BBA - General Subjects (v.1820, #7)

Microtubule assembly-derived by dimerization of TPPP/p25. Evaluation of thermodynamic parameters for multiple equilibrium system from ITC data by Judit Oláh; Ágnes Zotter; Emma Hlavanda; Sándor Szunyogh; Ferenc Orosz; Krisztián Szigeti; Judit Fidy; Judit Ovádi (785-794).
The disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system. In this paper the role of dimerization in its microtubule-related functions is established, and an approach is proposed to evaluate thermodynamic constants for multiple equilibrium systems from ITC measurements.For structural studies size exclusion chromatography, SDS-PAGE, chemical cross-linking, circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry were used; the functional effect was analyzed by tubulin polymerization assay. Numerical simulation of the multiple equilibrium was performed with Mathematica software.The dimerization of TPPP/p25 is promoted by elevation of the protein concentration and by GTP addition. The dimeric form displaying enhanced tubulin polymerization promoting activity is stabilized by disulfide bond or chemical cross-linking. The GTP binding to the dimeric form (Kd-GTP  = 200 μM) is tighter with one order of magnitude than to the monomeric one leading to the enrichment of the dimers. A mathematical model elaborated for the multiple equilibrium of the TPPP/p25-GTP system was validated by fitting the GTP-dependent changes of ellipticity and fluorescence signal in the course of TPPP/p25 titrations. The evaluation of the equilibrium constants rendered it possible to determine the thermodynamic parameters of the association of different TPPP/p25 forms with GTP from ITC measurements.The dimerization of TPPP/p25 with favorable physiological functional potency is proposed to play significant role in the fine tuning of TPPP/p25-mediated microtubule assembly; the unfolded monomers might be involved in the formation of pathological inclusions characteristic for Parkinson's disease and other synucleinopathies.► Tubulin Polymerization Promoting Protein/p25 occurs in monomeric and dimeric forms. ► The binding of GTP shifts the equilibrium towards the oligomerization of the protein. ► Mathematical model was elaborated to characterize the multiple equilibrium system. ► The dimeric form has higher tubulin polymerization promoting potency. ► The dimer may play a role in the fine tuning of microtubule assembly in vivo.
Keywords: Tubulin Polymerization Promoting Protein/p25; GTP binding; Oligomerization-dependent function; Mathematical modeling for multiple equilibrium;

Photodynamic therapy (PDT) that induces oxidative stress and cell death is used for tumor destruction in oncology. To characterize early molecular events in photosensitized glioblastoma cells, we studied expression of 224 proteins after sublethal PDT that doesn't kill but wounds cells.Cultured glioblastoma D54Mg cells were photosensitized with 5-aminolevulinic acid so that cell survival was 95–100%. At following 0.5–5.5 h protein expression and phosphorylation was assayed using proteomic antibody microarrays.Within the first post-treatment hour we observed phosphorylation of protein kinase Raf, adhesion-related kinases FAK and Pyk2, and microtubule-associated protein tau. Protein kinase Cγ and microtubule-associated protein MAP-1B were overexpressed. Dystrophin, calponin, and vinculin, components of the actin cytoskeleton scaffold, microtubule-associated proteins MAP2 and CNP, cytokeratins 4 and 7 were down-regulated that indicated changes in adhesion and cell shape. Down-regulation of cyclins A, D1 and D3, c-Myc, checkpoint proteins chk1/2 and up-regulation of Smad4 could arrest the cell cycle. Overexpression of Bcl-xL and down-regulation of caspase 9 demonstrated anti-apoptotic response. At 2 h post-treatment protein expression changed lesser but at 5.5 h levels of PKCγ and β-synuclein and phosphorylation of Raf, FAK, Pyk2, and tau increased again.Sub-lethal PDT induces complex response of glioblastoma cells including changes in activity and expression of proteins involved in adhesion-mediated signaling, signal transduction, cytoskeleton remodeling, cell cycle regulation and anti-apoptotic processes.Multiple reactions of various cellular subsystems including adhesion, cytoskeleton, signal transduction, cell cycle, and apoptosis are integrated into the general cell response to a sublethal impact.Display Omitted► Phosphorylation of protein kinases Raf, FAK and Pyk2 and overexpression of PKCγ. ► Down-regulation of actin skeleton-related proteins dystrophin, calponin and vinculin. ► Various changes of microtubule-associated proteins tau, MAP-1B, MAP2 and CNP. ► Down-regulation of cMyc, some cyclins and checkpoint proteins inhibits proliferation. ► Antiapoptotic response with Bcl-xL overexpression and caspase 9 down-regulation.
Keywords: Proteomics; Antibody microarray; Signaling; Cytoskeleton; Proliferation; Adhesion;

Galectin-3 endocytosis by carbohydrate independent and dependent pathways in different macrophage like cell types by Adriana Lepur; Michael C. Carlsson; Ruđer Novak; Jerka Dumić; Ulf J. Nilsson; Hakon Leffler (804-818).
Galectin-3 (the Mac-2 antigen) is abundantly expressed in both macrophage like cells and certain non-macrophage cells. We have studied endocytosis of galectin-3 as one important step relevant for its function, and compared it between variants of a macrophage like cell line, and non-macrophage cells.Endocytosis of galectin-3 was observed by fluorescence microscopy and measured by flow cytometry. The endocytosis mechanism was analysed using galectin-3 mutants, galectin-3 inhibitors and endocytic pathways inhibitors in the human leukaemia THP-1 cell line differentiated into naïve (M0), classical (M1) or alternatively activated (M2) macrophage like cells, and the non-macrophage cell lines HFL-1 fibroblasts and SKBR3 breast carcinoma.Galectin-3 endocytosis in non-macrophage cells and M2 cells was blocked by lactose and a potent galectin-3 inhibitor TD139, and also by the R186S mutation in the galectin-3 carbohydrate recognition domain (CRD). In M1 cells galectin-3 endocytosis could be inhibited only by chlorpromazine and by interference with the non-CRD N-terminal part of galectin-3. In all the cell types galectin-3 entered early endosomes within 5–10 min, to be subsequently targeted mainly to non-degradative vesicles, where it remained even after 24 h.Galectin-3 endocytosis in M1 cells is receptor mediated and carbohydrate independent, while in M2 cells it is CRD mediated, although the non-CRD galectin-3 domain is also involved.General significanceThe demonstration that galectin-3 endocytosis in M1 macrophages is carbohydrate independent and different from M2 macrophages and non-macrophage cells, suggests novel, immunologically significant interactions between phagocytic cells, galectin-3 and its ligands.► Galectin-3 is endocytosed in macrophage-like cells within minutes. ► Galectin-3 endocytosis in M1 cells can be carbohydrate independent. ► The uptake mechanism is different for M1, M2 and other cells. ► In macrophage-like cells galectin-3 enhances its own uptake. ► Most endocytosed galectin-3 is not targeted for degradation but remains after 24 h.
Keywords: Endocytosis; Galectin-3; M1 macrophages; M2 macrophages; THP-1 cell line;

Collagen adhesin–nanoparticle interaction impairs adhesin's ligand binding mechanism by Aribam Swarmistha Devi; Yohsuke Ogawa; Yoshihiro Shimoji; Subramanian Balakumar; Karthe Ponnuraj (819-828).
Pathogenic bacteria specifically recognize extracellular matrix (ECM) molecules of the host (e.g. collagen, fibrinogen and fibronectin) through their surface proteins known as MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) and initiate colonization. On implantation, biomaterials easily get coated with these ECM molecules and the MSCRAMMs mediate bacterial adherence to biomaterials. With the rapid rise in antibiotic resistance, designing alternative strategies to reduce/eliminate bacterial colonization is absolutely essential.The Rhusiopathiae surface protein B (RspB) is a collagen‐binding MSCRAMM of Erysipelothrix rhusiopathiae. It also binds to abiotic surfaces. The crystal structure of the collagen‐binding region of RspB (rRspB31–348) reported here revealed that RspB also binds collagen by a unique ligand binding mechanism called “Collagen Hug” which is a common theme for collagen‐binding MSCRAMMs of many Gram-positive bacteria. Here, we report the interaction studies between rRspB31–348 and silver nanoparticles using methods like gel shift assay, gel permeation chromatography and circular dichroism spectroscopy.The “Collagen Hug” mechanism was inhibited in the presence of silver nanoparticles as rRspB31–348 was unable to bind to collagen. The total loss of binding was likely because of rRspB31–348 and silver nanoparticle protein corona formation and not due to the loss of the structural integrity of rRspB31–348 on binding with nanoparticles as observed from circular dichroism experiments.Interaction of rRspB31–348 with silver nanoparticle impaired its ligand binding mechanism. Details of this inhibition mechanism may be useful for the development of antimicrobial materials and antiadhesion drugs.► The crystal structure of collagen‐binding region of RspB, rRspB31–348, was determined. ► rRspB31–348 binds to collagen by a unique mechanism called “Collagen Hug”. ► Bacterial adhesins were shown to be involved in biomaterial infection. ► Nanoparticle coated biomaterials showed reduced bacterial colonization on it. ► rRspB31–348–silver nanoparticle interaction impaired “Collagen Hug” mechanism.
Keywords: Rhusiopathiae surface protein B (RspB); Crystal structure; Collagen Hug model; Silver nanoparticle; Biomaterial; Antiadhesive property;

Different endocytic functions of AGEF-1 in C. elegans coelomocytes by Lichun Tang; Hanna Fares; Xingfu Zhao; Wei Du; Bi-Feng Liu (829-840).
ADP-ribosylation factors (ARFs) are a family of small GTP-binding proteins that play roles in membrane dynamics and vesicle trafficking. AGEF-1, which is thought to act as a guanine nucleotide exchange factor of class I ARFs, is required for caveolin-1 body formation and receptor-mediated endocytosis in oocytes of Caenorhabditis elegans. This study explores additional roles of AGEF-1 in endocytic transport. agef-1 expression was knocked down by using RNAi in C. elegans. Markers that allow analysis of endocytic transport in scavenger cells were investigated for studying the effect of AGEF-1 on different steps of membrane transport.Knockdown of AGEF-1 levels results in two apparent trafficking defects in coelomocytes of C. elegans. First, there is a delay in the uptake of solutes from the extracellular medium. Second, there is a dramatic enlargement of the sizes of lysosomes, even though lysosomal acidification is normal and degradation still occurs.Our results suggest that AGEF-1 regulates endosome/lysosome fusion or fission events, in addition to earlier steps in endocytic transport.AGEF-1 is the first identified GTPase regulator that functions at the lysosome fusion or fission stage of the endocytic pathway. Our study provides insight into lysosome dynamics in C. elegans.► Reduced AGEF-1 levels in C. elegans results in Cup and lysosomal enlargement in CC. ► ARL-8 functions upstream of AGEF-1 in lysosomes but downstream in uptake. ► AGEF-1 may function in lysosomes independent of its ARF exchange activity. ► ARF-1 and ARF-3 are candidate proteins as AGEF-1 substrates for the Cup defect.
Keywords: C. elegans; Lysosomes; Endosomes; ARF-GEF; AGEF-1; ARL-8;

Induction of 6-sulfated glycans with cell adhesion activity via T-bet and GATA-3 in human helper T cells by Keiichiro Sakuma; Guo-Yun Chen; Masahiro Aoki; Reiji Kannagi (841-848).
Cell surface 6-sulfated glycans play important roles in various immunological events through cell-to-cell interactions. The 6-sulfation process is mediated by 6-sulfotransferase family isoenzymes. We previously demonstrated that GlcNAc6ST-1, one of the isoenzyme genes, is induced by GATA-3 and NF-κB in human helper T (Th) cells. However, transcriptional regulation of HEC-GlcNAc6ST, another isoenzyme important in Th cells, remains unclear.5′-RACE analysis, chromatin immunoprecipitation, and reporter assays were performed to reveal transcriptional regulation of HEC-GlcNAc6ST. RNA-knockdown and forced expression experiments were performed to demonstrate the contribution of HEC-GlcNAc6ST to the 6-sulfated glycan expression.We identified potential binding sites of Sp1, T-bet, and GATA-3 in the HEC-GlcNAc6ST promoter. Reporter assays indicated that transfection of Sp1 enhanced the activity, whereas mithramycin A, an Sp1-specific inhibitor, repressed it. Transfection of T-bet increased the activity, which was inhibited by introducing a mutation into the potential T-bet binding site. GATA-3 alone could not elevate the activity, although co-transfection of protein kinase A, which is known to enhance IL-5 transcription in Th2 cells through phosphorylation of GATA-3, caused elevation. RNA-knockdown and forced expression of HEC-GlcNAc6ST in Jurkat cells down- and up-regulated α2,6-sialylated 6-sulfo N-acetyllactosamine, a preferential ligand for B-cell-specific CD22 antigen, respectively.From these results, we concluded that T-bet and GATA-3 as well as Sp1 control the expression of glycan with cell-adhesion activity by regulating HEC-GlcNAc6ST transcription in Th cells.These results may provide a clue to biological regulation of Th-cell interaction with selectins and other carbohydrate-recognition molecules by T-bet and GATA-3.► Transcription of HEC-GlcNAc6ST is induced by T-bet, GATA-3, and Sp1 in Jurkat cells. ► Induction of HEC-GlcNAc6ST by GATA-3 requires cooperation of protein kinase A. ► HEC-GlcNAc6ST contributes to the expression of 6-sulfated glycan ligands for CD22.
Keywords: HEC-GlcNAc6ST; 6-Sulfotransferase; Helper T cells; Transcriptional regulation; Glycan; CD22;

In yeast, 14-3-3 proteins bind to hundreds of phosphorylated proteins and play a role in the regulation of many processes including tolerance to NaCl. However, the mechanism of 14-3-3 involvement in the cell answer to salt or osmotic stresses is weakly understood.We studied the role of the Saccharomyces cerevisiae 14-3-3 homologs Bmh1 and Bmh2 in the regulation of alkali-metal-cation homeostasis using the genetic-interaction approach. Obtained results were confirmed with the Bimolecular-Fluorescence-Complementation method.Deletion of BMH1, encoding the major 14-3-3 isoform, resulted in an increased sensitivity to Na+, Li+ and K+ and to cationic drugs but did not affect membrane potential. This bmh1Δ phenotype was complemented by overexpression of BMH2. Testing the genetic interaction between BMH genes and genes encoding plasma-membrane cation transporters revealed, that 14-3-3 proteins neither interact with the potassium uptake systems, nor with the potassium-specific channel nor with the Na+(K+)-ATPases. Instead, a genetic interaction was identified between BMH1 and NHA1 which encodes an Na+(K+)/H+ antiporter. In addition, a physical interaction between 14-3-3 proteins and the Nha1 antiporter was shown. This interaction does not depend on the phosphorylation of the Nha1 antiporter by Hog1 kinase. Our results uncovered a previously unknown interaction partner of yeast 14-3-3 proteins and provided evidence for the previously hypothesized involvement of Bmh proteins in yeast salt tolerance.Our results showed for the first time that the yeast 14-3-3 proteins and an alkali-metal-cation efflux system interact and that this interaction enhances the cell survival upon salt stress.► Yeast 14-3-3p participates in cation homeostasis and cationic-drug resistance. ► A genetic interaction was identified between yeast BMH1 and NHA1. ► A physical interaction between 14-3-3 proteins and Nha1 antiporter was shown. ► Interaction of 14-3-3p and Nha1 enhances the yeast survival upon salt stress.
Keywords: 14-3-3 protein; Cation homeostasis; Nha1 antiporter; Saccharomyces cerevisiae; Salt stress;

Cellular retinol-binding protein, type 1 (Crbp1), chaperones retinyl ester (RE) biosynthesis catalyzed by lecithin:retinol acyltransferase (LRAT).We monitored the subcellular loci of LRAT and Crbp1 before and during RE biosynthesis, and compared the results to diacylglycerol:acyltransferase type 2 (DGAT2) during triacylglycerol biosynthesis in three cell lines: COS7, CHO and HepG2.Before initiation of RE biosynthesis, LRAT distributed throughout the endoplasmic reticulum (ER), similar to DGAT2, and Crpb1 localized with mitochondria associated membranes (MAM), surrounded by LRAT. Upon initiating RE biosynthesis in cells transfected with low amounts of vector to simulate physiological expression levels, Crpb1 remained with MAM, and both Crbp1 and MAM re-localized with LRAT. LRAT formed rings around the growing lipid droplets. LRAT activity was higher in these rings relative to the general ER. LRAT-containing rings colocalized with the lipid-droplet surface proteins, desnutrin/adipose triglyceride lipase and perilipin 2. Colocalization with lipid droplets required the 38 N-terminal amino acid residues of LRAT, and specifically K36 and R38. Formation of rings around the growing lipid droplets did not require functional microtubules.These data indicate a relationship between LRAT and Crbp1 during RE biosynthesis in which MAM-associated Crpb1 and LRAT colocalize, and both surround the growing RE-containing lipid droplet. The N-terminus of LRAT, especially K36 and R38, is essential to colocalization with the lipid droplet.► LRAT and Crbp1 co-localize during retinyl ester biosynthesis. ► LRAT and Crbp1 surround lipid droplets during retinyl ester formation. ► Crbp1 localizes in intact cells with mitochondrial associated membranes. ► The LRAT N-terminal thirty-eight residues direct it to lipid droplets.
Keywords: Acyltransferase; Lipid droplet; Retinol; Retinyl ester; Vitamin A;

Blebbistatin, a myosin inhibitor, is phototoxic to human cancer cells under exposure to blue light by Aliaksandr Mikulich; Simona Kavaliauskiene; Petras Juzenas (870-877).
Blebbistatin is a new inhibitor of cell motility. It is used to study dynamics of cytokinesis machinery in cells. However, the potential of this inhibitor as an anticancer agent has not been studied so far.Cytotoxicity of blebbistatin was evaluated in five human cell lines, FEMX-I melanoma, U87 glioma, androgen independent Du145 and androgen sensitive LNCaP prostate adenocarcinoma, and F11-hTERT immortalized fibroblasts. Phototoxicity of blebbistatin was assessed in these cell lines after their exposure to a blue light (390–470 nm). Photostability of blebbistatin and its reactive oxygen species (ROS) generating properties were measured during irradiation with the blue light.Blebbistatin at a concentration range of 10–200 μmol/L was toxic to all studied cells. Toxic concentrations (TC) were about 10–25 μmol/L corresponding to TC10, 50–100 μmol/L to TC50 and 140–190 μmol/L to TC90. Only for the U87 glioma cells TC90 could not be measured as the highest studied concentration of 200 μmol/L gave around 70% toxicity. However, after exposure to the blue light blebbistatin exhibited phototoxicity on the cells, with a cytotoxicity enhancement ratio that was greatest for the FEMX-I cells (about 9) followed by LNCaP (5), Du145 (3), U87 (2) and F11-hTERT (1.7) cells.Blebbistatin inhibits cell motility and viability. Under exposure to the blue light blebbistatin exhibits photodynamic action on human cancer cells. During the irradiation blebbistatin oxidizes dihydrorhodamine 123 but not Singlet Oxygen Sensor Green.Our findings offer new possibilities for blebbistatin as a potential anticancer and photodynamic agent.► The study shows blebbistatin as a potential photodynamic drug to kill cancer cells. ► Extent of the photodynamic effect varies for different cell lines. ► During light exposure blebbistatin generates ROS but not singlet oxygen. ► Blebbistatin is photodegraded along with the formation of its photoproducts.
Keywords: Free radical; Photosensitization; Photodynamic therapy (PDT); Reactive oxygen species (ROS); Scavenger; Singlet oxygen;

Corrigendum to “S-allyl cysteine in combination with clotrimazole downregulates Fas induced apoptotic events in erythrocytes of mice exposed to lead” [Biochim. Biophys. Acta 1820 (2012) 9–23] by Samir Mandal; Sudip Mukherjee; Kaustav Dutta Chowdhury; Avik Sarkar; Kankana Basu; Soumosish Paul; Debasish Karmakar; Mahasweta Chatterjee; Tuli Biswas; Gobinda Chandra Sadhukhan; Gargi Sen (878).

Honokiol, a naturally occurring biphenyl, possesses anti-neoplastic properties. We investigated activities of honokiol against adult T-cell leukemia (ATL) associated with human T-cell leukemia virus type 1 (HTLV-1).Cell viability was assessed using colorimetric assay. Propidium iodide staining was performed to determine cell cycle phase. Apoptotic effects were evaluated by 7A6 detection and caspases activity. Expressions of cell cycle- and apoptosis-associated proteins were analyzed by Western blot. We investigated the efficacy of honokiol in mice harboring tumors of HTLV-1-infected T-cell origin.Honokiol exhibited cytotoxic activity against HTLV-1-infected T-cell lines and ATL cells. We identified two different effects of honokiol on HTLV-1-infected T-cell lines: cell cycle inhibition and induction of apoptosis. Honokiol induced G1 cell cycle arrest by reducing the expression of cyclins D1, D2, E, CDK2, CDK4, CDK6 and c-Myc, while apoptosis was induced via reduced expression of cIAP-2, XIAP and survivin. The induced apoptosis was also associated with activation of caspases-3 and -9. In addition, honokiol suppressed the phosphorylation of IκBα, IKKα, IKKβ, STAT3, STAT5 and Akt, down-regulated JunB and JunD, and inhibited DNA binding of NF-κB, AP-1, STAT3 and STAT5. These effects resulted in the inactivation of survival signals including NF-κB, AP-1, STATs and Akt. Honokiol was highly effective against ATL in miceOur data suggested that honokiol is a systemically available, non-toxic inhibitor of ATL cell growth that should be examined for potential clinical application.Our findings provide a rationale for clinical evaluation of honokiol for the management of ATL.Display Omitted► Honokiol had cytotoxic activity against HTLV-1-infected T-cell lines and ATL cells. ► Honokiol reduced the expression of cyclins D1, D2, E, CDKs 2, 4, 6 and c-Myc. ► Apoptosis was induced via reduced expression of cIAP-2, XIAP and survivin. ► Honokiol was effective in mice harboring tumors of HTLV-1-infected T-cell origin. ► Inhibition of survival signals including NF-κB, AP-1, STATs and Akt confers effects.
Keywords: Honokiol; Adult T-cell leukemia; Human T-cell leukemia virus type 1; Cell survival; Signaling;

Plexin-B3 interacts with EB-family proteins through a conserved motif by Piret Laht; Kaie Pill; Elina Haller; Andres Veske (888-893).
Plexins are transmembrane receptors that are highly expressed in the central nervous system. They participate in the patterning of neural connections and regulation of cell adhesion and motility in many cell types. The aim of this study was to characterize novel protein–protein interactions of plexin-B3 intracellular portion.To identify new interactors of plexin-B3 yeast two-hybrid screen was performed. We used GST pull-down and co-immunoprecipitation to verify those results. Deletion mutants were used to map the interacting regions. The physiological relevance of this interaction was assessed with neurite outgrowth assay in Neuro2A cell line.We show that the N-terminal segment of intracellular domain of plexin-B3 interacts with microtubule plus end-binding proteins EB1, EB2 and EB3. The corresponding region in human plexin-A2, B1 and B3 contains the conserved EB-binding motif SxIP and these plexins also associate with EBs indicating the specificity of plexin-EB binding. As to the EB proteins, their N-terminal microtubule-binding domain is dispensable for plexin interaction. Plexin-EB interaction is involved in neurite growth as the synthetic peptide corresponding to the EB-binding region of plexin-B1 increases significantly the number of neurite tips in Neuro2A cells.Microtubule end-binding proteins EB1, EB2 and EB3 interact with plexin-A2, B1 and B3 through a conserved EB-binding motif, which is located in their intracellular domain N-terminal segment.The observed interaction between plexin intracellular domain and EBs suggests a novel function for plexins in regulating EB-mediated changes in microtubule dynamics and neurite growth.► Microtubule end-binding EB proteins are novel interactors for plexins. ► EB proteins form complexes with plexin-A2, B1 and B3 but not with plexin-B2. ► Plexins that interact with EBs contain the conserved EB-binding motif SxIP. ► Plexin-B1 peptide containing the EB-binding motif modulates neurite growth.
Keywords: Plexins; End-binding proteins; EB-binding motif;

Plant-derived polyphenols regulate expression of miRNA paralogs miR-103/107 and miR-122 and prevent diet-induced fatty liver disease in hyperlipidemic mice by Jorge Joven; Eugenia Espinel; Anna Rull; Gerard Aragonès; Esther Rodríguez-Gallego; Jordi Camps; Vicente Micol; María Herranz-López; Javier A. Menéndez; Isabel Borrás; Antonio Segura-Carretero; Carlos Alonso-Villaverde; Raúl Beltrán-Debón (894-899).
MicroRNAs have the potential for clinical application. Probable modulation by plant-derived polyphenols might open preventive measures using simple dietary recommendations.We assessed the ability of continuous administration of high-dose polyphenols to modulate hepatic metabolism and microRNA expression in diet-induced fatty liver disease in commercially available hyperlipidemic mice using well-established and accepted procedures that included the development of new antibodies against modified quercetin.Weight gain, liver steatosis, changes in the composition of liver tissue, and insulin resistance were all attenuated by the continuous administration of polyphenols. We also demonstrated that metabolites of polyphenols accumulate in immune cells and at the surface of hepatic lipid droplets indicating not only bioavailability but a direct likely action on liver cells. The addition of polyphenols also resulted in changes in the expression of miR-103, miR-107 and miR-122.Polyphenols prevent fatty liver disease under these conditions. The differential expression of mRNAs and miRNAs was also associated with changes in lipid and glucose metabolism and with the activation of 5′-adenosine monophosphate-activated protein kinase, effects that are not necessarily connected. miRNAs function via different mechanisms and miRNA–mRNA interactions are difficult to ascertain with current knowledge. Further, cell models usually elicit contradictory results with those obtained in animal models.Our data indicate that plant-derived polyphenols should be tested in humans as preventive rather than therapeutic agents in the regulation of hepatic fatty acid utilization. A multi-faceted mechanism of action is likely and the regulation of liver miRNA expression blaze new trails in further research.► MicroRNAs are modulated by polyphenols. ► Mice with diet-induced fatty liver disease were used to test interactions. ► Polyphenols accumulated in the liver and attenuated deleterious effects. ► Polyphenols induced changes in miR-103/107 and miR-122 expression. ► Regulation of liver fatty acid utilization is a probable common mechanism of action.
Keywords: Fatty acid synthase; Insulin sensitivity; Lipid metabolism; miRNA–mRNA interaction; PANK; PPARs;

In vivo up-regulation of the unfolded protein response after hypoxia by Luigina Tagliavacca; Anna Caretti; Paola Bianciardi; Michele Samaja (900-906).
Low oxygen (O2) availability, a condition called hypoxia, has different and profound consequences in tissues and organs. Besides the hypoxia-inducible response, mammalian cells induce a coordinated cytoprotective pathway called Unfolded Protein Response (UPR). We studied the molecular basis of UPR and apoptosis in animal models exposed to different hypoxic stresses and assessed the ability of liver and myocardium to respond to low oxygen by activating different arms of the UPR according to the severity of the insults in a tissue specific manner.We assessed the levels of several UPR markers in hypoxic animals by Real Time PCR and Western blotting.While the hepatocytes activate the apoptotic pathway mediated, in part, by CHOP and p-JNK, we could not detect an UPR-dependent apoptosis in myocytes. Moreover, severe hypoxia results in ATF4 translation, and induction of CHOP and GADD34 transcripts in liver, by contrast in the myocardium, the ATF4-CHOP-GADD34 signaling pathway is not detectably activated.Comparison of several UPR markers in liver and myocardium enabled to underscore the ability of hepatocytes and myocites to selectively activate and fine tune the UPR signaling pathway during hypoxia in vivo.► We monitored the UPR in liver and myocardium, tissues that differ in O2 requirement. ► Mice exposure to 6.5% O2 resulted in ATF4, CHOP and GADD34 activation in liver. ► In the myocardium the ATF4-CHOP-GADD34 markers were not detectably activated. ► The apoptotic pathway mediated by CHOP and p-JNK was activated in hepatocytes only. ► Hepatocytes and myocites fine tune the UPR signaling pathway in in vivo hypoxia.
Keywords: Hypoxia; Unfolded protein response; In vivo animal models; Apoptosis;

Cardiac cell apoptosis is the initiating factor of cardiac complications especially diabetic cardiomyopathy. Mitochondria are susceptible to the damaging effects of elevated glucose condition. Calcium overload and oxidative insult are the two mutually non-exclusive phenomena suggested to cause cardiac dysfunction. Here, we examined the effect of high-glucose induced calcium overload in calpain-1 mediated cardiac apoptosis in an in vitro setting.H9c2, rat ventricular myoblast cell line was treated with elevated glucose condition and the cellular consequences were studied. Intracellular calcium trafficking, ROS generation, calpain-1 activation and caspase-12 and caspase-9 pathway were studied using flow cytometry, confocal microscopy and Western blot analysis.High-glucose treatment resulted in increased intracellular calcium ([Ca2 +]i) which was mobilized to the mitochondria. Concomitant intra-mitochondrial calcium ([Ca2 +]m) increase resulted in enhanced reactive oxygen and nitrogen species generation. These events led to mitochondrial dysfunction and apoptosis. Cardiomyocyte death exhibited several classical markers of apoptosis, including activation of caspases, appearance of annexin V on the outer plasma membrane, increased population of cells with sub-G0/G1 DNA content and nuclear condensation. Key findings include elucidation of cell signaling mechanism of high-glucose induced calcium-dependent cysteine protease calpain-1 activation, which triggers non-conventional caspases as alternate mode of cell death.This information increases the understanding of cardiac cell death under hyperglycemic condition and can possibly be extended for designing new therapeutic strategies for diabetic cardiomyopathy.The novel findings of the study reveal that high glucose induces apoptosis by both mitochondria-dependent and independent pathways via concomitant rise in intracellular calcium.
Keywords: H9c2 cells; Mitochondrion; High glucose; Apoptosis; Calcium overload; Reactive oxygen species;

αB-crystallin/sHSP protects cytochrome c and mitochondrial function against oxidative stress in lens and retinal cells by Rebecca S. McGreal; Wanda Lee Kantorow; Daniel C. Chauss; Jianning Wei; Lisa A. Brennan; Marc Kantorow (921-930).
αB-crystallin/sHSP protects cells against oxidative stress damage. Here, we mechanistically examined its ability to preserve mitochondrial function in lens and retinal cells and protect cytochrome c under oxidative stress conditions.αB-crystallin/sHSP was localized in human lens (HLE-B3) and retinal (ARPE-19) cells. αB-crystallin/sHSP was stably over-expressed and its ability to preserve mitochondrial membrane potential under oxidative stress conditions was monitored. Interactions between αB-crystallin/sHSP and cytochrome c were examined by fluorescent resonance energy transfer (FRET) and by co-immune precipitation. The ability of αB-crystallin/sHSP to protect cytochrome c against methionine-80 oxidation was monitored.αB-crystallin/sHSP is present in the mitochondria of lens and retinal cells and is translocated to the mitochondria under oxidative conditions. αB-crystallin/sHSP specifically interacts with cytochrome c in vitro and in vivo and its overexpression preserves mitochondrial membrane potential under oxidative stress conditions. αB-crystallin/sHSP directly protects cytochrome c against oxidation.These data demonstrate that αB-crystallin/sHSP maintains lens and retinal cells under oxidative stress conditions at least in part by preserving mitochondrial function and by protecting cytochrome c against oxidation. Since oxidative stress and loss of mitochondrial function are associated with eye lens cataract and age-related macular degeneration, loss of these αB-crystallin/sHSP functions likely plays a key role in the development of these diseases.αB-crystallin/sHSP is expressed throughout the body and its ability to maintain mitochondrial function is likely important for the prevention of multiple degenerative diseases.► Increased αB-crystallin/sHSP levels occur in oxidative stress diseases. ► αB-crystallin/sHSP protects mitochondria and cyt c against oxidative inactivation. ► Mitochondrial protection by αB-crystallin/sHSP prevents oxidative stress diseases.
Keywords: Cytochrome c; Oxidation; αB-crystallin; Small heat shock protein; Mitochondrion;

Cytotoxic effects of copper overload on human-derived lung and liver cells in culture by Nathalie Arnal; María J. Tacconi de Alaniz; Carlos Alberto Marra (931-939).
Copper (Cu) is an essential trace metal used as a catalytic cofactor for many enzymes. However, it can have nocive effects when it participates in the Fenton reaction, producing reactive oxygen species (ROS). Excess Cu is present in the plasma of patients with diseases in which cell survival is crucial. In order to investigate the effect of Cu overload on the induction of cellular damage we chose two human cell lines derived from liver (HepG2) and lung (A-549) as representative cells exposed to exogenous (polluted air) and/or endogenous (systemic) Cu overload.We studied ROS production using thiobarbituric acid reactive substances (TBARS) and fluorimetric measurements with dichlorofluorescein, cell viability by the trypan dye exclusion test, the methyltetrazolium (MTT) and lactate dehydrogenase leakage (LDH) assays, various cytotoxic indexes, and caspasa-3 and calpain-dependent activation as the main signals involved in the apoptosis pathway.Cu overload induces cell death by a differential activation of calpains (m- and μ-) and caspase-3, and modifies various proliferative indexes in a cell-type and concentration-dependent manner. The involvement of these two protease systems and the response of the two main Cu homoestatic proteins ceruloplasmin and metallothioneins are specific to each cell type. We demonstrated that Cu can trigger cell death by activation of specific protease systems and modify various proliferative indexes in a cell-type and concentration-dependent manner.These findings contribute to understanding the diverse effects of Cu overload on the pathogenesis of human diseases like cancer, cirrhosis and degenerative disorders.► Cu overload induce oxidative stress in HepG2 and A-549 human-derived cells. ► The reaction of Cu homeostatic proteins is different in each kind of cells. ► Cell survival after Cu overload varied as a function of cell type. ► Caspase-3 and calpains are differentially activated as a function of Cu overload. ► Various proliferative indexes are affected by Cu uptake.
Keywords: Copper; Cell division; Oxidative stress; Lung; Liver; Apoptosis;

Exosomes: Current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials by Alexander V. Vlassov; Susan Magdaleno; Robert Setterquist; Rick Conrad (940-948).
Cells continuously secrete a large number of microvesicles, macromolecular complexes, and small molecules into the extracellular space. Of the secreted microvesicles, the nanoparticles called exosomes are currently undergoing intense scrutiny. These are small vesicles (30–120 nm) containing nucleic acid and protein, perceived to be carriers of this cargo between diverse locations in the body. They are distinguished in their genesis by being budded into endosomes to form multivesicular bodies (MVBs) in the cytoplasm. The exosomes are released to extracellular fluids by fusion of these multivesicular bodies with the cell surface, resulting in secretion in bursts. Exosomes are secreted by all types of cells in culture, and also found in abundance in body fluids including blood, saliva, urine, and breast milk.In this review, we summarize strategies for exosome isolation, our understanding to date of exosome composition, functions, and pathways, and discuss their potential for diagnostic and therapeutic applications.Currently, the control of exosome formation, the makeup of the “cargo”, biological pathways and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication — exosomes are thought to function as the messengers, delivering various effectors or signaling macromolecules between supposedly very specific cells.Both seasoned and newer investigators of nanovesicles have presented various viewpoints on what exosomes are, with some differences but a large common area. It would be useful to develop a codified definition of exosomes in both descriptive and practical terms. We hope this in turns leads to a consistent set of practices for their isolation, characterization and manipulation.► Exosomes are microvesicles containing nucleic acid and protein, secreted by all cells. ► Exosomes are found in abundance in all body fluids including blood, saliva, urine. ► Exosomes most intriguing role is intercellular communication. ► We describe exosomes composition, functions, and pathways. ► We discuss exosomes potential for diagnostic and therapeutic applications.
Keywords: Exosome; Microvesicle; Extracellular nucleic acid; Signaling;

CD36 promotes adipocyte differentiation and adipogenesis by Valerie Christiaens; Matthias Van Hul; H. Roger Lijnen; Ilse Scroyen (949-956).
CD36 is a membrane glycoprotein, contributing to the pathogenesis of metabolic disorders, like obesity, which has become a major health concern worldwide.A potential functional role of the scavenger receptor CD36 was investigated in in vitro adipocyte differentiation and in vivo adipogenesis.During differentiation of 3T3-F442A preadipocytes into mature adipocytes, expression of CD36 was upregulated and CD36 gene silencing resulted in impaired differentiation, as monitored by Oil Red O staining and expression of adipogenic markers. De novo fat pad formation in NUDE mice following injection of preadipocytes was significantly reduced upon CD36 gene silencing as compared to control. This was associated with marked adipocyte hypotrophy and reduced adipose tissue adipocyte content. Macrophage infiltration in de novo fat tissues derived from preadipocytes with CD36 gene silencing was not significantly different from controls. Collagen content was significantly higher in de novo fat with CD36 gene silencing. In a nutritionally induced obesity model, total body weight as well as subcutaneous and gonadal adipose tissue mass were significantly lower in CD36 deficient mice as compared to wild-type littermates.Thus, our data support a functional role of CD36 in promoting adipogenesis in vitro as well as in vivo. ► In vitro CD36 gene silencing in preadipocytes impairs differentiation. ► CD36 downregulation impairs de novo fat pad formation in vivo. ► CD36 deficiency impairs adipose tissue development.
Keywords: (Pre)adipocyte; Adipogenesis; Adipose tissue; CD36;

Protein crowding impedes pressure-induced unfolding of staphylococcal nuclease by Suntao Wang; Mark W. Tate; Sol M. Gruner (957-961).
In the cellular environment, macromolecules occupy about 30% of a cell's volume. In this crowded environment, proteins behave very differently than in dilute solution where scientists typically study the properties of proteins. For this reason, recent studies have investigated proteins in cell-like crowded conditions so as to understand if this changes their properties. The present study was performed to examine if molecular crowding impedes the protein unfolding process that is known to occur upon the application of high pressure.Crowding of staphylococcal nuclease (SNase) was induced by dissolving low concentrations of SNase in high concentrations of crowding agents (16 wt.% or 25 wt.% PEG 3000 or 16 wt.% Dextran T10). SNase unfolding was then monitored via tryptophan fluorescence as pressure was applied.Fluorescence spectra can be decomposed into the sum of two components indicative, respectively, of native and unfolded states, and the center of spectral mass was then used as a measure of the degree of protein unfolding. It was found that SNase unfolding as a function of pressure was impeded in crowded solutions. These results suggest that crowded environments, such as those found in the cellular cytoplasm, may also impede high-pressure protein unfolding in cells.This is the first report on the effect of crowding on the pressure-induced unfolding of a protein (staphylococcal nuclease) monitored via tryptophan fluorescence.► We measure high-pressure Trp-fluorescence spectra of staphylococcal nuclease (SNase). ► We examine three SNase samples in the presence of crowding agents. ► All the samples exhibit a two-state behavior (native and unfolded states). ► We find SNase unfolding as a function of pressure is impeded in crowded solutions. ► The result suggests this crowding effect may also apply to the proteins in cells.
Keywords: Crowding; Protein unfolding; Staphylococcal nuclease; High pressure; Fluorescence;

Evaluation of in vitro and in vivo anti-melanogenic activity of a newly synthesized strong tyrosinase inhibitor (E)-3-(2,4 dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP) by Ki Wung Chung; Yun Jung Park; Yeon Ja Choi; Min Hi Park; Young Mi Ha; Yohei Uehara; Jung Hyun Yoon; Pusoon Chun; Hyung Ryong Moon; Hae Young Chung (962-969).
Tyrosinase inhibitors have become increasingly important because of their ability to inhibit the synthesis of the pigment melanin. A search for new agents with strong tyrosinase activity led to the synthesis of the tyrosinase inhibitor (E)-3-(2,4-dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP).The inhibitory effect of 3-DBP on tyrosinase activity and melanin production was examined in murine melanoma B16F10 cells. Additional experiments were performed using HRM2 hairless mice to demonstrate the effects of 3-DBP in vivo.The novel compound, 3-DBP, showed an inhibitory effect against mushroom tyrosinase (IC50  = 0.53 μM), which indicated that it was more potent than the well-known tyrosinase inhibitor kojic acid (IC50  = 8.2 μM). When tested in B16F10 melanoma cells treated with α-melanocyte stimulating hormone (α-MSH), 3-DBP also inhibited murine tyrosinase activity, which in turn induced a decrease in melanin production in these cells. The anti-melanogenic effect of 3-DBP was further verified in HRM2 hairless mice. The skin-whitening index (L value) of HRM2 hairless mice treated with 3-DBP before irradiation with UVB was greater than that of UVB-irradiated mice that were not treated with 3-DBP.The newly synthesized 3-DBP has a potent inhibitory effect on tyrosinase. In addition to an in vitro investigation of the effects of 3-DBP on tyrosinase, in vivo studies using an HRM2 hairless mouse model demonstrated the anti-melanogenic potency of 3-DBP. Our newly synthesized 3-DBP showed efficient tyrosinase inhibitory effect in vivo and in vitro. Our finding suggests that 3-DBP can be an effective skin-whitening agent.► We designed and synthesized 3-DBP as a potent tyrosinase inhibitor. ► The depigmenting effect of 3-DBP was resulted from inhibition of tyrosinase non-competitively. ► 3-DBP suppressed α-MSH induced melanin synthesis in B16 melanoma cells. ► 3-DBP showed great efficiency in reducing UVB-induced melanin synthesis in HRM2 hairless mouse in vivo model. ► 3-DBP can be an effective skin-whitening agent.
Keywords: Melanogenesis; (E)-3-(2,4-dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP); Tyrosinase inhibitor; HRM2 hairless mouse;

Abnormally high activity of protein kinase CK2 is linked to various diseases including cancer. Therefore, the inhibition of CK2 is a promising therapeutic strategy to fight this disease.We screened a library of synthetic molecules concerning their capacity to inhibit CK2. The activity of CK2 and their IC50 and Ki values were determined by a capillary electrophoresis assay. The effects of the inhibitor in a cell culture model were analyzed by cell counting, a viability assay, cytofluorimetry and Western blot.The best CK2 inhibitor found in this screen was 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)methylen]dibenzo [b,d]furan-3(2H)-one, which we refer to as “TF”. TF showed tight binding to CK2 with low IC50 (29 nM) and Ki (15 nM) values. TF inhibited only seven out of 61 human kinases tested (> 70% inhibition). Incubation of LNCaP cells with 50 μM TF for 48 h decreased the intracellular CK2 activity by 50%, confirming that the inhibitor is membrane permeable. The decrease in activity was correlated with a severe reduction in cell viability. The reduction in cell viability is at least partly due to the induction of apoptosis.In many cancers the protein kinase CK2 is significantly up-regulated and supports the neoplastic phenotype. New therapeutic strategies should be based on diverse reliable inhibitors to reverse the abnormal high levels to normal settings.Display Omitted► A new dibenzofuranone derivative (TF) is a selective inhibitor of the kinase CK2. ► TF is characterized by low IC50 (29 nM) and Ki (15 nM) values. ► TF is effective in strongly reducing cell viability of prostate cancer cells. ► TF is a new lead structure for the development of more effective inhibitors.
Keywords: Dibenzo[b,d]furan; CK2; LNCaP; Drug discovery; Inhibitor;

Ecklonia cava polyphenol protects the liver against ethanol-induced injury in rats by Mai Takahashi; Naoko Satake; Haruka Yamashita; Akiko Tamura; Mio Sasaki; Isao Matsui-Yuasa; Masaki Tabuchi; Yasumitsu Akahoshi; Masaki Terada; Akiko Kojima-Yuasa (978-988).
The development of alcoholic liver disease is a complex process that involves both the parenchymal and non-parenchymal cells of the liver. We examined the effect of an Ecklonia cava extract on ethanol-induced liver injury.Isolated hepatocytes and hepatic stellate cells (HSCs) were incubated with ethanol. Ecklonia cava polyphenol (ECP) was added to the cultures that had been incubated with ethanol. Male Wistar rats were fed a diet that included 0.02% or 0.2% ECP or no ECP. For a period of 3 weeks, the animals were given drinking water containing 5% ethanol and were also treated with carbon tetrachloride (CCl4) (0.1 ml/kg of body weight).In the cultured hepatocytes, the ECP treatment suppressed the ethanol-induced increase in cell death by maintaining intracellular glutathione (GSH) levels. In HSCs, ECP treatment suppressed the ethanol-induced increases in type I collagen and α-smooth muscle actin expression by maintaining intracellular levels of reactive oxygen species and GSH. We examined the effects of ECP on serum AST and ALT activity, as well as the progression of liver fibrosis in rats treated with ethanol and CCl4. ECP treatment suppressed plasma AST and ALT activities in the ethanol- and CCl4-treated rats. ECP treatment fully protected the rats against ethanol- and CCl4-induced liver injury.ECP may be a candidate for preventing ethanol-induced liver injury.► ECP suppressed ethanol-induced hepatocyte cell death. ► ECP treatment maintained the levels of intracellular MDA below the levels of control cells. ► Ethanol-induced type I collagen and α-SMA upregulation was suppressed by ECP treatment. ► ECP treatment maintained intracellular ROS and GSH levels. ► ECP treatment fully protected rats against ethanol- and CCl4-induced liver injury.
Keywords: Ethanol-induced liver injury; Ecklonia cava; Glutathione; Reactive oxygen species; Rat hepatocytes; Hepatic stellate cells;

The aim is to analyze the structure, anticoagulant and antithrombotic activities of a sulfated fucan isolated from sea cucumber Isostichopus badionotus (fucan-Ib).Fucan-Ib was hydrolyzed under mild acid conditions. The oligosaccharide fragments were fractionated by gel-filtration chromatography and the structures were determined by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation and two-dimensional NMR. Anticoagulant activities were measured by activated partial thromboplastin, thrombin and prothrombin times, and by in vitro inhibition experiments with factors IIa and Xa. Antithrombotic activities were determined in vitro by measuring the length and weight of the thrombus generated.The linear polysaccharide sequence of fucan-Ib was deduced from the structures of its oligosaccharide fragments produced by acid hydrolysis. Under mild conditions, the glycosidic bonds between the non-sulfated and 2,4-O-disulfated fucose residues were selectively cleaved and highly ordered oligosaccharide fragments with a tetrasaccharide repeating unit [→3Fuc(2S,4S)α1→3Fuc(2S)α1→3Fuc(2S)α1→3Fucα1→]n were obtained. In in vitro assays fucan-Ib showed good anticoagulant and antithrombotic activities compared with heparin and the fucosylated chondroitin sulfate isolated from the same source (fCS-Ib). The two polysaccharides, fucan-Ib and fCS-Ib, differ in the mechanism of action; the former exhibited activity mainly by potentiation of antithrombin acted on thrombin and factor Xa whereas the latter mainly through heparin cofactor II.Fucan-Ib has a well defined structure with tetrasaccharide tandem repeats and good anticoagulant and antithrombotic activities.Fucan-Ib has a well defined structure and can be readily quality-controlled, and therefore has potential therapeutic value as an affective antithrombotic drug with low risk of bleeding.► The structure fucan-Ib was determined by ES-MS/MS together with NMR. ► fucan-Ib had high antithrombotic activity but low anticoagulant activity. ► fucan-Ib has a well defined structure and can be readily for quality control. ► The therapeutic potential of fucan-Ib deserves further investigation.
Keywords: Sea cucumber; Fucan; ES-MS; NMR; Anticoagulant; Antithrombotic;

Na+-dependent and Na+-independent mechanisms for inorganic phosphate uptake in Trypanosoma rangeli by C.F. Dick; A.L.A. Dos-Santos; D. Majerowicz; K.C. Gondim; C. Caruso-Neves; I.V. Silva; A. Vieyra; J.R. Meyer-Fernandes (1001-1008).
Trypanosoma rangeli is dependent on the presence of exogenous orthophosphate (Pi) for maximal growth and ecto-phosphatase activity is responsible for Pi supply under low Pi. Here we investigated the mechanisms of Pi uptake.We investigated the kinetics of 32 Pi transport, its Na+ and H+ dependence, its correlation with the Na+-ATPase and H+-ATPase, and gene expression of the Na+:Pi cotransporter and Na+-ATPase. T. rangeli grown under limiting Pi transports this anion to the cytosol in the absence and presence of Na+, suggesting that influx is mediated by both Na+-independent and Na+-dependent transporters. Cloning studies demonstrated that this parasite expresses a Pi transporter not previously studied in trypanosomatids. The H+ ionophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, decreased both components of 32 Pi influx by 80–95%. The H+-ATPase inhibitor, bafilomycin A1, inhibited the Na+-independent mechanism. Furosemide, an inhibitor of ouabain-insensitive Na+-ATPase, decreased both uptake mechanisms of 32 Pi to the same extent, whereas ouabain had no effect, indicating that the former is the pump responsible for inwardly directed Na+ and the electric gradients required by the transporters. Parasite growth in high Pi had a lower Pi influx than that found in those grown in low Pi, without alteration in TrPho89 expression, showing that turnover of the transporters is stimulated by Pi starvation.Two modes of Pi transport, one coupled to Na+-ATPase and other coupled to H+-ATPase seem to be responsible for Pi acquisition during development of T. rangeli.This study provides the first description of the mechanism of Pi transport across the plasma membrane of trypanosomatids.► Trypanosoma rangeli expresses a Na+:Pi cotransporter. ► TrENA Na+-ATPase is responsible for the Na+ gradient required by Pi transport. ► Low Pi stimulates Pi uptake allowing its acquisition during parasite development.
Keywords: Trypanosoma rangeli; Inorganic phosphate transport; Sodium-independent phosphate uptake; Sodium-dependent phosphate uptake; Inorganic phosphate starvation;

Changes in glycosaminoglycans and proteoglycans of normal breast and fibroadenoma during the menstrual cycle by Cilene Rebouças de Lima; José de Arimatéa dos Santos; Afonso Celso Pinto Nazário; Yara M. Michelacci (1009-1019).
Fibroadenoma is the most common breast tumor in young women, and its growth and metabolism may be under hormonal control. In the present paper we described the proteoglycan (PG) composition and synthesis rate of normal breast and fibroadenoma during the menstrual cycle.Samples of fibroadenoma and adjacent normal breast tissue were obtained at surgery. PGs were characterized by agarose gel electrophoresis and enzymatic degradation with glycosaminoglycan (GAG) lyases, and immunolocalized by confocal microscopy. To assess the synthesis rate, PGs were metabolic labeled by 35S-sulfate.The concentration of PGs in normal breast was higher during the secretory phase. Fibroadenoma contained and synthesized more PGs than their paired controls, but the PG concentrations varied less with the menstrual cycle and, in contrast to normal tissue, peaked in the proliferative phase. The main mammary GAGs are heparan sulfate (HS, 71%–74%) and dermatan sulfate (DS, 26%–29%). The concentrations of both increased in fibroadenoma, but DS increased more, becoming 35%–37% of total. The DS chains contained more β‐d-glucuronic acid (IdoUA/GlcUA ratios were > 10 in normal breast and 2–7 in fibroadenoma). The 35S-sulfate incorporation rate revealed that the in vitro synthesis rate of DS was higher than HS. Decorin was present in both tissues, while versican was found only in fibroadenoma.In normal breast, the PG concentration varied with the menstrual cycle. It was increased in fibroadenoma, especially DS.General significancePGs are increased in fibroadenoma, but their concentrations may be less sensitive to hormonal control.► The concentration of PGs in normal human breast varies with the menstrual cycle. ► In fibroadenoma, PGs are increased and less sensitive to hormonal conditions. ► Dermatan sulfate and heparan sulfate are the main GAGs in breast and fibroadenoma. ► Fibroadenoma contains more dermatan sulfate, with higher GlcUA/IdoUA ratios. ► Versican was detected only in tumor.
Keywords: Fibroadenoma; Proteoglycan; Menstrual cycle; Normal breast; Decorin; Versican;

Corrigendum to “Hydogen peroxide-dependent photocytotoxicity by phloxine B, a xanthene-type food colorant” [Biochim. Biophys. Acta 1810 (2011) 704-712] by Hang Qi; Hiroshi Takano; Yoji Kato; Qian Wu; Chiharu Ogata; Beiwei Zhu; Yoshiyuki Murata; Yoshimasa Nakamura (1020).

Liver X receptor and peroxisome proliferator-activated receptor agonist from Cornus alternifolia by Yang-Qing He; Guo-Yi Ma; Jiang-nan Peng; Zhan-Ying Ma; Mark T. Hamann (1021-1026).
Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptors superfamily and are transcription factors activated by specific ligands. Liver X receptors (LXR) belong to the nuclear hormone receptors and have been shown to play an important role in cholesterol homeostasis. From the previous screening of several medicinal plants for potential partial PPARγ agonists, the extracts of Cornus alternifolia were found to exhibit promising bioactivity. In this paper, we report the isolation and structural elucidation of four new compounds and their potential as ligands for PPAR.The new compounds were extracted from the leaves of C. alternifolia and fractionated by high-performance liquid chromatography. Their structures were elucidated on the basis of spectroscopic evidence and analysis of their hydrolysis products.Three new iridoid glycosides including an iridolactone, alternosides A–C (13), a new megastigmane glycoside, cornalternoside (4) and 10 known compounds, were obtained from the leaves of C. alternifolia. Kaempferol-3-O-β-glucopyranoside (5) exhibited potent agonistic activities for PPARα, PPARγ and LXR with EC50 values of 0.62, 3.0 and 1.8 μM, respectively.We isolated four new and ten known compounds from C. alternifolia, and one known compound showed agonistic activities for PPARα, PPARγ and LXR.Compound 1 is the first example of a naturally occurring iridoid glycoside containing a β-glucopyranoside moiety at C-6.►The metabolites isolated show potential active agonists for PPAR and LXR receptors. ►Four new natural products 14 were isolated from the leaves of Cornus alternifolia. ►Metabolite 1 is the first iridoid glycoside containing a β-glucopyranoside at C-6. ►Metabolite 5 showed potent agonistic activities for PPARα, PPARγ and LXR. ►The results presented can be used for further synthetic and pharmacological studies.
Keywords: Peroxisome proliferator-activated receptors; Liver X receptors; Cornus alternifolia; Iridoid glycosides;

Sanguisorba minor extract suppresses plasmin-mediated mechanisms of cancer cell migration by Massimiliano Cuccioloni; Laura Bonfili; Matteo Mozzicafreddo; Valentina Cecarini; Anna Maria Eleuteri; Mauro Angeletti (1027-1034).
Sanguisorba minor, as well as several other edible herbs and vegetables, has been used extensively in traditional medicine. The observed beneficial effects can be attributed at least in part to the direct modulation of several enzymatic activities by its polyphenolic constituents.The ethanol extract of Sanguisorba minor was characterized by reversed-phase liquid chromatography, and most relevant analytes were identified by multiple stage mass spectrometry. The whole extract and the most relevant isolated constituents were tested for their ability to modulate the activity of human plasmin both toward a synthetic substrate and in human breast cancer cell culture models. Kinetic and equilibrium parameters were obtained by a concerted spectrophotometric and biosensor-based approach.Quercetin-3-glucuronide was recognized as the compound mainly responsible for the in vitro plasmin inhibition by S. minor extract, with an inhibition constant in the high nanomolar range; in detail, our approach based on bioinformatic, enzymatic and binding analyses classified the inhibition as competitive. Most interestingly, cell-based assays showed that this flavonoid was effective in suppressing plasmin-induced loss of cancer cell adhesion.Our results show that the extract from Sanguisorba minor limits plasmin-mediated tumor cell motility in vitro, mostly due to quercetin-3-glucuronide. This glucuronated flavonoid is a promising template for rational designing of anticancer drugs to be used in the treatment of pathological states involving the unregulated activity of plasmin.► Q3G is one of the major monomeric flavonoid of the S.minor extract. ► Q3G competitively targets the catalytic pocket of human plasmin with high affinity. ► Q3G efficiently down-regulates plasmin-mediated adhesion and motility of tumor cells.
Keywords: Natural extract; Quercetin-3-glucuronide; Plasmin inhibition; Kinetics; Cancer cell migration;

Mechanism of YB-1-mediated translational induction of GluR2 mRNA in response to neural activity through nAChR by Toru Tanaka; Sachiyo Ohashi; Masamitsu Moue; Shunsuke Kobayashi (1035-1042).
We have reported previously that YB-1 induces translation of GluR2 mRNA in response to neural activity, and that HSP60 affects the association of YB-1 with polysomes. Here we examined the mechanism of YB-1-mediated translational activation of GluR2 mRNA through the nAChR.Expression of nAChRs in NG108-15 cells was verified. Translation of GluR2 mRNA and YB-1/HSP60 interaction were examined in nicotine-treated NG108-15 cells. Effects of inhibition of α7-nAChR and the PI3K/Akt pathway were investigated. The ratios of YB-1 to GluR2 mRNA and to HSP60 were explored in polysomal and non-polysomal fractions, respectively, and the role of HSP60 in cytoplasmic retention of YB-1 was evaluated.Nicotine treatment transiently induced translation of GluR2 mRNA and Akt phosphorylation with a concomitant increase of YB-1/HSP60 interaction. Both α-bungarotoxin and LY294002 abolished the effects of nicotine. On a sucrose gradient, nicotine treatment shifted the distribution of YB-1 to much heavier-sedimenting polysome fractions. In these fractions, the ratio of YB-1 to its binding GluR2 mRNA was decreased, and ribosome association with the YB-1-bound GluR2 mRNA was increased. HSP60 was distributed only in the non-polysomal fractions as its binding to YB-1 increased. In HSP60-depleted cells, nicotine treatment induced nuclear localization of YB-1.YB-1 is released from GluR2 mRNA during α7-nAChR-mediated neurotransmission, causing the PI3K/Akt pathway to recruit ribosomes into the translational machinery, and HSP60 is involved in cytoplasmic retention of polysome-free YB-1.Activation of the PI3K/Akt pathway through the α7-nAChR and YB-1/HSP60 interaction are important for YB-1-mediated translational activation of GluR2 mRNA.► Nicotine induces the translation of GluR2 mRNA through α7-nAChR in NG108-15 cells. ► A transient increase of YB-1/HSP60 interaction is observed in nicotine-treated cells. ► PI3K/Akt pathway is necessary for translational increase and YB-1/HSP60 interaction. ► Neural activity alters the molar ratio of YB-1 bound to GluR2 mRNA in polysomes. ► In HSP60-decreased cells, YB-1 nuclear localization occurs with nicotine treatment.
Keywords: YB-1; GluR2 mRNA; HSP60; Translational regulation; Neural activity;

Biocompatibility of mannan nanogel—safe interaction with plasma proteins by Sílvia A. Ferreira; Cecilia Oslakovic; Risto Cukalevski; Birgitta Frohm; Björn Dahlbäck; Sara Linse; Francisco M. Gama; Tommy Cedervall (1043-1051).
Self-assembled mannan nanogels are designed to provide a therapeutic or vaccine delivery platform based on the bioactive properties of mannan to target mannose receptor expressed on the surface of antigen-presenting cells, combined with the performance of nanogels as carriers of biologically active agents.Proteins in the corona around mannan nanogel formed in human plasma were identified by mass spectrometry after size exclusion chromatography or centrifugation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Structural changes and time dependent binding of human apolipoprotein A-I (apoA-I) and human serum albumin (HSA) to mannan nanogel were studied using intrinsic tryptophan fluorescence and circular dichroism spectroscopy. The mannan nanogel effect on blood coagulation and fibrillation of Alzheimer's disease-associated amyloid β peptide and hemodialysis-associated amyloidosis β2 microglobulin was evaluated using thrombin generation assay or thioflavin T fluorescence assay, respectively.The protein corona around mannan nanogel is formed through a slow process, is quite specific comprising apolipoproteins B-100, A-I and E and HSA, evolves over time, and the equilibrium is reached after hours to days. Structural changes and time dependent binding of apoA-I and HSA to mannan nanogel are minor. The mannan nanogel does not affect blood coagulation and retards the fibril formation.Mannan nanogel has a high biosafety and biocompatibility, which is mandatory for nanomaterials to be used in biomedical applications.Our research provides a molecular approach to evaluate the safety aspects of nanomaterials, which is of general concern in society and science.Display Omitted► Protein corona is quite specific comprising apolipoproteins B-100, A-I and E and HSA. ► Protein corona evolves over time, and the equilibrium is reached after hours to days. ► Mannan nanogel does not affect blood coagulation. ► Mannan nanogel retards the fibrillation of Aβ(M1-40) and β2m.
Keywords: Mannan nanogel; Protein corona; Coagulation; Fibrillation; Biosafety; Biocompatibility;

Deinococcus radiodurans survives extreme doses of radiations contributed by efficient DNA repair pathways. DR2417 (DncA) was detected separately both in a pool of nucleotide binding proteins and multiprotein complex isolated from cells undergoing DNA repair.DR_2417m ORF was sequenced and amino acid sequence of DncA was search for structural similarities with other proteins and functional motifs. Recombinant DncA was characterized for its DNA metabolic functions in vitro and its role in radiation resistance.Sequencing of DR_2417m did not show the reported frame shift at 996th nucleotide position of this gene. DncA showed similarities with β-CASP family nucleases. Recombinant protein acted efficiently on dsDNA and showed an Mn2+ dependent 3′ → 5′ exonuclease and ssDNA/dsDNA junction endonuclease activities while a very low level activity on RNA. The DNase activity of this protein was inhibited in presence of ATP. Its transcription was induced upon γ radiation exposure and a reduction in its copy number resulted in reduced growth rate and loss of γ radiation resistance in Deinococcus. Conclusion — our results suggest that DncA was a novel nuclease of β CASP family having a strong dsDNA end processing activity and it seems to be an essential gene required for both growth and γ radiation resistance of this bacterium.Traditionally DncA should have shown both DNase and RNase functions as other members of β CASP family nucleases. A strong DNase and poor RNase activity possibly made it functionally significant in the radioresistance of D. radiodurans, which would be worth investigating independently.► DR2417 (DncA) was a β CASP family DNase with poor RNase activity. ► DncA has end processing activity required in DSB repair. ► DncA is an essential protein for survival and gamma resistance in D. radiodurans.
Keywords: β-CASP family nuclease; Deinococcus; DNA processing; DSB repair; Junction endonuclease; Radioresistance;

Characterization of dual effects induced by antimicrobial peptides: Regulated cell death or membrane disruption by Edgar J. Paredes-Gamero; Marta N.C. Martins; Fábio A.M. Cappabianco; Jaime S. Ide; Antonio Miranda (1062-1072).
Some reports describe lysis mechanisms by antimicrobial peptides (AMPs), while others describe the activation of regulated cell death. In this study, we compare the cell death-inducing activities of four β-hairpin AMPs (gomesin, protegrin, tachyplesin and polyphemusin II) along with their linear analogs in the human erythroleukemia K562 cell line to investigate the relationship between their structure and activity.K562 cells were exposed to AMPs. Morphological and biochemistry alterations were evaluated using light microscopy, confocal microscopy and flow cytometry.Gomesin and protegrin displayed cytotoxic properties that their linear counterparts did not. Tachyplesin and polyphemusin II and also their linear analogs induced cell death. We were able to distinguish two ways in which these AMPs induced cell death. Lower concentrations of AMPs induced controlled cell death mechanisms. Gomesin, tachyplesin and linear-tachyplesin promoted apoptosis that was characterized by annexin labeling, sensitivity to Z-VAD, and caspase-3 activation, but was also inhibited by necrostatin-1. Gomesin and protegrin induced cell death was dependent on intracellular Ca2+ mechanisms and the participation of free radicals was observed in protegrin induced cell death. Polyphemusin II and its linear analog mainly induced necrosis. Conversely, treatment with higher concentrations of AMPs primarily resulted in cell membrane disruption, but with clearly different patterns of action for each AMP tested.Different actions by β-hairpin AMPs were observed at low concentrations and at higher concentrations despite the structure similarity.Controlled intracellular mechanism and direct membrane disruption were clearly distinguished helping to understand the real action of AMPs in mammalian cells.Display Omitted► AMPs exhibit cytotoxic ability by mechanism not well understood. ► Cell death-inducing activities by four β-hairpin AMPs were compared. ► At low concentration AMPs promoted cell death by different intracellular mechanisms. ► At high concentration AMPs promoted membrane disruption with different features. ► Different actions by β-hairpin AMPs are observed despite their similar structure.
Keywords: Antimicrobial peptide; Cell death; Membrane permeabilization; Intracellular mechanism;

Adequate evidence mounts to the fact that several bacteria and their toxins have protective or curative roles in colorectal cancers. Thermostable direct hemolysin (TDH), produced by Vibrio parahaemolyticus, down regulates cell proliferation in colon carcinoma cell lines. TDH induces Ca2+ influx from an extracellular environment accompanied by protein kinase C phosphorylation. Activated protein kinase C inhibits the tyrosine kinase activity of epidermal growth factor receptor (EGFR), the rational target of anti-colorectal cancer therapy.Immunoblotting analyses were performed to ascertain protein kinase C activation, EGFR status, EGFR phosphorylation and mitogen activated protein kinase (MAPK) activity. Flow cytometry analysis and ELISA reconfirmed tyrosine phosphorylation of EGFR and ERK activations, respectively. PKC-α siRNA knockdown was done to corroborate the involvement of PKC-α in the undertaken study.Our study showed the translocation of PKC-α from cytosol to the membrane fraction in colon carcinoma cell lines on incubation with TDH. The EGFR tyrosine kinase activity exhibited a down regulation on TDH treatment which involved PKC-α, as confirmed by siRNA knockdown. Also ERK phosphorylation occurred on PKC-α activation. Conclusion: TDH activated PKC‐α down regulates EGFR tyrosine kinase activity by MEK dependent mechanism involving MAPK.In this study we have seen that TDH has an implication in EGFR based therapeutic approach in colorectal cancer via PKC mediated mechanism.► TDH phosphorylates PKC-α in colon carcinoma cell line and causes its activation. ► Activated PKC-α downregulates tyrosine phosphorylation of EGFR. ► PKC-α exerts its effect on EGFR through MAPK activation.
Keywords: Colorectal cancer; Thermostable direct hemolysin; PKC-α; EGFR;

Apigenin, a natural plant flavone, may have chemopreventive and therapeutic potentials for anti-inflammatory, antioxidant, and anti-cancer. Nevertheless, the anti-tumor effect of apigenin on human head and neck squamous cell carcinoma (HNSCC) is not fully understood.The antioxidant capacity and protective effects of apigenin against oxidative stress in murine normal embryonic liver BNLCL2 cells are examined. Cell viability, morphologic change, clonogenic survival, cell cycle distribution, reactive oxygen species (ROS) production, glutathione formation, and death receptors- and Bcl-2-mediated caspase pathways of HNSCC SCC25 cells and A431 cells with apigenin are investigated.Apigenin inhibits the growth of SCC25 and A431 cells and induces cell cycle arrest in the G2/M phase. Apigenin has an antioxidant capacity as well as the ability to inhibit lipid peroxidation. It protects BNLCL2 cells against oxidative damage, and is potentially able to prevent cancer. Apigenin increases intracellular ROS levels and reduces levels of glutathione; it also induces cell apoptosis via tumor necrosis factor receptor (TNF-R)-, TNF-related apoptosis-inducing ligand receptor (TRAIL-R)-, and Bcl-2-mediated caspase-dependent cell death pathways in SCC25 cells. The combination of apigenin with 5-fluorouracil (5-Fu) or cisplatin induces the dramatic death of SCC25 cells.Apigenin induces SCC25 cell apoptosis via the up-regulation of both TNF-R and TRAIL-R signaling pathways, and has a synergistic effect on the inhibition of cell proliferation in combination with 5-Fu or cisplatin.These analytical findings suggest that apigenin may be a good therapeutic agent against HNSCC cells.Apigenin induced SCC25 cell apoptosis via up-regulation of both TNF-R and TRAIL-R signaling pathways, down-regulated Bcl-2, activated caspase-3, and showed synergistic effect on inhibiting cell proliferation as combination with 5-Fu or cisplatin.Display Omitted► Apigenin protected BNLCL2 cells against oxidative damage. ► Apigenin induced HNSCC cell apoptosis via TNF-R and TRAIL-R signaling pathways. ► Apigenin induced cell apoptosis by generating oxidative stress. ► The combinatory use of apigenin with 5-Fu or cisplatin accelerated apoptotic cell death.
Keywords: Apigenin; Human head and neck squamous cell carcinoma; Death receptor; 5-Fluorouracil; Cisplatin;

PFOS-induced hepatic steatosis, the mechanistic actions on β-oxidation and lipid transport by H.T. Wan; Y.G. Zhao; X. Wei; K.Y. Hui; J.P. Giesy; Chris K.C. Wong (1092-1101).
Perfluorooctane sulfonate (PFOS) was produced by various industries and was widely used in diverse consumer products. Human sample analysis indicated PFOS contamination in body fluids. Animal studies revealed that PFOS tends to accumulate in livers and is able to induce hepatomegaly. However the underlying mechanism of PFOS-elicited hepatotoxicity has not yet been fully addressed. The objective of this study is to identify the cellular target of PFOS and to reveal the mechanisms of PFOS-induced toxicity.In this study, mature 8-week old male CD-1 mice were administered 0, 1, 5 or 10 mg/kg/day PFOS for 3, 7, 14 or 21 days. Histological analysis of liver sections, and biochemical/molecular analysis of biomarkers for hepatic lipid metabolism were assessed.PFOS-induced steatosis was observed in a time- and dose-dependent manner. The gene expression levels of fatty acid translocase (FAT/CD36) and lipoprotein lipase (Lpl) were significantly increased by 10 and/or 5 mg/kg PFOS. Serum levels of very-low density lipoprotein were decreased by 14 days of PFOS exposure (p < 0.05). The rate of mitochondrial β-oxidation was also found to be significantly reduced, leading to the restriction of fatty acid oxidation for energy production.Taken together, the disturbance of lipid metabolism leads to the accumulation of excessive fatty acids and triglycerides in hepatocytes.Since PFOS-elicited pathological manifestation resembles one of the most common human liver diseases—nonalcoholic fatty liver disease, environmental exposure to PFOS may attribute to the disease progression.► PFOS exposure induced hepatomegaly and lipid accumulation in mice. ► The mechanisms involved the modulation of hepatic b-oxidation and lipid transport. ► PFOS-induced steatosis may play a role in the initiation and progression of NAFLD.
Keywords: Perfluorooctane sulfonate; Lipid metabolism; β-oxidation; Fatty acid uptake; Very-low density lipoprotein; Liver metabolism;

Mitochondrial dysfunction promotes cell migration via reactive oxygen species-enhanced β5-integrin expression in human gastric cancer SC-M1 cells by Wen-Yi Hung; Kuo-Hung Huang; Chew-Wun Wu; Chin-Wen Chi; Hwa-Li Kao; Anna Fen-Yau Li; Pen-Hui Yin; Hsin-Chen Lee (1102-1110).
Mitochondrial dysfunction has been shown to promote cancer cell migration. However, molecular mechanism by which mitochondrial dysfunction enhances gastric cancer (GC) cell migration remains unclear.Mitochondria specific inhibitors, oligomycin and antimycin A, were used to induce mitochondrial dysfunction and to enhance cell migration of human gastric cancer SC-M1 cells. Antioxidant N-acetylcysteine (NAC) was used for evaluating the effect of reactive oxygen species (ROS). Protein expressions of epithelial-to-mesenchymal transition (EMT) markers and the cell–extracellular matrix (ECM) adhesion molecules, the integrin family, were analyzed. A migratory subpopulation of SC-M1 cells (SC-M1-3rd) was selected using a transwell assay for examining the association of mitochondrial bioenergetic function, intracellular ROS content and β5-integrin expression. Clinicopathologic characteristics of β5-integrin expression were analyzed in GC specimens by immunohistochemical staining.Treatments with mitochondrial inhibitors elevated mitochondria-generated ROS and cell migration of SC-M1 cells. The protein expression of β5-integrin and cell surface expression of αvβ5-integrin were upregulated, and which were suppressed by NAC. Pretreatments with NAC and anti-αvβ5-integrin neutralizing antibody respectively prevented the mitochondrial dysfunction-induced cell migration. The selected migratory SC-M1-3rd cells showed impaired mitochondrial function, higher mitochondria-generated ROS, and increased β5-integrin expression. The migration ability was also repressed by anti-αvβ5-integrin neutralizing antibody. In clinical specimens, GCs with higher β5-integrin protein expression had more aggressive behavior. In conclusion, mitochondrial dysfunction may lead to GC progression by enhancing migration through mitochondria-generated ROS mediated β5-integrin expression.These results support the role of mitochondrial dysfunction in GC progression.► Mitochondrial dysfunction-generated ROS promotes gastric cancer cell migration. ► Mitochondrial dysfunction-generated ROS increases β5-integrin protein expression. ► β5-integrin upregulation is required for the enhanced migration. ► Migratory cells exhibit mitochondrial dysfunction and high β5-integrin expression. ► Gastric cancers with higher β5-integrin expression show more aggressive behavior.
Keywords: Mitochondrial dysfunction; Reactive oxygen species; β5-Integrin; Migration; Gastric cancer;

AFM nano-mechanics and calcium dynamics of prostate cancer cells with distinct metastatic potential by Lyndon Bastatas; Dalia Martinez-Marin; James Matthews; Jood Hashem; Yong J. Lee; Souad Sennoune; Stephanie Filleur; Raul Martinez-Zaguilan; Soyeun Park (1111-1120).
Despite recent advances, it is not clear to correlate the mechanical compliances and the metastatic potential of cancer cells. In this study, we investigated combined signatures of mechanical compliances, adhesions, and calcium dynamics correlated with the metastatic potential of cancer cells.We used the lowly (LNCaP) and highly (CL-1, CL-2) metastatic human prostate cancer cells. The AFM-based nanomechanics was performed to determine the elastic moduli and the cell-to-substrate adhesion. The intracellular calcium dynamics was evaluated by fluorescence spectroscopy. Cell migration and the distribution of cytoskeleton were evaluated using the wounded monolayer model and immunofluorescence, respectively. The elastic moduli, the calcium dynamics, and the migratory ability are greater in CL-1 and CL-2 than LNCaP. CL-1 and CL-2 also display a significantly larger area of cell-to-substrate adhesions while the LNCaP displays a limited adhesion. These properties were slightly reduced in CL-2 compared with CL-1 cells. The enhanced elastic moduli and calcium dynamics found in CL-1 and CL-2 can be consistently explained by the intensified tensile stress generated by actin cytoskeletons anchored at more focal adhesion sites.Although the suppressed mechanical compliance of highly metastatic cells may not support the enhanced cancer metastasis, the enhanced adhesion and calcium dynamics are favorable for invasion and extra-vasation required for malignant progression.Our results suggest that the mechanical compliance alone may fail to indicate the metastatic progression, but the combined biomechanical signatures of mechanical compliance, adhesion, and calcium dynamics can provide critical clues to determine the metastatic potential of cells.►The elastic moduli are greater for more metastatic cancer cells. ►The substrate adhesion is enhanced for more metastatic cancer cells. ►The calcium dynamics and the migration are enhanced in more metastatic cells. ►Changes in the tensile stress generated by adhesions explain the above results. ►Enhanced adhesion and calcium dynamics are beneficial for metastasis.
Keywords: Elastic moduli; Adhesion; Atomic Force Microscope; Calcium dynamics; Metastatic potential;

Chronic exposure to natural uranium via drinking water affects bone in growing rats by Ndéye Marième Wade-Gueye; Olivia Delissen; Patrick Gourmelon; Jocelyne Aigueperse; Isabelle Dublineau; Maâmar Souidi (1121-1127).
Bone is the main site of uranium accumulation after long term contamination. Several studies describe that at high dose of exposure, uranium impairs bone growth. Nevertheless little is known about the effects of chronic exposure at low doses of this radionuclide on bone, especially when ingested via drinking water, which is considered as the main exposure pathway for the public.In this study, male rats were exposed to natural uranium in drinking water for a 9 month period, either at 40 mg l− 1 starting just after birth (post-natal model) or starting at 3 months of age (adult model).In the post-natal model at 40 mg l− 1, three-dimensional microtomography analysis showed that NU decreased significantly the cortical bone diameter in NU-contaminated rats. Bone histomorphometry analysis also showed a significant increase of the osteoid thickness in trabecular bone of the femur of NU-contaminated rats. In addition, mRNA expression in trabecular bone of genes involved in osteoblast differentiation (OSX, BMP2, RUNX2), bone remodeling (TRAP, OCN), bone mineralization (BSP, OPN, DMP1), calcium transport (TRPV5) as well as vitamin D receptor (VDR) was significantly decreased in this model. In contrast, in the adult model, no morphometric, cellular and molecular changes were observed in bone.This study showed for the first time that NU at this concentration has no detectable effect in adult bone while it significantly affects growing bone, which thus appears more sensitive to low dose contamination by this radionuclide.► Bone is a target of uranium toxicity after chronic low dose exposure in growing rats. ► Uranium decreases cortical bone diameter of growing rats after chronic exposure. ► Growing bone is more sensitive than adult bone after chronic exposure of uranium.
Keywords: Natural uranium; Drinking water; Chronic exposure; Bone; Growing rat;

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves by Fredy D.A. Silva; Ilka M. Vasconcelos; Marina D.P. Lobo; Patrícia G. de Castro; Vladimir G. Magalhães; Cléverson D.T. de Freitas; Célia R.R.S. Carlini; Paulo M. Pinto; Leila M. Beltramini; José H.A. Filho; Eduardo B. Barros; Luciana M.R. Alencar; Thalles B. Grangeiro; José T.A. Oliveira (1128-1140).
Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx).Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography.Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56–4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% β-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx.The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.► A 2-Cys-Prx (Vu-2-Cys-Prx) was purified from cowpea (Vigna unguiculata) leaves. ► Vu-2-Cys-Prx binds to chitin, is a 44/46 kDa protein with pI of 4.56 – 4.72. ► Its Cys52, Pro45, Thr49, and Arg128 residues are conserved as in all other 2-Cys-Prx. ► Vu-2-Cys-Prx prevented DNA degradation, forms decamers, and has chaperone activity.
Keywords: 2-Cys-peroxiredoxin; Vigna unguiculata; Amino acid sequence; Circular dichroism; cDNA sequence;

Globoside promotes activation of ERK by interaction with the epidermal growth factor receptor by Seung-Yeol Park; Chan-Yeong Kwak; James A. Shayman; Jung Hoe Kim (1141-1148).
Globoside (Gb4), a globo-series glycosphingolipid (GSL), has been characterized as a stage-specific embryonic antigen (SSEA), and is highly expressed during embryogenesis as well as in cancer tissues. However, the functional role and molecular mechanism of Gb4 are so far unknown.GSLs were preferentially inhibited by treatment with D-threo-1-ethylenedioxyphenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4), a nanomolar inhibitor of GSL synthesis, in two carcinoma cell lines, HCT116 and MCF7. The effect of EtDO-P4 was examined by MTT assay, FACS, wound assay, western blotting, and RTK array analysis. The functional role of Gb4 was determined by the exogenous addition of various GSLs, and an assay utilizing GSL-coated latex beads.Both cell lines contained higher levels of neutral GSLs than of sialic acid-containing GSLs. Gb4 was one of the major neutral GSLs. The depletion of total GSLs caused significant reduction of cell proliferation, but had less effect on cell apoptosis or motility. EtDO-P4 treatment also suppressed activation of the epidermal growth factor receptor (EGFR)-induced ERK pathway and various receptor tyrosine kinases (RTKs). The reduced activation of ERK was restored by the exogenous addition of Gb4, but not by the addition of gangliosides (GM1, GM2, GM3, and GD1a). The GSL-coated bead assay indicated that Gb4 forms a complex with EGFR, but not with other RTKs. Taken together, Gb4 promotes activation of EGFR-induced ERK signaling through direct interaction with EGFR.A globo-series GSL, Gb4, promotes EGFR-induced MAPK signaling, resulting in cancer cell proliferation. These findings suggest a possible application of Gb4 in cancer diagnostics and drug targeting.► Two carcinoma cells, HCT116 and MCF7, have similar GSL profile and Gb4 is one of the major neutral GSL. ► The depletion of total GSL suppressed cell proliferation and RTK-induced ERK signaling. ► Exogeneous addition of Gb4 restored phosphorylation of EGFR and ERK. ► Gb4 forms a complex with EGFR.
Keywords: Glycosphingolipid; Globoside; Mitogen-activated protein kinase; Epidermal growth factor receptor;

5-epi-Sinuleptolide induces cell cycle arrest and apoptosis through tumor necrosis factor/mitochondria-mediated caspase signaling pathway in human skin cancer cells by Chia-Hua Liang; Guey-Horng Wang; Tzung-Han Chou; Shih-Hao Wang; Rong-Jyh Lin; Leong-Perng Chan; Edmund Cheung So; Jyh-Horng Sheu (1149-1157).
Skin cancers are reportedly increasing worldwide. Developing novel anti-skin cancer drugs with minimal side effects is necessary to address this public health issue. Sinuleptolide has been demonstrated to possess anti-cancer cell activities; however, the mechanisms underlying the anti-skin cancer effects of 5-epi-sinuleptolide and sinuleptolide remain poorly understood.Apoptosis cell, cell-cycle-related regulatory factors, and mitochondria- and death receptor-dependent caspase pathway in 5-epi-sinuleptolide-induced cell apoptosis were examined using SCC25 cells.5-epi-Sinuleptolide inhibited human skin cancer cell growth more than did sinuleptolide. Treatment of SCC25 cells with 5-epi-sinuleptolide increased apoptotic body formation, and induced cell-cycle arrest during the G2/M phase. Notably, 5-epi-sinuleptolide up-regulated p53 and p21 expression and inhibited G2/M phase regulators of cyclin B1 and cyclin-dependent kinease 1 (CDK1) in SCC25 cells. Additionally, 5-epi-sinuleptolide induced apoptosis by mitochondria-mediated cytochrome c and Bax up-expression, down-regulated Bcl-2, and activated caspase-9 and -3. 5-epi-Sinuleptolide also up-regulated tBid, which is associated with up-regulation of tumor necrosis factor-α (TNF-α) and Fas ligand (FasL) and their cognate receptors (i.e., TNF-RI, TNF-R2 and Fas), downstream adaptor TNF-R1-associated death domain (TRADD) and Fas-associated death domain (FADD), and activated caspase-8 in SCC25 cells.The analytical results indicate that the death receptor- and mitochondria-mediated caspase pathway is critical in 5-epi-sinuleptolide-induced apoptosis of skin cancer cells.This is the first report suggesting that the apoptosis mediates the anti-tumor effect of 5-epi-sinuleptolide. The results of this study might provide useful suggestions for designing of anti-tumor drugs for skin cancer patients.This study is the first to identify the cell growth inhibition activity of 5-epi-sinuleptolide and sinuleptolide and examine their effect on cell cycle distribution and apoptosis in human skin cancer cells.Display Omitted► Inhibition of cell growth by 5-epi-sinuleptolide exceeded that by sinuleptolide. ► 5-epi-sinuleptolide inhibited G2/M phase regulators of cyclin B1 and CDK1. ► 5-epi-Sinuleptolide induced SCC25 cells apoptosis by TNFR and Fas-mediated pathways. ► 5-epi-Sinuleptolide induced apoptosis via mitochondria-mediated caspase pathway.
Keywords: 5-epi-Sinuleptolide; Sinuleptolide; Skin cancer; Death receptor; Mitochondria death pathway;