BBA - General Subjects (v.1830, #3)

β-Catenin signaling induces CYP1A1 expression by disrupting adherens junctions in Caco-2 human colon carcinoma cells by Shuya Kasai; Takanori Ishigaki; Ryo Takumi; Toru Kamimura; Hideaki Kikuchi (2509-2516).
The aryl hydrocarbon (Ah) receptor is one of the best known ligand-activated transcription factors. The present study has focused on the wound-healing process on Ah receptor function.Depletion of calcium from culture medium of Caco-2 human colon carcinoma cells by transfer to Minimal Essential Medium (Spinner Modification; S-MEM) destroyed adherens junctions and the cells were used as the model of wound-healing process.Calcium depletion induced both nuclear translocation of the Ah receptor, and increased expression of CYP1A1 and Slug mRNAs in Caco-2 cells. However, expression of Slug mRNA was not significantly induced by treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Knockdown of the Ah receptor and treatment with Ah receptor antagonists decreased level of CYP1A1 mRNA. The fragment of E-cadherin released by γ-secretase was not involved in induction of CYP1A1 mRNA following S-MEM treatment. Knockdown of β-catenin increased levels of Ah receptor mRNA, which may be attributable to direct or indirect involvement of β-catenin in suppression of the Ah receptor gene.Our results suggest that mRNA induction of some genes by destruction of adherens junctions depends on the Ah receptor. β-Catenin, one of the components of the adherens junction, was released from the E-cadherin complex, which resulted in its increased interaction with the Ah receptor, and was translocated into the nucleus, and consequently the target genes would be transcribed.Our observations suggest that some aspects of the molecular mechanism of wound healing involve the Ah receptor.► Depletion of calcium by transfer of cells to S-MEM destroyed adherens junctions. ► Nuclear translocation of the Ah receptor induced CYP1A1 and Slug mRNAs. ► β-Catenin was released from E-cadherin complexes by destruction of adherens junctions. ► Interaction between β-catenin and the Ah receptor increased after S-MEM treatment. ► Knockdown of β-catenin increased the level of Ah receptor mRNA.
Keywords: Aryl hydrocarbon receptor; β-Catenin; Cytochrome P-450 1A1; E-cadherin; E-cadherin carboxy terminal fragment 2; S-MEM;

Monogalactosyl diacylglycerol, a replicative DNA polymerase inhibitor, from spinach enhances the anti-cell proliferation effect of gemcitabine in human pancreatic cancer cells by Hiroaki Akasaka; Ryohei Sasaki; Kenji Yoshida; Izumi Takayama; Toyofumi Yamaguchi; Hiromi Yoshida; Yoshiyuki Mizushina (2517-2525).
Gemcitabine (GEM) is used to treat various carcinomas and represents an advance in pancreatic cancer treatment. In the screening for DNA polymerase (pol) inhibitors, a glycoglycerolipid, monogalactosyl diacylglycerol (MGDG), was isolated from spinach.Phosphorylated GEM derivatives were chemically synthesized. In vitro pol assay was performed according to our established methods. Cell viability was measured using MTT assay.Phosphorylated GEMs inhibition of mammalian pol activities assessed, with the order of their effect ranked as: GEM-5′-triphosphate (GEM-TP) > GEM-5′-diphosphate > GEM-5′-monophosphate > GEM. GEM suppressed growth in the human pancreatic cancer cell lines BxPC-3, MIAPaCa2 and PANC-1 although phosphorylated GEMs showed no effect. MGDG suppressed growth in these cell lines based on its selective inhibition of replicative pol species. Kinetic analysis showed that GEM-TP was a competitive inhibitor of pol α activity with nucleotide substrates, and MGDG was a noncompetitive inhibitor with nucleotide substrates. GEM combined with MGDG treatments revealed synergistic effects on the inhibition of DNA replicative pols α and γ activities compared with GEM or MGDG alone. In cell growth suppression by GEM, pre-addition of MGDG significantly enhanced cell proliferation suppression, and the combination of these compounds was found to induce apoptosis. In contrast, GEM-treated cells followed by MGDG addition did not influence cell growth.GEM/MGDG enhanced the growth suppression of cells based on the inhibition of pol activities.Spinach MGDG has great potential for development as an anticancer food compound and could be an effective clinical anticancer chemotherapy in combination with GEM.► Gemcitabine (GEM) suppressed growth in the human pancreatic cancer cell lines. ► Spinach monogalactosyl diacylglycerol (MGDG) suppressed cancer cell growth. ► GEM–MGDG combination revealed synergistic effects on the cancer cell growth. ► These enhancements might cause the inhibition of mammalian DNA polymerase. ► MGDG from spinach could be an effective clinical anticancer chemotherapy of GEM.
Keywords: Monogalactosyl diacylglycerol (MGDG); Gemcitabine (GEM); DNA polymerase (pol); Enzyme inhibitor; Anticancer; Apoptosis;

The antileukemic activity of modified fibrinogen–methotrexate conjugate by Tomasz Goszczyński; Dmitry Nevozhay; Joanna Wietrzyk; Mohamed Salah Omar; Janusz Boratyński (2526-2530).
The search for new, innovative methods to treat all types of diseases, especially cancer-related ones, is a challenge taken by pharmaceutical companies and academic institutions. The use of conjugates containing widely-known and widely-used bioactive substances is one of the ways to solve this problem. Research into drug binding with macromolecular carrier systems has joined the search for new therapeutic strategies.The main goal of this paper is the potential offered by the use of fibrinogen derivatives as an antileukemic drug carrier. Physicochemical properties of the obtained conjugate were analyzed, characterizing alterations in relation to the starting carrier and analyzing biological implications. The intraperitoneally (i.p.) inoculated P388 mouse leukemia model for in vivo studies was used.Conjugates consisting of a fibrinogen derivative with a covalently bound anticancer drug were developed. Carrier preparation and a conjugate synthesis in aqueous solution were formulated, as well as purification of the conjugate was performed. The study showed that the survival of leukemia mice treated with FH–MTX conjugate was indeed significantly longer than survival in both untreated animals (control) and mice treated with unbound MTX. A significant increase in the antileukemic activity of MTX conjugated with hydrolysed fibrinogen was observed as compared with the unconjugated drug. Reported data suggest that hydrolysed fibrinogen can serve as a carrier molecule for the MTX drug with the aim of enhancing its antileukemic activity.Conjugates consisting of a fibrinogen derivative with a covalently bound anticancer drug seem to be a promising anticancer drug.► Covalent conjugation of methotrexate with fibrinogen was performed. ► Conjugate is more active against leukemia in the in vivo model vs free drug. ► Partial hydrolysis of fibrinogen provides a promising anti-cancer drug carrier.
Keywords: Fibrinogen; Methotrexate; Protein–drug conjugate; Antileukemic activity;

Exhaled breath and fecal volatile organic biomarkers of chronic kidney disease by Simone Meinardi; Kyu-Bok Jin; Barbara Barletta; Donald R. Blake; Nosratola D. Vaziri (2531-2537).
While much is known about the effect of chronic kidney disease (CKD) on composition of body fluids little is known regarding its impact on the gases found in exhaled breath or produced by intestinal microbiome. We have recently shown significant changes in the composition of intestinal microbiome in humans and animals with CKD. This study tested the hypothesis that uremia-induced changes in cellular metabolism and intestinal microbiome may modify the volatile organic metabolites found in the exhaled breath or generated by intestinal flora.SD rats were randomized to CKD (5/6 nephrectomy) or control (sham operation) groups. Exhaled breath was collected by enclosing each animal in a glass chamber flushed with clean air, then sealed for 45 min and the trapped air collected. Feces were collected, dissolved in pure water, incubated at 37 °C in glass reactors for 24 h and the trapped air collected. Collected gases were analyzed by gas chromatography.Over 50 gases were detected in the exhaled breath and 36 in cultured feces. Four gases in exhaled breath and 4 generated by cultured feces were significantly different in the two groups. The exhaled breath in CKD rats showed an early rise in isoprene and a late fall in linear aldehydes. The CKD animals' cultured feces released larger amounts of dimethyldisulfide, dimethyltrisulfide, and two thioesters.CKD significantly changes the composition of exhaled breath and gaseous products of intestinal flora.Analysis of breath and bowel gases may provide useful biomarkers for detection and progression of CKD and its complications.► Uremia changes the gut microbiome which is a major source of several toxic solutes. ► Effect of uremia on gaseous byproducts of microbiome is unknown and was studied here. ► Over 50 gases were detected in rats' exhaled breath and 36 gases in rats' cultured feces. ► Several fecal and exhaled breath gases were different between uremic and control rats. ► Value of these gases as biomarkers of kidney disease requires further investigation.
Keywords: Intestinal microbial flora; Microbiome; Uremia; Inflammation; Gastrointestinal tract;

Brain lipid peroxidation has long been considered a potential therapeutic target for Alzheimer's disease (AD). β-sitosterol (BS), a plant sterol that is prevalent in plant plasma membrane, has been suggested to have antioxidant activity. Previous studies have demonstrated that dietary BS can enter the brain and accumulates in the plasma membrane of brain cells. However, it is unknown whether and how BS exerts its antioxidant activity in plasma membrane.To incorporate BS into the plasma membrane in vitro, HT22 cells and primarily cultured hippocampal cells were supplemented with BS using 2-hydroxypropyl-β-cyclodextrin (HPβCD) as a carrier. The present study then tested the antioxidant effect of membrane BS against glucose oxidase (GOX)-induced oxidative stress and lipid peroxidation, and whether the antioxidant effect of membrane BS was associated with estrogen receptor (ER)-mediated phosphatidyl inositol 3-kinase (PI3K)/glycogen synthase kinase 3 (GSK3β) signaling.Incorporation of BS into cell membrane prevented GOX-induced oxidative stress and lipid peroxidation, which could be suppressed by the ER antagonists and PI3K inhibitor. Additional experiments showed that incorporation of BS into cell membrane induced an up-regulation of PI3K activity and a recruitment of PI3K to lipid rafts, which could be inhibited by the ER antagonist. Membrane BS also increased the expression of p-GSK3β, which could be suppressed in the presence of the ER antagonist and PI3K inhibitor.Given that BS is prevalent in foods such as plant oil, the results provide a better understanding of the beneficial effects of these BS-enriched nutrients on neurodegenerative diseases such as AD.► The antioxidant effect of BS in plasma membrane. ► Incorporation of BS into cell membrane prevented GOX-induced oxidative stress and lipid peroxidation. ► Membrane BS exerted antioxidant activity via ER-induced PI3K/GSK3β.
Keywords: Lipid peroxidation; β-sitosterol; Estrogen receptor; Phosphatidyl inositol 3-kinase;

Impaired cardiac mitochondrial function and contractile reserve following an acute exposure to environmental particulate matter by T. Marchini; N. Magnani; V. D'Annunzio; D. Tasat; R.J. Gelpi; S. Alvarez; P. Evelson (2545-2552).
It has been suggested that mitochondrial function plays a central role in cardiovascular diseases associated with particulate matter inhalation. The aim of this study was to evaluate this hypothesis, with focus on cardiac O2 and energetic metabolism, and its impact over cardiac contractility.Swiss mice were intranasally instilled with either residual oil fly ash (ROFA) (1.0 mg/kg body weight) or saline solution. After 1, 3 or 5 h of exposure, O2 consumption was evaluated in heart tissue samples. Mitochondrial respiration, respiratory chain complexes activity, membrane potential and ATP content and production rate were assessed in isolated mitochondria. Cardiac contractile reserve was evaluated according to the Langendorff technique.Three hours after ROFA exposure, tissue O2 consumption was significantly decreased by 35% (from 1180 ± 70 to 760 ± 60 ng-at O/min g tissue), as well as mitochondrial rest (state 4) and active (state 3) respiration, by 30 and 24%, respectively (control state 4: 88 ± 5 ng-at O/min mg protein; state 3: 240 ± 20 ng-at O/min mg protein). These findings were associated with decreased complex II activity, mitochondrial depolarization and deficient ATP production. Even though basal contractility was not modified (control: 75 ± 5 mm Hg), isolated perfused hearts failed to properly respond to isoproterenol in ROFA-exposed mice. Tissue O2 consumption rates positively correlated with cardiac contractile state in controls (r 2  = 0.8271), but not in treated mice (r 2  = 0.1396).The present results show an impaired mitochondrial function associated with deficient cardiac contractility, which could represent an early cardiovascular alteration after the exposure to environmental particulate matter.► Exposure to PM is associated with increased cardiovascular morbidity and mortality. ► Altered oxidative and energetic metabolism is suggested as an underlying mechanism. ► Mitochondrial dysfunction and impaired oxidative phosphorylation was induced by PM. ► Altered oxidative metabolism correlated with deficient cardiac performance. ► The findings comprise an early cardiovascular alteration triggered by PM exposure.
Keywords: Air pollution; Heart; Mitochondrion; Particulate matter; Residual oil fly ash (ROFA);

Overexpression of human Argonaute2 inhibits cell and tumor growth by Xiaoxiao Zhang; Paul Graves; Yan Zeng (2553-2561).
Argonaute (Ago) proteins are essential for the biogenesis and function of ~ 20–30 nucleotide long RNAs such as microRNAs (miRNAs). Ago expression increases or decreases under various physiological conditions, although the functional consequences are unknown. In addition, while reduced global miRNA production was shown to enhance cellular transformation and tumorigenesis, how Ago proteins contribute to human diseases has not been reported.Ago2, an essential Ago isoform in mammals, was stably expressed in 293 T, the human embryonic kidney cell line, and H1299, the human lung adenocarcinoma cell line. miRNA and mRNA expression was investigated by quantitative PCR and microarray profiling. Cell proliferation and migration was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scratch assay in the cell cultures, respectively. How Ago2 affected cell growth in vivo was determined by H1299 xenograft tumor growth in mice. Changes in Ago2 expression in human lung cancer samples were investigated by quantitative PCR and immunohistochemistry.Stable Ago2 overexpression elicited specific changes in miRNA and mRNA expression in both 293 T and H1299 cells. It also inhibited cell proliferation and migration in cell cultures as well as xenograft tumor growth in nude mice. Ago2 expression was lower in human lung adenocarcinomas than in the paired, non-cancerous tissues.We concluded that changes in Ago2 expression might have significant physiological and pathological consequences in vivo.► Stable overexpression of Ago2 regulates gene expression specifically in human cells. ► Ago2 overexpression inhibits cell proliferation and migration in human cell cultures. ► Ago2 overexpression in H1299 cells inhibits xenograft tumor growth in nude mice.
Keywords: Argonaute2; microRNA; Gene expression; let-7; Tumorigenesis;

Site-specifically 11C-labeled Sel-tagged annexin A5 and a size-matched control for dynamic in vivo PET imaging of protein distribution in tissues prior to and after induced cell death by Qing Cheng; Li Lu; Jonas Grafström; Maria Hägg Olofsson; Jan-Olov Thorell; Erik Samén; Katarina Johansson; Hanna-Stina Ahlzén; Stig Linder; Elias S.J. Arnér; Sharon Stone-Elander (2562-2573).
Radiolabeled annexin A5 (AnxA5) is widely used for detecting phosphatidylserine exposed on cell surfaces during apoptosis. We describe here a new method for labeling AnxA5 and a size-matched control protein with short-lived carbon-11, for probing the specificity of in vivo cell death monitoring using positron emission tomography (PET) imaging.AnxA5 and the control protein were recombinantly expressed with a C-terminal “Sel-tag”, the tetrapeptide –Gly-Cys-Sec-Gly–COOH. The proteins were then labeled either fluorescently for in vitro corroborations of binding behaviors or with 11C for dynamic in vivo PET studies.AnxA5 demonstrated retained calcium-dependent binding to apoptotic cells after the C-terminus modification. The control protein showed no functional binding. The 11C-ligands demonstrated similar in vivo pharmacokinetic behavior in healthy mice except for higher uptake in kidney and higher intact elimination to urine of AnxA5. After inducing hepatic apoptosis, however, the uptake of labeled AnxA5 in the targeted tissue increased compared to baseline levels while that of the control protein tended to decrease.These data suggest that the combined use of these two tracers can facilitate differentiating specific AnxA5 binding and its changes caused by induced cell death from uptake due to non-specific permeability and retention effects at baseline or after therapy.The Sel-tag enables rapid and mild reactions with electrophilic agents giving site-specifically labeled proteins for multi-probe analyses. The combined use of 11C-labeled AnxA5 and a size-matched control protein with dynamic PET can be useful for evaluating drug effects on target as well as off-target tissues.Display Omitted► first site-specific 11C-labeling of AnxA5 in the C-terminus for PET studies of induced cell death ► first use of a size-matched protein to estimate non-specific uptake and retention ► use of Sel-tag to identically label both proteins, with retained Ca-dependent binding of AnxA5-ST to apoptotic cells ► comparable pharmacokinetics of AnxA5 and its control except in the kidney and urine ► uptake of AnxA5 increased but control protein decreased in induced cell death.
Keywords: Positron emission tomography; Molecular imaging; Annexin A5; Carbon-11; Cell death; Sel-tag;

The conserved role of the AKT/GSK3 axis in cell survival and glycogen metabolism in Rhipicephalus (Boophilus) microplus embryo tick cell line BME26 by Leonardo Araujo de Abreu; Christiano Calixto; Camila Fernanda Waltero; Bárbara Pitta Della Noce; Naftaly Wang'ombe Githaka; Adriana Seixas; Luís Fernando Parizi; Satoru Konnai; Itabajara da Silva Vaz; Kazuhiko Ohashi; Carlos Logullo (2574-2582).
Tick embryogenesis is a metabolically intensive process developed under tightly controlled conditions and whose components are poorly understood.In order to characterize the role of AKT (protein kinase B) in glycogen metabolism and cell viability, glycogen determination, identification and cloning of an AKT from Rhipicephalus microplus were carried out, in parallel with experiments using RNA interference (RNAi) and chemical inhibition.A decrease in glycogen content was observed when AKT was chemically inhibited by 10-DEBC treatment, while GSK3 inhibition by alsterpaullone had an opposing effect. RmAKT ORF is 1584-bp long and encodes a polypeptide chain of 60.1 kDa. Phylogenetic and sequence analyses showed significant differences between vertebrate and tick AKTs. Either AKT or GSK3 knocked down cells showed a 70% reduction in target transcript levels, but decrease in AKT also reduced glycogen content, cell viability and altered cell membrane permeability. However, the GSK3 reduction promoted an increase in glycogen content. Additionally, either GSK3 inhibition or gene silencing had a protective effect on BME26 viability after exposure to ultraviolet radiation. R. microplus AKT and GSK3 were widely expressed during embryo development. Taken together, our data support an antagonistic role for AKT and GSK3, and strongly suggest that such a signaling axis is conserved in tick embryos, with AKT located upstream of GSK3.The AKT/GSK3 axis is conserved in tick in a way that integrates glycogen metabolism and cell survival, and exhibits phylogenic differences that could be important for the development of novel control methods.Display Omitted► PI3K/AKT pathway is highly conserved in growth factor signal transduction. ► RmAKT ORF was phylogenetically and functionally analyzed. ► AKT inhibition or gene silencing reduces glycogen and viability of BME26 cells. ► GSK3 inhibition and gene silencing cause opposing effects. ► AKT/GSK3 signaling axis may be present during all tick stages.
Keywords: Tick cell line; Glycogen metabolism; Cell survival; Gene silencing; Protein kinase B (AKT/PKB); Glycogen synthase kinase 3 (GSK3);

Metabolomic analysis of pancreatic beta cells following exposure to high glucose by Martina Wallace; Helena Whelan; Lorraine Brennan (2583-2590).
Chronic exposure to hyperglycaemic conditions has been shown to have detrimental effects on beta cell function. The resulting glucotoxicity is a contributing factor to the development of type 2 diabetes. The objective of this study was to combine a metabolomics approach with functional assays to gain insight into the mechanism by which glucotoxicity exerts its effects.The BRIN-BD11 and INS-1E beta cell lines were cultured in 25 mM glucose for 20 h to mimic glucotoxic effects. PDK-2 protein expression, intracellular glutathione levels and the change in mitochondrial membrane potential and intracellular calcium following glucose stimulation were determined. Metabolomic analysis of beta cell metabolite extracts was performed using GC–MS, 1H NMR and 13C NMR.Conditions to mimic glucotoxicity were established and resulted in no loss of cellular viability in either cell line while causing a decrease in insulin secretion. Metabolomic analysis of beta cells following exposure to high glucose revealed a change in amino acids, an increase in glucose and a decrease in phospho-choline, n−3 and n−6 PUFAs during glucose stimulated insulin secretion relative to cells cultured under control conditions. However, no changes in calcium handling or mitochondrial membrane potential were evident.Results indicate that a decrease in TCA cycle metabolism in combination with an alteration in fatty acid composition and phosphocholine levels may play a role in glucotoxicity induced impairment of glucose stimulated insulin secretion.Alterations in certain metabolic pathways play a role in glucotoxicity in the pancreatic beta cell.► Metabolomic analysis of exposure to high glucose ► Understanding the molecular mechanisms involved in glucotoxicity ► Metabolic alterations play a role in impairment of insulin secretion.
Keywords: Metabolomics; Glucotoxicity; Insulin secretion;

Asteropsin A: An unusual cystine-crosslinked peptide from porifera enhances neuronal Ca2 + influx by Huayue Li; John J. Bowling; Frank R. Fronczek; Jongki Hong; Sairam V. Jabba; Thomas F. Murray; Nam-Chul Ha; Mark T. Hamann; Jee H. Jung (2591-2599).
Herein we report the discovery of a cystine-crosslinked peptide from Porifera along with high-quality spatial details accompanied by the description of its unique effect on neuronal calcium influx.Asteropsin A (ASPA) was isolated from the marine sponge Asteropus sp., and its structure was independently determined using X-ray crystallography (0.87 Å) and solution NMR spectroscopy.An N-terminal pyroglutamate modification, uncommon cis proline conformations, and absence of basic residues helped distinguish ASPA from other cystine-crosslinked knot peptides. ASPA enhanced Ca2 + influx in murine cerebrocortical neuron cells following the addition of the Na+ channel activator veratridine but did not modify the oscillation frequency or amplitude of neuronal Ca2 + currents alone. Allosterism at neurotoxin site 2 was not observed, suggesting an alternative to the known Na+ channel interaction.Together with a distinct biological activity, the origin of ASPA suggests a new subclass of cystine-rich knot peptides associated with Porifera.The discovery of ASPA represents a distinctive addition to an emerging subclass of cystine-crosslinked knot peptides from Porifera.Display Omitted► Considerable yield of unusual cystine knot from sponge. ► Enhanced Ca2 + influx during veratridine-induced Na+ channel activation. ► Uncommon X-ray crystal and NMR solution structure for bioactive knot peptide. ► Peptide sequence, structure and bioactivity suggest new peptide subclass from sponges.
Keywords: Porifera; Ion channelopathy; Natural product; Bioactive peptide; X-ray crystallography; NMR spectroscopy;

The proteolytic processing of amelogenin by enamel matrix metalloproteinase (MMP-20) is controlled by mineral ions by Feroz Khan; Haichuan Liu; Aileen Reyes; H. Ewa Witkowska; Olga Martinez-Avila; Li Zhu; Wu Li; Stefan Habelitz (2600-2607).
Enamel synthesis is a highly dynamic process characterized by simultaneity of matrix secretion, assembly and processing during apatite mineralization. MMP-20 is the first protease to hydrolyze amelogenin, resulting in specific cleavage products that self-assemble into nanostructures at specific mineral compositions and pH. In this investigation, enzyme kinetics of MMP-20 proteolysis of recombinant full-length human amelogenin (rH174) under different mineral compositions is elucidated.Recombinant amelogenin was cleaved by MMP-20 under various physicochemical conditions and the products were analyzed by SDS-PAGE and MALDI-TOF MS.It was observed that mineral ions largely affect cleavage pattern, and enzyme kinetics of rH174 hydrolysis. Out of the five selected mineral ion compositions, MMP-20 was most efficient at high calcium concentration, whereas it was slowest at high phosphate, and at high calcium and phosphate concentrations. In most of the compositions, N- and C-termini were cleaved rapidly at several places but the central region of amelogenin was protected up to some extent in solutions with high calcium and phosphate contents.These in vitro studies showed that the chemistry of the protein solutions can significantly alter the processing of amelogenin by MMP-20, which may have significant effects in vivo matrix assembly and subsequent calcium phosphate mineralization.This study elaborates the possibilities of the processing of the organic matrix into mineralized tissue during enamel development.► Enzyme kinetics of MMP-20 over amelogenin ► Enzyme kinetics is highly affected by calcium and phosphate. ► Cleavage positions depend on calcium and phosphate concentrations. ► Few new in vitro cleavage sites were also reported.
Keywords: Enamelysin; Amelogenin; Enamel; Tooth; Proteolysis; Enzyme kinetics;

Multiple roles of glucose-6-phosphatases in pathophysiology by Paola Marcolongo; Rosella Fulceri; Alessandra Gamberucci; Ibolya Czegle; Gabor Banhegyi; Angelo Benedetti (2608-2618).
The endoplasmic reticulum enzyme glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate to glucose and inorganic phosphate. The enzyme is a part of a multicomponent system that includes several integral membrane proteins; the catalytic subunit (G6PC) and transporters for glucose-6-phosphate, inorganic phosphate and glucose. The G6PC gene family presently includes three members, termed as G6PC, G6PC2, and G6PC3. Although the three isoforms show a moderate amino acid sequence homology, their membrane topology and catalytic site are very similar. The isoforms are expressed differently in various tissues. Mutations in all three genes have been reported to be associated with human diseases.The present review outlines the biochemical features of the G6PC gene family products, the regulation of their expression, their role in the human pathology and the possibilities for pharmacological interventions.G6PCs emerge as integrators of extra- and intracellular glucose homeostasis. Beside the well known key role in blood glucose homeostasis, the members of the G6PC family seem to play a role as sensors of intracellular glucose and of intraluminal glucose/glucose-6-phosphate in the endoplasmic reticulum.Since mutations in the three G6PC genes can be linked to human pathophysiological conditions, the better understanding of their functioning in connection with genetic alterations, altered expression and tissue distribution has an eminent importance.► The glucose-6-phosphatase (G6PC) gene family presently includes three members. ► G6PC isoforms show similar intracellular location, membrane topology and catalytic site. ► G6PC isoforms function as glucose producer, glucose sensor or antiapoptotic factor. ► Mutations of G6PCs are associated with human diseases.
Keywords: Glucose-6-phosphatase; Endoplasmic reticulum; Glycemia; von Gierke disease; Diabetes; Congenital neutropenia;

Endovascular injury induces switching of contractile phenotype of vascular smooth muscle cells (VSMCs) to synthetic phenotype, thereby causing proliferation of VSMCs leading to intimal thickening. The purpose of this study was to assess the effect of magnolol on the proliferation of VSMCs in vitro and neointima formation in vivo, as well as the related cell signaling mechanisms.Tumor necrosis factor alpha (TNF-α) induced proliferation of VSMCs was assessed using colorimetric assay. Cell cycle progression and mRNA expression of cell cycle associated molecules were determined by flow cytometry and reverse transcription polymerase chain reaction (RT-PCR) respectively. The signaling molecules such as ERK1/2, JNK, P38 and NF-κB were determined by Western blot analysis. In addition, rat carotid artery balloon injury model was performed to assess the effect of magnolol on neointima formation in vivo.Oral administration of magnolol significantly inhibited intimal area and intimal/medial ratio (I/M). Our in vitro assays revealed magnolol dose dependently induced cell cycle arrest at G0/G1. Also, magnolol inhibited mRNA and protein expression of cyclin D1, cyclin E, CDK4 and CDK2 in vitro and in vivo. The cell cycle arrest was associated with inhibition of ERK1/2 phosphorylation and NF-κB translocation.Magnolol suppressed proliferation of VSMCs in vitro and attenuated neointima formation in vivo by inducing cell cycle arrest at G0/G1 through modulation of cyclin D1, cyclin E, CDK4 and CDK2 expression.Thus, the results suggest that magnolol could be a potential therapeutic candidate for the prevention of restenosis and atherosclerosis.Display Omitted► Magnolol was isolated from Magnoliae Cortex using column chromatography. ► Magnolol inhibited neo-intima formation in vivo. ► Magnolol induced cell cycle arrest at G0/G1 phase. ► Proliferation of VSMCs was ERK1/2 and NF-κB dependent.
Keywords: Magnolol; Neointima; Vascular smooth muscle cell; Cell cycle regulator; Restenosis;

Integrin participates in the effect of thyroxine on plasma membrane in immature rat testis by Ana Paula Zanatta; Leila Zanatta; Renata Gonçalves; Ariane Zamoner; Fátima Regina Mena Barreto Silva (2629-2637).
The secretory activity of Sertoli cells (SC) is dependent on ion channel functions and protein synthesis and is critical to ongoing spermatogenesis. The aim of this study was to investigate the mechanism of action associated with a non-metabolizable amino acid [14C]-MeAIB (α-(methyl-amino)isobutyric acid) accumulation stimulated by T4 and the role of the integrin receptor in this event, and also to clarify whether the T4 effect on MeAIB accumulation and on Ca2+ influx culminates in cell secretion.We have studied the rapid and plasma membrane initiated effects of T4 by using 45Ca2+ uptake and [45C]-MeAIB accumulation assays, respectively. Thymidine incorporation into DNA was used to monitor nuclear activity and quinacrine to analyze the secretory activity on SC.The stimulation of MeAIB accumulation by T4 appears to be mediated by the integrin receptor in the plasma membrane since tetrac and RGD peptide were able to nullify the effect of this hormone. In addition, T4 increases extracellular Ca2+ uptake and Ca2+ from intracellular stocks to enhance nuclear activity, but this genomic action seems not to influence SC secretion mediated by T4. Also, the cytoskeleton and ClC-3 chloride channel contribute to the membrane-associated responses of SC.T4 integrin receptor activation ultimately determines the plasma membrane responses on amino acid transport in SC, but it is not involved in calcium influx, cell secretion or the nuclear effect of the hormone.The integrin receptor activation by T4 may take a role in plasma membrane processes involved in the male reproductive system.► T4 stimulates Sertoli cell exocytosis. ► The effect of T4 on amino acid accumulation is mediated by integrin receptor. ► T4 integrin receptor determines the plasma membrane responses in Sertoli cells.
Keywords: Thyroxine; Integrin; MeAIB accumulation; Exocytosis; Calcium; Rat testis;

The radio-sensitizing effect of xanthohumol is mediated by STAT3 and EGFR suppression in doxorubicin-resistant MCF-7 human breast cancer cells by Youra Kang; Min-A Park; Se-Woong Heo; Su-Young Park; Keon Wook Kang; Pil-Hoon Park; Jung-Ae Kim (2638-2648).
Chemotherapeutic drug resistance remains a clinical obstacle in cancer management. Drug-resistant cancer cells usually exhibit cross-resistance to ionizing radiation, which has devastating consequences for patients. With a better understanding of the molecular mechanisms, it will be possible to develop strategies to overcome this cross-resistance and to increase therapeutic sensitivity.Natural and synthetic flavonoid compounds including xanthohumol, the principal flavonoid in hops, were investigated for its radio-sensitizing activity on human breast cancer MCF-7 and adriamycin-resistant MCF-7 (MCF-7/ADR) cells. Chemo-sensitizing or radio-sensitizing effect was analyzed by tetrazolium-based colorimetric assay and flow cytometry. Western blot analysis, confocal microscopy, gene silencing with siRNA transfection and luciferase reporter gene assay were performed to examine signaling molecule activation.Among the tested flavonoid compounds, pretreatment of the cells with xanthohumol significantly sensitized MCF-7/ADR cells to the radiation treatment by inducing apoptosis. In MCF-7/ADR cells, treatment with xanthohumol alone or with gamma-rays significantly decreased levels of anti-apoptotic proteins. Multi-drug resistance 1 (MDR1), epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription 3 (STAT3) expression levels in MCF-7/ADR cells were suppressed by xanthohumol treatment. In addition, xanthohumol treatment increased death receptor (DR)-4 and DR5 expression. The xanthohumol-induced changes of these resistance-related molecules in MCF-7/ADR cells were synergistically increased by gamma-ray treatment.Xanthohumol restored sensitivity of MCF-7/ADR cells to doxorubicin and radiation therapies.Our results suggest that xanthohumol may be a potent chemo- and radio-sensitizer, and its actions are mediated through STAT3 and EGFR inhibition.Display Omitted► Xanthohumol sensitizes MCF-7/ADR cells to the radiation treatment. ► Xanthohumol significantly decreased anti-apoptotic proteins Bcl-2, Bcl-x/L and survivin. ► Xanthohumol increased DR4 and DR5 expression. ► Xanthohumol suppressed activity and expression levels of STAT3 and EGFR. ► The xanthohumol-induced changes were synergistically increased by gamma-ray treatment.
Keywords: Xanthohumol; Flavonoid; Radio-sensitizer; Cancer cell cross-resistance; STAT3; EGFR;

High glucose induces vascular endothelial growth factor production in human synovial fibroblasts through reactive oxygen species generation by Chun-Hao Tsai; Yi-Chun Chiang; Hsien-Te Chen; Po-Hao Huang; Horng-Chaung Hsu; Chih-Hsin Tang (2649-2658).
Diabetes is an independent risk factor of osteoarthritis (OA). Angiogenesis is essential for the progression of OA. Here, we investigated the intracellular signaling pathways involved in high glucose (HG)-induced vascular endothelial growth factor (VEGF) expression in human synovial fibroblast cells.HG-mediated VEGF expression was assessed with qPCR and ELISA. The mechanisms of action of HG in different signaling pathways were studied using Western blotting. Knockdown of proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of c-Jun to the VEGF promoter.Stimulation of OA synovial fibroblasts (OASF) with HG induced concentration- and time-dependent increases in VEGF expression. Treatment of OASF with HG increased reactive oxygen species (ROS) generation. Pretreatment with NADPH oxidase inhibitor (APO or DPI), ROS scavenger (NAC), PI3K inhibitor (Ly294002 or wortmannin), Akt inhibitor, or AP-1 inhibitor (curcumin or tanshinone IIA) blocked the HG-induced VEGF production. HG also increased PI3K and Akt activation. Treatment of OASF with HG increased the accumulation of phosphorylated c-Jun in the nucleus, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the VEGF promoter.Our results suggest that the HG increases VEGF expression in human synovial fibroblasts via the ROS, PI3K, Akt, c-Jun and AP-1 signaling pathway.We link high glucose on VEGF expression in osteoarthritis.► High glucose-induced VEGF expression in human synovial fibroblast cells. ► HG activates ROS, PI3K, Akt ,and AP-1 pathways, leading to up-regulation of VEGF expression. ► Involvement of AP-1 in HG-induced VEGF expression.
Keywords: Glucose; VEGF; ROS; OA;

Tyrosin kinase inhibitors (TKIs) and monoclonal antibodies aimed to target epidermal growth factor receptor (EGFR) have shown limited effect as monotherapies and drug resistance is a major limitation for therapeutic success. Adjuvant therapies to EGFR targeting therapeutics are therefore of high clinical relevance.Three EGFR targeting drugs, Cetuximab, Erlotinib and Tyrphostin AG1478 were used in combination with photodynamic therapy (PDT) in two EGFR positive cell lines, A-431 epidermoid skin carcinoma and WiDr colorectal adenocarcinoma cells. The amphiphilic meso-tetraphenylporphine with 2 sulphonate groups on adjacent phenyl rings (TPPS2a) was utilized as a photosensitizer for PDT. The cytotoxic outcome of the combined treatments was evaluated by cell counting and MTT. Cellular signalling was explored by Western blotting.PDT as neoadjuvant to Tyrphostin in A-431 cells as well as to Tyrphostin or Erlotinib in WiDr cells revealed synergistic cytotoxicity. In contrast, Erlotinib or Cetuximab combined with neoadjuvant PDT induced an antagonistic effect on cell survival of A-431 cells. Neoadjuvant PDT and EGFR targeting therapies induced a synergistic inhibition of ERK as well as synergistic cytotoxicity only when the EGFR targeting monotherapies caused a prolonged ERK inhibition. There were no correlation between EGFR inhibition by the EGFR targeting monotherapies or the combined therapies and the cytotoxic outcome combination-therapies.The results suggest that sustained ERK inhibition by EGFR targeting monotherapies is a predictive factor for synergistic cytotoxicity when combined with neoadjuvant PDT.The present study provides a rationale for selecting anticancer drugs which may benefit from PDT as adjuvant therapy.► We here evaluate PDT as neoadjuvant to EGFR targeting therapeutics. ► Cetuximab, Erlotinib and Tyrphostin were combined with PDT in two cancer cell lines. ► Synergy was observed when the EGFR targeting drug exerted prolonged ERK inhibition. ► Synergistic cytotoxicity was linked to synergistic ERK inhibition.
Keywords: Photodynamic; Tyrphostin AG1478; Erlotinib; Cetuximab; epidermal growth factor receptor (EGFR); extracellular signal-regulated kinase (ERK);

Green tea catechins can bind and modify ERp57/PDIA3 activity by Lucie Trnková; Daniela Ricci; Caterina Grillo; Gianni Colotti; Fabio Altieri (2671-2682).
Green tea is a rich source of polyphenols, mainly catechins (flavanols), which significantly contribute to the beneficial health effects of green tea in the prevention and treatment of various diseases. In this study the effects of four green tea catechins on protein ERp57, also known as protein disulfide isomerase isoform A3 (PDIA3), have been investigated in an in vitro model.The interaction of catechins with ERp57 was explored by fluorescence quenching and surface plasmon resonance techniques and their effect on ERp57 activities was investigated.A higher affinity was observed for galloylated cathechins, which bind close to the thioredoxin-like redox-sensitive active sites of the protein, with a preference for the oxidized form. The effects of these catechins on ERp57 properties were also investigated and a moderate inhibition of the reductase activity of ERp57 was observed as well as a strong inhibition of ERp57 DNA binding activity.Considering the high affinity of galloylated catechins for ERp57 and their capability to inhibit ERp57 binding to other macromolecular ligands, some effects of catechins interaction with this protein on eukaryotic cells may be expected.This study provides information to better understand the molecular mechanisms underlying the biological activities of catechins and to design new polyphenol-based ERp57-specific inhibitors.► Green tea catechins can bind ERp57 and modify its properties. ► The presence of a galloyl moiety is a key structural feature for the binding to ERp57. ► Galloylated catechins interact with ERp57 near the thioredoxin-like active sites. ► Galloylated catechins strongly inhibit ERp57 DNA binding capability.
Keywords: Green tea catechins; ERp57/PDIA3; Fluorescence analysis; Surface plasmon resonance; Ligand–protein binding; Protein activity;

Transport of inorganic phosphate in Leishmania infantum and compensatory regulation at low inorganic phosphate concentration by T. Russo-Abrahão; M. Alves-Bezerra; D. Majerowicz; A.L. Freitas-Mesquita; C.F. Dick; K.C. Gondim; J.R. Meyer-Fernandes (2683-2689).
Proliferation of Leishmania infantum depends on exogenous inorganic phosphate (Pi) but little is known about energy metabolism and transport of Pi across the plasma membrane in Leishmania sp.We investigated the kinetics of 32Pi transport, the influence of H+ and K+ ionophores and inhibitors, and expression of the genes for the Na+:Pi and H+:Pi cotransporters.The proton ionophore FCCP, bafilomycin A1 (vacuolar ATPase inhibitor), nigericin (K+ ionophore) and SCH28080 (an inhibitor of H+, K+–ATPase) all inhibited the transport of Pi. This transport showed Michaelis–Menten kinetics with K0.5 and Vmax values of 0.016 ± 0.002 mM and 564.9 ± 18.06 pmol × h− 1  × 10− 7  cells, respectively. These values classify the Pi transporter of L. infantum among the high-affinity transporters, a group that includes Pho84 of Saccharomyces cerevisiae. Two sequences were identified in the L. infantum genome that code for phosphate transporters. However, transcription of the PHO84 transporter was 10-fold higher than the PHO89 transporter in this parasite. Accordingly, Pi transport and LiPho84 gene expression were modulated by environmental Pi variations.These findings confirm the presence of a Pi transporter in L. infantum, similar to PHO84 in S. cerevisiae, that contributes to the acquisition of inorganic phosphate and could be involved in growth and survival of the promastigote forms of L. infantum. This work provides the first description of a PHO84-like Pi transporter in a Trypanosomatide parasite of the genus Leishmania, responsible for many infections worldwide.► The Pi transporter in L. infantum is a high-affinity transporter. ► The Pi transporter in L. infantum is regulated by extracellular Pi concentration. ► The Pi transporter in L. infantum is similar to PHO84 in S. cerevisiae.
Keywords: Leishmania infantum; Pi transporter; Pi starvation;

E-cadherin and adherens-junctions stability in gastric carcinoma: Functional implications of glycosyltransferases involving N-glycan branching biosynthesis, N-acetylglucosaminyltransferases III and V by Salomé S. Pinho; Joana Figueiredo; Joana Cabral; Sandra Carvalho; Joana Dourado; Ana Magalhães; Fátima Gärtner; Ana Maria Mendonça; Tomoya Isaji; Jianguo Gu; Fátima Carneiro; Raquel Seruca; Naoyuki Taniguchi; Celso A. Reis (2690-2700).
E-cadherin is a cell–cell adhesion molecule and the dysfunction of which is a common feature of more than 70% of all invasive carcinomas, including gastric cancer. Mechanisms behind the loss of E-cadherin function in gastric carcinomas include mutations and silencing at either the DNA or RNA level. Nevertheless, in a high percentage of gastric carcinoma cases displaying E-cadherin dysfunction, the mechanism responsible for E-cadherin dysregulation is unknown. We have previously demonstrated the existence of a bi-directional cross-talk between E-cadherin and two major N-glycan processing enzymes, N-acetylglucosaminyltransferase-III or -V (GnT-III or GnT-V).In the present study, we have characterized the functional implications of the N-glycans catalyzed by GnT-III and GnT-V on the regulation of E-cadherin biological functions and in the molecular assembly and stability of adherens-junctions in a gastric cancer model. The results were validated in human gastric carcinoma samples.We demonstrated that GnT-III induced a stabilizing effect on E-cadherin at the cell membrane by inducing a delay in the turnover rate of the protein, contributing for the formation of stable and functional adherens-junctions, and further preventing clathrin-dependent E-cadherin endocytosis. Conversely, GnT-V promotes the destabilization of E-cadherin, leading to its mislocalization and unstable adherens-junctions with impairment of cell–cell adhesion.This supports the role of GnT-III on E-cadherin-mediated tumor suppression, and GnT-V on E-cadherin-mediated tumor invasion.These results contribute to fill the gap of knowledge of those human carcinoma cases harboring E-cadherin dysfunction, opening new insights into the molecular mechanisms underlying E-cadherin regulation in gastric cancer with potential translational clinical applications.► E-cadherin is often dysregulated in invasive gastric carcinomas. ► We characterized the functional role of N-glycans on E-cadherin in gastric cancer. ► GnT-III-mediated glycosylation has a stabilizer effect on E-cadherin functions. ► GnT-V-mediated glycosylation has a destabilizer effect on E-cadherin functions. ► Gastric carcinoma cases exhibit E-cadherin modifications with deleterious N-glycans.
Keywords: Gastric cancer; E-cadherin; Adherens-junctions; N-glycosylation; Glycosyltransferases;

IGF dependent modulation of IGF binding protein (IGFBP) proteolysis by pregnancy-associated plasma protein-A (PAPP-A): Multiple PAPP-A–IGFBP interaction sites by Ervinas Gaidamauskas; Claus Gyrup; Henning B. Boldt; Vivien R. Schack; Michael T. Overgaard; Lisbeth S. Laursen; Claus Oxvig (2701-2709).
Pregnancy-associated plasma protein-A (PAPP-A) is a local regulator of insulin-like growth factor (IGF) bioavailability in physiological systems, but many structural and functional aspects of the metzincin metalloproteinase remain to be elucidated. PAPP-A cleaves IGF binding protein (IGFBP)-4 and IGFBP-5. Cleavage of IGFBP-4, but not IGFBP-5, depends on the binding of IGF before proteolysis by PAPP-A can occur. The paralogue PAPP-A2 has two substrates among the six IGFBPs: IGFBP-3 and IGFBP-5.Sets of chimeric proteins between IGFBP-4 and -5, and IGFBP-3 and -5 were constructed to investigate the structural requirements for IGF modulation. At the proteinase level, we investigated the importance of individual acidic amino acids positioned in the proteolytic domain of PAPP-A for proteolytic activity against IGFBP-4 and -5. Interaction between PAPP-A and its substrates was analyzed by surface plasmon resonance.We provide data suggesting that the C-terminal domain of the IGFBPs is responsible for IGF-dependent modulation of access to the scissile bond. Loss or reduction of IGFBP proteolysis by PAPP-A was observed upon mutation of residues positioned in the unique 63-residue stretch separating the zinc and Met-turn motifs, and in the short sequence following the Met-turn methionine. A model of the proteolytic domain of PAPP-A suggests the presence of structural calcium ions in the C-terminal subdomain, implicated in IGFBP substrate interactions.Detailed knowledge of interactions between PAPP-A and its substrates is required to understand the modulatory role of PAPP-A on IGF receptor stimulation.► Acidic residues of PAPP-A are required for substrate cleavage. ► Structural calcium ions are suggested to be implicated in interaction with IGFBPs. ► IGF–IGFBP interaction determines protease–substrate binding. ► The central and the C-terminal domains of IGFBPs mediate IGF dependent proteolysis.
Keywords: Pregnancy-associated plasma protein-A (PAPP-A); Pappalysins; Proteolysis; IGF-binding protein (IGFBP); IGF dependent modulation; Ulilysin;

Forces measured with micro-fabricated cantilevers during actomyosin interactions produced by filaments containing different myosin isoforms and loop 1 structures by Albert Kalganov; Nabil Shalabi; Nedjma Zitouni; Linda Hussein Kachmar; Anne-Marie Lauzon; Dilson E. Rassier (2710-2719).
There is evidence that the actin-activated ATP kinetics and the mechanical work produced by muscle myosin molecules are regulated by two surface loops, located near the ATP binding pocket (loop 1), and in a region that interfaces with actin (loop 2). These loops regulate force and velocity of contraction, and have been investigated mostly in single molecules. There is a lack of information of the work produced by myosin molecules ordered in filaments and working cooperatively, which is the actual muscle environment.We use micro-fabricated cantilevers to measure forces produced by myosin filaments isolated from mollusk muscles, skeletal muscles, and smooth muscles containing variations in the structure of loop 1 (tonic and phasic myosins). We complemented the experiments with in-vitro assays to measure the velocity of actin motility.Smooth muscle myosin filaments produced more force than skeletal and mollusk myosin filaments when normalized per filament overlap. Skeletal muscle myosin propelled actin filaments in a higher sliding velocity than smooth muscle myosin. The values for force and velocity were consistent with previous studies using myosin molecules, and suggest a close correlation with the myosin isoform and structure of surface loop 1.The technique using micro-fabricated cantilevers to measure force of filaments allows for the investigation of the relation between myosin structure and contractility, allowing experiments to be conducted with an array of different myosin isoforms. Using the technique we observed that the work produced by myosin molecules is regulated by amino-acid sequences aligned in specific loops.► Myosin filaments isolated from mollusk or polymerized using skeletal and smooth muscles ► Filament forces measured with micro-fabricated cantilevers ► Velocity of actin measured with in-vitro motility assay
Keywords: Actin filament; Myosin filament; Force; Cantilevers; Muscle contraction; Loop 1;

Cancer is one of the leading worldwide causes of death. It may be induced by a variety of factors, including carcinogens, radiation, genetic factors, or DNA and RNA viruses. The early detection of cancer is critical for its successful therapy, which can result in complete recovery from some types of cancer.Raman spectroscopy has been widely used in medicine and biology. It is a noninvasive, nondestructive, and water-insensitive technique that can detect changes in cells and tissues that are caused by different disorders, such as cancer.In this study, Raman spectroscopy was used for the identification and characterization of murine fibroblast cell lines (NIH/3T3) and malignant fibroblast cells transformed by murine sarcoma virus (NIH-MuSV) cells.Using principal component analysis and LDA it was possible to differentiate between the NIH/3T3 and NIH-MuSV cells with an 80–85% success rate based on their Raman shift spectra.The best results for differentiation were achieved from spectra that were obtained from the rich membrane sites.Because of its homogeneity and complete control of most factors affecting its growth, cell culture is a preferred model for the detection and identification of specific biomarkers related to cancer transformation or other cellular modifications.► Raman spectroscopy may be used to detect malignant cells transformed by MuSV. ► 80–85% success was achieved in classifying the control NIH/3T3 and NIH-MuSV cells. ► Measurements from membranes' region sites are more effective for differentiation. ► Membrane changes caused by malignancy may contribute to classification procedure.
Keywords: Raman spectroscopy; NIH/3T3; NIH-MuSV; Statistical analysis;

Translational repression of the McKusick–Kaufman syndrome transcript by unique upstream open reading frames encoding mitochondrial proteins with alternative polyadenylation sites by Chizuru Akimoto; Eiji Sakashita; Katsumi Kasashima; Kenji Kuroiwa; Kaoru Tominaga; Toshiro Hamamoto; Hitoshi Endo (2728-2738).
Upstream open reading frames (uORFs) are commonly found in the 5′-untranslated region (UTR) of many genes and function in translational control. However, little is known about the existence of the proteins encoded by uORFs, and the role of the proteins except translational control. There was no report about uORFs of the McKusick–Kaufman syndrome (MKKS) gene that causes a genetic disorder.Northern blotting, 3′-RACE, and bioinformatics were used for determining the length of transcripts and their 3′ ends. Luciferase assay and in vitro translation were used for evaluation of translational regulatory activity of uORFs. Immunoblotting and immunocytochemical analyses were used for detection of uORF-derived protein products and their subcellular localization.The MKKS gene generates two types of transcripts: a canonical long transcript that encodes both uORFs and MKKS, and a short transcript that encodes only uORFs by using alternative polyadenylation sites at the 5′-UTR. The simultaneous disruption of the uORF initiation codons increased the translation of the downstream ORF. Furthermore, both protein products from the two longest uORFs were detected in the mitochondrial membrane fraction of HeLa cells. Database searches indicated that such uORFs with active alternative polyadenylation sites at the 5′-UTR are atypical but surely exist in human transcripts.Multiple uORFs at the 5′-UTR of the MKKS long transcript function as translational repressor for MKKS. Two uORFs are translated in vivo and imported onto the mitochondrial membrane.Our findings provide unique insights into production of uORF-derived peptides and functions of uORFs.► MKKS mRNA has multiple uORFs and alternative polyadenylation sites at the 5′-UTR. ► The presence of uORFs at the MKKS 5′-UTR represses the translation of the main ORF. ► Two uORFs of the MKKS mRNA are translated as mitochondrial proteins in human cells.
Keywords: Upstream open reading frame; Polyadenylation signal; mRNA processing; Translational regulation; McKusick–Kaufman syndrome; Mitochondrion;

Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) β-glucosidase from Aspergillus niger ASKU28 by Preeyanuch Thongpoo; Lauren S. McKee; Ana Catarina Araújo; Prachumporn T. Kongsaeree; Harry Brumer (2739-2749).
The commercially important glycoside hydrolase family 3 (GH3) β-glucosidases from Aspergillus niger are anomeric-configuration-retaining enzymes that operate through the canonical double-displacement glycosidase mechanism. Whereas the catalytic nucleophile is readily identified across all GH3 members by sequence alignments, the acid/base catalyst in this family is phylogenetically variable and less readily divined.In this report, we employed three-dimensional structure homology modeling and detailed kinetic analysis of site-directed mutants to identify the catalytic acid/base of a GH3 β-glucosidase from A. niger ASKU28.In comparison to the wild-type enzyme and other mutants, the E490A variant exhibited greatly reduced kcat and kcat /Km values toward the natural substrate cellobiose (67,000- and 61,000-fold, respectively). Correspondingly smaller kinetic effects were observed for artificial chromogenic substrates p-nitrophenyl β-d-glucoside and 2,4-dinitrophenyl β-d-glucoside, the aglycone leaving groups of which are less dependent on acid catalysis, although changes in the rate-determining catalytic step were revealed for both. pH-rate profile analyses also implicated E490 as the general acid/base catalyst. Addition of azide as an exogenous nucleophile partially rescued the activity of the E490A variant with the aryl β-glucosides and yielded β-glucosyl azide as a product.These results strongly support the assignment of E490 as the acid/base catalyst in a β-glucosidase from A. niger ASKU28, and provide crucial experimental support for the bioinformatic identification of the homologous residue in a range of related GH3 subfamily members.► The catalytic acid/base residue in a glycoside hydrolase family 3 β-glucosidase was identified. ► Site-directed mutagenesis, enzyme kinetic analysis, and nucleophile rescue were used. ► Prediction of the corresponding residue among other GH3 enzymes is enabled. ► These results are crucial to understanding catalysis and active-site structure in GH3.
Keywords: β-Glucosidase; Acid/base catalyst; Aspergillus niger; Glycoside hydrolase family 3; Azide rescue;

Factors implicated in the assessment of aminolevulinic acid-induced protoporphyrin IX fluorescence by Beata Čunderlíková; Qian Peng; Anton Mateašík (2750-2762).
Photodynamic therapy and photodiagnosis of cancer requires preferential accumulation of fluorescent photosensitizers in tumors. Clinical evidence documents feasibility of ALA-based photodiagnosis for tumor detection. However, false positive results and large variations in fluorescence intensities are also reported. Furthermore, selective accumulation of fluorescent species of photosensitizers in tumor cell lines, as compared to normal ones, when cultured in vitro, is not always observed. To understand this discrepancy we analyzed the impact of various factors on the intensity of detected PpIX fluorescence.Impacts of cell type, mitochondrial potential, cell–cell interactions and relocalization of PpIX among different cell types in co-cultures of different cell lines were analyzed by confocal microscopy and flow cytometry. Fluorescence spectroscopy was used to estimate absolute amounts of ALA-induced PpIX in individual cell lines. Immunofluorescence staining was applied to evaluate the ability of cell lines to produce collagen.Higher ALA-induced PpIX fluorescence in cancer cell lines as compared to normal ones was not detected by all the methods used. Mitochondrial activity was heterogeneous throughout the cell monolayers and could not be clearly correlated with PpIX fluorescence. Positive collagen staining was detected in all cell lines tested.Contrary to in vivo situation, ALA-induced PpIX production by cell lines in vitro may not result in higher PpIX fluorescence signals in tumor cells than in normal ones. We suggest that a combination of several properties of tumor tissue, instead of tumor cells only, is responsible for increased ALA-induced PpIX fluorescence in solid tumors.Understanding the reasons of increased ALA-induced PpIX fluorescence in tumors is necessary for reliable ALA-based photodiagnosis, which is used in various oncological fields.► Analysis of ALA-induced PpIX fluorescence in co-cultures of tumor and normal esophageal cell lines ► Comparable ALA-induced PpIX fluorescence intensities in tumor and normal cell lines ► Heterogeneous mitochondrial activity throughout the monolayers of tumor and normal esophageal cell lines ► Positive collagen immunostaining in all tumor and normal esophageal cell lines tested
Keywords: Aminolevulinic acid; Protoporphyrin IX; Collagen; Mitochondrial potential;

Development of peptide inhibitor as a therapeutic agent against head and neck squamous cell carcinoma (HNSCC) targeting p38α MAP kinase by Kamaldeep Gill; Abhay K. Singh; Vaishali Kapoor; Lokesh Nigam; Rahul Kumar; Prasida Holla; Satya N. Das; Savita Yadav; Naidu Subbarao; Bidhu K. Mohanti; Sharmistha Dey (2763-2769).
The p38α MAP kinase pathway is involved in inflammation, cell differentiation, growth, apoptosis and production of pro-inflammatory cytokines TNF-α and IL-1β. The overproduction of these cytokines plays an important role in cancer. The aim of this work was to design a peptide inhibitor on the basis of structural information of the active site of p38α.A tetrapeptide, VWCS as p38α inhibitor was designed on the basis of structural information of the ATP binding site by molecular modeling. The inhibition study of peptide with p38α was performed by ELISA, binding study by Surface Plasmon Resonance and anti-proliferative assays by MTT and flow cytometry.The percentage inhibition of designed VWCS against pure p38α protein and serum of HNSCC patients was 70.30 and 71.5%, respectively. The biochemical assay demonstrated the KD and IC50 of the selective peptide as 7.22 × 10− 9  M and 20.08 nM, respectively. The VWCS as inhibitor significantly reduced viability of oral cancer KB cell line with an IC50 value of 10 μM and induced apoptosis by activating Caspase 3 and 7.VWCS efficiently interacted at the ATP binding pocket of p38α with high potency and can be used as a potent inhibitor in case of HNSCC.VWCS can act as an anticancer agent as it potentially inhibits the cell growth and induces apoptosis in oral cancer cell-line in a dose as well as time dependent manner. Hence, p38α MAP kinase inhibitor can be a potential therapeutic agent for human oral cancer.► p38α, an inflammatory MAP kinase plays an important role in Head and Neck Squamous Cell Carcinoma (HNSCC). ► A tetrapeptide, VWCS was designed and synthesized against the ATP binding site of the p38 α kinase. ► The peptide VWCS showed an efficient interaction with the ATP binding site as determined by the KD and IC50 value. ► VWCS also exhibited anti-cancerous activity and induced apoptosis.
Keywords: p38α; HNSCC; Tetrapeptide; Anticancer; Apoptosis;

Mitochondrial and sarcoplasmic reticulum abnormalities in cancer cachexia: Altered energetic efficiency? by Cibely Cristine Fontes-Oliveira; Sílvia Busquets; Míriam Toledo; Fabio Penna; Maria Paz Aylwin; Sònia Sirisi; Ana Paula Silva; Marcel Orpí; Albert García; Angelica Sette; Maria Inês Genovese; Mireia Olivan; Francisco J. López-Soriano; Josep M. Argilés (2770-2778).
Cachexia is a wasting condition that manifests in several types of cancer, and the main characteristic is the profound loss of muscle mass.The Yoshida AH-130 tumor model has been used and the samples have been analyzed using transmission electronic microscopy, real-time PCR and Western blot techniques.Using in vivo cancer cachectic model in rats, here we show that skeletal muscle loss is accompanied by fiber morphologic alterations such as mitochondrial disruption, dilatation of sarcoplasmic reticulum and apoptotic nuclei. Analyzing the expression of some factors related to proteolytic and thermogenic processes, we observed in tumor-bearing animals an increased expression of genes involved in proteolysis such as ubiquitin ligases Muscle Ring Finger 1 (MuRF-1) and Muscle Atrophy F-box protein (MAFBx). Moreover, an overexpression of both sarco/endoplasmic Ca2 +-ATPase (SERCA1) and adenine nucleotide translocator (ANT1), both factors related to cellular energetic efficiency, was observed. Tumor burden also leads to a marked decreased in muscle ATP content.In addition to muscle proteolysis, other ATP-related pathways may have a key role in muscle wasting, both directly by increasing energetic inefficiency, and indirectly, by affecting the sarcoplasmic reticulum–mitochondrial assembly that is essential for muscle function and homeostasis.The present study reports profound morphological changes in cancer cachectic muscle, which are visualized mainly in alterations in sarcoplasmic reticulum and mitochondria. These alterations are linked to pathways that can account for energy inefficiency associated with cancer cachexia.► Skeletal muscle from cachectic animals showed fiber morphologic alterations. ► These alterations are mitochondrial disruption and dilatation of sarcoplasmic reticulum. ► An overexpression of both sarco/endoplasmic Ca2 +-ATPase (SERCA1) and adenine nucleotide translocator (ANT1) was reported. ► Tumor burden also leads to a marked decreased in muscle ATP content.
Keywords: Cancer cachexia; Muscle wasting; Sarcoplasmic reticulum; Mitochondri; SERCA; ANT1;

Celastrol, a quinine methide triterpene extracted from a Chinese medicine (Trypterygium wilfordii Hook F.), has the potential to become an anticancer drug with promising prospects. Cell culture metabolomics has been a powerful method to study metabolic profiles in cell line after drug treatment, which can be used for discovery of drug targets and investigation of drug effects.We analyzed the metabolic modifications induced by celastrol treatment in human cervical cancer cells, using an ion-trap gas chromatography–mass spectrometry based metabolomics combined with multivariate statistical analysis, which allows simultaneous screening of multiple characteristic metabolic pathways related to celastrol treatment. Three representative apoptosis-inducing cytotoxic agents, namely cisplatin, doxorubicin hydrochloride and paclitaxel, were selected as positive control drugs to validate reasonableness and accuracy of our metabolomic investigation on celastrol.Anti-proliferation and apoptotic effects of celastrol were demonstrated by CCK-8 assay, Annexin-V/PI staining method, mitochondrial membrane potential (ΔΨm) assay and caspase-3 assay. Several significant metabolites involved in energy, amino acid and nucleic acid metabolism in HeLa cells induced by celastrol and positive drugs were reported. Our method is proved to be effective and robust to provide new evidence of pharmacological mechanism of celastrol.The metabolic alterations induced by drug treatment showed the impaired physiological activity of HeLa cells, which also indicated anti-proliferative and apoptotic effects of celastrol and these positive drugs.GC/MS-based metabolomic approach applied to cell culture could give valuable information on the systemic effects of celastrol in vitro and help us to further study its anticancer mechanism.► Celastrol induces apoptosis in HeLa cells by ΔΨm↓ and capase-3 activation. ► Celastrol down-regulates HeLa cells' energy, amino acids and nucleic acid metabolism. ► Metabolomic investigation on celastrol is validated using positive control drugs. ► GC/MS-based metabolomic approach elucidates celastrol's pharmacological mechanism.
Keywords: Celastrol; Human cervical cancer cell; Apoptosis; Gas chromatography–mass spectrometry; Metabolic profiling; Metabolomics;

Functional characterisation of p53 mutants identified in breast cancers with suboptimal responses to anthracyclines or mitomycin by Elisabet O. Berge; Johanna Huun; Johan R. Lillehaug; Per E. Lønning; Stian Knappskog (2790-2797).
Approximately 4300 different TP53 mutations have been reported in human cancers. TP53 mutations, in particular those affecting the L2/L3 domains, are associated with resistance to anthracycline or mitomycin treatment in breast cancer patients. While many mutations have been characterised functionally, novel TP53 mutations are continuously reported. Here, we characterise 10 p53 protein variants encoded by mutated TP53 (5 within and 5 outside L2/L3) detected in locally advanced or metastatic breast cancers. Each tumour was previously characterised for response to therapy, allowing comparison between in vivo and in vitro findings.Mutated p53 variants were analysed for their ability to oligomerise with the wild-type protein and their subcellular localisation by immunoprecipitation and immunofluorescence, respectively. Their ability to induce transcription of target genes was determined by qPCR. Cellular growth rate, apoptosis and senescence were monitored by WST-1, TUNEL and beta-galactosidase assays, respectively.Immunoprecipitation assays revealed each mutant protein to retain binding capacity for wild-type p53, thus potentially acting in a dominant negative manner. Even though each p53 variant located predominantly in the nucleus, the percentage of cells with only nuclear p53 localisation varied between 60% and 90%. None of the p53 variants were able to induce target genes to levels similar to wild-type p53, nor where they able to reduce cellular growth rate, induce apoptosis or senescence similar to wild-type p53 after anthracycline treatment in vitro.All the 10 variants studied displayed inferior p53 functionality compared to the wild-type protein.Our data add further information characterising the effects of somatic TP53 mutations on p53 protein function and anthracycline resistance in breast cancer.► 10 p53 variants encoded by mutated TP53 in breast cancers were characterised. ► Each tumour was characterised for response to anthracycline or mitomycin therapy. ► All mutants bound wild-type p53, potentially acting in a dominant negative manner. ► All mutants showed reduced induction of apoptosis/senescence ► P53 mutations both within and outside the L2/L3 domains may cause treatment failure.
Keywords: Breast; Cancer; Mutation; Resistance; p53;

Electrochemistry of cytochrome c immobilized on cardiolipin-modified electrodes: A probe for protein–lipid interactions by Antoine Perhirin; Edouard Kraffe; Yanic Marty; François Quentel; Philippe Elies; Frederic Gloaguen (2798-2803).
Electrochemistry of cytochrome c (cyt c) immobilized on a cardiolipin (CL)/phosphatidylcholine (PC) film supported on a glassy carbon electrode was investigated using variable-frequency AC voltammetry. At low ionic strength, we observed two redox-active subpopulations characterized by distinct values of potential (E 1/2) and electron transfer rate constant (k ET). At high ionic strength, only one subpopulation was detected, consistent with the existence of very stable cyt c–CL adducts, most probably formed by hydrophobic interactions between the protein and the fatty acid (FA) chains carried by CL. This subpopulation exhibits a comparatively high k ET value (> 300 s− 1) apparently changing with the structure of the FA chains of CL, i.e. 18:2(n − 6) or 14:0. Our study suggests that electrochemistry can be a useful technique for probing protein–lipid interactions, and more particularly the role played by the specific structure of the FA chains of CL on cyt c binding.► PC/CL supported films were used to study electrochemistry of immobilized cyt c. ► AC voltammetry experiments showed two subpopulations of immobilized cyt c. ► At high ionic strength, only hydrophobically bound cyt c is detected. ► Redox behavior of cyt c depends on the structure of the FA chains carried by CL.
Keywords: Cardiolipin; Cytochrome c; Electron transfer; Fatty acid chain; Lipid anchorage; Voltammetry;

Monoclonal antibody against the poly-γ-d-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge by Jeyoun Jang; Minhui Cho; Hae-Ri Lee; Kiweon Cha; Jeong-Hoon Chun; Kee-Jong Hong; Jungchan Park; Gi-eun Rhie (2804-2812).
The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT.To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with Kd values of 0.8 μM and 2.6 μM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed.Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen.Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.► We characterized two new mouse anti-poly-γ-d-glutamic acid (PGA) antibodies. ► In silico three dimensional models of mAbs with PGA complex were analyzed. ► The enhancement of PGA on LT-mediated cytotoxicity was inhibited by anti-PGA mAb. ► mAb partially rescued mice from anthrax spore challenge through opsonophagocytosis. ► Data supports the role of anti-PGA antibody in prevention and treatment of anthrax.
Keywords: Bacillus anthracis; Monoclonal antibody; Modeling; Lethal toxin; Poly-γ-d-glutamic acid capsule;

The small GTPase Ran, Ras-related nuclear protein, plays important roles in multiple fundamental cellular functions such as nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation, by binding to either GTP or GDP as a molecular switch. Although it has been clinically demonstrated that Ran is highly expressed in multiple types of cancer cells and specimens, the physiological significance of Ran expression levels is unknown.During the long-term culture of normal mammalian cells, we found that the endogenous Ran level gradually reduced in a passage-dependent manner. To examine the physiological significance of Ran reduction, we first performed small interfering RNA (siRNA)-mediated abrogation of Ran in human diploid fibroblasts.Ran-depleted cells showed several senescent phenotypes. Furthermore, we found that nuclear accumulation of importin α, which was also observed in cells treated with siRNA against CAS, a specific export factor for importin α, occurred in the Ran-depleted cells before the cells showed senescent phenotypes. Further, the CAS-depleted cells also exhibited cellular senescence. Indeed, importin α showed predominant nuclear localisation in a passage-dependent manner.Reduction in Ran levels causes cytoplasmic decrease and nuclear accumulation of importin α leading to cellular senescence in normal cells.The amount of intracellular Ran may be critically related to cell fate determination, such as malignant transformation and senescence. The cellular ageing process may proceed through gradual regression of Ran-dependent nucleocytoplasmic transport competency.► Small GTPase Ran decreases in normal mammalian cells in a passage-dependent manner. ► siRNA-mediated depletion of Ran causes cellular senescence in human fibroblasts. ► Ran-depletion causes nuclear accumulation of importin α. ► Nuclear accumulation of importin α occurs in old cells and cells lacking CAS. ► CAS-depleted cells also exhibit cellular senescence.
Keywords: Cellular ageing; Cellular senescence; Small GTPase Ran; CAS; Importin α; Cell fate determination;

The JNK/NFκB pathway is required to activate murine lymphocytes induced by a sulfated polysaccharide from Ecklonia cava by Ginnae Ahn; So Jin Bing; Sung-Myung Kang; Won-Woo Lee; Seung-Hong Lee; Hiroshi Matsuda; Akane Tanaka; Ik-Hyun Cho; You-Jin Jeon; Youngheun Jee (2820-2829).
The proven immunomodulatory and immune system activating properties of Ecklonia cava (E. cava) have been attributed to its plentiful polysaccharide content. Therefore, we investigated whether the sulfated polysaccharide (SP) of E. cava specifically activates the protein kinases (MAPKs) and nuclear factor-κB (NFκB) to incite immune responses.To assess immune responsiveness, lymphocytes were isolated from spleens of ICR mice and cultured with SP and its inhibitors. Assays included 3H-thymidine incorporation, flow cytometry, real time polymerase chain reaction (rtPCR), enzyme linked immunosorbent assay (ELISA), intracellular cytokine assay, Western blot, and electrophoretic mobility shift assay (EMSA).SP dose-dependently increased the proliferation of lymphocytes without cytotoxicity. In particular, SP markedly enhanced the proliferation and differentiation of CD3+ mature T cells and CD45R/B220+ pan B cells. Additionally, SP increased the expression and/or production of IL-2, IgG1a, and IgG2b compared to that in untreated cells. The subsequent application of JNK (SP600125), NFκB (PDTC), and serine protease (TPCK) inhibitors significantly inhibited the proliferation and IL-2 production of SP-treated lymphocytes as well as the phosphorylation of JNK and IκB, the activation of nuclear NFκB p65, and binding of NFκB p65 DNA. Moreover, co-application of both JNK and NFκB inhibitors completely blocked the proliferation of lymphocytes even in the presence of SP.These results suggest that SP induced T and B cell responses via both JNK and NFκB pathways.The effect of SP on splenic lymphocyte activation was assayed here for the first time and indicated the underlying functional mechanism.► SP enhances the numerical and functional responses in lymphocytes. ► JNK/NFκB signal pathway is required for SP-induced lymphocyte activation. ► Immunological functions of SP in lymphocytes were evaluated for the first time.
Keywords: Ecklonia cava (E. cava); Sulfated polysaccharide (SP); Lymphocytes; JNK; NFκB p65;

Nitric oxide depletion alters hematopoietic stem cell commitment toward immunogenic dendritic cells by Roberto Tiribuzi; Lucia Crispoltoni; Francesco Tartacca; Antonio Orlacchio; Sabata Martino; Carlo Alberto Palmerini; Aldo Orlacchio (2830-2838).
NO is a key molecule involved in the regulation of cell survival, proliferation and differentiation in many cell types. In this study we investigated the contribution of NO during the differentiation of human peripheral blood hemopoietic stem cells (CD34+HSCs) toward immunogenic dendritic cells (i-DCs).We depleted autocrine NO production, using NG-monomethyl-l-arginine monoacetate (l-NMMA) and paracrine NO, using oxy-hemoglobin (HbO2) as a NO scavenger during in vitro differentiation of CD34+HSCs to i-DCs. We monitored the NO level, cell proliferation, phenotype and differentiation potential.We found that the depletion of paracrine or autocrine NO correlated with (I) an active proliferation state at the end of differentiation, when control cells were not proliferating; (II) a significant reduction in the expression levels of differentiative markers (CD1a and HLA-DR) with a parallel high expression of the CD34 marker (III) with a retrieved clonogenic ability compared to control cells.On the whole, our data indicate that the depletion of NO during the commitment stage blocks CD34+HSC differentiation into i-DCs and maintains an undifferentiated, highly proliferating cell population, indicating/revealing a novel role for NO in the commitment of CD34+HSCs into i-DCs.The essential finding of the present study is that NO, produced in HSCs by NOS enzymes, may act as autocrine and paracrine effectors regulating the in vitro differentiation process of CD34+-HSCs toward i-DCs.Display Omitted► We investigated the contribution of NO during the differentiation of human CD34+HSCs toward i-DCs. ► We found a peak of NO production at the time of stem cell commitment. ► The inhibition of NOS isoforms impaired the differentiation process of i-DCs from CD34+HSCs. ► The depletion of NO preserved the stem cell in a commitment stage for a long time in culture. ► The depletion of NO retrieved the clonogenic ability of cytokine induced CD34+HSCs.
Keywords: CD34 + hemopoietic stem cell; Nitric oxide; Lysosomal enzyme; Hemoglobin; NG-monomethyl-l-arginine monoacetate;

A good understanding of the mechanism of gene regulation that is involved in bone mineralization is critical for the design of anabolic treatments for bone deficiency diseases. Alkaline phosphatase (ALP) expressed by osteoblasts plays an important role in promoting bone mineralization by hydrolyzing pyrophosphate. However, the mechanism by which the expression of ALP is regulated during osteoblast differentiation has not been thoroughly investigated.Chromatin immunoprecipitation, EMSA and mutagenesis were used to identify the Runx2 binding sites on ALP gene and to analyze the role of nuclear matrix-localization of Runx2 on the recognition and activation of ALP gene.Using chromatin immunoprecipitation, we determined that both ectopic and endogenous Runx2 bound to ALP intron 1 in a region containing a cluster of five putative core-sites. The third one (I1C3) among those fives was bound most strongly in vitro by Runx2 and acted as a Runx2-dependent transcriptional enhancer. Furthermore, a Runx2 mutant lacking the nuclear matrix-targeting sequence (Runx2ΔNMTS) bound to the ALP gene less efficiently than the wild-type protein and a Runx2 mutant that is deficient in its ability to bind to DNA (Runx2K120A) accumulated largely in the nuclear matrix.Nuclear matrix-localization of Runx2 influences its ALP gene recognition.Our results showed for the first time that ALP is a direct target gene of Runx2 and illustrated that the recognition/binding and activation of the ALP by this transcription factor are dependent on its nuclear matrix-targeting.► Alkaline phosphatase (ALP) is a direct target gene of Runx2. ► Nuclear matrix-targeting of Runx2 is essential for its recognition of ALP. ► Nuclear matrix-targeting of Runx2 is essential for its activation of ALP. ► Runx2K120A accumulates largely in the nuclear matrix fraction. ► Runx2K120A hampers the activation of ALP by wild-type Runx2.
Keywords: Alkaline phosphatase; Runx2; Nuclear matrix; Osteoblast differentiation; Protein DNA-interaction;

Optical control of calcium-regulated exocytosis by Mercè Izquierdo-Serra; Dirk Trauner; Artur Llobet; Pau Gorostiza (2853-2860).
Neurons signal to each other and to non-neuronal cells as those in muscle or glands, by means of the secretion of neurotransmitters at chemical synapses. In order to dissect the molecular mechanisms of neurotransmission, new methods for directly and reversibly triggering neurosecretion at the presynaptic terminal are necessary. Here we exploit the calcium permeability of the light-gated channel LiGluR in order to reversibly manipulate cytosolic calcium concentration, thus controlling calcium-regulated exocytosis.Bovine chromaffin cells expressing LiGluR were stimulated with light. Exocytic events were detected by amperometry or by whole-cell patch-clamp to quantify membrane capacitance and calcium influx.Amperometry reveals that optical stimulation consistently triggers exocytosis in chromaffin cells. Secretion of catecholamines can be adjusted between zero and several Hz by changing the wavelength of illumination. Differences in secretion efficacy are found between the activation of LiGluR and native voltage-gated calcium channels (VGCCs). Our results show that the distance between sites of calcium influx and vesicles ready to be released is longer when calcium influx is triggered by LiGluR instead of native VGCCs.LiGluR activation directly and reversibly increases the intracellular calcium concentration. Light-gated calcium influx allows for the first time to control calcium-regulated exocytosis without the need of applying depolarizing solutions or voltage clamping in chromaffin cells.LiGluR is a useful tool to study the secretory mechanisms and their spatiotemporal patterns in neurotransmission, and opens a window to study other calcium-dependent processes such as muscular contraction or cell migration.► LiGluR enables reversibly triggering regulated exocytosis with light. ► Vesicle fusion is directly induced by calcium influx and not by depolarization. ► LiGluR secretion efficacy is compared to that of native calcium channels. ► Applications include manipulating neurotransmission and muscle contraction with light.
Keywords: Optical control; Calcium; Exocytosis; Light-gated glutamate receptor (LiGluR); Neurotransmission; Optogenetics;