved in EMT and CSCs.To our knowledge, this is the first study revealing that Bmi-1 deregulation is associated with enhanced migration, invasion and poor prognosis in salivary adenoid cystic carcinoma.
Keywords: Bmi-1; Salivary adenoid cystic carcinoma; Metastasis;

Binding kinetics, potency, and selectivity of the hepatitis C virus NS3 protease inhibitors GS-9256 and vedroprevir by Ona Barauskas; Amoreena C. Corsa; Ruth Wang; Scott Hluhanich; Debi Jin; Magdeleine Hung; Huiling Yang; William E. Delaney; Brian E. Schultz (3292-3298).
GS-9256 and vedroprevir are inhibitors of the hepatitis C virus NS3 protease enzyme, an important drug target. The potency, selectivity, and binding kinetics of the two compounds were determined using in vitro biochemical assays.Potency of the compounds against NS3 protease and selectivity against a panel of mammalian proteases were determined through steady-state enzyme kinetics. Binding kinetics were determined using stopped-flow techniques. Dissociation rates were measured using dilution methods.GS-9256 and vedroprevir had measured K i values of 89 pM and 410 pM, respectively, against genotype 1b NS3 protease; K i values were higher against genotype 2a (2.8 nM and 39 nM) and genotype 3 proteases (104 nM and 319 nM) for GS-9256 and vedroprevir, respectively. Selectivity of GS-9256 and vedroprevir was > 10,000-fold against all tested off-target proteases. Association rate constants of 4 × 105  M− 1  s− 1 and 1 × 106  M− 1  s− 1, respectively, were measured, and dissociation rate constants of 4.8 × 10− 5  s− 1 and 2.6 × 10− 4  s− 1 were determined.GS-9256 and vedroprevir are potent inhibitors of NS3 protease with high selectivity against off-target proteases. They have rapid association kinetics and slow dissociation kinetics.The NS3 protease is a key drug target for the treatment of hepatitis C. The potency, selectivity, and binding kinetics of GS-9256 and vedroprevir constitute a biochemical profile that supports the evaluation of these compounds in combination with other direct-acting antivirals in clinical trials for hepatitis C.
Keywords: Hepatitis C virus; NS3 protease; Inhibition; Binding kinetics; Selectivity; Rate constant;

A comparison of ceruloplasmin to biological polyanions in promoting the oxidation of Fe2 + under physiologically relevant conditions by Bruce X. Wong; Scott Ayton; Linh Q. Lam; Peng Lei; Paul A. Adlard; Ashley I. Bush; James A. Duce (3299-3310).
Iron oxidation is thought to be predominantly handled enzymatically in the body, to minimize spontaneous combustion with oxygen and to facilitate cellular iron export by loading transferrin. This process may be impaired in disease, and requires more accurate analytical assays to interrogate enzymatic- and auto-oxidation within a physiologically relevant environment.A new triplex ferroxidase activity assay has been developed that overcomes the previous assay limitations of measuring iron oxidation at a physiologically relevant pH and salinity.Revised enzymatic kinetics for ceruloplasmin (Vmax  ≈ 35 μM Fe3 +/min/μM; Km  ≈ 15 μM) are provided under physiological conditions, and inhibition by sodium azide (Ki for Ferric Gain 78.3 μM, Ki for transferrin loading 8.1 × 104  μM) is quantified. We also used this assay to characterize the non-enzymatic oxidation of iron that proceeded linearly under physiological conditions.These findings indicate that the requirement of an enzyme to oxidize iron may only be necessary under conditions of adverse pH or anionic strength, for example from hypoxia. In a normal physiological environment, Fe3 + incorporation into transferrin would be sufficiently enabled by the biological polyanions that are prevalent within extracellular fluids.
Keywords: Ferroxidase; Ceruloplasmin; Transferrin; Iron; Oxidation;

Tiliroside is a dietary glycosidic flavonoid which has shown in vivo anti-inflammatory activity. This study is aimed at evaluating the effect of tiliroside on neuroinflammation in BV2 microglia, and to identify its molecular targets of anti-neuroinflammatory action.BV2 cells were stimulated with LPS + IFNγ in the presence or absence of tiliroside. TNFα, IL-6, nitrite and PGE2 production was determined with ELISA, Griess assay and enzyme immunoassay, respectively. iNOS, COX-2, phospho-p65, phospho-IκBα, phospho-IKKα, phospho-p38, phospho-MK2, phosopho-MKK3/6 and TRAF-6 were determined by western blot analysis. NF-κB activity was also investigated using a reporter gene assay in HEK293 cells. LPS-induced microglia ROS production was tested using the DCFDA method, while HO-1 and Nrf2 activation was determined with western blot.Tiliroside significantly suppressed TNFα, IL-6, nitrite and PGE2 production, as well as iNOS and COX-2 protein expression from LPS + IFNγ-activated BV2 microglia. Further mechanistic studies showed that tiliroside inhibited neuroinflammation by targeting important steps in the NF-κB and p38 signalling in LPS + IFNγ-activated BV2 cells. This compound also inhibited LPS-induced TRAF-6 protein expression in BV2 cells. Antioxidant activity of tiliroside in BV2 cells was demonstrated through attenuation of LPS + IFNγ-induced ROS production and activation of HO-1/Nrf2 antioxidant system.Tiliroside inhibits neuroinflammation in BV2 microglia through a mechanism involving TRAF-6-mediated activation of NF-κB and p38 MAPK signalling pathways. These activities are possibly due, in part, to the antioxidant property of this compound.Tiliroside is a potential novel natural compound for inhibiting neuroinflammation in neurodegenerative disorders.Display Omitted
Keywords: Tiliroside; NF-κB; p38; TRAF-6; Neuroinflammation; Antioxidant;

Methyl pyruvate rescues mitochondrial damage caused by SIGMAR1 mutation related to amyotrophic lateral sclerosis by Hideaki Tagashira; Yasuharu Shinoda; Norifumi Shioda; Kohji Fukunaga (3320-3334).
Amyotrophic lateral sclerosis (ALS) is a disease caused by motor neuron degeneration. Recently, a novel SIGMAR1 gene variant (p.E102Q) was discovered in some familial ALS patients.We address mechanisms underlying neurodegeneration caused by the mutation using Neuro2A cells overexpressing σ1RE102Q, a protein of a SIGMAR1 gene variant (p.E102Q) and evaluate potential amelioration by ATP production via methyl pyruvate (MP) treatment.σ1RE102Q overexpression promoted dissociation of the protein from the endoplasmic reticulum (ER) membrane and cytoplasmic aggregation, which in turn impaired mitochondrial ATP production and proteasome activity. Under ER stress conditions, overexpression of wild-type σ1R suppressed ER stress-induced mitochondrial injury, whereas σ1RE102Q overexpression aggravated mitochondrial damage and induced autophagic cell death. Moreover, σ1RE102Q-overexpressing cells showed aberrant extra-nuclear localization of the TAR DNA-binding protein (TDP-43), a condition exacerbated by ER stress. Treatment of cells with the mitochondrial Ca2 + transporter inhibitor Ru360 mimicked the effects of σ1RE102Q overexpression, indicating that aberrant σ1R-mediated mitochondrial Ca2 + transport likely underlies TDP-43 extra-nuclear localization, segregation in inclusion bodies, and ubiquitination. Finally, enhanced ATP production promoted by methyl pyruvate (MP) treatment rescued proteasome impairment and TDP-43 extra-nuclear localization caused by σ1RE102Q overexpression.Our observations suggest that neurodegeneration seen in some forms of ALS are due in part to aberrant mitochondrial ATP production and proteasome activity as well as TDP-43 mislocalization resulting from the SIGMAR1 mutation.ATP supplementation by MP represents a potential therapeutic strategy to treat ALS caused by SIGMAR1 mutation.Display Omitted
Keywords: Sigma-1 receptor; Endoplasmic reticulum stress; Autophagy; Neurodegeneration; Amyotrophic lateral sclerosis;

SH2B1 increases the numbers of IRSp53-induced filopodia by Shao-Jing Hong; Szu-Ting Liu; Chien-Jen Chen; Linyi Chen (3335-3344).
Filopodia are actin-rich membrane protrusions that play instrumental roles in development, cell migration, pathogen detection, and wound healing. During neurogenesis, filopodium formation precedes the formation of dendrites and spines. The insulin receptor substrate protein of 53 kDa (IRSp53) has been implicated in regulating the formation of filopodia. Our previous results suggest that a signaling adaptor protein SH2B1β is required for neurite outgrowth of hippocampal neurons and neurite initiation of PC12 cells. Thus, we hypothesize that IRSp53 and SH2B1β may act together to regulate filopodium formation.To determine the contribution of IRSp53 and SH2B1β in the formation of filopodia, we transiently transfect IRSp53 and/or SH2B1β to 293T cells. Cell morphology and protein distribution are assessed via confocal microscopy and subcellular fractionation. Total numbers of filopodia and filopodium numbers per perimeter are calculated to show the relative contribution of IRSp53 and SH2B1β.In this study, we show that SH2B1β interacts with IRSp53 and increases the number of IRSp53-induced filopodia. One mechanism for this enhancement is that IRSp53 recruits SH2B1β to the plasma membrane to actively promote membrane protrusion. The increased numbers of filopodia likely result from SH2B1-mediated cytoplasmic extension and thus increased cell perimeter as well as IRSp53-mediated filopodium formation.Taken together, this study provides a novel finding that SH2B1β interacts with IRSp53-containing complexes to increase the number of filopodia.A better understanding of how SH2B1β and IRSp53 promote filopodium formation may have clinical implication in neurogenesis and regeneration.
Keywords: Filopodia; Membrane protrusion; IRSp53; SH2B1;

Scavenger receptor CL-P1 mainly utilizes a collagen-like domain to uptake microbes and modified LDL by Kenichiro Mori; Katsuki Ohtani; SeongJae Jang; YounUck Kim; Insu Hwang; Nitai Roy; Yasuyuki Matsuda; Yasuhiko Suzuki; Nobutaka Wakamiya (3345-3356).
Collectins are considered to play a role in host defense via complement activation and opsonization, and are composed of a collagen-like domain and a carbohydrate recognition domain (CRD). Collectin placenta 1 (CL-P1) showed scavenger receptor activity as functions in vitro, and has three candidate domains: a coiled-coil domain, a collagen-like domain and CRD.We constructed seven types of CL-P1 deletion mutants to determine the site of each ligand binding domain, and observed whether the specific binding to sugar ligand, microbes, or oxidized LDL decreases or not in cells with CL-P1 deletion mutants and CL-P1 containing mutations of amino acid, respectively.CL-P1 mainly interacted with ligands of microbes through the collagen-like domain and it binds a sugar ligand through the CRD. Additionally it could bind oxidized low density lipoprotein (OxLDL) due to the coiled-coil domain as well as the collagen-like domain. This binding study using mutants at three positively charged sites in the collagen-like domain reveals that the site of R496 K499 K502 plays the most important role in ligand binding functions for microbes and OxLDL.CL-P1 has three unique functional domains: the collagen-like domain mainly acts against most negatively charged ligands, and the CRD specifically does against sugar substances, while the coiled-coil domain additionally acts on modified LDL.We considered that the binding activity for various ligands due to the association of a coiled-coil domain, a collagen-like domain and/or a CRD in CL-P1, might play a role in physiological functions in the animal body.
Keywords: Collectin; Scavenger receptor; Endocytosis; Phagocytosis; Collagen; OxLDL;

One of the activities of histidine triad nucleotide-binding protein 1 (Hint1) under in vitro conditions is the conversion of nucleoside 5′-O-phosphorothioate (NMPS) to its 5′-O-phosphate (NMP), which is accompanied by the release of hydrogen sulfide.Non-hydrolyzable derivatives of AMPS and dCMPS, each containing the residue able to form a covalent bond in nucleic acid–protein complexes via photocrosslinking (at 308 nm), were applied at the complexing experiments with recombinant and cellular Hint1.The cellular lysates prepared after RNAi-mediated knockdown of Hint1 were incubated with AMPS and the level of desulfuration was measured.Recombinant Hint1 and Hint1 present in the cellular lysate of A549 cells, formed complexes with the used substrate analogs.Computer modeling experiments, in which the ligand was docked at the binding pocket, confirmed that direct interactions between Hint1 and the screened analogs are possible.Using RNAi technology, we demonstrated lowered levels of AMPS substrate desulfuration in reactions that employed the cell lysates with a reduced Hint1 level.The enzymatic conversion of AMPS to AMP occurred with the participation of cellular Hint1, the protein, which is present in all organisms.The intracellular Hint1 could be responsible for the in vivo desulfuration of nucleosides-5′-monophosphorothioate, thus it can contribute to the phosphorothioate oligonucleotides metabolism. H2S released during this process may participate in several physiological processes, thus NMPSs can be precursors/donors of H2S in vivo and can be used to study the effects of this gas in biological systems. Moreover, the controlled delivery of (d)NMPSs into cells may be of medicinal utility.
Keywords: Hint1; Photocrosslink; siRNA; (d)NMPS; PS-oligo metabolism; Hydrogen sulfide;

Allosteric inhibitors of calpains: Reevaluating inhibition by PD150606 and LSEAL by Kristin E. Low; Sarathy Karunan Partha; Peter L. Davies; Robert L. Campbell (3367-3373).
The mercaptoacrylate calpain inhibitor, PD150606, has been shown by X-ray crystallography to bind to a hydrophobic groove in the enzyme's penta-EF-hand domains far away from the catalytic cleft and has been previously described as an uncompetitive inhibitor of calpains. The penta-peptide LSEAL has been reported to be an inhibitor of calpain and was predicted to bind in the same hydrophobic groove. The X-ray crystal structure of calpain-2 bound to its endogenous calpain inhibitor, calpastatin, shows that calpastatin also binds to the hydrophobic grooves in the two penta-EF-hand domains, but its inhibitory domain binds to the protease core domains and blocks the active site cleft directly.The mechanisms of inhibition by PD150606 and LSEAL were investigated using steady-state kinetics of cleavage of a fluorogenic substrate by calpain-2 and the protease core of calpain1, as well as by examining the inhibition of casein hydrolysis by calpain and the autoproteolysis of calpain.PD150606 inhibits both full-length calpain-2 and the protease core of calpain-1 with an apparent noncompetitive kinetic model. The penta-peptide LSEAL failed to inhibit either whole calpain or its protease core in vitro.PD150606 cannot inhibit cleavage by calpain-2 of small substrates via binding to the penta-EF-hand domain.PD150606 is often described as a calpain-specific inhibitor due to its ability to target the penta-EF-hand domains of calpain, but we show that it must be acting at a site on the protease core domain instead.
Keywords: Allosteric inhibitor; Calcium-activated protease; Calpain; Penta-EF-hand; PD150606; Enzyme kinetics;

Gambogic acid is a potent anticancer agent and has been found effective against various types of cancer cells. The present study was addressed to explore the cytotoxic potential of Gambogic acid and the modulation of autophagy and apoptosis in bladder cancer cells T24 and UMUC3.Bladder cancer cell lines T24 and UMUC3 were treated with Gambogic acid, apoptosis was checked by flow-cytometry and expression of various autophagy and apoptosis related proteins was monitored by Western blotting. Confocal microscope was used for colocalization of p62 and Beclin-1.Gambogic acid induces reactive oxygen species, and elicits a strong autophagic response by activating JNK at earlier time points, which is inhibited at later time points with the activation of caspases. Reactive oxygen species mediated caspase activation causes degradation of autophagic proteins, cleavage of molecular chaperones (Hsp90 and GRP-78) and adaptor proteins (p62 and NBR1). Gambogic acid treatment results in mitochondrial hyperpolarization and cytochrome c release and activates caspases involved in both extrinsic and intrinsic apoptotic pathways. Gambogic acid abrogates NF-κB activation by ROS mediated inhibition of IκB-α phosphorylation. Functionally Gambogic acid induced autophagy acts as a strong cell survival response and delays caspase activation.Our study provides the new insights about the mechanism of Gambogic acid induced modulation of autophagy and apoptosis in bladder cancer cells. All the molecular events responsible for Gambogic acid induced autophagy and apoptosis are mediated by reactive oxygen species.Since Gambogic acid targets various cell survival molecules therefore, it may be considered as a potential anticancer agent against bladder cancer.
Keywords: Gambogic acid; Bladder cancer; Apoptosis; Autophagy; p62;

dl-trans-3,4-Dihydroxy-1-selenolane (DHSred) heals indomethacin-mediated gastric ulcer in mice by modulating arginine metabolism by Saikat Chakraborty; Sudhir K. Yadav; Mahesh Subramanian; Michio Iwaoka; Subrata Chattopadhyay (3385-3392).
The importance of the arginine metabolism in gastric ulcer-healing is given relatively less attention. Hence the role of controlling this pathway by dl-trans-3,4-dihydroxy-1-selenolane (DHSred) and omeprazole against indomethacin-induced stomach ulceration in mouse was investigated.Swiss albino mice were ulcerated with indomethacin followed by treatment with the test samples, and the activities of myeloperoxidase (MPO), total nitric oxide synthase (NOS) and arginase, the expressions of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS), and the pro-/anti-inflammatory cytokine levels were assayed. NOS-inhibitors were also used to establish the biochemical mechanism.Indomethacin induced maximum ulceration in mice on the 3rd day, associated with reduced arginase activity, eNOS expression, along with increased MPO and total NOS activities, nitric oxide (NO) generation, iNOS expression, and pro-/anti-inflammatory (Th1/Th2) cytokine ratio. Treatment with DHSred (2.5 mg kg− 1  × 3 days) restored the cytokine balance to shift the iNOS/NO axis to the arginase/polyamine axis as revealed from the increased arginase activity and eNOS expression, and reduced iNOS expression, total NOS activity and NO level.The ulcer-healing property of DHSred, but not of omeprazole was due to a favorable pro-/anti-inflammatory cytokine ratio that shifted the arginine metabolism to the polyamine pathway and increased the eNOS/iNOS ratio. The healing action of omeprazole was not significantly associated with these parameters.The shift in the ariginine-metabolism from the iNOS/NO axis to the arginase/polyamine axis is guided by Th1/Th2 cytokines ratio and plays an important role in gastric ulcer-healing. The favourable effects of the non-toxic and water-soluble compound, DHSred on these pathways and other COX-dependent and antioxidative parameters suggested it to be a promising anti-ulcer formulation for further studies.
Keywords: Arginase; Cytokine balance; Gastric ulcer healing; Indomethacin; NOS;

Diatomite biosilica nanocarriers for siRNA transport inside cancer cells by Ilaria Rea; Nicola M. Martucci; Luca De Stefano; Immacolata Ruggiero; Monica Terracciano; Principia Dardano; Nunzia Migliaccio; Paolo Arcari; Rosarita Taté; Ivo Rendina; Annalisa Lamberti (3393-3403).
Diatomite is a natural porous biomaterial of sedimentary origin, formed by fragments of diatom siliceous skeletons, called “frustules”. Due to large availability in many areas of the world, chemical stability, and non-toxicity, these fossil structures have been widespread used in lot of industrial applications, such as food production, water extracting agent, production of cosmetics and pharmaceutics. However, diatomite is surprisingly still rarely used in biomedical applications. In this work, we exploit diatomite nanoparticles for small interfering ribonucleic acid (siRNA) transport inside human epidermoid cancer cells (H1355).Morphology and composition of diatomite microfrustules (average size lower than 40 μm) are investigated by scanning electron microscopy equipped by energy dispersive X-ray spectroscopy, Fourier transform infrared analysis, and photoluminescence measurements. Nanometric porous particles (average size lower than 450 nm) are obtained by mechanical crushing, sonication, and filtering of micrometric frustules. siRNA bioconjugation is performed on both micrometric and nanometric fragments by silanization. In-vitro experiments show very low toxicity on exposure of the cells to diatomite nanoparticle concentration up to 300 μg/ml for 72 h. Confocal microscopy imaging performed on cancer cells incubated with siRNA conjugated nanoparticles demonstrates a cytoplasmatic localization of vectors. Gene silencing by delivered siRNA is also demonstrated.Our studies endorse diatomite nanoparticles as non-toxic nanocarriers for siRNA transport in cancer cells.siRNA is a powerful molecular tool for cancer treatment but its delivery is inefficient due to the difficulty to penetrate the cell membrane. siRNA-diatomite nanoconjugate may be well suited for delivery of therapeutic to cancer cells.
Keywords: Diatomite; Porous material; Surface modification; Photoluminescence; Nanovector; Drug delivery;

Mitochondrial PKA mediates sperm motility by Rashel Mizrahi; Haim Breitbart (3404-3412).
Mitochondria are the major source of ATP to power sperm motility. Phosphorylation of mitochondrial proteins has been proposed as a major regulatory mechanism for mitochondrial bioenergetics.Sperm motility was measured by a computer-assisted analyzer, protein detection by western blotting, membrane potential by tetramethylrhodamine, cellular ATP by luciferase assay and localization of PKA by immuno-electron microscopy.Bicarbonate is essential for the creation of mitochondrial electro-chemical gradient, ATP synthesis and sperm motility. Bicarbonate stimulates PKA-dependent phosphorylation of two 60 kDa proteins identified as Tektin and glucose-6-phosphate isomerase. This phosphorylation was inhibited by respiration inhibition and phosphorylation could be restored by glucose in the presence of bicarbonate. However, this effect of glucose cannot be seen when the mitochondrial ATP/ADP exchanger was inhibited indicating that glycolytic-produced ATP is transported into the mitochondria and allows PKA-dependent protein phosphorylation inside the mitochondria.Bicarbonate activates mitochondrial soluble adenylyl cyclase (sAC) which catalyzes cAMP production leading to the activation of mitochondrial PKA. Glucose can overcome the lack of ATP in the absence of bicarbonate but it cannot affect the mitochondrial sAC/PKA system, therefore the PKA-dependent phosphorylation of the 60 kDa proteins does not occur in the absence of bicarbonate.Production of CO2 in Krebs cycle, which is converted to bicarbonate is essential for sAC/PKA activation leading to mitochondrial membrane potential creation and ATP synthesis.
Keywords: Spermatozoa; Motility; Mitochondria; PKA; Soluble adenylyl cyclase;

Jasmonates are plant lipid-derived oxylipins that act as key signaling compounds when plants are under oxidative stress, but little is known about their functions in mammalian cells. Here we investigated whether jasmonates could protect human neuroblastoma SH-SY5Y cells against oxidative stress-induced toxicity.The cells were pretreated with individual jasmonates for 24 h and exposed to hydrogen peroxide (H2O2) for 24 h. Before the resulting cytotoxicity, intracellular reactive oxygen species (ROS) levels, and mitochondrial membrane potential were measured. We also measured intracellular glutathione (GSH) levels and investigated changes in the signaling cascade mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) in cells treated with 12-oxo phytodienoic acid (OPDA).Among the jasmonates, only OPDA suppressed H2O2-induced cytotoxicity. OPDA pretreatment also inhibited the H2O2-induced ROS increase and mitochondrial membrane potential decrease. In addition, OPDA induced the nuclear translocation of Nrf2 and increased intracellular GSH level and the expression of the Nrf2-regulated phase II antioxidant enzymes heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutathione reductase. Finally, the cytoprotective effects of OPDA were reduced by siRNA-induced knockdown of Nrf2.These results demonstrated that among jasmonates, only OPDA suppressed oxidative stress-induced death of human neuroblastoma cells, which occurred via activation of the Nrf2 pathway.Plant-derived oxylipin OPDA may have the potential to provide protection against oxidative stress-related diseases.
Keywords: Plant oxylipins; Jasmonates; OPDA; Nrf2; Oxidative stress; SH-SY5Y;

The diversity of platelet functions implies multiple activation states arising in response to different stimuli. Distinguishing between these states has been challenging.We used fluorescently labelled carbohydrate binding proteins lectins to investigate agonist-induced changes in platelet surface glycosylation.Each of the seven agonists we used caused a unique set of changes in platelet surface glycosylation, eliciting a unique functional state. Some of these changes could be correlated with the expression of granule-specific markers CD62P and CD63, but lectins proved much more sensitive to differences between agonists than antibodies against those markers. This sensitivity appears to arise from the relation between the surface glycosylation changes and the signalling pathways through which various agonists act. In this context it is interesting that the effects of calcium ionophore were significantly different from those of other agonists. We also found that that P-selectin (CD62P) contains haptens for lectins VFA and PTII, because these lectins compete with the anti-CD62P antibody binding and vice a versa.We report for the first time that changes in platelet surface glycosylation are agonist-specific and can be distinguished using lectin-binding assays. Lectin fingerprinting represents a new research and diagnostic tool for studying platelet activation.The observation of agonist-specific platelet surface glycosylation changes is interesting in the context of the diversity of platelet function, because surface glycans mediate contact interactions between platelets and other cells and serve as binding sites for some of the agonists (galectins).Display Omitted
Keywords: Platelets; Lectins; Platelet activation; Haemostasis; Glycobiology;

Neuroprotective effect of glutamate-substituted analog of gramicidin A is mediated by the uncoupling of mitochondria by Denis N. Silachev; Ljudmila S. Khailova; Valentina A. Babenko; Mikhail V. Gulyaev; Sergey I. Kovalchuk; Ljubava D. Zorova; Egor Y. Plotnikov; Yuri N. Antonenko; Dmitry B. Zorov (3434-3442).
Reactive oxygen species are grossly produced in the brain after cerebral ischemia and reperfusion causing neuronal cell death. Mitochondrial production of reactive oxygen species is nonlinearly related to the value of the mitochondrial membrane potential with significant increment at values exceeding 150 mV. Therefore, limited uncoupling of oxidative phosphorylation could be beneficial for cells exposed to deleterious oxidative stress-associated conditions by preventing excessive generation of reactive oxygen species.Protonophoric and uncoupling activities of different peptides were measured using pyranine-loaded liposomes and isolated mitochondria. To evaluate the effect of glutamate-substituted analog of gramicidin A ([Glu1]gA) administration on the brain ischemic damage, we employed the in vitro model of neuronal hypoxia using primary neuronal cell cultures and the in vivo model of cerebral ischemia induced in rats by the middle cerebral artery occlusion.[Glu1]gA was the most effective in proton-transferring activity among several N-terminally substituted analogs of gramicidin A tested in liposomes and rat brain and liver mitochondria. The peptides were found to be protective against ischemia-induced neuronal cell death and they lowered mitochondrial membrane potential in cultured neurons and diminished reactive oxygen species production in isolated brain mitochondria. The intranasal administration of [Glu1]gA remarkably diminished the infarct size indicated in MR-images of a brain at day 1 after the middle cerebral artery occlusion. In [Glu1]gA-treated rats, the ischemia-induced brain swelling and behavioral dysfunction were significantly suppressed.The glutamate-substituted analogs of gramicidin A displaying protonophoric and uncoupling activities protect neural cells and the brain from the injury caused by ischemia/reperfusion.[Glu1]gA may be potentially used as a therapeutic agent to prevent neuron damage after stroke.Display Omitted
Keywords: Stroke; Mitochondrion; Membrane transport; Peptide; Gramicidin A; Uncoupler;

Release and inhibitory effects of prostaglandin D2 in guinea pig urinary bladder and the role of urothelium by Na N. Guan; Kristofer F. Nilsson; Peter N. Wiklund; Lars E. Gustafsson (3443-3451).
While studying a urothelium-derived inhibitory factor in guinea pig urinary bladders we observed considerable release of prostanoids, including PGD2-like activity. The present study was carried out to identify the prostanoids and to study their roles in modulating guinea pig urinary bladder motility.Release of PGE2 and PGD2 in isolated guinea pig urinary bladder preparations was analyzed by high performance liquid chromatography (HPLC) combined with bioassay on bladder strips. Isolated urothelium-intact (UI) or -denuded (UD) bladder strips were subjected to electrical field stimulation (EFS) and applications of PGE2 and PGD2.A resting release of 95 ± 9 (n = 5) ng g tissue− 1  h− 1 PGE2-like activity and 210 ± 34 (n = 4) ng g tissue− 1  h− 1 PGD2-like activity was found, where PGD2-like was subject to marked spontaneous inactivation during isolation. Prostanoids release was decreased by 70–90% by the cyclo-oxygenase inhibitor diclofenac in UI preparations. Urothelium removal decreased prostanoids release by more than 90%.PGE2 increased basal tone and spontaneous contractions, whereas PGD2 had little or no effect on these. Exogenous PGE2 enhanced and PGD2 inhibited contractile responses to EFS, exogenous acetylcholine- and ATP, whereas PGD2 caused marked dose-dependent inhibition. PGE2 and PGD2 effects were more pronounced in diclofenac-treated UD tissues.PGD2 and PGE2 are released from guinea pig bladder urothelium and PGD2 has inhibitory effects on bladder motility, mainly through a postjunctional action on smooth muscle responsiveness.The release and inhibitory effects merit further studies in relation to normal biological function as well as overactive bladder syndrome.Display Omitted
Keywords: Lower urinary tract; Prostaglandin D2; HPLC; Urothelium; Detrusor;

The delay of dermal burn wound healing caused by vascular disorders is a critical problem for many diabetic patients. Thymosin β4 (Tβ4), identified by subtractive cloning of endothelial cells on plastic versus basement membrane substrates, has been found to promote angiogenesis and dermal wound repair in rats, aged mice, and db/db diabetic mice. However, previous studies involving the role of Tβ4 in wound repair were limited to mechanical damage and dermal impairment. Thus, this study aimed to evaluate the improvement of healing of burn wounds by Tβ4 in relation to advanced glycation end products (AGE), which are pathological factors in diabetes.We adapted a dermal burn wound in vivo model in which the dorsal skin of db/db mice was exposed for 10 s to 100 °C heated water to produce a deep second-degree burn 10 mm in diameter. Five mg/kg of Tβ4 was then injected intradermally near the burn wound twice a week for 2 weeks.After treatment, Tβ4 improved wound healing markers such as wound closure, granulation, and vascularization. Interestingly, Tβ4 reduced levels of receptor of AGE (RAGE) during the wound healing period.Tβ4 exerts effects to remedy burn wounds via downregulation of RAGE.Our results suggest the potential importance of Tβ4 as a new therapy for impaired burn wound healing that is associated with diabetes.
Keywords: Thymosin β4; Diabetes; Burn wound; RAGE; Vascularization;

The utility of metabolomics in natural product and biomarker characterization by Daniel G. Cox; Joonseok Oh; Adam Keasling; Kim L. Colson; Mark T. Hamann (3460-3474).
Metabolomics is a well-established rapidly developing research field involving quantitative and qualitative metabolite assessment within biological systems. Recent improvements in metabolomics technologies reveal the unequivocal value of metabolomics tools in natural products discovery, gene-function analysis, systems biology and diagnostic platforms.We review here some of the prominent metabolomics methodologies employed in data acquisition and analysis of natural products and disease-related biomarkers.This review demonstrates that metabolomics represents a highly adaptable technology with diverse applications ranging from environmental toxicology to disease diagnosis. Metabolomic analysis is shown to provide a unique snapshot of the functional genetic status of an organism by examining its biochemical profile, with relevance toward resolving phylogenetic associations involving horizontal gene transfer and distinguishing subgroups of genera possessing high genetic homology, as well as an increasing role in both elucidating biosynthetic transformations of natural products and detecting preclinical biomarkers of numerous disease states.This review expands the interest in multiplatform combinatorial metabolomic analysis. The applications reviewed range from phylogenetic assignment, biosynthetic transformations of natural products, and the detection of preclinical biomarkers.
Keywords: Integrated approach; PCA; Plant metabolomics; Diagnostic biomarker; NMR; Targeted metabolomics;

Islet NADPH oxidase activity modulates β-cell mass and endocrine function in rats with fructose-induced oxidative stress by Carolina L. Román; Luis E. Flores; Bárbara Maiztegui; María A. Raschia; Héctor Del Zotto; Juan J. Gagliardino (3475-3482).
Islet NADPH oxidase activity is modulated by glucose and other insulin secretagogues and it might be part of the regulatory mechanism of insulin secretion. We studied its modulatory role of islet NADPH oxidase upon β-cell function in rats with fructose-induced oxidative stress.Normal rats were fed for 3 weeks with a standard diet, a fructose-rich diet or both diets plus apocynin. We measured plasma glucose, insulin, triacylglycerol and lipid peroxidation levels and the homeostasis model assessment-insulin resistance (HOMA-IR) and HOMA-β indexes, and performed an oral glucose tolerance test. β-cell volume density and the number of islets per mm2 were determined by immunomorphometric analysis of the pancreas. Insulin secretion, glucose metabolism, glucokinase and NADPH oxidase activities were studied in islets isolated from each experimental group.Fructose-fed rats had increased plasma triacylglycerol, insulin and lipid peroxidation levels associated with an insulin resistance state; the reactive higher secretion was unable to cope with the increased demand of insulin, leading to an impaired glucose tolerance. They also have a lower number of islets per area unit with a decreased β-cell volume density. All these alterations were prevented by blocking NADPH oxidase activity with apocynin.Fructose-induced changes are partly mediated by modulation of NADPH oxidase activity.The metabolic dysfunctions and enhanced oxidative stress measured in fructose-fed rats resemble those recorded in human prediabetes; thus, successful strategies employed in this model could be later used to prevent the progression of this state towards type 2 diabetes in human beings.
Keywords: NADPH oxidase; β-cell function; Oxidative stress; Insulin resistance;

Leptin increases VEGF expression and enhances angiogenesis in human chondrosarcoma cells by Wei-Hung Yang; Jui-Chieh Chen; Kai-Hsiang Hsu; Chih-Yang Lin; Shih-Wei Wang; Shoou-Jyi Wang; Yung-Sen Chang; Chih-Hsin Tang (3483-3493).
Leptin, 16 kDa product of obese gene, is adipocytokine playing critical role in regulation of body weight. In recent years, leptin is also defined as potent angiogenic factor involving in tumorigenesis, angiogenesis, and metastasis. However, it is unknown whether leptin regulates VEGF production in human chondrosarcoma and contributing the tumor-associated angiogenesis.We analyzed protein level of leptin and VEGF in human chondrosarcoma tissues. Effects of leptin on chondrosarcoma cells were examined by in vitro and in vivo assays. In addition, intracellular signal pathways were investigated by pharmacological and genetic approaches.We found that both leptin and VEGF are highly expressed in human chondrosarcoma tissues, and positively correlated with tumor stage. Leptin increases VEGF production by activating OBRl receptor and MAPKs (p38, ERK, and JNK), which in turn enhances binding of AP-1 transcription factor to VEGF promoter, resulting in the transactivation of VEGF expression and subsequently promoting migration and tube formation in endothelial progenitor cells (EPCs). In vivo, knockdown leptin significantly reduces angiogenesis and tumor growth.Leptin may be a therapeutic target of angiogenesis and metastasis in chondrosarcoma.These findings provide better understanding of pathogenesis of chondrosarcoma and can utilize this knowledge to design new therapeutic strategy.
Keywords: Chondrosarcoma; Leptin; VEGF; Tumor angiogenesis; Endothelial progenitor cells;

Epigenetics of cancer stem cells: Pathways and therapeutics by Samriddhi Shukla; Syed Musthapa Meeran (3494-3502).
Epigenetic alterations including DNA methylation and histone modifications are the key factors in the differentiation of stem cells into different tissue subtypes. The generation of cancer stem cells (CSCs) in the process of carcinogenesis may also involve similar kind of epigenetic reprogramming where, in contrast, it leads to the loss of expression of genes specific to the differentiated state and regaining of stem cell-specific characteristics. The most important predicament with treatment of cancers includes the non-responsive quiescent CSC.The distinctive capabilities of the CSCs make cancer treatment even more difficult as this population of cells tends to remain quiescent for longer intervals and then gets reactivated leading to tumor relapse. Therefore, the current review is aimed to focus on recent advances in understanding the relation of epigenetic reprogramming to the generation, self-renewal and proliferation of CSCs.CSC-targeted therapeutic approaches would improve the chances of patient survival by reducing the frequency of tumor relapse. Differentiation therapy is an emerging therapeutic approach in which the CSCs are induced to differentiate from their quiescent state to a mature differentiated form, through activation of differentiation-related signalling pathways, miRNA-mediated alteration and epigenetic differentiation therapy. Thus, understanding the origin of CSC and their epigenetic regulation is crucial to develop treatment strategy against not only for the heterogeneous population of cancer cells but also to CSCs.Characterizing the epigenetic marks of CSCs and the associated signalling cascades might help in developing therapeutic strategies against chemo-resistant cancers.
Keywords: Cancer stem cell; Epigenetic reprogramming; DNA methylation; Chromatin remodelling; Chemo-resistance; Chemotherapy;

Role of G-quadruplex located at 5ʹ end of mRNAs by Prachi Agarwala; Satyaprakash Pandey; Souvik Maiti (3503-3510).
Secondary structures in 5′ UTR of mRNAs play a critical role in regulating protein synthesis. Though studies have indicated the role of secondary structure G-quadruplex in translational regulation, position-specific effect of G-quadruplex in naturally occurring mRNAs is still not understood. As a pre-initiation complex recognises 5′ cap of the mRNA and scans along the untranslated region (UTR) before initiating translation, the presence of G-quadruplex in 5′ region may have a significant contribution in regulating translation. Here, we investigate the role of G-quadruplex located at the 5′ end of an mRNA.Biophysical characterisation of putative G-quadruplexes was performed using UV and CD spectroscopy. Functional implication of G-quadruplex in the context of their location was assessed in cellulo using qRT-PCR and dual luciferase assay system.PG4 sequences in 5′ UTR of AKT interacting protein (AKTIP), cathepsin B (CTSB) and forkhead box E3 (FOXE3) mRNAs form G-quadruplex whereas it is unable to form G-quadruplex in apolipoprotein A-I binding protein (APOA1BP). Our results demonstrated diverse roles of G-quadruplex located at 5′ end of mRNAs. Though G-quadruplex in AKTIP and CTSB mRNA act as inhibitory modules, it activates translation in FOXE3 mRNA.Our works suggests that G-quadruplex present at the 5′ terminal of an mRNA behaves differently in a different gene context. It can activate or inhibit gene expression.This study demonstrated that it is difficult to predict the role of G-quadruplex on the basis of its position in 5′ UTR. The neighbouring nucleotide sequence, the intracellular milieu and the interacting partners might render diverse functions to this secondary structure.
Keywords: G-quadruplex; UTR; AKTIP; CTSB; FOXE3; APOA1BP;