BBA - General Subjects (v.1830, #10)

In recent years, there has been a growing interest to explore the association between liver injury and diabetes. Advanced glycated end product (AGE) formation which characterizes diabetic complications is formed through hyperglycemia mediated oxidative stress and is itself a source for ROS. Further, in VL-17A cells over-expressing ADH and CYP2E1, greatly increased oxidative stress and decreased viability have been observed with high glucose exposure.In VL-17A cells treated with high glucose and pretreated with the different inhibitors of ADH and CYP2E1, the changes in cell viability, oxidative stress parameters and formation of AGE, were studied.Inhibition of CYP2E1 with 10 μM diallyl sulfide most effectively led to decreases in the oxidative stress and toxicity as compared with ADH inhibition with 2 mM pyrazole or the combined inhibition of ADH and CYP2E1 with 5 mM 4-methyl pyrazole. AGE formation was decreased in VL-17A cells when compared with HepG2 cells devoid of the enzymes. Further, AGE formation was decreased to the greatest extent with the inhibitor for CYP2E1 suggesting that high glucose inducible CYP2E1 and the consequent ROS aid AGE formation.Thus, CYP2E1 plays a pivotal role in the high glucose induced oxidative stress and toxicity in liver cells as observed through direct evidences obtained utilizing the different inhibitors for ADH and CYP2E1.The study demonstrates the role of CYP2E1 mediated oxidative stress in aggravating hyperglycemic insult and suggests that CYP2E1 may be a vital component of hyperglycemia mediated oxidative injury in liver.
Keywords: Hyperglycemia; Liver cell; Cytochrome P4502E1 (CYP2E1); Inhibition; Oxidative stress; Advanced glycated end product;

Low ATP level is sufficient to maintain the uncommitted state of multipotent mesenchymal stem cells by L.B. Buravkova; Y.V. Rylova; E.R. Andreeva; A.V. Kulikov; M.V. Pogodina; B. Zhivotovsky; V. Gogvadze (4418-4425).
Multipotent mesenchymal stromal cells (MMSCs) are minimally differentiated precursors with great potential to transdifferentiate. These cells are quite resistant to oxygen limitation, suggesting that a hypoxic milieu can be physiological for MMSCs.Human MMSCs isolated from adipose tissue were grown at various oxygen concentrations. Alteration in cell immunophenotype was determined by flow cytometry after staining with specific antibodies. Concentrations of glucose and lactate were determined using the Biocon colorimetric test. Cellular respiration was assessed using oxygen electrode. The modes of cell death were analyzed by flow cytometry after staining with Annexin V and propidium iodide.We found that permanent oxygen deprivation attenuated cellular ATP levels in these cells, diminishing mitochondrial ATP production but stimulating glycolytic ATP production. At the same time, permanent hypoxia did not affect MMSCs' viability, stimulated their proliferation and reduced their capacity to differentiate. Further, permanent hypoxia decreased spontaneous cell death by MMSCs.Under hypoxic conditions glycolysis provides sufficient energy to maintain MMSCs in an uncommitted state.These findings are of interest not only for scientific reasons, but also in practical terms. Oxygen concentration makes an essential contribution to MMSC physiology and should be taken into account in the setting of protocols for cellular therapy.
Keywords: Multipotent mesenchymal stromal cells; Hypoxia; Cell death; ATP;

ATP interacts with the CPVT mutation-associated central domain of the cardiac ryanodine receptor by Lynda Blayney; Konrad Beck; Ewan MacDonald; Leon D'Cruz; Michail Nomikos; Julia Griffiths; Angelos Thanassoulas; George Nounesis; F. Anthony Lai (4426-4432).
This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6).Wild-type (WT) RyR2 central domain constructs (G2236to G2491) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation.The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC50 of ~ 200–400 μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found.The RyR2 central domain, expressed as a ‘correctly’ folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP.Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding.
Keywords: Cardiac ryanodine receptor; ATP binding site; ATP binding motifs; FKBP12.6; Catecholaminergic polymorphic ventricular tachycardia (CPVT) mutations; Caffeine;

The oncogenic transcription factors NF-κB and β-catenin, constitutively activated by upstream serine/threonine kinases control several cellular processes implicated in malignant transformation including apoptosis evasion. The aim of this study was to investigate the chemopreventive effects of astaxanthin, an antioxidant carotenoid, in the hamster buccal pouch (HBP) carcinogenesis model based on its ability to modulate NF-κB and Wnt signaling pathways and induce apoptosis.We determined the effect of dietary supplementation of astaxanthin on the oncogenic signaling pathways — NF-κB and Wnt/β-catenin, their upstream activator kinases — Erk/MAPK and PI-3K/Akt, and the downstream event — apoptosis evasion by real-time quantitative RT-PCR, western blot, and immunohistochemical analyses.We found that astaxanthin inhibits NF-κB and Wnt signaling by downregulating the key regulatory enzymes IKKβ and GSK-3β. Analysis of gene expression and docking interactions revealed that inhibition of these pathways may be mediated via inactivation of the upstream signaling kinases Erk/Akt by astaxanthin. Astaxanthin also induced caspase-mediated mitochondrial apoptosis by downregulating the expression of antiapoptotic Bcl-2, p-Bad, and survivin and upregulating proapoptotic Bax and Bad, accompanied by efflux of Smac/Diablo and cytochrome-c into the cytosol, and induced cleavage of poly (ADP-ribose) polymerase (PARP).The results provide compelling evidence that astaxanthin exerts chemopreventive effects by concurrently inhibiting phosphorylation of transcription factors and signaling kinases and inducing intrinsic apoptosis.Astaxanthin targets key molecules in oncogenic signaling pathways and induces apoptosis and is a promising candidate agent for cancer prevention and therapy.
Keywords: Astaxanthin; Intrinsic apoptosis; Extracellular signal-regulated kinase/Mitogen activated protein kinase; Nuclear factor kappa B; Phosphatidyl inositol-3-kinase/Akt; Wnt/β-catenin;

A disintegrin and metalloproteinase-12 (ADAM12): Function, roles in disease progression, and clinical implications by Erin K. Nyren-Erickson; Justin M. Jones; D.K. Srivastava; Sanku Mallik (4445-4455).
A disintegrin and metalloproteinase-12 (ADAM12) is a member of the greater ADAM family of enzymes: these are multifunctional, generally membrane-bound, zinc proteases for which there are forty genes known (21 of these appearing in humans). ADAM12 has been implicated in the pathogenesis of various cancers, liver fibrogenesis, hypertension, and asthma, and its elevation or decrease in human serum has been linked to these and other physiological/pathological conditions.In this review, we begin with a brief overview of the ADAM family of enzymes and protein structure. We then discuss the role of ADAM12 in the progression and/or diagnosis of various disease conditions, and we will conclude with an exploration of currently known natural and synthetic inhibitors.ADAM12 has potential to emerge as a successful drug target, although targeting the metalloproteinase domain with any specificity will be difficult to achieve due to structural similarity between the members of the ADAM and MMP family of enzymes. Overall, more research is required to establish ADAM12 being as a highly desirable biomarker and drug target of different diseases, and their selective inhibitors as potential therapeutic agents.Given the appearance of elevated levels of ADAM12 in various diseases, particularly breast cancer, our understanding of this enzyme both as a biomarker and a potential drug target could help make significant inroads into both early diagnosis and treatment of disease.Display Omitted
Keywords: ADAM12; Disintegrin and metalloproteinase; Structure; Function; Inhibitors;

Forkhead Box-O (FoxO) transcription factors regulate the expression of many genes involved in suppression. Released nucleotides can regulate intracellular signaling pathways through membrane-bound purinergic receptors, to promote or prevent malignant cell transformation. We studied the role of extracellular ATP in the modulation of Forkhead Box O (FoxO) transcription factors and of cell cycle progression in MCF-7 breast cancer cells.Western blot analysis, cell transfections with siRNA against Akt, immunocytochemistry, subcellular fractionation studies and flow cytometry analysis were performed.ATP induced the phosphorylation of FoxO1/3a at threonine 24/32, whereas reduced the expression of FoxO1. In addition, ATP increased the expression of the cyclins D1 and D3 and down-regulated the cell cycle inhibitory proteins p21Cip1 and p27Kip1. The use of the phosphatidylinositol 3 kinase (PI3K) inhibitor, Ly294002, and/or of siRNA to reduce the expression of the serine/threonine kinase Akt showed that these effects are mediated by the PI3K/Akt signaling pathway. ATP induced the translocation of FoxO3a from the nucleus to the cytoplasm. Also, ATP increased the number of cells in the S phase of cell cycle; this effect was reverted by the use of Ly294002 and the proteasome inhibitor bortezomib.Extracellular ATP induces the inactivation of FoxO transcription factors and cell cycle progression through the PI3K/Akt pathway in MCF-7 cells.These findings provide new molecular basis for further understanding the mechanisms involved in ATP signal transduction in breast cancer cells, and should be considered for the development of effective breast cancer therapeutic strategies.
Keywords: Extracellular ATP; FoxO transcription factors; Cell cycle; PI3K/Akt; MCF-7 cells;

Induction of epithelial–mesenchymal transition (EMT) is essential for the metastasis of tumor cells and maintaining their stemness. This study aimed to examine whether endothelial cells, which are most closely located to tumor cells in vivo, play a role in inducing EMT in tumor cells or not.Concentrated culture medium of bovine aortic endothelial cells (BAECs) was applied to tumor cell lines (A549 and PANC-1) and epithelial cell line (NMuMg). Cadherin conversion, expressions of α-smooth muscle actin and ZO-1, actin fiber formation and cell migration were examined as hallmarks of the induction of EMT in these cell lines. Transforming growth factor β (TGFβ) antibodies were used to neutralize TGFβ1, TGFβ2 and TGFβ3. Expression and release of TGFβ proteins in BAECs as well as in porcine and human endothelial cells were assessed by Western blotting and ELISA, respectively.Conditioned medium of BAEC induced EMT in the examined cell lines. All endothelial cells from various species and locations expressed TGFβ1 and TGFβ2 proteins and much lower level of TGFβ3 protein. Conditioned medium from these endothelial cells contained TGFβ1 and TGFβ2, but TGFβ3 could not be detected. Neutralizing antibody against each of TGFβ1 or TGFβ2 did not reverse endothelium-dependent EMT, but simultaneous neutralization of both TGFβ1 and TGFβ2 completely abolished it.Endothelial cells may play a role in the induction and maintenance of EMT in tumor cells by constitutively releasing TGFβ1 and TGFβ2.The present results provide a novel strategy of the inhibition of tumor metastasis by targeting vascular endothelium.
Keywords: Vascular endothelial cell; Tumor cell; Epithelial–mesenchymal transition; Tumor metastasis; Transforming growth factor β1; Transforming growth factor β2;

Difucosylation of chitooligosaccharides by eukaryote and prokaryote α1,6-fucosyltransferases by Hideyuki Ihara; Shinya Hanashima; Hiroki Tsukamoto; Yoshiki Yamaguchi; Naoyuki Taniguchi; Yoshitaka Ikeda (4482-4490).
The synthesis of eukaryotic N-glycans and the rhizobia Nod factor both involve α1,6-fucosylation. These fucosylations are catalyzed by eukaryotic α1,6-fucosyltransferase, FUT8, and rhizobial enzyme, NodZ. The two enzymes have similar enzymatic properties and structures but display different acceptor specificities: FUT8 and NodZ prefer N-glycan and chitooligosaccharide, respectively. This study was conducted to examine the fucosylation of chitooligosaccharides by FUT8 and NodZ and to characterize the resulting difucosylated chitooligosaccharides in terms of their resistance to hydrolysis by glycosidases.The issue of whether FUT8 or NodZ catalyzes the further fucosylation of chitooligosaccharides that had first been monofucosylated by the other. The oligosaccharide products from the successive reactions were analyzed by normal-phase high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. The effect of difucosylation on sensitivity to glycosidase digestion was also investigated.Both FUT8 and NodZ are able to further fucosylate the monofucosylated chitooligosaccharides. Structural analyses of the resulting oligosaccharides showed that the reducing terminal GlcNAc residue and the third GlcNAc residue from the non-reducing end are fucosylated via α1,6-linkages. The difucosylation protected the oligosaccharides from extensive degradation to GlcNAc by hexosamidase and lysozyme, and also even from defucosylation by fucosidase.The sequential actions of FUT8 and NodZ on common substrates effectively produce site-specific-difucosylated chitooligosaccharides. This modification confers protection to the oligosaccharides against various glycosidases.The action of a combination of eukaryotic and bacterial α1,6-fucosyltransferases on chitooligosaccharides results in the formation of difucosylated products, which serves to stabilize chitooligosaccharides against the action of glycosidases.Display Omitted
Keywords: Fucosyltransferase; Fucosylation; Chitooligosaccharide; Glycosidase; Lysozyme;

Structural basis of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1 by Eva Macakova; Miroslava Kopecka; Zdenek Kukacka; Dana Veisova; Petr Novak; Petr Man; Tomas Obsil; Veronika Obsilova (4491-4499).
Trehalases are highly conserved enzymes catalyzing the hydrolysis of trehalose in a wide range of organisms. The activity of yeast neutral trehalase Nth1 is regulated in a 14-3-3- and a calcium-dependent manner. The Bmh proteins (the yeast 14-3-3 isoforms) recognize phosphorylated Nth1 and enhance its enzymatic activity through an unknown mechanism.To investigate the structural basis of interaction between Nth1 and Bmh1, we used hydrogen/deuterium exchange coupled to mass spectrometry, circular dichroism spectroscopy and homology modeling to identify structural changes occurring upon the complex formation.Our results show that the Bmh1 protein binding affects structural properties of several regions of phosphorylated Nth1: the N-terminal segment containing phosphorylation sites responsible for Nth1 binding to Bmh, the region containing the calcium binding domain, and segments surrounding the active site of the catalytic trehalase domain. The complex formation between Bmh1 and phosphorylated Nth1, however, is not accompanied by the change in the secondary structure composition but rather the change in the tertiary structure.The 14-3-3 protein-dependent activation of Nth1 is based on the structural change of both the calcium binding domain and the catalytic trehalase domain. These changes likely increase the accessibility of the active site, thus resulting in Nth1 activation.The results presented here provide a structural view of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1, which might be relevant to understand the process of Nth1 activity regulation as well as the role of the 14-3-3 proteins in the regulation of other enzymes.Display Omitted
Keywords: 14-3-3; Bmh; Neutral trehalase; H/D exchange; Molecular modeling; Circular dichroism;

The expression and proangiogenic effect of nucleolin during the recovery of heat-denatured HUVECs by Pengfei Liang; Bimei Jiang; Chunliu Lv; Xu Huang; Li Sun; Pihong Zhang; Xiaoyuan Huang (4500-4512).
The present study aims to examine the expression patterns and roles of nucleolin during the recovery of heat-denatured human umbilical vein endothelial cells (HUVECs).Deep partial thickness burn model in Sprague–Dawley rats and the heat denatured cell model (52 °C, 35 s) were used. The expression of nucleolin was measured using Western blot analysis and real-time PCR. Angiogenesis was assessed using in vitro parameters including endothelial cell proliferation, transwell migration assay, and scratched wound healing. Gene transfection and RNA interference approaches were employed to investigate the roles of nucleolin.Nucleolin mRNA and protein expression showed a time-dependent increase during the recovery of heat-denatured dermis and HUVECs. Heat-denaturation time-dependently promoted cell growth, adhesion, migration, scratched wound healing and formation of tube-like structures in HUVECs. These effects of heat denaturation on endothelial wound healing and formation of tube-like structures were prevented by knockdown of nucleolin, whereas over-expression of nucleolin increased cell growth, migration, and formation of tube-like structures in cultured HUVEC endothelial cells. In addition, we found that the expression of vascular endothelial growth factor (VEGF) increased during the recovery of heat-denatured dermis and HUVECs, and nucleolin up-regulated VEGF in HUVECs.The present study reveals that the expression of nucleolin is up-regulated, and plays a pro-angiogenic role during the recovery of heat-denatured dermis and its mechanism is probably dependent on production of VEGF.We find a novel and important pro-angiogenic role of nucleolin during the recovery of heat-denatured dermis.
Keywords: Nucleolin; Heat-denatured; Human umbilical vein endothelial cells; Angiogenesis; Vascular endothelial growth factor;

Structural determinants of specificity and catalytic mechanism in mammalian 25-kDa thiamine triphosphatase by David Delvaux; Frédéric Kerff; Mamidanna R.V.S. Murty; Bernard Lakaye; Jan Czerniecki; Gregory Kohn; Pierre Wins; Raphaël Herman; Valérie Gabelica; Fabien Heuze; Xavier Tordoir; Raphaël Marée; André Matagne; Paulette Charlier; Edwin De Pauw; Lucien Bettendorff (4513-4523).
Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates.Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase.Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine–tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds.The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals.We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins.Display Omitted
Keywords: CyaB, ThTPase; Triphosphate tunnel metalloenzyme; Thiamine triphosphate; Tripolyphosphate; Divalent cation;

The potentiation of myeloperoxidase activity by the glycosaminoglycan-dependent binding of myeloperoxidase to proteins of the extracellular matrix by Lukáš Kubala; Hana Kolářová; Jan Víteček; Silvie Kremserová; Anna Klinke; Denise Lau; Anna L.P. Chapman; Stephan Baldus; Jason P. Eiserich (4524-4536).
Myeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood.We investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested.MPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed.Collectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins.Our findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins.
Keywords: Endothelium; Enzyme activity; Collagen IV; Fibronectin; Inflammation; Cardiovascular disease;

Reverse electron flow-mediated ROS generation in ischemia-damaged mitochondria: Role of complex I inhibition vs. depolarization of inner mitochondrial membrane by Thomas Ross; Karol Szczepanek; Elizabeth Bowler; Ying Hu; Andrew Larner; Edward J. Lesnefsky; Qun Chen (4537-4542).
The reverse electron flow-induced ROS generation (RFIR) is decreased in ischemia-damaged mitochondria. Cardiac ischemia leads to decreased complex I activity and depolarized inner mitochondrial membrane potential (ΔΨ) that are two key factors to affect the RFIR in isolated mitochondria. We asked if a partial inhibition of complex I activity without alteration of the ΔΨ is able to decrease the RFIR.Cardiac mitochondria were isolated from mouse heart (C57BL/6) with and without ischemia. The rate of H2O2 production from mitochondria was determined using amplex red coupled with horseradish peroxidase. Mitochondria were isolated from the mitochondrial-targeted STAT3 overexpressing mouse (MLS-STAT3E) to clarify the role of partial complex I inhibition in RFIR production.The RFIR was decreased in ischemia-damaged mouse heart mitochondria with decreased complex I activity and depolarized ΔΨ. However, the RFIR was not altered in the MLS-STAT3E heart mitochondria with complex I defect but without depolarization of the ΔΨ. A slight depolarization of the ΔΨ in wild type mitochondria completely eliminated the RFIR.The mild uncoupling but not the partially decreased complex I activity contributes to the observed decrease in RFIR in ischemia-damaged mitochondria.The RFIR is less likely to be a key source of cardiac injury during reperfusion.
Keywords: Ischemia; Electron transport chain; STAT3; Reactive oxygen species;

Analysis of SEMA6B gene expression in breast cancer: Identification of a new isoform by Luciana D'Apice; Valerio Costa; Carmen Valente; Maria Trovato; Anna Pagani; Stefania Manera; Lea Regolo; Alberto Zambelli; Alfredo Ciccodicola; Piergiuseppe De Berardinis (4543-4553).
SEMA6B is a member of the semaphorins axon-guidance family. A growing body of evidence has been accumulated describing the role of semaphorin molecules in cancer development and the involvement of SEMA6B in cancer progression has recently been proposed.Our analysis, based on real-time PCR, focused on the expression of SEMA6B in a panel of breast cancer tissues, compared to the normal counterpart.In cancer tissues we found a significantly strong down-modulation of this transcript. Moreover we identified and characterized a novel SEMA6B isoform, named SEMA6Ba. This isoform has a novel splice junction, created by the usage of alternative donor and acceptor splice sites internal to the exon 17. By in silico analysis we found that the new transcript 3′ UTR lacks some highly-conserved miRNA binding sites, suggesting possible consequences on both spatial and temporal expression of SEMA6Ba. The translated sequence of SEMA6Ba lacks the cytoplasmic tail, crucial for triggering the reverse signaling described for the transmembrane semaphorins. We also demonstrated, by immunofluorescence analysis of endogenous and overexpressed SEMA6Ba, that the protein clearly localized to the endoplasmic reticulum and plasma membrane. In conclusion, SEMA6B gene products are strongly down modulated in breast cancer tissues and a new isoform named SEMA6Ba has been described and characterized.Our work states a clear relation among breast cancer and SEMA6B expression; moreover we describe for the first time the SEMA6Ba protein and report here the analysis of SEMA6Ba RNA messenger, the protein expression and the cellular localization.
Keywords: Breast cancer; Semaphorin; Alternative splicing; Subcellular localization;

Kinetic uptake profiles of cell penetrating peptides in lymphocytes and monocytes by Margarida Rodrigues; Beatriz G. de la Torre; David Andreu; Nuno C. Santos (4554-4563).
Nucleolar targeting peptides (NrTPs), resulting from structural minimization of the rattlesnake toxin crotamine, are a novel family of cell-penetrating peptides (CPPs) shown to internalize and deliver cargos into different cell types.In this study, we address NrTP kinetics of translocation into primary cells. We used flow cytometry to measure the intracellular uptake of rhodamine B-labeled NrTPs in peripheral blood mononucleated cells (PBMCs).The kinetic profiles for each peptide are concentration-independent but significantly different among NrTPs, pointing out for the amino acid sequence importance. Arginine-containing peptides (NrTP7 and Tat48–60, used for comparison) were found to be more toxic than lysine-containing ones, as expected. On the other hand, one same peptide behaves differently in each of the lymphocyte and monocyte cell populations, suggesting differences in entry mechanism that in turn reflect diversity in cell functionality. Uptake results obtained at 4 °C or using chemical endocytosis inhibitors support the importance of non-endocytic mechanisms in the cellular internalization of NrTP1 and NrTP5, while confirming endocytosis as the main mechanism of NrTPs entry.Overall, both direct translocation and endocytosis mechanisms play a role in NrTP entry. Yet, there is predominance of endocytosis-mediated mechanisms. NrTPs (especially NrTP6) are an excellent intracellular delivery tool, with efficient internalization and no toxicity.This work validates NrTPs as potential therapeutic tools for, e.g., cancer or inhibition of viral replication and establishes a new comparative and quantitative method to test CPP efficiency.Display Omitted
Keywords: Nucleolar targeting peptide; Cell penetrating peptide; Lymphocyte; Monocyte; Uptake; Kinetics;

Hofmeister ions control protein dynamics by Balázs Szalontai; Gergely Nagy; Sashka Krumova; Elfrieda Fodor; Tibor Páli; Stefka G. Taneva; Győző Garab; Judith Peters; András Dér (4564-4572).
Recently, we have elaborated a thermodynamic theory that could coherently interpret the diverse effects of Hofmeister ions on proteins, based on a single physical parameter, the protein–water interfacial tension (Dér et al., Journal of Physical Chemistry B. 2007, 111, 5344–5350). This theory, implying a “liquid drop model”, predicts changes in protein conformational fluctuations upon addition of Hofmeister salts (containing either kosmotropic or chaotropic anions) to the medium.Here, we report experimental tests of this prediction using a complex approach by applying methods especially suited for the detection of protein fluctuation changes (neutron scattering, micro-calorimetry, and Fourier-transform infrared spectroscopy).It is demonstrated that Hofmeister salts, via setting the hydrophobic/hydrophilic properties of the protein–water interface, control conformational fluctuations even in the interior of the typical membrane transport protein bacteriorhodopsin, around its temperature-induced, unusual α(II) → α(I) conformational transition between 60 and 90 °C. We found that below this transition kosmotropic (COOCH3 ), while above it chaotropic (ClO4 ) anions increase structural fluctuations of bR. It was also shown that, in each case, an onset of enhanced equilibrium fluctuations presages this phase transition in the course of the thermotropic response of bR.These results are in full agreement with the theory, and demonstrate that predictions based on protein–water interfacial tension changes can describe Hofmeister effects and interpret protein dynamics phenomena even in unusual cases.This approach is expected to provide a useful guide to understand the principles governing the interplay between protein interfacial properties and conformational dynamics, in general.
Keywords: Hofmeister effect; Differential scanning calorimetry; Neutron scattering; Fourier transform infrared spectroscopy; Protein structural fluctuation; Bacteriorhodopsin;

O-acetyl serine sulfhydrylase (OASS) is a pyridoxal phosphate (PLP) dependent enzyme catalyzing the last step of the cysteine biosynthetic pathway. Here we analyze and investigate the factors responsible for recognition and different conformational changes accompanying the binding of various ligands to OASS.X ray crystallography was used to determine the structures of OASS from Entamoeba histolytica in complex with methionine (substrate analog), isoleucine (inhibitor) and an inhibitory tetra-peptide to 2.00 Å, 2.03 Å and 1.87 Å resolutions, respectively. Molecular dynamics simulations were used to investigate the reasons responsible for the extent of domain movement and cleft closure of the enzyme in presence of different ligands.Here we report for the first time an OASS-methionine structure with an unmutated catalytic lysine at the active site. This is also the first OASS structure with a closed active site lacking external aldimine formation. The OASS-isoleucine structure shows the active site cleft in open state. Molecular dynamics studies indicate that cofactor PLP, N88 and G192 form a triad of energy contributors to close the active site upon ligand binding and orientation of the Schiff base forming nitrogen of the ligand is critical for this interaction.Methionine proves to be a better binder to OASS than isoleucine. The β branching of isoleucine does not allow it to reorient itself in suitable conformation near PLP to cause active site closure.Our findings have important implications in designing better inhibitors against OASS across all pathogenic microbial species.
Keywords: Cysteine biosynthetic pathway; O-acetyl serine sulfhydrylase; Active site cleft; Inhibition; Ligand binding; Conformational change;

Osmotic stress transcription factor 1/transforming growth factor-β-stimulated clone 22 domain 3 (Ostf1/Tsc22d3) is a transcription factor that plays an osmoregulatory role in euryhaline fishes. Its mRNA and protein levels are up-regulated under hyperosmotic stress. However, its osmoregulatory and developmental functions have not been studied in any stenohaline freshwater fishes. Zebrafish is an excellent model to perform such study to unfold the functional role of Tsc22d3.We identified the zebrafish Tsc22d3 and performed knockdown studies using morpholino antisense oligonucleotide (MO).Zebrafish Tsc22d3 did not response to hypertonic stress and ts22d3 knockdown or overexpression by injecting MO or capped RNA did not change the transcriptional levels of any of the known ionocyte markers. To reveal the unknown function of zebrafish Tsc22d3, we performed several in situ molecular marker studies on tsc22d3 morphants and found that Tsc22d3 plays multi-functional roles in dorsoventral (DV) patterning, segmentation, and brain development. We then aimed to identify the mechanism of Tsc22d3 in the earliest stages of DV patterning. Our results demonstrated that tsc22d3 is a ventralizing gene that can stimulate the transcription of bone morphogenetic protein 4 (bmp4) and, thus, has a positive effect on the Bmp signaling pathway. Furthermore, we showed that Tsc22d3 interacts with deubiquitylating enzymes, ubiquitin-specific protease 15 (Usp15) and ovarian tumor domain containing protein 4 (Otud4). In addition, the interruption of Bmp4 signaling by double knockdown of usp15 and otud4 reduced the ventralized effects in tsc22d3-overexpressing embryos.This is the first study to identify new developmental functions of Tsc22d3 in zebrafish.Zebrafish tsc22d3 is a ventralizing gene and plays a role in early embryogenesis.
Keywords: Embryogenesis; Osmoregulation; Osmotic stress transcription factor 1;

Vascular targeting to the SST2 receptor improves the therapeutic response to near-IR two-photon activated PDT for deep-tissue cancer treatment by Jean R. Starkey; Elizabeth M. Pascucci; Mikhail A. Drobizhev; Aleisha Elliott; Aleksander K. Rebane (4594-4603).
Broader clinical acceptance of photodynamic therapy is currently hindered by (a) poor depth efficacy, and (b) predisposition towards establishment of an angiogenic environment during the treatment. Improved depth efficacy is being sought by exploiting the NIR tissue transparency window and by photo-activation using two-photon absorption (2PA). Here, we use two-photon activation of PDT sensitizers, untargeted and targeted to SST2 receptors or EGF receptors, to achieve deep tissue treatment.Human tumor lines, positive or negative for SST2r expression were used, as well as murine 3LL cells and bovine aortic endothelial cells. Expression of SST2 receptors on cancer cells and tumor vasculature was evaluated in vitro and frozen xenograft sections. PDT effects on tumor blood flow were followed using in vivo scanning after intravenous injection of FITC conjugated dextran 150 K. Dependence of the PDT efficacy on the laser pulse duration was evaluated. Effectiveness of targeting to vascular SST2 receptors was compared to that of EGF receptors, or no targeting.Tumor vasculature stained for SST2 receptors even in tumors from SST2 receptor negative cell lines, and SST2r targeted PDT led to tumor vascular shutdown. Stretching the pulse from ~ 120 fs to ~ 3 ps led to loss of the PDT efficacy especially at greater depth. PDT targeted to SST2 receptors was much more effective than untargeted PDT or PDT targeted to EGF receptors.The use of octreotate to target SST2 receptors expressed on tumor vessels is an excellent approach to PDT with few recurrences and some long term cures.
Keywords: Photodynamic therapy; Somatostatin receptor 2; Vascular shutdown; Laser pulse;

Prostasomes from four different species are able to produce extracellular adenosine triphosphate (ATP) by K. Göran Ronquist; Bo Ek; Jane Morrell; Anneli Stavreus-Evers; Bodil Ström Holst; Patrice Humblot; Gunnar Ronquist; Anders Larsson (4604-4610).
Prostasomes are extracellular vesicles. Intracellularly they are enclosed by another larger vesicle, a so called “storage vesicle” equivalent to a multivesicular body of late endosomal origin. Prostasomes in their extracellular context are thought to play a crucial role in fertilization.Prostasomes were purified according to a well worked-out schedule from seminal plasmas obtained from human, canine, equine and bovine species. The various prostasomes were subjected to SDS-PAGE separation and protein banding patterns were compared. To gain knowledge of the prostasomal protein systems pertaining to prostasomes of four different species proteins were analyzed using a proteomic approach. An in vitro assay was employed to demonstrate ATP formation by prostasomes of different species.The SDS-PAGE banding pattern of prostasomes from the four species revealed a richly faceted picture with most protein bands within the molecular weight range of 10–150 kDa. Some protein bands seemed to be concordant among species although differently expressed and the number of protein bands of dog prostasomes seemed to be distinctly fewer. Special emphasis was put on proteins involved in energy metabolic turnover. Prostasomes from all four species were able to form extracellular adenosine triphosphate (ATP). ATP formation was balanced by ATPase activity linked to the four types of prostasomes.These potencies of a possession of functional ATP-forming enzymes by different prostasome types should be regarded against the knowledge of ATP having a profound effect on cell responses and now explicitly on the success of the sperm cell to fertilize the ovum.This study unravels energy metabolic relationships of prostasomes from four different species.
Keywords: Prostasomes; Seminal plasma; Microvesicles; Energy metabolism; Extracellular ATP;

Liposomal temocene (m-THPPo) photodynamic treatment induces cell death by mitochondria-independent apoptosis by Jorge Soriano; María García-Díaz; Margarita Mora; Maria Lluïsa Sagristá; Santi Nonell; Angeles Villanueva; Juan Carlos Stockert; Magdalena Cañete (4611-4620).
The cell death pathway activated after photodynamic therapy (PDT) is controlled by a variety of parameters including the chemical structure of the photosensitizer, its subcellular localization, and the photodynamic damage induced. The present study aims to characterize a suitable m-THPPo liposomal formulation, to determine its subcellular localization in HeLa cells and to establish the cell death mechanisms that are activated after photodynamic treatments.Liposomes containing m-THPPo were prepared from a mixture of DPPC and DMPG at a 9:1 molar ratio. In order to procure the best encapsulation efficiency, the m-THPPo/lipid molar ratio was considered. HeLa cells were incubated with liposomal m-THPPo and the subcellular localization of m-THPPo was studied. Several assays such as TUNEL, annexin V/propidium iodide and Hoechst-33258 staining were performed after photodynamic treatments. The apoptotic initiation was assessed by cytochrome c and caspase-2 immunofluorescence. m-THPPo encapsulated in liposomes showed a decrease of the fluorescence and singlet oxygen quantum yields, compared to those of m-THPPo dissolved in tetrahydrofuran. Liposomal m-THPPo showed colocalization with LysoTracker® and it induced photoinactivation of HeLa cells by an apoptotic mechanism. In apoptotic cells no relocalization of cytochrome c could be detected, but caspase-2 was positive immediately after photosensitizing treatments.Photodynamic treatment with liposomal m-THPPo leads to a significant percentage of apoptotic morphology of HeLa cells. The activation of caspase-2, without the relocalization of cytochrome c, indicates a mitochondrial-independent apoptotic mechanism.These results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment.
Keywords: Apoptosis; Liposome; Photosensitizer; Temocene;

Although microcalcifications of hydroxyapatite can be found in both benign and malignant osteotropic tumors, they are mostly seen in proliferative lesions, including carcinoma. The aim of this present study is to develop a molecular imaging contrast agent for selective identification of hydroxyapatite calcification in human osteotropic tumor tissues ex vivo and in human osteosarcoma cells in vitro.A bioinspired biomarker, hydroxyapatite binding peptide (HABP), was designed to mimic natural protein osteocalcin property in vivo. A fluorescein isothiocyanate dye conjugated HABP (HABP-19) was utilized to characterize hydroxyapatite on human osteotropic tumor tissue sections ex vivo and to selectively image hydroxyapatite calcifications in human osteosarcoma cells in vitro.Using a HABP-19 molecular imaging probe, we have shown that it is possible to selectively image hydroxyapatite calcifications in osteotropic cancers ex vivo and in human SaOS-2 osteosarcoma cells in vitro.Hydroxyapatite calcifications were selectively detected in osteotropic tissues ex vivo and in the early stage of the calcification process of SaOS-2 human osteosarcoma in vitro using our HABP-19 molecular imaging probe. This new target-selective molecular imaging probe makes it possible to study the earliest events associated with hydroxyapatite deposition in various osteotropic cancers at the cellular and molecular levels.It potentially could be used to diagnose and treat osteotropic cancer or to anchor therapeutic agents directing the local distribution of desired therapy at calcified sites.
Keywords: Osteotropic; Calcification; Hydroxyapatite; Molecular imaging; Osteocalcin;

Collagen based magnetic nanobiocomposite as MRI contrast agent and for targeted delivery in cancer therapy by A. Mandal; S. Sekar; M. Kanagavel; N. Chandrasekaran; A. Mukherjee; T.P. Sastry (4628-4633).
In this study, an attempt has been made with the advent of technology to prepare a multifunctional nanobiocomposite (NBC) for targeted drug delivery in cancer therapy.Collagen (C) was fabricated as nanofibers with multifunctional moieties viz. CFeAb*D by incorporating iron oxide nanoparticles (Fe), coupling with fluorescein isothiocyanate (FITC) labeled antibody (Ab*) and loading an anticancer gemcitabine drug (D). This NBC was characterized by conventional methods and evaluated for its biological activities.The UV–vis and FTIR spectroscopic studies revealed the fluorescein to protein ratio and revealed the presence of iron oxide nanoparticles and their interaction with the collagen molecules, respectively. While SDS-PAGE showed the proteinaceous nature of collagen, VSM and TEM studies revealed magnetic saturation as 54.97 emu/g and a magnetic nanoparticle with a diameter in the range of 10–30 nm and the dimension of nanofiber ranging from 97 to 270 nm. A MRI scan has shown a super paramagnetic effect, which reveals that the prepared NBC can be used as a MRI contrast agent. The MTT assay has shown biocompatibility and an apoptotic effect while phase contrast microscopy exhibited receptor mediated uptake of endocytosis.The novelty in the prepared NBC lies in the collagen nanofibers, which have a higher penetrating property without causing much cell damage, biocompatibility and multifunctional properties and is able to carry multifunctional agents.The study has demonstrated the possible use of CFeAb*D as a multifunctional NBC for biomedical applications.Display Omitted
Keywords: Nanobiocomposite; Magnetic resonance imaging; Targeted delivery; Labeled monoclonal antibody; Cancer cell;

Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin by Horia Nicolae Roman; Nedjma B. Zitouni; Linda Kachmar; Gijs IJpma; Lennart Hilbert; Oleg Matusovsky; Andrea Benedetti; Apolinary Sobieszek; Anne-Marie Lauzon (4634-4641).
Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin.To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb ) in the absence and presence of calponin or phosphorylated calponin. Funb from F-actin alone (0.12 ± 0.01 pN; mean ± SE) was significantly increased in the presence of calponin (0.20 ± 0.02 pN). This enhancement was lost when calponin was phosphorylated (0.12 ± 0.01 pN). To further verify that this enhancement of Funb was due to the cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25 mM (0.21 ± 0.02 pN; mean ± SE) was significantly decreased at a [KCl] of 150 mM, (0.13 ± 0.01 pN).This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state.This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.
Keywords: Latch-state; Actin regulatory proteins; In vitro motility assay; Laser trap;

PLIN2, the major perilipin regulated during sebocyte differentiation, controls sebaceous lipid accumulation in vitro and sebaceous gland size in vivo by Maik Dahlhoff; Emanuela Camera; Mauro Picardo; Christos C. Zouboulis; Lawrence Chan; Benny Hung-Junn Chang; Marlon R. Schneider (4642-4649).
Lipid synthesis and storage are accomplished by lipid droplets (LDs). The perilipin family of LD-associated proteins, comprising 5 members (PLIN1–PLIN5), has been well characterized in adipocytes but not in sebocytes, epithelial cells in which LD formation is a key feature of the cellular differentiation.Perilipin expression in the sebaceous gland cell line SZ95 and in human sebaceous glands was studied by qRT-PCR, Western blots, and immunohistochemistry. Lipid accumulation was evaluated by Nile red staining and mass spectrometry.PLIN2 and PLIN3 are the most abundant perilipins in undifferentiated sebocytes. Induction of lipogenesis by linoleic acid (LA) resulted in increased transcript levels of all perilipins except for PLIN3 and in a time-dependent increase of PLIN2 protein. Nile red staining revealed that siRNA-mediated downregulation of PLIN2 significantly impaired basal and LA-induced lipid accumulation. Mass spectrometry revealed PLIN2 deficiency to cause a reduction in the amount of several specific lipid fractions, including di- and triacyl-glycerol esters, phosphatidylcholine lipids, and ceramides in sebocytes under basal conditions. In contrast, PLIN2 downregulation exerted a statistically significant inhibitory effect only on the accumulation of specific LA-induced triglycerides. PLIN2-deficient mice showed normal morphology of sebaceous glands. However, their sebaceous glands were significantly reduced in size and showed less cell proliferation.PLIN2 is the major perilipin regulated during sebocyte differentiation in vitro. PLIN2 is also important for sebaceous lipid accumulation in vitro and regulates sebaceous gland size in vivo.Our study provides the first systematic analysis of LD-associated proteins in sebocytes.
Keywords: Sebaceous glands; Lipid droplets; Perilipins;

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2 by Guadalupe Martel-Gallegos; Griselda Casas-Pruneda; Filiberta Ortega-Ortega; Sergio Sánchez-Armass; Jesús Alberto Olivares-Reyes; Becky Diebold; Patricia Pérez-Cornejo; Jorge Arreola (4650-4659).
Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.Display Omitted
Keywords: Calcium; Purinergic receptor; Oxidative stress; Mitogen-activated protein kinase; Macrophage;

G-quadruplexes are polymorphic non-canonical nucleic acid conformations involved both in physiological and pathological processes. Given the high degree of folding heterogeneity and comparable conformational stabilities, different G-quadruplex forms can occur simultaneously, hence rendering the use of basic instrumental methods for structure determination, like X-ray diffraction or NMR, hardly useful. Footprinting techniques represent valuable and relatively rapid alternative to characterize DNA folding. The natural diterpenoid clerocidin is an alkylating agent that specifically reacts at single-stranded DNA regions, with different mechanisms depending on the exposed nucleotide.Clerocidin was used to footprint G-quadruplex structures formed by telomeric and oncogene promoter sequences (c-myc, bcl-2, c-kit2), and by the thrombin binding aptamer.The easy modulability of CL reactivity towards DNA bases permitted to discriminate fully and partially protected sites, highlights stretched portions of the G-quadruplex conformation, and discriminate among topologies adopted by one sequence in different environmental conditions. Importantly, CL displayed the unique property to allow detection of G-quadruplex folding within a duplex context.CL is a finely performing new tool to unveil G-quadruplex arrangements in DNA sequences under genomically relevant conditions.Nucleic acid G-quadruplex structures are an emerging research field because of the recent indication of their involvement in a series of key biological functions, in particular in regulation of proliferation-associated gene expression. The use of clerocidin as footprinting agent to identify G-quadruplex structures under genomically relevant conditions may allow detection of new G-quadruplex-based regulatory regions.Display Omitted
Keywords: G-quadruplex; Clerocidin; Footprinting; Protection assay; Alkylating agent; DNA conformation;

Iron release from ferritin by flavin nucleotides by Galina Melman; Fadi Bou-Abdallah; Eleanor Vane; Poli Maura; Paolo Arosio; Artem Melman (4669-4674).
Extensive in-vitro studies have focused on elucidating the mechanism of iron uptake and mineral core formation in ferritin. However, despite a plethora of studies attempting to characterize iron release under different experimental conditions, the in-vivo mobilization of iron from ferritin remains poorly understood.Several iron-reductive mobilization pathways have been proposed including, among others, flavin mononucleotides, ascorbate, glutathione, dithionite, and polyphenols. Here, we investigate the kinetics of iron release from ferritin by reduced flavin nucleotide, FMNH2, and discuss the physiological significance of this process in-vivo.Iron release from horse spleen ferritin and recombinant human heteropolymer ferritin was followed by the change in optical density of the Fe(II)–bipyridine complex using a Cary 50 Bio UV–Vis spectrophotometer. Oxygen consumption curves were followed on a MI 730 Clark oxygen microelectrode.The reductive mobilization of iron from ferritin by the nonenzymatic FMN/NAD(P)H system is extremely slow in the presence of oxygen and might involve superoxide radicals, but not FMNH2. Under anaerobic conditions, a very rapid phase of iron mobilization by FMNH2 was observed.Under normoxic conditions, FMNH2 alone might not be a physiologically significant contributor to iron release from ferritin.There is no consensus on which iron release pathway is predominantly responsible for iron mobilization from ferritin under cellular conditions. While reduced flavin mononucleotide (FMNH2) is one likely candidate for in-vivo ferritin iron removal, its significance is confounded by the rapid oxidation of the latter by molecular oxygen.Display Omitted
Keywords: Reductive mobilization; Iron core; Ferritin; FMN/NAD(P)H; Oxygen consumption; Iron release;

Calorimetric analysis of the plasma proteome: Identification of type 1 diabetes patients with early renal function decline by Nichola C. Garbett; Michael L. Merchant; Jonathan B. Chaires; Jon B. Klein (4675-4680).
Microalbuminuria (MA) has been questioned as a predictor of progressive renal dysfunction in patients with type 1 diabetes (T1D). Consequently, new clinical end points are needed that identify or predict patients that are at risk for early renal function decline (ERFD). The potential clinical utility of differential scanning calorimetry (DSC) analysis of blood plasma and other biofluids has recently been reported. This method provides an alternate physical basis with which to study disease-associated changes in the bulk plasma proteome.DSC analysis of blood plasma was applied to identify unique signatures of ERFD in subjects enrolled in the 1st Joslin Study of the Natural History of Microalbuminuria in Type 1 Diabetes, a prospective cohort study of T1D patients. Recent data suggests that differences in the plasma peptidome of these patients correlate with longitudinal measures of renal function. Differences in DSC profile (thermogram) features were evaluated between T1D MA individuals exhibiting ERFD (n = 15) and matched control subjects (n = 14).The average control group thermogram resembled a previously defined healthy thermogram. Differences were evident between ERFD and control individuals. Heat capacity values of the main two transitions were found to be significant discriminators of patient status.Results from this pilot study suggest the potential utility of DSC proteome analysis to prognostic indicators of renal disease in T1D.DSC shows sensitivity to changes in the bulk plasma proteome that correlate with clinical status in T1D providing additional support for the utility of DSC profiling in clinical diagnostics.
Keywords: Plasma proteome; Differential scanning calorimetry; Thermogram; Clinical diagnostics; Type 1 diabetes; Renal function decline;

Anti-HIV-1 activity and structure–activity-relationship study of a fucosylated glycosaminoglycan from an echinoderm by targeting the conserved CD4 induced epitope by Wu Lian; Mingyi Wu; Ning Huang; Na Gao; Chuang Xiao; Zi Li; Zhigang Zhang; Yongtang Zheng; Wenlie Peng; Jinhua Zhao (4681-4691).
Fucosylated glycosaminoglycan (FG) is a novel glycosaminoglycan with a chondroitin sulfate-like backbone and fucose sulfate branches. The aim of this study is to investigate the mechanism and structure–activity relationships (SAR) of FG for combating HIV-1 infection.Anti-HIV activities of FGs were assessed by a cytopathic effect assay and an HIV-1 p24 detection assay. The biomolecule interactions were explored via biolayer interferometry technology. The SAR was established by comparing its anti-HIV-1 activities, conserved CD4 induced (CD4i) epitope-dependent interactions and anticoagulant activities.FG efficiently and selectively inhibited the X4- and R5X4-tropic HIV-1 infections in C8166 cells with little cytotoxicity against C8166 cells and PBMCs. Our data indicated that FG bound to gp120 with nanomolar affinity and may interact with CD4i of gp120. Additionally, the CD4i binding affinity of FG was higher than that of dextran sulfate. SAR studies suggested that the unique sulfated fucose branches account for the anti-HIV-1 activity. The molecular size and present carboxyl groups of FG may also play important roles in various activities. Notably, several FG derivatives showed higher anti-HIV-1 activities and much lower anticoagulant activities than those of heparin.FG exhibits strong activity against X4- and R5X4-tropic HIV-1 infections. The mechanism may be related to targeting CD4i of gp120, which results in inhibition of HIV-1 entry. The carboxyl group substituted derivatives of FG (8.5–12.8 kDa), might display high anti-HIV-1 activity and low anticoagulant activity.Our data supports further the investigation of FG derivatives as novel HIV-1 entry inhibitors targeting CD4i.Display Omitted
Keywords: Glycosaminoglycan; Anti-human immunodeficiency virus activity; Envelope glycoprotein 120; Cluster of Differentiation 4 induced epitope; Biolayer interferometry; Structure–activity relationship;

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes by María Florencia Leal Denis; J. Jeremías Incicco; María Victoria Espelt; Sandra V. Verstraeten; Omar P. Pignataro; Eduardo R. Lazarowski; Pablo J. Schwarzbaum (4692-4707).
The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics).Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin–luciferase based real-time luminometry.Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%.Erythrocytes pre-exposure to 10 μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux.Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers.MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers.Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.
Keywords: Extracellular ATP; ATPases; Erythrocyte; Pannexin 1;

Interaction of the plant alkaloid aristololactam-β-d-glucoside and the antitumor drug daunomycin with single stranded RNAs poly(G), poly(I), poly(C) and poly(U) has been investigated.Biophysical techniques of absorption, fluorescence, competition dialysis, circular dichroism, and microcalorimetry have been used.Absorption and fluorescence studies have revealed noncooperative binding of ADG and DAN to the single stranded RNAs. The binding affinity of ADG varied as poly(G) > poly(I) > > poly(C) > poly(U). The affinity of DAN was one order higher than that of ADG and varied as poly(G) > poly(I) > poly(U) > poly(C). This binding preference was further confirmed by competition dialysis assay. The thermodynamics of the binding was characterised to be favourable entropy and enthalpic terms but their contributions were different for different systems. The major non-polyelectrolytic contribution to the binding revealed from salt dependent data appears to be arising mostly from stacking of DAN and ADG molecules with the bases leading to partial intercalation to single stranded RNA structures. Small negative heat capacity values have been observed in all the four cases.This study presents the comparative structural and thermodynamic profiles of the binding of aristololactam-β-d-glucoside and daunomycin to single stranded polyribonucleotides.These results suggest strong, specific but differential binding of these drug molecules to the single stranded RNAs and highlight the role of their structural differences in the interaction profile.Display Omitted
Keywords: Aristololactam-β-d-glucoside; Daunomycin; Single stranded RNA; Interaction; Spectroscopy; Calorimetry;

Biosynthesis and function of chondroitin sulfate by Tadahisa Mikami; Hiroshi Kitagawa (4719-4733).
Chondroitin sulfate proteoglycans (CSPGs) are principal pericellular and extracellular components that form regulatory milieu involving numerous biological and pathophysiological phenomena. Diverse functions of CSPGs can be mainly attributed to structural variability of their polysaccharide moieties, chondroitin sulfate glycosaminoglycans (CS-GAG). Comprehensive understanding of the regulatory mechanisms for CS biosynthesis and its catabolic processes is required in order to understand those functions.Here, we focus on recent advances in the study of enzymatic regulatory pathways for CS biosynthesis including successive modification/degradation, distinct CS functions, and disease phenotypes that have been revealed by perturbation of the respective enzymes in vitro and in vivo.Fine-tuned machineries for CS production/degradation are crucial for the functional expression of CS chains in developmental and pathophysiological processes.Control of enzymes responsible for CS biosynthesis/catabolism is a potential target for therapeutic intervention for the CS-associated disorders.
Keywords: Chondroitin sulfate; Glycosaminoglycan; Glycosyltransferase; Proteoglycan; Sulfotransferase; Biosynthesis/catabolism;

PEGylation of lysine residues reduces the pro-migratory activity of IGF-I by Manaswini Sivaramakrishnan; Abhishek S. Kashyap; Beat Amrein; Stefanie Saenger; Sonja Meier; Christian Staudenmaier; Zee Upton; Friedrich Metzger (4734-4742).
The insulin-like growth factor (IGF) system is composed of ligands and receptors which regulate cell proliferation, survival, differentiation and migration. Some of these functions involve regulation by the extracellular milieu, including binding proteins and other extracellular matrix proteins. However, the functions and exact nature of these interactions remain incomplete.IGF-I variants PEGylated at lysines K27, K65 and K68, were assessed for binding to IGFBPs using BIAcore, and for phosphorylation of the IGF-IR. Furthermore, functional consequences of PEGylation were investigated using cell viability and migration assays. In addition, downstream signaling pathways were analyzed using phospho-AKT and phospho-ERK1/2 assays.IGF-I PEGylated at lysines 27 (PEG-K27), 65 (PEG-K65) or 68 (PEG-K68) was employed. Receptor phosphorylation was similarly reduced 2-fold with PEG-K65 and PEG-K68 in 3T3 fibroblasts and MCF-7 breast cancer cells, whereas PEG-K27 showed a more than 10- and 3-fold lower activation for 3T3 and MCF-7 cells, respectively. In addition, all PEG-IGF-I variants had a 10-fold reduced association rate to IGF binding proteins (IGFBPs). Functionally, all PEG variants lost their ability to induce cell migration in the presence of IGFBP-3/vitronectin (VN) complexes, whereas cell viability was fully preserved. Analysis of downstream signaling revealed that AKT was preferentially affected upon treatment with PEG-IGF-I variants whereas MAPK signaling was unaffected by PEGylation.PEGylation of IGF-I has an impact on cell migration but not on cell viability.PEG-IGF-I may differentially modulate IGF-I mediated functions that are dependent on receptor interaction as well as key extracellular proteins such as VN and IGFBPs.
Keywords: Insulin-like growth factor 1; IGF-I; PEG-IGF-I; Migration; Viability;

Mitochondrial dysfunction represses HIF-1α protein synthesis through AMPK activation in human hepatoma HepG2 cells by Chia-Chi Hsu; Chun-Hui Wang; Ling-Chia Wu; Cheng-Yuan Hsia; Chin-Wen Chi; Pen-Hui Yin; Chun-Ju Chang; Ming-Ta Sung; Yau-Huei Wei; Shing-Hwa Lu; Hsin-Chen Lee (4743-4751).
Hypoxia-inducible factor-1α (HIF-1α) is an important transcription factor that modulates cellular responses to hypoxia and also plays critical roles in cancer progression. Recently, somatic mutations and decreased copy number of mitochondrial DNA (mtDNA) were detected in hepatocellular carcinoma (HCC). These mutations were shown to have the potential to cause mitochondrial dysfunction. However, the effects and mechanisms of mitochondrial dysfunction on HIF-1α function are not fully understood. This study aims to explore the underlying mechanism by which mitochondrial dysfunction regulates HIF-1α expression.Human hepatoma HepG2 cells were treated with various mitochondrial respiration inhibitors and an uncoupler, respectively, and the mRNA and protein expressions as well as transactivation activity of HIF-1α were determined. The role of AMP-activated protein kinase (AMPK) was further analyzed by compound C and AMPK knock-down.Treatments of mitochondrial inhibitors and an uncoupler respectively reduced both the protein level and transactivation activity of HIF-1α in HepG2 cells under normoxia or hypoxia. The mitochondrial dysfunction-repressed HIF-1α protein synthesis was associated with decreased phosphorylations of p70S6K and 4E-BP-1. Moreover, mitochondrial dysfunction decreased intracellular ATP content and elevated the phosphorylation of AMPK. Treatments with compound C, an AMPK inhibitor, and knock-down of AMPK partially rescued the mitochondrial dysfunction-repressed HIF-1α expression.Mitochondrial dysfunctions resulted in reduced HIF-1α protein synthesis through AMPK-dependent manner in HepG2 cells.Our results provided a mechanism for communication from mitochondria to the nucleus through AMPK-HIF-1α. Mitochondrial function is important for HIF-1α expression in cancer progression.
Keywords: Mitochondrial dysfunction; Hypoxia-inducible factor-1α; Adenosine monophosphate-activated protein kinase; Protein synthesis;

Characterization of a small molecule inhibitor of melanogenesis that inhibits tyrosinase activity and scavenges nitric oxide (NO) by Ki Wung Chung; Hyoung Oh Jeong; Eun Ji Jang; Yeon Ja Choi; Dae Hyun Kim; So Ra Kim; Kyung Jin Lee; Hye Jin Lee; Pusoon Chun; Youngjoo Byun; Hyung Ryong Moon; Hae Young Chung (4752-4761).
Excessive melanin production and accumulation are characteristics of a large number of skin diseases, including melasma, and post-inflammatory hyperpigmentation. During our on-going search for new agents with an inhibitory effect on tyrosinase, we synthesized a new type of tyrosinase inhibitor, 4-(thiazolidin-2-yl)benzene-1,2-diol (MHY-794), which directly inhibits mushroom tyrosinase.The inhibitory effect of MHY-794 on tyrosinase activity and nitric oxide (NO) scavenging activity was evaluated in cell free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of MHY-794 in vitro. HRM2 hairless mice were used to evaluate anti-melanogenic effects of MHY-794 in vivo.MHY-794 effectively inhibited mushroom tyrosinase activity in cell free system. In silico docking simulation also supported the inhibitory effects of MHY-794 on mushroom tyrosinase. MHY-794 also proved to be effective at scavenging nitric oxide (NO), which serves as an important modulator in the melanogenesis signaling pathway. In addition, MHY-794 effectively inhibited SNP (NO donor)-induced melanogenesis by directly inhibiting tyrosinase and diminishing NO-mediated melanogenesis signaling in B16 melanoma cells. The anti-melanogenic effects of MHY-794 were further confirmed in HRM2 hairless mice. Ultraviolet light (UV) significantly up-regulated NO-mediated melanogenesis signaling in HRM2 hairless mice, but MHY-794 effectively inhibited both melanogenesis and diminished UV-induced NO-signaling.Our results indicate that MHY-794 is highly effective at inhibiting NO-mediated melanogenesis in vitro and in vivo by direct NO scavenging and directly inhibiting tyrosinase activity, and suggest that MHY-794 be considered a new developmental candidate for the treatment of hyper-pigmentation disorders.MHY-794, which showed great efficacy on NO-mediated melanogenesis by direct NO scavenging as well as direct inhibition of tyrosinase catalytic activity, might be utilized for the development of a new candidate for treatment of the hyper-pigmentation disorders.
Keywords: Melanogenesis; Tyrosinase inhibitor; Nitric oxide (NO); HRM2 hairless mouse;

Protein mechanics: How force regulates molecular function by Christian Seifert; Frauke Gräter (4762-4768).
Regulation of proteins is ubiquitous and vital for any organism. Protein activity can be altered chemically, by covalent modifications or non-covalent binding of co-factors. Mechanical forces are emerging as an additional way of regulating proteins, by inducing a conformational change or by partial unfolding.We review some advances in experimental and theoretical techniques to study protein allostery driven by mechanical forces, as opposed to the more conventional ligand driven allostery. In this respect, we discuss recent single molecule pulling experiments as they have substantially augmented our view on the protein allostery by mechanical signals in recent years. Finally, we present a computational analysis technique, Force Distribution Analysis, that we developed to reveal allosteric pathways in proteins.Any kind of external perturbation, being it ligand binding or mechanical stretching, can be viewed as an external force acting on the macromolecule, rendering force-based experimental or computational techniques, a very general approach to the mechanics involved in protein allostery.This unifying view might aid to decipher how complex allosteric protein machineries are regulated on the single molecular level.
Keywords: Allostery; Cooperativity; Force distribution analysis; Functional regulation; Protein mechanics;

Mechanistic studies for the role of cellular nucleic-acid-binding protein (CNBP) in regulation of c-myc transcription by Siqi Chen; Lijuan Su; Jun Qiu; Nannan Xiao; Jing Lin; Jia-heng Tan; Tian-miao Ou; Lian-quan Gu; Zhi-shu Huang; Ding Li (4769-4777).
Guanine-rich sequence of c-myc nuclease hypersensitive element (NHE) III1 is known to fold in G-quadruplex and subsequently serves as a transcriptional silencer. Cellular nucleic-acid-binding protein (CNBP), a highly conserved zinc-finger protein with multiple biological functions, could bind to c-myc NHE III1 region, specifically to the single strand G-rich sequence.In the present study, a variety of methods, including cloning, expression and purification of protein, EMSA, CD, FRET, Ch-IP, RNA interference, luciferase reporter assay, SPR, co-immunoprecipitation, and co-transfection, were applied to investigate the mechanism for the role of CNBP in regulating c-myc transcription.We found that human CNBP specifically bound to the G-rich sequence of c-myc NHE III1 region both in vitro and in cellulo, and subsequently promoted the formation of G-quadruplex. CNBP could induce a transient decrease followed by an increase in c-myc transcription in vivo. The interaction of CNBP with NM23-H2 was responsible for the increase of c-myc transcription.Based on above experimental results, a new mechanism, involving G-quadruplex related CNBP/NM23-H2 interaction, for the regulation of c-myc transcription was proposed.These findings indicated that the regulation of c-myc transcription through NHE III1 region might be governed by mechanisms involving complex protein–protein interactions, and suggested a new possibility of CNBP as a potential anti-cancer target based on CNBP's biological function in c-myc transcription.
Keywords: Cellular nucleic-acid-binding protein; c-myc; G-quadruplex; Transcriptional regulation; Nuclease hypersensitive element III1;

Molecular insights into 4-nitrophenol-induced hepatotoxicity in zebrafish: Transcriptomic, histological and targeted gene expression analyses by Siew Hong Lam; Choong Yong Ung; Mya Myintzu Hlaing; Jing Hu; Zhi-Hua Li; Sinnakaruppan Mathavan; Zhiyuan Gong (4778-4789).
4-Nitrophenol (4-NP) is a prioritized environmental pollutant and its toxicity has been investigated using zebrafish, advocated as an alternative toxicological model. However, molecular information of 4-NP induced hepatotoxicity is still limited. This study aimed to obtain molecular insights into 4-NP-induced hepatotoxicity using zebrafish as a model.Adult male zebrafish were exposed to 4-NP for 8, 24, 48 and 96 h. Livers were sampled for microarray experiment, qRT-PCR and various histological analyses.Transcriptomic analysis revealed that genes associated with oxidative phosphorylation and electron transport chain were significantly up-regulated throughout early and late stages of 4-NP exposure due to oxidative phosphorylation uncoupling by 4-NP. This in turn induced oxidative stress damage and up-regulated pathways associated with tumor suppressors Rb and p53, cell cycle, DNA damage, proteasome degradation and apoptosis. Pathways associated with cell adhesion and morphology were deregulated. Carbohydrate and lipid metabolisms were down-regulated while methionine and aromatic amino acid metabolisms as well as NFKB pathway associated with chronic liver conditions were up-regulated. Up-regulation of NFKB, NFAT and interleukin pathways suggested hepatitis. Histological analyses with specific staining methods and qRT-PCR analysis of selected genes corroborated with the transcriptomic analysis suggesting 4-NP induced liver injury.Our findings allowed us to propose a plausible model and provide a broader understanding of the molecular events leading to 4-NP induced acute hepatotoxicity for future studies involving other nitrophenol derivatives.This is the first transcriptomic report on 4-NP induced hepatotoxicity.
Keywords: 4-Nitrophenol; Zebrafish; Liver; Transcriptomic; Microarray; Toxicity;

Conformational mobility of active and E-64-inhibited actinidin by Milica M. Grozdanović; Branko J. Drakulić; Marija Gavrović-Jankulović (4790-4799).
Actinidin, a protease from kiwifruit, belongs to the C1 family of cysteine proteases. Cysteine proteases were found to be involved in many disease states and are valid therapeutic targets. Actinidin has a wide pH activity range and wide substrate specificity, which makes it a good model system for studying enzyme–substrate interactions.The influence of inhibitor (E-64) binding on the conformation of actinidin was examined by 2D PAGE, circular dichroism (CD) spectroscopy, hydrophobic ligand binding assay, and molecular dynamics simulations.Significant differences were observed in electrophoretic mobility of proteolytically active and E-64-inhibited actinidin. CD spectrometry and hydrophobic ligand binding assay revealed a difference in conformation between active and inhibited actinidin. Molecular dynamics simulations showed that a loop defined by amino-acid residues 88–104 had greater conformational mobility in the inhibited enzyme than in the active one. During MD simulations, the covalently bound inhibitor was found to change its conformation from extended to folded, with the guanidino moiety approaching the carboxylate.Conformational mobility of actinidin changes upon binding of the inhibitor, leading to a sequence of events that enables water and ions to protrude into a newly formed cavity of the inhibited enzyme. Drastic conformational mobility of E-64, a common inhibitor of cysteine proteases found in many crystal structures stored in PDB, was also observed.The analysis of structural changes which occur upon binding of an inhibitor to a cysteine protease provides a valuable starting point for the future design of therapeutic agents.Display Omitted
Keywords: Actinidin; E-64; Molecular dynamics; Cysteine protease;

It is well documented that poly(sulfate) and poly(sulfonate) anions suppress protein thermal aggregation much more efficiently than poly(carboxylic) anions, but as a rule, they denature protein molecules. In this work, a polymer of different nature, i.e. poly(phosphate) anion (PP) was used to elucidate the influence of phosphate groups on stability and thermal aggregation of the model enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).Isothermal titration calorimetry and differential scanning calorimetry were used for studying the protein–polyanion interactions and the influence of bound polyanions on the protein structure. The enzymatic activity of GAPDH and size of the complexes were measured. The aggregation level was determined from the turbidity.Highly polymerized PP chains were able to suppress the aggregation completely, but at significantly higher concentrations as compared with poly(styrenesulfonate) (PSS) or dextran sulfate chains of the same degree of polymerization. The effect of PP on the enzyme structure and activity was much gentler as opposed to the binding of dextran sulfate or, especially, PSS that denatured GAPDH molecules with the highest efficacy caused by short PSS chains. These findings agreed well with the enhanced affinity of polysulfoanions to GAPDH.The revealed trends might help to illuminate the mechanism of control of proteins functionalities by insertion of charged groups of different nature through posttranslational modifications.Practical implementation of the results could be the use of PP chains as promising tools to suppress the proteins aggregation without noticeable loss in the enzymatic activity.Display Omitted
Keywords: Protein aggregation; Polyanions; Polyphosphate; Poly(styrenesulfonate); Dextran sulfate; Protein sulfation;

Previously, we identified two missense mutations in the chondroitin N-acetylgalactosaminyltransferase-1 gene in patients with neuropathy. These mutations are associated with a profound decrease in chondroitin N-acetylgalactosaminyltransferase-1 enzyme activity. Here, we describe a patient with neuropathy who is heterozygous for a chondroitin synthase-1 mutation. Chondroitin synthase-1 has two glycosyltransferase activities: it acts as a GlcUA and a GalNAc transferase and is responsible for adding repeated disaccharide units to growing chondroitin sulfate chains.Recombinant wild-type chondroitin synthase-1 enzyme and the F362S mutant were expressed. These enzymes and cells expressing them were then characterized.The mutant chondroitin synthase-1 protein retained approximately 50% of each glycosyltransferase activity relative to the wild-type chondroitin synthase-1 protein. Furthermore, unlike chondroitin polymerase comprised of wild-type chondroitin synthase-1 protein, the non-reducing terminal 4-O-sulfation of GalNAc residues synthesized by chondroitin N-acetylgalactosaminyltransferase-1 did not facilitate the elongation of chondroitin sulfate chains when chondroitin polymerase that consists of the mutant chondroitin synthase-1 protein was used as the enzyme source.The chondroitin synthase-1 F362S mutation in a patient with neuropathy resulted in a decrease in chondroitin polymerization activity and the mutant protein was defective in regulating the number of chondroitin sulfate chains via chondroitin N-acetylgalactosaminyltransferase-1. Thus, the progression of peripheral neuropathies may result from defects in these regulatory systems.The elongation of chondroitin sulfate chains may be tightly regulated by the cooperative expression of chondroitin synthase-1 and chondroitin N-acetylgalactosaminyltransferase-1 in peripheral neurons and peripheral neuropathies may result from synthesis of abnormally truncated chondroitin sulfate chains.
Keywords: Chondroitin sulfate; Glycosaminoglycan; Glycosyltransferase; Neuropathy; Proteoglycan;

Honokiol: A non-adipogenic PPARγ agonist from nature by Atanas G. Atanasov; Jian N. Wang; Shi P. Gu; Jing Bu; Matthias P. Kramer; Lisa Baumgartner; Nanang Fakhrudin; Angela Ladurner; Clemens Malainer; Anna Vuorinen; Stefan M. Noha; Stefan Schwaiger; Judith M. Rollinger; Daniela Schuster; Hermann Stuppner; Verena M. Dirsch; Elke H. Heiss (4813-4819).
Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are clinically used to counteract hyperglycemia. However, so far experienced unwanted side effects, such as weight gain, promote the search for new PPARγ activators.We used a combination of in silico, in vitro, cell-based and in vivo models to identify and validate natural products as promising leads for partial novel PPARγ agonists.The natural product honokiol from the traditional Chinese herbal drug Magnolia bark was in silico predicted to bind into the PPARγ ligand binding pocket as dimer. Honokiol indeed directly bound to purified PPARγ ligand-binding domain (LBD) and acted as partial agonist in a PPARγ-mediated luciferase reporter assay. Honokiol was then directly compared to the clinically used full agonist pioglitazone with regard to stimulation of glucose uptake in adipocytes as well as adipogenic differentiation in 3T3-L1 pre-adipocytes and mouse embryonic fibroblasts. While honokiol stimulated basal glucose uptake to a similar extent as pioglitazone, it did not induce adipogenesis in contrast to pioglitazone. In diabetic KKAy mice oral application of honokiol prevented hyperglycemia and suppressed weight gain.We identified honokiol as a partial non-adipogenic PPARγ agonist in vitro which prevented hyperglycemia and weight gain in vivo.This observed activity profile suggests honokiol as promising new pharmaceutical lead or dietary supplement to combat metabolic disease, and provides a molecular explanation for the use of Magnolia in traditional medicine.
Keywords: Peroxisome proliferator-activated receptor; Natural product; Metabolic disease;

Calorie restriction (CR) is accepted as an experimental anti-aging paradigm. Several important signal transduction pathways including AMPK and SIRT1 are implicated in the regulation of physiological processes of CR. However, the mechanisms responsible for adaptations remain unclear in humans.Four overweight male participants were enrolled and treated with 25% CR of their baseline energy requirements for 7 weeks. Characteristics, including body weight (BW), body mass index (BMI), %fat, visceral fat area (VFA), mean blood pressure (MBP) and VO2 max, as well as metabolic parameters, such as insulin, lipid profiles and inflammatory makers and the expression of phosphorylated AMPK and SIRT1 in peripheral blood mononuclear cells (PBMNCs), were determined at baseline and then after 7 weeks. In addition, we assessed the effects of the serum collected from the participants on AMPK and SIRT1 activation and mitochondrial biogenesis in cultured human skeletal muscle cells.After CR, BW, BMI, %fat, VFA and MBP all significantly decreased, while VO2 max increased, compared to those at baseline. The levels of fasting insulin, free fatty acid, and inflammatory makers, such as interleukin-6 and visfatin, were significantly reduced, whereas the expression of phosphorylated AMPK and SIRT1 was significantly increased in PBMNCs collected after CR, compared to those at baseline. The skeletal muscle cells that were cultured in serum collected after CR showed an increase in AMPK and SIRT1 activity as well as mitochondrial biogenesis.CR is beneficial for obesity-related metabolic alterations and induces cellular adaptations against aging, possibly through AMPK and SIRT1 activation via circulating factors.
Keywords: Calorie restriction; AMPK; SIRT1; Mitochondrial biogenesis;

Akt signaling and freezing survival in the wood frog, Rana sylvatica by Jing Zhang; Kenneth B. Storey (4828-4837).
The wood frog (Rana sylvatica) exhibits well-developed natural freeze tolerance supported by multiple mechanisms of biochemical adaptation. The present study investigated the role and regulation of the Akt signaling pathway in wood frog tissues (with a focus on liver) responding to freezing stress.Immunoblotting was used to assess total and phospho-Akt levels, total and phospho-PDK1, PTEN protein level, as well as total and phospho-FOXO1 levels. RT-PCR was used to investigate transcript levels of PTEN and microRNAs.Akt was inhibited in skeletal muscle, kidney and heart after 24 h freezing exposure with a reversal after thawing. The responses of the main kinase (PDK-1) and phosphatase (PTEN) that regulate Akt were consistent with freeze activation of Akt in liver; freezing exposure activated PDK-1 via enhanced Ser-241 phosphorylation whereas PTEN protein levels were reduced. Levels of three microRNAs (miR-26a, miR-126 and miR-217) that regulate pten expression were elevated in liver during freezing. One well-known role of Akt is in anti-apoptosis, mediated in part by Akt phosphorylation of Ser-256 on FOXO1. Freezing triggered an increase in liver phospho-FOXO1 Ser-256 content, suggesting that an important action of Akt may be apoptosis inhibition.Akt activation in wood frog is stress and tissue specific, with multi-facet regulations (posttranslational and posttranscriptional) involved in supporting this specific signal transduction response.This study implicates the Akt pathway in the metabolic reorganization of cellular metabolism in support of freezing survival.
Keywords: Freeze tolerance; miRNA; FOXO1; PDK-1; PTEN; Apoptosis;

Cell response to PEGylated poly(dopamine) coated liposomes considering shear stress by Boon M. Teo; Rebecca van der Westen; Leticia Hosta-Rigau; Brigitte Städler (4838-4847).
Liposomes have gained immerse attention in the field of drug delivery as carriers of therapeutic molecules. Their modification with a polymer either to make them stealth (e.g. using PEG) and/or more stable (e.g. using poly(dopamine) (PDA)) is a crucial aspect to improve their performance e.g. the blood circulation time. Despite their potential, there are only a few commercialized liposome-based formulations for intravenous drug delivery. Hence, there is still considerable need to address the challenges involved in the design and characterization of liposomal therapeutics. In the latter case, it is of paramount importance to consider the dynamic in vivo environment, e.g. the interstitial fluidic pressure in tumors, blood flow, or bile flow in the liver.The PEGylation of PDA films was characterized by quartz crystal microbalance with dissipation monitoring, and the optimized protocol was used to assemble PEGylated PDA coated liposomes (LPDA_PEG). Dynamic light scattering, a plate reader, a flow cytometer and a cytotoxicity assay were used to characterize the liposomes and quantify cellular association/uptake and cell viability in the presence and absence of shear stress after 30 min and 4 h. The immortalized skeletal mouse myoblast (C2C12) cell line was chosen as model cancer cells, and a hepatic cell line (HepG2) was selected due to their importance in nanosized drug carrier clearance from the system in the liver.The presence of hydrophilic cargo did not affect the PDA assembly process. In the absence of shear stress, there was no difference in cellular uptake/association of both PDA coated liposomes (LPDA) and LPDA_PEG for hepatocytes while myoblasts preferentially internalized/associated with LPDA. In the presence of shear stress, hepatocytes preferentially internalized/associated with LPDA after 30 min, while there was only a significant difference for myoblasts after 4 h. The cell viability remained unaffected in all cases.LPDA_PEG are a promising platform towards drug delivery. The nature of cells and fluidic flow are important factors to be considered in their characterization using cell cultures.These findings will contribute in the better understanding of polymer coated liposomes with cells. The importance of microfluidics in cell culture based characterization is demonstrated, and this will eventually affect the way advanced drug delivery vehicles are designed and characterized prior to animal experiments.
Keywords: Liposomes; PEGylated liposomes; Polydopamine; Shear stress; Microfluidics;

The activation of HO-1/Nrf-2 contributes to the protective effects of diallyl disulfide (DADS) against ethanol-induced oxidative stress by Tao Zeng; Cui-Li Zhang; Fu-Yong Song; Xiu-Lan Zhao; Li-Hua Yu; Zhen-Ping Zhu; Ke-Qin Xie (4848-4859).
Diallyl disulfide (DADS) is a garlic-derived organosulfur compound. The current study is designed to evaluate the protective effects of DADS against ethanol-induced oxidative stress, and to explore the underlying mechanisms by examining the HO-1/Nrf-2 pathway.We investigated whether or not DADS could activate the HO-1 in normal human liver cell LO2, and then evaluated the protective effects of DADS against ethanol-induced damage in LO2 cells and in acute ethanol-intoxicated mice. The biochemical parameters were measured using commercial kits. HO-1 mRNA level was determined by RT-PCR. Histopathology and immunofluorescence assay were performed with routine methods. Protein levels were measured by western blot.DADS significantly increased the mRNA and protein levels of HO-1, stimulated the nuclear translocation of Nrf-2 and increased the phosphorylation of MAPK in LO2 cells. The nuclear translocation of Nrf-2 was abrogated by MAPK inhibitors. DADS significantly suppressed ethanol-induced elevation of lactate dehydrogenase (LDH) and aspartate transaminase (AST) activities, decrease of glutathione (GSH) level, increase of malondialdehyde (MDA) levels, and apoptosis of LO2 cells, which were all blocked by ZnPPIX. In mice, DADS effectively suppressed acute ethanol-induced elevation of aminotransferase activities, and improved liver histopathological changes, which might be associated with HO-1 activation.These results demonstrate that DADS could induce the activation of HO-1/Nrf-2 pathway, which may contribute to the protective effects of DADS against ethanol-induced liver injury.DADS may be beneficial for the prevention and treatment of ALD due to significant activation of HO-1/Nrf-2 pathway.
Keywords: Alcoholic liver disease; Oxidative stress; Diallyl disulfide; HO-1; Nrf-2;

Inhibiting toxic aggregation of amyloidogenic proteins: A therapeutic strategy for protein misfolding diseases by Biao Cheng; Hao Gong; Hongwen Xiao; Robert B. Petersen; Ling Zheng; Kun Huang (4860-4871).
The deposition of self-assembled amyloidogenic proteins is associated with multiple diseases, including Alzheimer's disease, Parkinson's disease and type 2 diabetes mellitus. The toxic misfolding and self-assembling of amyloidogenic proteins are believed to underlie protein misfolding diseases. Novel drug candidates targeting self-assembled amyloidogenic proteins represent a potential therapeutic approach for protein misfolding diseases.In this perspective review, we provide an overview of the recent progress in identifying inhibitors that block the aggregation of amyloidogenic proteins and the clinical applications thereof.Compounds such as polyphenols, certain short peptides, and monomer- or oligomer-specific antibodies, can interfere with the self-assembly of amyloidogenic proteins, prevent the formation of oligomers, amyloid fibrils and the consequent cytotoxicity.Some inhibitors have been tested in clinical trials for treating protein misfolding diseases. Inhibitors that target the aggregation of amyloidogenic proteins bring new hope to therapy for protein misfolding diseases.Display Omitted
Keywords: Amyloid; Inhibitor; Protein misfolding disease; Cytotoxicity; Polyphenols;

Double-tailed lipid modification as a promising candidate for oligonucleotide delivery in mammalian cells by Begoña Ugarte-Uribe; Santiago Grijalvo; Jon V. Busto; César Martín; Ramón Eritja; Félix M. Goñi; Itziar Alkorta (4872-4884).
The potential use of nucleic acids as therapeutic drugs has triggered the quest for oligonucleotide conjugates with enhanced cellular permeability. To this end, the biophysical aspects of previously reported potential lipid oligodeoxyribonucleotide conjugates were studied including its membrane-binding properties and cellular uptake.These conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography. Their ability to insert into lipid model membrane systems was evaluated by Langmuir balance and confocal microscopy followed by the study of the internalization of a lipid oligodeoxyribonucleotide conjugate bearing a double-tail lipid modification (C28) into different cell lines by confocal microscopy and flow cytometry. This compound was also compared with other lipid containing conjugates and with the classical lipoplex formulation using Transfectin as transfection reagent.This double-tail lipid modification showed better incorporation into both lipid model membranes and cell systems. Indeed, this lipid conjugation was capable of inserting the oligodeoxyribonucleotide into both liquid-disordered and liquid-ordered domains of model lipid bilayer systems and produced an enhancement of oligodeoxyribonucleotide uptake in cells, even better than the effect caused by lipoplexes. In addition, in β2 integrin (CR3) expressing cells this receptor was directly involved in the enhanced internalization of this compound.All these features confirm that the dual lipid modification (C28) is an excellent modification for enhancing nucleic acid delivery without altering their binding properties.Compared to the commercial lipoplex approach, oligodeoxyribonucleotide conjugation with C28 dual lipid modification seems to be promising to improve oligonucleotide delivery in mammalian cells.
Keywords: Lipid oligonucleotide conjugates; Model membrane systems; Cellular uptake and localization;

Ovarian cancer ascites-derived vitronectin and fibronectin: Combined purification, molecular features and effects on cell response by Ludovic Carduner; Rémy Agniel; Sabrina Kellouche; Cédric R. Picot; Cécile Blanc-Fournier; Johanne Leroy-Dudal; Franck Carreiras (4885-4897).
Intra-abdominal ascites is a complication of ovarian cancers and constitutes a permissive microenvironment for metastasis. Since fibronectin and vitronectin are key actors in ovarian cancer progression, we investigated their occurrence and molecular characteristics in various ascites fluids and the influence of these ascites-derived proteins on cell behavior.Fibronectin and vitronectin were investigated by immunoblotting within various ascites fluids. A combined affinity-based protocol was developed to purify both proteins from the same sample. Each purified protein was characterized with regard to its molecular features (molecular mass of isoforms, tryptophan intramolecular environment, hydrodynamic radii), and its influence on cell adhesion.Fibronectin and vitronectin were found in all tested ascites. Several milligrams of purified proteins were obtained from ascites of varying initial volumes. Molecular mass isoforms and conformational lability of proteins differed according to the ascites of origin. When incorporated into the cancer cell environment, ascites-derived fibronectin and vitronectin supported cell adhesion and migration with various degrees of efficiency, and induced the recruitment of integrins into focal contacts.To our knowledge, this is the first combined purification of two extracellular matrix proteins from a single pathological sample containing a great variety of bioactive molecules. This study highlights that ascites-derived fibronectin and vitronectin exhibit different properties depending on the ascites.Investigating the relationships between the molecular properties of ascites components and ovarian cancer cell phenotype according to the ascites may be critical for a better understanding of the recurrence of this lethal disease and for further biomarker identification.
Keywords: Ascites; Microenvironment; Vitronectin; Fibronectin; Affinity chromatography;

Corrigendum to “Rejoining of cut wounds by engineered gelatin-keratin glue” [Biochim. Biophys. Acta, Gen. Subj. (2013) 4030–4039] by S. Thirupathi Kumara Raja; T. Thiruselvi; G. Sailakshmi; S. Ganesh; A. Gnanamani (4898).

H19 inhibits RNA polymerase II-mediated transcription by disrupting the hnRNP U–actin complex by Hai-shan Bi; Xiang-yue Yang; Ji-hang Yuan; Fu Yang; Dan Xu; Ying-jun Guo; Ling Zhang; Chuan-chuan Zhou; Fang Wang; Shu-han Sun (4899-4906).
H19 was one of the earliest identified, and is the most studied, long noncoding RNAs. It is presumed that H19 is essential for regulating development and disease conditions, and it is associated with carcinogenesis for many types. However the biological function and regulatory mechanism of this conserved RNA, particularly with respect to its effect on transcription, remain largely unknown.We performed RNA pulldown, RNA immunoprecipitation and deletion mapping to identify the proteins that are associated with H19. In addition, we employed EU (5-ethynyl uridine) incorporation, immunoprecipitation and Western blotting to investigate the functional aspects of H19.Our research further verifies that H19 is bound to hnRNP U, and this interaction is located within the 5′ 882 nt region of H19. Moreover, H19 disrupts the interaction between hnRNP U and actin, which inhibits phosphorylation at Ser5 of the RNA polymerase II (Pol II) C-terminal domain (CTD), consequently preventing RNA Pol II-mediated transcription. We also showed that hnRNP U is essential for H19-mediated transcription repression.In this study, we demonstrate that H19 inhibits RNA Pol II-mediated transcription by disrupting the hnRNP U–actin complex.These data suggest that H19 regulates general transcription and exerts wide-ranging effects in organisms.
Keywords: C-terminal domain phosphorylation; 5-Ethynyl uridine incorporation; H19; hnRNP U; Transcription repression;

RETRACTED: Acetyl-11-keto-beta-boswellic acid (AKBA) prevents human colonic adenocarcinoma growth through modulation of multiple signaling pathways by Yi Yuan; Shu-Xiang Cui; Yan Wang; Han-Ni Ke; Rui-Qi Wang; Hong-Xiang Lou; Zu-Hua Gao; Xian-Jun Qu (4907-4916).
This article has been retracted at the request of the authors. It contained multiple inaccurate and inappropriately processed Western Blots. Some Figure panels of this article were also found in a second paper from the same group (Yuan et al. BBA General Subjects (2013) 1830, 4907–4916) that has also been retracted. The corresponding author has taken full responsibility and apologizes to the readers of BBA General Subjects for submitting and publishing the erroneous article and any inconvenience caused.

CCL2 increases αvβ3 integrin expression and subsequently promotes prostate cancer migration by Tien-Huang Lin; Hsin-Ho Liu; Tsung-Hsun Tsai; Chi-Cheng Chen; Teng-Fu Hsieh; Shang-Sen Lee; Yuan-Ju Lee; Wen-Chi Chen; Chih-Hsin Tang (4917-4927).
Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), belongs to the CC chemokine family which is associated with the disease status and outcomes of cancers. Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to the bone. However, the effect of CCL2 on human prostate cancer cells is largely unknown. The aim of this study was to examine the role of CCL2 in integrin expression and migratory activity in prostate cancers.Prostate cancer migration was examined using Transwell, wound healing, and invasion assay. The PKCδ and c-Src phosphorylations were examined by using western blotting. The qPCR was used to examine the mRNA expression of integrins. A transient transfection protocol was used to examine AP-1 activity.Stimulation of prostate cancer cell lines (PC3, DU145, and LNCaP) induced migration and expression of integrin αvβ3. Treatment of cells with αvβ3 antibody or siRNA abolished CCL2-increased cell migration. CCL2-increased migration and integrin expression were diminished by CCR2 but not by CCR4 inhibitors, suggesting that the CCR2 receptor is involved in CCL2-promoted prostate cancer migration. CCL2 activated a signal transduction pathway that includes PKCδ, c-Src, and AP-1. Reagents that inhibit specific components of this pathway each diminished the ability of CCL2 to effect cell migration and integrin expression.Interaction between CCL2 and CCR2 enhances migration of prostate cancer cells through an increase in αvβ3 integrin production.CCL2 is a critical factor of prostate cancer metastasis.
Keywords: CCL2; CCR2; Prostate cancer; Migration; Integrin;

In the present study, we have investigated the possibility that cartilage oligomeric matrix protein angiopoietin1 (COMP-Ang1), important factor in angiogenesis, osteogenesis and the survival of mesenchymal stem cells (MSCs) through the Ang1/Tie2 pathway has beneficial effects on osteogenic differentiated cells (ODCs) from MSCs treated by advanced glycation end products (AGE), which are pathological factors of diabetes.Primary culture of MSCs was used. For comparison analysis of AGE and COMP-Ang1 effects, we performed cell viability assay with each treated variety concentration for 24 h. Apoptosis rate and Caspase-3 activity were measured by each ELISA assay. To make sure with Ang1/Tie2 pathway, we performed small interfering RNA transfected to MSCs. Real-time RT-PCR was performed to identify ODCs marker genes. Immunoblotting was used to evaluate the expression of Tie2, AKT, p38 and ERK.Our results clearly demonstrate that COMP-Ang1 upregulates the phosphorylation of AKT and p38 by activating the Ang1/Tie2 signaling pathway, indicating that COMP-Ang1 affects both AGE-induced apoptosis and the attenuated osteogenic differentiation of MSCs through the p38/MAPK and PI3K/AKT pathways.COMP-Ang1 improves cell viability and differentiation function of ODCs against AGE via Ang/Tie2 signaling pathway.Our results suggest the potential importance of COMP-Ang1 as a new therapy for impaired bone formation that is associated with diabetes and advanced age.
Keywords: Diabetes; COMP-Ang1; Advanced glycation end products; Mesenchymal stem cells; Tie2 signaling;

G-Quadruplex conformational change driven by pH variation with potential application as a nanoswitch by Yi-Yong Yan; Jia-Heng Tan; Yu-Jing Lu; Siu-Cheong Yan; Kwok-Yin Wong; Ding Li; Lian-Quan Gu; Zhi-Shu Huang (4935-4942).
G-Quadruplex is a highly polymorphic structure, and its behavior in acidic condition has not been well studied.Circular dichroism (CD) spectra were used to study the conformational change of G-quadruplex. The thermal stabilities of the G-quadruplex were measured with CD melting. Interconversion kinetics profiles were investigated by using CD kinetics. The fluorescence of the inserted 2-Aminopurine (Ap) was monitored during pH change and acrylamide quenching, indicating the status of the loop. Proton NMR was adopted to help illustrate the change of the conformation.G-Quadruplex of specific loop was found to be able to transform upon pH variation. The transformation was resulted from the loop rearrangement. After screening of a library of diverse G-quadruplex, a sequence exhibiting the best transformation property was found. A pH-driven nanoswitch with three gears was obtained based on this transition cycle.Certain G-quadruplex was found to go through conformational change at low pH. Loop was the decisive factor controlling the interconversion upon pH variation. G-Quadruplex with TT central loop could be converted in a much milder condition than the one with TTA loop. It can be used to design pH-driven nanodevices such as a nanoswitch.These results provide more insights into G-quadruplex polymorphism, and also contribute to the design of DNA-based nanomachines and logic gates.Display Omitted
Keywords: G-Quadruplex; Acidic pH; Conformational interconversion; Nanoswitch; pH-driven; Thiazole orange (TO);

Glutathione metabolism modeling: A mechanism for liver drug-robustness and a new biomarker strategy by Suzanne Geenen; Franco B. du Preez; Jacky L. Snoep; Alison J. Foster; Sunil Sarda; J. Gerry Kenna; Ian D. Wilson; Hans V. Westerhoff (4943-4959).
Glutathione metabolism can determine an individual's ability to detoxify drugs. To increase understanding of the dynamics of cellular glutathione homeostasis, we have developed an experiment-based mathematical model of the kinetics of the glutathione network. This model was used to simulate perturbations observed when human liver derived THLE cells, transfected with human cytochrome P452E1 (THLE-2E1 cells), were exposed to paracetamol (acetaminophen).Human liver derived cells containing extra human cytochrome P4502E1 were treated with paracetamol at various levels of methionine and in the presence and absence of an inhibitor of glutamyl-cysteine synthetase (GCS). GCS activity was also measured in extracts. Intracellular and extracellular concentrations of substances involved in glutathione metabolism were measured as was damage to mitochondria and proteins. A bottom up mathematical model was made of the metabolic pathways around and including glutathione.Our initial model described some, but not all the metabolite-concentration and flux data obtained when THLE-2E1 cells were exposed to paracetamol at concentrations high enough to affect glutathione metabolism. We hypothesized that the lack of correspondence could be due to upregulation of expression of glutamyl cysteine synthetase, one of the enzymes controlling glutathione synthesis, and confirmed this experimentally. A modified model which incorporated this adaptive response adequately described the observed changes in the glutathione pathway. Use of the adaptive model to analyze the functioning of the glutathione network revealed that a threshold input concentration of methionine may be required for effective detoxification of reactive metabolites by glutathione conjugation. The analysis also provided evidence that 5-oxoproline and ophthalmic acid are more useful biomarkers of glutathione status when analyzed together than when analyzed in isolation, especially in a new, model-assisted integrated biomarker strategy.A robust mathematical model of the dynamics of cellular changes in glutathione homeostasis in cells has been developed and tested in vitro.Mathematical models of the glutathione pathway that help examine mechanisms of cellular protection against xenobiotic toxicity and the monitoring thereof, can now be made.
Keywords: Gene expression-metabolism hybrid model; Glutathione metabolism; Acetaminophen toxicity; Biomarker; Adaptation;

Extensive evaluations of the cytotoxic effects of gold nanoparticles by Show-Mei Chuang; Yi-Hui Lee; Ruei-Yue Liang; Gwo-Dong Roam; Zih-Ming Zeng; Hsin-Fang Tu; Shi-Kwun Wang; Pin Ju Chueh (4960-4973).
Many in vitro studies have revealed that the interference of dye molecules in traditional nanoparticle cytotoxicity assays results in controversial conclusions. The aim of this study is to establish an extensive and systematic method for evaluating biological effects of gold nanoparticles in mammalian cell lines.We establish the cell-impedance measurement system, a label-free, real-time cell monitoring platform that measures electrical impedance, displaying results as cell index values, in a variety of mammalian cell lines. Cytotoxic effects of gold nanoparticles are also evaluated with traditional in vitro assays.Among the six cell lines, gold nanoparticles induce a dose-dependent suppression of cell growth with different levels of severity and the suppressive effect of gold nanoparticles was indirectly associated with their sizes and cellular uptake. Mechanistic studies revealed that the action of gold nanoparticles is mediated by apoptosis induction or cell cycle delay, depending on cell type and cellular context. Although redox signaling is often linked to the toxicity of nanoparticles, in this study, we found that gold nanoparticle-mediated reactive oxygen species generation was not sustained to notably modulate proteins involved in antioxidative defense system.The cell-impedance measurement system, a dye-free, real-time screening platform, provides a reliable analysis for monitoring gold nanoparticle cytotoxicity in a variety of mammalian cell lines. Furthermore, gold nanoparticles induce cellular signaling and several sets of gene expression to modulate cellular physical processes.The systematic approach, such as cell-impedance measurement, analyzing the toxicology of nanomaterials offers convincing evidence of the cytotoxicity of gold nanomaterials.Display Omitted
Keywords: Cytotoxicity; Cell-impedance measurement; DNA microarray; Gold nanoparticle; In vitro assay;

The impact of high hydrostatic pressure on structure and dynamics of β-lactoglobulin by Daniela Russo; Maria Grazia Ortore; Francesco Spinozzi; Paolo Mariani; Camille Loupiac; Burkhard Annighofer; Alessandro Paciaroni (4974-4980).
Combining small-angle X-ray and neutron scattering measurements with inelastic neutron scattering experiments, we investigated the impact of high hydrostatic pressure on the structure and dynamics of β-lactoglobulin (βLG) in aqueous solution.βLG is a relatively small protein, which is predominantly dimeric in physiological conditions, but dissociates to monomer below about pH 3.High-pressure structural results show that the dimer–monomer equilibrium, as well as the protein–protein interactions, are only slightly perturbed by pressure, and βLG unfolding is observed above a threshold value of 3000 bar. In the same range of pressure, dynamical results put in evidence a slowing down of the protein dynamics in the picosecond timescale and a loss of rigidity of the βLG structure. This dynamical behavior can be related to the onset of unfolding processes, probably promoted from water penetration in the hydrophobic cavity.Results suggest that density and compressibility of water molecules in contact with the protein are key parameters to regulate the protein flexibility.
Keywords: Hydrostatic pressure; Protein folding; Protein dynamics; Neutron scattering; Small angle X-ray and neutron scattering;