BBA - General Subjects (v.1840, #1)

Interplay between autophagy and apoptosis mediated by copper oxide nanoparticles in human breast cancer cells MCF7 by Dipranjan Laha; Arindam Pramanik; Jyotirindra Maity; Ananda Mukherjee; Panchanan Pramanik; Aparna Laskar; Parimal Karmakar (1-9).
Metal oxide nanoparticles are well known to generate oxidative stress and deregulate normal cellular activities. Among these, transition metals copper oxide nanoparticles (CuO NPs) are more compelling than others and able to modulate different cellular responses.In this work, we have synthesized and characterized CuO NPs by various biophysical methods. These CuO NPs (~ 30 nm) induce autophagy in human breast cancer cell line, MCF7 in a time- and dose-dependent manner. Cellular autophagy was tested by MDC staining, induction of green fluorescent protein-light chain 3 (GFP-LC3B) foci by confocal microscopy, transfection of pBABE-puro mCherry-EGFP-LC3B plasmid and Western blotting of autophagy marker proteins LC3B, beclin1 and ATG5. Further, inhibition of autophagy by 3-MA decreased LD50 doses of CuO NPs. Such cell death was associated with the induction of apoptosis as revealed by FACS analysis, cleavage of PARP, de-phosphorylation of Bad and increased cleavage product of caspase 3. siRNA mediated inhibition of autophagy related gene beclin1 also demonstrated similar results. Finally induction of apoptosis by 3-MA in CuO NP treated cells was observed by TEM.This study indicates that CuO NPs are a potent inducer of autophagy which may be a cellular defense against the CuO NP mediated toxicity and inhibition of autophagy switches the cellular response into apoptosis.A combination of CuO NPs with the autophagy inhibitor is essential to induce apoptosis in breast cancer cells.CuO NP induced autophagy is a survival strategy of MCF7 cells and inhibition of autophagy renders cellular fate to apoptosis.Copper oxide NPs induce autophagy in human breast cancer cell line MCF7.Display Omitted
Keywords: Copper oxide nanoparticle; Autophagy; Apoptosis; Small interfering RNA;

Chaperone-mediated native folding of a β-scorpion toxin in the periplasm of Escherichia coli by A.O. O'Reilly; A.R. Cole; J.L.S. Lopes; A. Lampert; B.A. Wallace (10-15).
Animal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria.The β-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the Escherichia coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined.Western blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold.The chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method.We report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors.
Keywords: Chaperones; Disulfide-linked toxin; Protein folding; Protein expression; Crystal structure; Synchrotron radiation circular dichroism spectroscopy;

The details of the functional interaction between G proteins and the G protein coupled receptors (GPCRs) have long been subjected to extensive investigations with structural and functional assays and a large number of computational studies.The nature and sites of interaction in the G-protein/GPCR complexes, and the specificities of these interactions selecting coupling partners among the large number of families of GPCRs and G protein forms, are still poorly defined.Many of the contact sites between the two proteins in specific complexes have been identified, but the three dimensional molecular architecture of a receptor-Gα interface is only known for one pair. Consequently, many fundamental questions regarding this macromolecular assembly and its mechanism remain unanswered.In the context of current structural data we review the structural details of the interfaces and recognition sites in complexes of sub-family A GPCRs with cognate G-proteins, with special emphasis on the consequences of activation on GPCR structure, the prevalence of preassembled GPCR/G-protein complexes, the key structural determinants for selective coupling and the possible involvement of GPCR oligomerization in this process.
Keywords: G protein coupled receptor/G-protein coupling; Specificity; Oligomerization; Coupling determinant;

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli by Chunli Zhang; Matteo Allegretti; Janet Vonck; Julian D. Langer; Marco Marcia; Guohong Peng; Hartmut Michel (34-40).
F1FO ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F1FO ATP synthases.Our system now allows performing previously impossible structural and functional studies on A. aeolicus F1FO ATP synthase.More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.Display Omitted
Keywords: Membrane protein complex; Hyperthermophilic organism; Heterologous expression; Artificial operon; Protein assembly; Respiratory enzyme;

Solution equilibria of cytosine- and guanine-rich sequences near the promoter region of the n-myc gene that contain stable hairpins within lateral loops by Sanae Benabou; Rubén Ferreira; Anna Aviñó; Carlos González; Sébastien Lyonnais; Maria Solà; Ramon Eritja; Joaquim Jaumot; Raimundo Gargallo (41-52).
Cytosine- and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34-base long cytosine-rich sequence and its complementary guanine-rich strand corresponding to the first intron of the n-myc gene were studied. Both sequences, not yet studied, contain a 12-base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively.Spectroscopic, mass spectrometry and separation techniques, as well as multivariate data analysis methods, were used to unravel the species and conformations present.The cytosine-rich sequence forms two i-motifs that differ in the protonation of bases located in the loops. A stable Watson–Crick hairpin is formed by the bases in the first loop, stabilizing the i-motif structure. The guanine-rich sequence adopts a parallel G-quadruplex structure that is stable throughout the pH range 3–7, despite the protonation of cytosine and adenine bases at lower pH values. The presence of G-quadruplex aggregates was confirmed using separation techniques. When mixed, G-quadruplex and i-motif coexist with the Watson–Crick duplex across a pH range from approximately 3.0 to 6.5.Two cytosine- and guanine-rich sequences in n-myc gene may form stable i-motif and G-quadruplex structures even in the presence of long loops. pH modulates the equilibria involving the intramolecular structures and the intermolecular Watson–Crick duplex.Watson–Crick hairpins located in the intramolecular G-quadruplexes and i-motifs in the promoter regions of oncogenes could play a role in stabilizing these structures.
Keywords: G-quadruplex; i-Motif; Quadruplex/duplex competition; n-myc; Chromatography; Multivariate analysis;

Adaptive responses to glucose restriction enhance cell survival, antioxidant capability, and autophagy of the protozoan parasite Trichomonas vaginalis by Kuo-Yang Huang; Yi-Ywan Margaret Chen; Yi-Kai Fang; Wei-Hung Cheng; Chih-Chieh Cheng; Yu-Chuen Chen; Tiffany E. Wu; Fu-Man Ku; Shih-Chieh Chen; Rose Lin; Petrus Tang (53-64).
To establish an infection in the vagina, Trichomonas vaginalis must adapt to various environmental cues for survival and further replication. Nutrient competition by lactobacilli, the major normal vaginal flora, is one of the mechanisms to limit the growth of other microorganisms. Additionally, lactobacilli produce H2O2 that can reduce the genital infections caused by other pathogens. Thus, the ability to overcome the metabolic stresses, such as glucose restriction (GR), as well as the oxidative stresses, is critical for T. vaginalis to establish an infection.To gain insights into the molecular mechanisms of adaptation to GR, we utilized next-generation RNA sequencing (RNA-seq) to quantify the gene expression changes upon GR. Autophagy, a cytoprotective response to starvation, was monitored by using autophagy-specific staining, autophagy inhibition assay, and co-localization of autophagosomes with lysosomes.We demonstrated that GR promotes the survival of T. vaginalis. Besides, GR-cultivated cells exhibit higher H2O2 resistance. Our RNA-seq data revealed that genes involved in general energy metabolism were downregulated, whereas genes encoding glutamate metabolism-related aminotransferases were strikingly upregulated under GR. Furthermore, autophagy was first identified and characterized in T. vaginalis under GR.These data suggest that GR induces a metabolic reprogramming, enhancing antioxidant ability and autophagy for cellular homeostasis to maintain survival.Our work not only led to significant advances in understanding the transcriptional changes in response to GR but also provided possible strategies elicited by GR for T. vaginalis to adapt to the vaginal microenvironment.Display Omitted
Keywords: T. vaginalis; Glucose restriction; RNA sequencing; Glutamate dehydrogenase; Autophagy;

Epigenetic regulation of glycosylation is the quantum mechanics of biology by Gordan Lauc; Aleksandar Vojta; Vlatka Zoldoš (65-70).
Most proteins are glycosylated, with glycans being integral structural and functional components of a glycoprotein. In contrast to polypeptides, which are fully encoded by the corresponding gene, glycans result from a dynamic interaction between the environment and a network of hundreds of genes.Recent developments in glycomics, genomics and epigenomics are discussed in the context of an evolutionary advantage for higher eukaryotes over microorganisms, conferred by the complexity and adaptability which glycosylation adds to their proteome.Inter-individual variation of glycome composition in human population is large; glycome composition is affected by both genes and environment; epigenetic regulation of “glyco-genes” has been demonstrated; and several mechanisms for transgenerational inheritance of epigenetic marks have been documented.Epigenetic recording of acquired characteristics and their transgenerational inheritance could be important mechanisms used by higher organisms to compete or collaborate with microorganisms.
Keywords: Glycosylation; Epigenetics; Adaptation; Evolution;

Modulated immune signal (CD14–TLR and TNF) in leishmaniasis can be linked to EGFR pathway involved in wound healing, through crosstalk points. This signaling network can be further linked to a synthetic gene circuit acting as a positive feedback loop to elicit a synchronized intercellular communication among the immune cells which may contribute to a better understanding of signaling dynamics in leishmaniasis.Network reconstruction with positive feedback loop, simulation (ODE 15s solver) and sensitivity analysis of CD14–TLR, TNF and EGFR was done in SimBiology (MATLAB 7.11.1). Cytoscape and adjacency matrix were used to calculate network topology. PCA was extracted by using sensitivity coefficient in MATLAB. Model reduction was done using time, flux and sensitivity score.Network has five crosstalk points: NIK, IκB–NFκB and MKK (4/7, 3/6, 1/2) which show high flux and sensitivity. PI3K in EGFR pathway shows high flux and sensitivity. PCA score was high for cytoplasmic ERK1/2, PI3K, Atk, STAT1/3 and nuclear JNK. Of the 125 parameters, 20% are crucial as deduced by model reduction.EGFR can be linked to CD14–TLR and TNF through the MAPK crosstalk points. These pathways may be controlled through Ras and Raf that lie upstream of signaling components ERK ½ (c) and JNK (n) that have a high PCA score via a synthetic gene circuit for activating cell–cell communication to elicit an inflammatory response. Also a disease resolving effect may be achieved through PI3K in the EGFR pathway.The reconstructed signaling network can be linked to a gene circuit with a positive feedback loop, for cell–cell communication resulting in synchronized response in the immune cell population, for disease resolving effect in leishmaniasis.Display Omitted
Keywords: Cross-talk; Signaling; Leishmania; Mathematical modeling; Systems biology; ODE;

Expanding the paradigm of thiol redox in the thermophilic root of life by Joshua Heinemann; Timothy Hamerly; Walid S. Maaty; Navid Movahed; Joseph D. Steffens; Benjamin D. Reeves; Jonathan K. Hilmer; Jesse Therien; Paul A. Grieco; John W. Peters; Brian Bothner (80-85).
The current paradigm of intracellular redox chemistry maintains that cells establish a reducing environment maintained by a pool of small molecule and protein thiol to protect against oxidative damage. This strategy is conserved in mesophilic organisms from all domains of life, but has been confounded in thermophilic organisms where evidence suggests that intracellular proteins have abundant disulfides.Chemical labeling and 2-dimensional gel electrophoresis were used to capture disulfide bonding in the proteome of the model thermophile Sulfolobus solfataricus. The redox poise of the metabolome was characterized using both chemical labeling and untargeted liquid chromatography mass spectrometry. Gene annotation was undertaken using support vector machine based pattern recognition.Proteomic analysis indicated the intracellular protein thiol of S. solfataricus was primarily in the disulfide form. Metabolic characterization revealed a lack of reduced small molecule thiol. Glutathione was found primarily in the oxidized state (GSSG), at relatively low concentration. Combined with genetic analysis, this evidence shows that pathways for synthesis of glutathione do exist in the archaeal domain.In observed thermophilic organisms, thiol abundance and redox poise suggest that this system is not directly utilized for protection against oxidative damage. Instead, a more oxidized intracellular environment promotes disulfide bonding, a critical adaptation for protein thermostability.Based on the placement of thermophilic archaea close to the last universal common ancestor in rRNA phylogenies, we hypothesize that thiol-based redox systems are derived from metabolic pathways originally tasked with promoting protein stability.Display Omitted
Keywords: Thiol; Glutathione; Thermophile; Disulfide; Proteomics; Metabolomics;

FoxO is required for the activation of hypertrehalosemic hormone expression in cockroaches by Songül Süren-Castillo; Marc Abrisqueta; José L. Maestro (86-94).
FoxO proteins are a subgroup of the Forkhead-box family of transcription factors, which function as the main transcriptional effectors of the insulin receptor pathway. This pathway, activated by the binding of insulin or IGFs (or insect insulin-like peptides), promotes the phosphorylation and inactivation of FoxO because of its export from the nucleus to the cytoplasm. The homolog of FoxO in the cockroach Blattella germanica works in a situation of nutrient shortage by inhibiting the endocrine induction of reproduction.Using Blattella germanica as a model, we studied the functions of FoxO using RNA interference methodologies. We analyzed the mRNA levels of hypertrehalosemic hormone (HTH) and genes related to lipolysis, glycogenolysis and gluconeogenesis and quantified triacylglycerides, glycogen and trehalose.FoxO knockdown eliminates the starvation-induced expression of HTH in the corpora cardiaca. In addition, FoxO knockdown prevents the activation of the expression of Brummer lipase, glycogen phosphorylase and phosphoenolpyruvate carboxylase in the fat body of starved females.Starvation-induced activation of FoxO stimulates the transcription of different genes related to catabolic processes, including HTH and genes involved in lipolysis, glycogenolysis and gluconeogenesis.Our results show conservation in the action of the transcription factor FoxO in the activation of catabolic processes from basal insects to vertebrates. The results also describe a new and essentially different mode of action of transcription factor FoxO, which works through the activation of neuropeptide HTH expression, which will subsequently produce its own catabolic stimulatory function.
Keywords: FoxO; Insulin; Catabolism; Hypertrehalosemic/adipokinetic hormone; Glucagon; Blattella germanica;

Resveratrol enhances TNF-α production in human monocytes upon bacterial stimulation by Guido A. Gualdoni; Johannes J. Kovarik; Johannes Hofer; Franziska Dose; Marc Pignitter; Daniel Doberer; Peter Steinberger; Veronika Somoza; Michael Wolzt; Gerhard J. Zlabinger (95-105).
Resveratrol is a key component of red wine that has been reported to have anti-carcinogenic and anti-aging properties. Additional studies conducted in vitro and in animal models suggested anti-inflammatory properties. However, data from primary human immune cells and in vivo studies are limited.A pilot study was performed including 10 healthy volunteers. Plasma cytokine levels were measured over 48 h after oral application of 5 g resveratrol.To verify the in vivo findings, cytokine release and gene expression in human peripheral blood mononuclear cells (PBMC) and/or monocytes was assessed after treatment with resveratrol or its metabolites and stimulation with several toll-like receptor (TLR)-agonists. Additionally, the impact on intracellular signaling pathways was analyzed using a reporter cell line and Western blotting.Resveratrol treated individuals showed a significant increase in tumor necrosis factor-α (TNF-α) levels 24 h after treatment compared to baseline. Studies using human PBMC or isolated monocytes confirmed potentiation of TNF-α production with different TLR agonists, while interleukin (IL)-10 was inhibited. Moreover, we observed significantly enhanced nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB) activation using a reporter cell line and found increased phosphorylation of p105, which is indicative of alternative NF-κB pathway activation.By administering resveratrol to healthy humans and utilizing primary immune cells we were able to detect TNF-α enhancing properties of the agent. In parallel, we found enhanced alternative NF-κB activation. We report on a novel pro-inflammatory property of resveratrol which has to be considered in concepts of its biologic activity.
Keywords: Resveratrol; Tumor necrosis factor-α; Monocyte; Toll-like receptor; Immunomodulation; Signaling;

Modulation of Dcytb (Cybrd 1) expression and function by iron, dehydroascorbate and Hif-2α in cultured cells by Xiaomin Luo; Melanie Hill; Anna Johnson; Gladys O. Latunde-Dada (106-112).
Duodenal cytochrome b (Dcytb) is a mammalian plasma ferric reductase enzyme that catalyses the reduction of ferric to ferrous ion in the process of iron absorption. The current study investigates the relationship between Dcytb, iron, dehydroascorbate (DHA) and Hif-2α in cultured cell lines.Dcytb and Hif-2α protein expression was analysed by Western blot technique while gene regulation was determined by quantitative PCR. Functional analyses were carried out by ferric reductase and 59Fe uptake assays.Iron and dehydroascorbic acid treatment of cells inhibited Dcytb mRNA and protein expression. Desferrioxamine also enhanced Dcytb mRNA level after cells were treated overnight. Dcytb knockdown in HuTu cells resulted in reduced mRNA expression and lowered reductase activity. Preloading cells with DHA (to enhance intracellular ascorbate levels) did not stimulate reductase activity fully in Dcytb-silenced cells, implying a Dcytb-dependence of ascorbate-mediated ferrireduction. Moreover, Hif-2α knockdown in HuTu cells led to a reduction in reductase activity and iron uptake.Taken together, this study shows the functional regulation of Dcytb reductase activity by DHA and Hif-2α.Dcytb is a plasma membrane protein that accepts electrons intracellularly from DHA/ascorbic acid for ferrireduction at the apical surface of cultured cells and enterocytes.
Keywords: Dcytb; DHA; Hif-2α; Cells; Gene silencing;

Tracking sulfur and phosphorus within single starch granules using synchrotron X-ray microfluorescence mapping by Alain Buléon; Marine Cotte; Jean-Luc Putaux; Christophe d'Hulst; Jean Susini (113-119).
Native starch accumulates as granules containing two glucose polymers: amylose and amylopectin. Phosphate (0.2–0.5%) and proteins (0.1–0.7%) are also present in some starches. Phosphate groups play a major role in starch metabolism while granule-bound starch synthase 1 (GBSS1) which represents up to 95% of the proteins bound to the granule is responsible for amylose biosynthesis.Synchrotron micro-X-ray fluorescence (μXRF) was used for the first time for high-resolution mapping of GBSS1 and phosphate groups based on the XRF signal of sulfur (S) and phosphorus (P), respectively. Wild-type starches were studied as well as their related mutants lacking GBSS1 or starch-phosphorylating enzyme.Wild-type potato and maize starch exhibited high level of phosphorylation and high content of sulfur respectively when compared to mutant potato starch lacking glucan water dikinase (GWD) and mutant maize starch lacking GBSS1. Phosphate groups are mostly present at the periphery of wild-type potato starch granules, and spread all over the granule in the amylose-free mutant. P and S XRF were also measured within single small starch granules from Arabidopsis or Chlamydomonas not exceeding 3–5 μm in diameter.Imaging GBSS1 (by S mapping) in potato starch sections showed that the antisense technique suppresses the expression of GBSS1 during biosynthesis. P mapping confirmed that amylose is mostly present in the center of the granule, which had been suggested before.μXRF is a potentially powerful technique to analyze the minor constituents of starch and understand starch structure/properties or biosynthesis by the use of selected genetic backgrounds.Display Omitted
Keywords: Micro X-ray fluorescence; Synchrotron; Starch; Sulfur; Phosphorus; Granule bound starch synthase;

1-Aminocyclopropane-1-carboxylate oxidase (ACO) is a key enzyme that catalyses the final step in the biosynthesis of the plant hormone ethylene. Recently, the first ACO homologue gene was isolated in Agaricus bisporus, whereas information concerning the nature of the ethylene-forming activity of this mushroom ACO is currently lacking.Recombinant ACO from A. bisporus (Ab-ACO) was purified and characterised for the first time. Molecular modelling combined with site-directed mutagenesis and kinetic and spectral analysis were used to investigate the property of Ab-ACO.Ab-ACO has eight amino acid residues that are conserved in the Fe (II) ascorbate family of dioxygenases, including four catalytic residues in the active site, but Ab-ACO lacks a key residue, S289. In comparison to plant ACOs, Ab-ACO requires ACC and Fe (II) but does not require ascorbate. In addition, Ab-ACO had relatively low activity and was completely dependent on bicarbonate, which could be ascribed to the replacement of S289 by G289. Moreover, the ferrous ion could induce a change in the tertiary, but not the secondary, structure of Ab-ACO.These results provide crucial experimental support for the ability of Ab-ACO to catalyse ethylene formation in a similar manner to that of plant ACOs, but there are differences between the biochemical and catalytic characteristics of Ab-ACO and plant ACOs.This work enhances the understanding of the ethylene biosynthesis pathways in fungi and could promote profound physiological research of the role of ethylene in the regulation of mushroom growth and development.Display Omitted
Keywords: Ethylene; Agaricus bisporus (J.E. Lange) Imbach; Mushroom; 1-Aminocyclopropane-1-carboxylate oxidase (ACO); Ethylene forming enzyme; Active site;

Crystal structures of CbpF complexed with atropine and ipratropium reveal clues for the design of novel antimicrobials against Streptococcus pneumoniae by Noella Silva-Martín; M. Gracia Retamosa; Beatriz Maestro; Sergio G. Bartual; María J. Rodes; Pedro García; Jesús M. Sanz; Juan A. Hermoso (129-135).
Streptococcus pneumoniae is a major pathogen responsible of important diseases worldwide such as pneumonia and meningitis. An increasing resistance level hampers the use of currently available antibiotics to treat pneumococcal diseases. Consequently, it is desirable to find new targets for the development of novel antimicrobial drugs to treat pneumococcal infections. Surface choline-binding proteins (CBPs) are essential in bacterial physiology and infectivity. In this sense, esters of bicyclic amines (EBAs) such as atropine and ipratropium have been previously described to act as choline analogs and effectively compete with teichoic acids on binding to CBPs, consequently preventing in vitro pneumococcal growth, altering cell morphology and reducing cell viability.With the aim of gaining a deeper insight into the structural determinants of the strong interaction between CBPs and EBAs, the three-dimensional structures of choline-binding protein F (CbpF), one of the most abundant proteins in the pneumococcal cell wall, complexed with atropine and ipratropium, have been obtained.The choline analogs bound both to the carboxy-terminal module, involved in cell wall binding, and, unexpectedly, also to the amino-terminal module, that possesses a regulatory role in pneumococcal autolysis.Analysis of the complexes confirmed the importance of the tropic acid moiety of the EBAs on the strength of the binding, through π–π interactions with aromatic residues in the binding site.These results represent the first example describing the molecular basis of the inhibition of CBPs by EBA molecules and pave the way for the development of new generations of antipneumococcal drugs.
Keywords: Antibiotic resistance; CBP family; Crystallography; Streptococcus pneumoniae; Atropine; Ipratropium;

Mechanisms of toxicity by proinflammatory cytokines in a novel human pancreatic beta cell line, 1.1B4 by Srividya Vasu; Neville H. McClenaghan; Jane T. McCluskey; Peter R. Flatt (136-145).
Molecular mechanisms of toxicity and cell damage were investigated in the novel human beta cell line, 1.1B4, after exposure to proinflammatory cytokines — IL-1β, IFN-γ, TNF-α.MTT assay, insulin radioimmunoassay, glucokinase assay, real time reverse transcription PCR, western blotting, nitrite assay, caspase assay and comet assay were used to investigate mechanisms of cytokine toxicity.Viability of 1.1B4 cells decreased after 18 h cytokine exposure. Cytokines significantly reduced cellular insulin content and impaired insulin secretion induced by glucose, alanine, KCl, elevated Ca2 +, GLP-1 or forskolin. Glucokinase enzyme activity, regulation of intracellular Ca2 + and PDX1 protein expression were significantly reduced by cytokines. mRNA expression of genes involved in secretory function — INS, GCK, PCSK2 and GJA1 was downregulated in cytokine treated 1.1B4 cells. Upregulation of transcription of genes involved in antioxidant defence — SOD2 and GPX1 was observed, suggesting involvement of oxidative stress. Cytokines also upregulated transcriptions of NFKB1 and STAT1, which was accompanied by a significant increase in NOS2 transcription and accumulation of nitrite in culture medium, implicating nitrosative stress. Oxidative and nitrosative stresses induced apoptosis was evident from increased % tail DNA, DNA fragmentation, caspase 3/7 activity, apoptotic cells and lower BCL2 protein expression.This study delineates molecular mechanisms of cytokine toxicity in 1.1B4 cells, which agree with earlier observations using human islets and rodent beta cells.This study emphasizes the potential usefulness of this cell line as a human beta cell model for research investigating autoimmune destruction of pancreatic beta cells.
Keywords: Cytokine; Toxicity; 1.1B4; Insulin secretion;

Dielectric characterization of costal cartilage chondrocytes by Michael W. Stacey; Ahmet C. Sabuncu; Ali Beskok (146-152).
Chondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enable the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy.Dielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are compared with other cell types in order to comparatively assess the electrical nature of chondrocytes.The results suggest that electrical cell membrane characteristics of chondrocyte cells are close to cardiomyoblast cells, cells known to possess an array of active ion channels. The blocking effect of the non-specific ion channel blocker gadolinium is tested on chondrocytes with a significant reduction in both membrane capacitance and conductance.We have utilized a microfluidic chamber to mimic biomechanical events through changes in bioelectrochemistry and described the dielectric properties of chondrocytes to be closer to cells derived from electrically excitably tissues.The study describes dielectric characterization of human costal chondrocyte cells using physical tools, where results and methodology can be used to identify potential anomalies in bioelectrochemical responses that may lead to cartilage disorders.
Keywords: Dielectric spectroscopy; Chondrocyte; Cartilage; Microfluidics;

Snake venoms are rich in Kunitz-type protease inhibitors that may have therapeutic applications. However, apart from trypsin or chymotrypsin inhibition, the functions of most of these inhibitors have not been elucidated. A detailed functional characterization of these inhibitors may lead to valuable drug candidates.A Kunitz-type protease inhibitor, named DrKIn-II, was tested for its ability to inhibit plasmin using various approaches such as far western blotting, kinetic analyses, fibrin plate assay and euglobulin clot lysis assay. In addition, the antifibrinolytic activity of DrKIn-II was demonstrated in vivo.DrKIn-II potently decreased the amidolytic activity of plasmin in a dose-dependent manner, with a global inhibition constant of 0.2 nM. Inhibition kinetics demonstrated that the initial binding of DrKIn-II causes the enzyme to isomerize, leading to the formation of a much tighter enzyme-inhibitor complex. DrKIn-II also demonstrated antifibrinolytic activity in fibrin plate assay and significantly prolonged the lysis of the euglobulin clot. Screening of DrKIn-II against a panel of serine proteases indicated that plasmin is the preferential target of DrKIn-II. Furthermore, DrKIn-II treatment prevented the increase of FDP in coagulation-stimulated mice and significantly reduced the bleeding time in a murine tail bleeding model.DrKIn-II is a potent, slow and tight-binding plasmin inhibitor that demonstrates antifibrinolytic activity both in vitro and in vivo.This is the first in-depth functional characterization of a plasmin inhibitor from a viperid snake. The potent antifibrinolytic activity of DrKIn-II makes it a potential candidate for the development of novel antifibrinolytic agents.
Keywords: Kunitz-type protease inhibitor; Plasmin; Russell's viper venom; Tight-binding inhibition; Time-dependent inhibition; Antifibrinolytic agent;

Synthesis and toxicity characterization of carbon coated iron oxide nanoparticles with highly defined size distributions by Rafael Gregorio Mendes; Britta Koch; Alicja Bachmatiuk; Ahmed Aboud El-Gendy; Yulia Krupskaya; Armin Springer; Rüdiger Klingeler; Oliver Schmidt; Bernd Büchner; Samuel Sanchez; Mark Hermann Rümmeli (160-169).
Iron oxide nanoparticles hold great promise for future biomedical applications. To this end numerous studies on iron oxide nanoparticles have been conducted. One aspect these studies reveal is that nanoparticle size and shape can trigger different cellular responses through endocytic pathways, cell viability and early apoptosis. However, systematic studies investigating the size dependence of iron oxide nanoparticles with highly defined diameters across multiple cells lines are not available yet.Iron oxide nanoparticles with well-defined size distributions were prepared. All samples were thoroughly characterized and the cytotoxicity for four standard cell lines (HeLa Kyoto, human osteosarcoma (U2OS), mouse fibroblasts (NIH 3T3) and mouse macrophages (J7442)) where investigated.Our findings show that small differences in size distribution (ca. 10 nm) of iron oxide nanoparticles do not influence cytotoxicity, while uptake is size dependent. Cytotoxicity is dose-dependent. Broad distributions of nanoparticles are more easily internalized as compared to the narrow distributions for two of the cell lines tested (HeLa Kyoto and mouse macrophages (J7442)).The data indicate that it is not feasible to probe changes in cytotoxicity within a small size range (10 nm). However, TEM investigations of the nanoparticles indicate that cellular uptake is size dependent.The present work compares narrow and broad distributions for various samples of carbon-coated iron oxide nanoparticles. The data highlights that cells differentiate between nanoparticle sizes as indicated by differences in cellular uptake. This information provides valuable knowledge to better understand the interaction of nanoparticles and cells.In this work, systematic studies on the size and clustering effects of carbon coated iron oxide nanoparticles on cell uptake across multiple cell lines are conducted. Our findings show that despite cytotoxicity remaining unaltered between different sizes of nanoparticles, the cellular uptake is size dependent in the size range investigated. Furthermore the data show that the clustering affects cytotoxicity, but its influence is limited.Display Omitted
Keywords: Iron oxide nanoparticle; Carbon coating; Cancer cell; (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay; Trypan blue assay; Nanotoxicity;

Structure–function relationships of the peptide Paulistine: A novel toxin from the venom of the social wasp Polybia paulista by Paulo Cesar Gomes; Bibiana Monson de Souza; Nathalia Baptista Dias; Patrícia Brigatte; Danilo Mourelle; Helen Andrade Arcuri; Marcia Perez dos Santos Cabrera; Rodrigo Guerino Stabeli; João Ruggiero Neto; Mario Sergio Palma (170-183).
The peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised — with an intra-molecular disulphide bridge; and reduced — in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now.Both forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge.Paulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway.The acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine.The peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies.Display Omitted
Keywords: Wasp venom; Molecular structure; Disulphide bridge; Pain; Hyperalgesia; Inflammation;

The diterpene cyclase ent-copalyl diphosphate synthase (CPS) catalyzes the first committed step in the biosynthesis of gibberellins. The previously reported 2.25 Å resolution crystal structure of CPS complexed with (S)-15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (1) established the αβγ domain architecture, but ambiguities regarding substrate analog binding remained.Use of crystallization additives yielded CPS crystals diffracting to 1.55 Å resolution. Additionally, active site residues that hydrogen bond with D379, either directly or through hydrogen bonded water molecules, were probed by mutagenesis.This work clarifies structure–function relationships that were ambiguous in the lower resolution structure. Well-defined positions for the diphosphate group and tertiary ammonium cation of 1, as well as extensive solvent structure, are observed.Two channels involving hydrogen bonded solvent and protein residues lead to the active site, forming hydrogen bonded “proton wires” that link general acid D379 with bulk solvent. These proton wires may facilitate proton transfer with the general acid during catalysis. Activity measurements made with mutant enzymes indicate that N425, which donates a hydrogen bond directly to D379, and T421, which hydrogen bonds with D379 through an intervening solvent molecule, help orient D379 for catalysis. Residues involved in hydrogen bonds with the proton wire, R340 and D503, are also important. Finally, conserved residue E211, which is located near the diphosphate group of 1, is proposed to be a ligand to Mg2 + required for optimal catalytic activity.This work establishes structure–function relationships for class II terpenoid cyclases.Display Omitted
Keywords: Protein crystallography; Terpene cyclase; Enzyme mechanism; Gibberellin biosynthesis;

Implications of the O-GlcNAc modification in the regulation of nuclear apoptosis in T cells by Bruno Johnson; Marlyse Opimba; Jacques Bernier (191-198).
O-linked β-N-acetylglucosamine (O-GlcNAc) is a nutrient-/stress-sensitive post-translational modification that affects nucleocytoplasmic proteins. The enzyme O-N-acetylglucosamine transferase (OGT) catalyzes the addition of O-GlcNAc, whereas O-N-acetylglucosaminidase (OGA) removes it. O-GlcNAcylation plays a role in fundamental regulatory mechanisms through the modification of proteins involved in cell division, metabolism, transcription, cell signaling and apoptosis. The effects of O-GlcNAcylation on apoptosis appear to be cell-dependent, as elevated levels played a protective role in primary neonatal rat ventricular myocytes but had a cytotoxic effect in rat pancreatic β-cells. The aim of the current study was to determine the implications of the O-GlcNAc modification on T cell apoptosis.Human T lymphoblastic HPB-ALL cells were treated with the OGA inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc), or with glucosamine (GlcN), to increase O-GlcNAcylation. Apoptosis was induced in the presence of tributyltin (TBT). DNA fragmentation was observed by cell cycle analysis and corresponded to the sub G0/G1 population. O-GlcNAcylated proteins were detected by immunoblot using a specific antibody (ctd110.6) and were precipitated using succinylated wheat germ agglutinin (sWGA).HPB-ALL cells treated with PUGNAc displayed a significant reduction in DNA fragmentation after TBT-induced apoptosis. DFF45, the protein that inhibits the endonuclease DFF40, was identified to be O-GlcNAc modified. O-GlcNAcylated DFF45 appeared to be more resistant to caspase cleavage during apoptosis. Our results suggest that a decrease in the O-GlcNAc modification on DFF45 occurs before its cleavage by caspase.Our results indicate that the O-GlcNAcylation of DFF45 may represent a mechanism to control the accidental activation of DFF.
Keywords: Apoptosis; O-linked β-N-acetylglucosamine; O-(2-acetamidO-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate; TBT; DFF; DFF45;

The influence of 5-lipoxygenase on cigarette smoke-induced emphysema in mice by Emanuel Kennedy-Feitosa; Rômulo Fonseca Santos Pinto; Karla Maria Pereira Pires; Ana Paula Teixeira Monteiro; Mariana Nascimento Machado; Juliana Carvalho Santos; Marcelo Lima Ribeiro; Walter Araújo Zin; Cláudio Azevedo Canetti; Bruna Romana-Souza; Luís Cristóvão Porto; Samuel Santos Valenca (199-208).
Pulmonary emphysema is characterized by the loss of lung architecture. Our hypothesis is that the inhibition of 5-lipoxygenase (5-LO) production may be an important strategy to reduce inflammation, oxidative stress, and metalloproteinases in lung tissue resulting from cigarette smoke (CS)-induced emphysema.5-LO knockout (129S2-Alox5tm1Fun/J) and wild-type (WT) mice (129S2/SvPas) were exposed to CS for 60 days. Mice exposed to ambient air were used as Controls. Oxidative, inflammatory, and proteolytic markers were analyzed.The alveolar diameter was decreased in CS 5-LO−/− mice when compared with the WT CS group. The CS exposure resulted in less pronounced pulmonary inflammation in the CS 5-LO−/− group. The CS 5-LO−/− group showed leukotriene B4 values comparable to those of the Control group. The expression of MMP-9 was decreased in the CS 5-LO−/− group when compared with the CS WT group. The expression of superoxide dismutase, catalase, and glutathione peroxidase were decreased in the CS 5-LO−/− group when compared with the Control group. The protein expression of nuclear factor (erythroid-derived 2)-like 2 was reduced in the CS 5-LO−/− group when compared to the CS WT group.In conclusion, we show for the first time that 5-LO deficiency protects 129S2 mice against emphysema caused by CS. We suggest that the main mechanism of pathogenesis in this model involves the imbalance between proteases and antiproteases, particularly the association between MMP-9 and TIMP-1.General significanceThis study demonstrates the influence of 5-LO mediated oxidative stress, inflammation, and proteolytic markers in CS exposed mice.
Keywords: 5-lipoxygenase; Cigarette smoke; Emphysema; MMP-9; Oxidative stress;

The mechanism(s) by which alcohol causes cell injury are still not clear but a major mechanism appears to be the role of lipid peroxidation and oxidative stress in alcohol toxicity. CYP2E1-generated ROS contributes to the ethanol-induced oxidant stress and inhibition of CYP2E1 activity decreases ethanol-induced fatty liver. The transcription factor Nrf2 regulates the expression of many cytoprotective enzymes which results in cellular protection against a variety of toxins.The current study was designed to evaluate the ability of sulforaphane, an activator of Nrf2, to blunt CYP2E1-dependent, ethanol-induced steatosis in vivo and in vitro.The sulforaphane treatment activated Nrf2, increased levels of the Nrf2 target heme oxygenase-1 and subsequently lowered oxidant stress as shown by the decline in lipid peroxidation and 3-nitrotyrosine protein adducts and an increase in GSH levels after the acute ethanol treatment. It decreased ethanol-elevated liver levels of triglycerides and cholesterol and Oil Red O staining. Similar results were found in vitro as addition of sulforaphane to HepG2 E47 cells, which express CYP2E1, elevated Nrf2 levels and decreased the accumulation of lipid in cells cultured with ethanol. Sulforaphane treatment had no effect on levels of or activity of CYP2E1.Sulforaphane proved to be an effective in vivo inhibitor of acute ethanol-induced fatty liver in mice.The possible amelioration of liver injury which occurs under these conditions by chemical activators of Nrf2 is of clinical relevance and worthy of further study.
Keywords: Sulforaphane; Nrf2; CYP2E1:cytochrome p4502E1; Binge alcohol; Liver steatosis;

Berberine and S allyl cysteine mediated amelioration of DEN + CCl4 induced hepatocarcinoma by Dipanwita Sengupta; Kaustav Dutta Chowdhury; Avik Sarkar; Soumosish Paul; Gobinda Chandra Sadhukhan (219-244).
Diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) have been used as initiator and promoter respectively to establish an animal model for investigating molecular events appear to be involved in development of liver cancer. Use of herbal medicine in therapeutics to avoid the recurrence of hepatocarcinoma has already generated considerable interest among oncologists. In this context studies involving S-allyl-cysteine (SAC) and berberine have come up with promising results. Here we have determined the individual effect of SAC and berberine on the biomolecules associated with DEN + CCl4 induced hepatocarcinoma. Effective therapeutic value of combined treatment has also been estimated.ROS accumulation was analyzed by FACS following DCFDA incubation. Bcl2-Bax and HDAC1‐pMdm2 interaction were demonstrated by co-immunoprecipitation. Immunosorbent assay was performed to analyze PP2A and caspase3 activities. MMP was determined cytofluorimetrically by investigating JC-1 fluorescence. AnnexinV binding was demonstrated by labeling the cells with AnV-FITC followed by flow cytometry.CytochromeP4502E1 mediated bioactivation of DEN + CCl4 induced Akt dependent pMdm2‐HDAC1 interaction that led to p53 deacetylation, probable cause of its degradation. In parallel, oxidative stress dependent Nrf2‐HO1 activation increased Bcl2 expression which in turn stimulated cell proliferation. SAC in combination with berberine inhibited Akt mediated cell proliferation. Activation of PP2A as well as inhibition of JNK resulted in induction of apoptosis after 30 days of treatment. Extension of combined treatment reverted tissue physiology towards control. Co-treated group displayed normal tissue structure.SAC and berberine mediated HDAC1/Akt inhibition implicates the efficacy of combined treatment in the amelioration of DEN + CCl4 induced hepatocarcinoma.
Keywords: S allyl cysteine; Berberine; p53 acetylation; Di ethyl nitrosamine; Carbon tetrachloride; Oxidative stress;

Small molecule inhibitors of protein interaction with glycosaminoglycans (SMIGs), a novel class of bioactive agents with anti-inflammatory properties by Nicholas Harris; Faina Yurgenzon Kogan; Gabriela Il'kova; Stefan Juhas; Orly Lahmy; Yevgeniya I. Gregor; Juraj Koppel; Regina Zhuk; Paul Gregor (245-254).
Small molecule inhibitors of biologically important protein–glycosaminoglycan (GAG) interactions have yet to be identified.Compound libraries were screened in an assay of L-selectin–IgG binding to heparin (a species of heparan sulfate [HS-GAG]). Hits were validated, IC-50s established and direct binding of hits to HS-GAGs was investigated by incubating compounds alone with heparin. Selectivity of inhibitors was assessed in 11 different protein-GAG binding assays. Anti-inflammatory activity of selected compounds was evaluated in animal models.Screening identified a number of structurally-diverse planar aromatic cationic amines. Scaffolds similar to known GAG binders, chloroquine and tilorone, were also identified. Inhibitors displayed activity also against bovine kidney heparan sulfate. Direct binding of compounds to GAGs was verified by incubating compounds with heparin alone. Selectivity of inhibitors was demonstrated in a panel of 11 heparin binding proteins, including selectins, chemokines (IL-8, IP-10), Beta Amyloid and cytokines (VEGF, IL-6). A number of selected lead compounds showed dose-dependent efficacy in peritonitis, paw edema and delayed type hypersensitivity.A new class of compounds, SMIGs, inhibits protein–GAG interaction by direct binding to GAGs. Although their IC-50s were in the low micro-molar range, SMIGs binding to HS-GAGs appeared to be stable in physiological conditions, indicating high avidity binding. SMIGs may interfere with major checkpoints for inflammatory and autoimmune events.SMIGs are a class of structurally-diverse planar aromatic cationic amines that have an unusual mode of action — inhibiting protein–GAG interactions via direct and stable binding to GAGs. SMIGs may have therapeutic potential in inflammatory and autoimmune disorders.
Keywords: Heparan sulfate; Heparin binding protein; Glycosaminoglycan; L-selectin; Inflammation; Cytokine;

Glutaredoxin 1 is a major player in copper metabolism in neuroblastoma cells by Maria Lisa De Benedetto; Concetta Rosa Capo; Alberto Ferri; Cristiana Valle; Renato Polimanti; Maria Teresa Carrì; Luisa Rossi (255-261).
Glutaredoxin 1 (Grx1), a small protein belonging to the thioredoxin family, is involved in redox-regulation since it catalyzes the reduction of protein disulfides and that of mixed disulfides. It was reported to modulate active copper extrusion from cells, by affecting the function of the pumps ATP7A and B. These are components of the network of protein chaperones involved in the control of the homeostasis of copper, an essential, though harmful, metal. However, the effect of Grx1 on copper levels, copper chaperones and copper-elicited cell toxicity was never investigated.In order to investigate the effect of Grx1 on copper metabolism, we constitutively overexpressed Grx1 in human neuroblastoma SH-SY5Y cells (SH-Grx1 cells) and assessed a number of copper-related parameters.SH-Grx1 cells show a basal intracellular copper level higher than control cells, accumulate more copper upon CuSO4 treatment, but are more resistant to copper-induced toxicity. Grx1 shows copper-binding properties and copper overload produces a decrease of Grx1 enzyme activity in SH-Grx1 cells. Finally, Grx1 overexpression decreases copper accumulation in mitochondria upon copper overload and modulates the expression of copper transporter 1 (Ctr1).Altogether, these data demonstrate that Grx1 is a major player in copper metabolism in neuronal cells.
Keywords: Copper; Ctr1; Grx1; SH-SY5Y;

PDT-induced epigenetic changes in the mouse cerebral cortex: A protein microarray study by S.V. Demyanenko; A.B. Uzdensky; S.A. Sharifulina; T.O. Lapteva; L.P. Polyakova (262-270).
Photodynamic therapy (PDT) is used for cancer treatment including brain tumors. But the role of epigenetic processes in photodynamic injury of normal brain tissue is unknown.5-Aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PpIX), was used to photosensitize mouse cerebral cortex. PpIX accumulation in cortical tissue was measured spectrofluorometrically. Hematoxylin/eosin, gallocyanin–chromalum and immunohistochemical staining were used to study morphological changes in PDT-treated cerebral cortex. Proteomic antibody microarrays were used to evaluate expression of 112 proteins involved in epigenetic regulation.ALA administration induced 2.5-fold increase in the PpIX accumulation in the mouse brain cortex compared to untreated mice. Histological study demonstrated PDT-induced injury of some neurons and cortical vessels. ALA-PDT induced dimethylation of histone H3, upregulation of histone deacetylases HDAC-1 and HDAC-11, and DNA methylation-dependent protein Kaiso that suppressed transcriptional activity. Upregulation of HDAC-1 and H3K9me2 was confirmed immunohistochemically. Down-regulation of transcription factor FOXC2, PABP, and hBrm/hsnf2a negatively regulated transcription. Overexpression of phosphorylated histone H2AX indicated activation of DNA repair, but down-regulation of MTA1/MTA1L1 and PML — impairment of DNA repair. Overexpression of arginine methyltransferase PRMT5 correlated with up-regulation of transcription factor E2F4 and importin α5/7.ALA-PDT injures and kills some but not all neurons and caused limited microvascular alterations in the mouse cerebral cortex. It alters expression of some proteins involved in epigenetic regulation of transcription, histone modification, DNA repair, nuclear protein import, and proliferation.These data indicate epigenetic markers of photo-oxidative injury of normal brain tissue.Display Omitted
Keywords: ALA-PDT; Brain cortex; Proteomic; Epigenetic; Antibody microarray;

The oxygen flow in humans and other higher animals depends on the erythrocyte-to-blood volume ratio, the hematocrit. Since it is physiologically favourable when the flow of oxygen transport is maximum it can be assumed that this situation has been achieved during evolution. If the hematocrit was too low, too few erythrocytes could transport oxygen. If it was too high, the blood would be very viscous, so that oxygen supply would again be reduced.The theoretical optimal hematocrit can be calculated by considering the dependence of blood viscosity on the hematocrit. Different approaches to expressing this dependence have been proposed in the literature. Here, we discuss early approaches in hydrodynamics proposed by Einstein and Arrhenius and show that especially the Arrhenius equation is very appropriate for this purpose.We show that despite considerable simplifications such as neglecting the deformation, orientation and aggregation of erythrocytes, realistic hematocrit values of about 40% can be derived based on optimality considerations. Also the prediction that the ratio between the viscosities of the blood and blood plasma at high shear rates nearly equals Euler's constant (2.718) is in good agreement with observed values. Finally, we discuss possible extensions of the theory. For example, we derive the theoretical optimal hematocrit for persevering divers among marine mammals to be 65%, in excellent agreement with the values observed in several species.These considerations are very important for human and animal physiology since oxygen transport is an important factor for medicine and physical performance.
Keywords: Blood flow; Einstein's viscosity equation; Hagen–Poiseuille law; Marine mammals; Optimal hematocrit; Viscosity of suspensions;

Dynamics of α-Hb chain binding to its chaperone AHSP depends on heme coordination and redox state by Laurent Kiger; Corinne Vasseur; Elisa Domingues-Hamdi; Gilles Truan; Michael C. Marden; Véronique Baudin-Creuza (277-287).
AHSP is an erythroid molecular chaperone of the α-hemoglobin chains (α-Hb). Upon AHSP binding, native ferric α-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6th coordination bond with His(E7).Recombinant AHSP, WT α-Hb:AHSP and α-HbHE7Q:AHSP complexes were expressed in Escherichia coli. Thermal denaturation curves were measured by circular dichroism for the isolated α-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of α-Hb bound to AHSP as well as α-Hb release from the α-Hb:AHSP complex were measured by time-resolved absorption spectroscopy.AHSP binding to α-Hb is kinetically controlled to prevail over direct binding with β-chains and is also thermodynamically controlled by the α-Hb redox state and not the liganded state of the ferrous α-Hb. The dramatic instability of isolated ferric α-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in α-HbH58(E7)Q:AHSP complex reduces the stabilizing effect of AHSP binding. Once the ferric α-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to α-Hb within the Hb-tetramer.α-Hb reduction could trigger its release from AHSP toward its final Hb β-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis.The cellular redox balance in Hb-synthesis should be considered as important as the relative proportional synthesis of both Hb-subunits and their heme cofactor. The in vivo role of AHSP is discussed in the context of the molecular disorders observed in thalassemia.
Keywords: Erythropoiesis; Alpha-hemoglobin stabilizing protein (AHSP); Chaperone; Hemoglobin; Heme hexacoordination;

Non-adsorbing macromolecules promote endothelial adhesion of erythrocytes with reduced sialic acids by Yang Yang; Stephanie Koo; Li Tze Heng; Herbert J. Meiselman; Björn Neu (288-293).
Abnormal adhesion of red blood cells (RBCs) to vascular endothelium is often associated with reduced levels of sialic acids on RBC membranes and with elevated levels of pro-adhesive plasma proteins. However, the synergistic effects of these two factors on the adhesion are not clear. In this work, we tested the hypothesis that macromolecular depletion interaction originating from non-adsorbing macromolecules can promote the adhesion of RBCs with reduced sialic acid content to the endothelium.RBCs are treated with neuraminidase to specifically remove sialic acids from their surface followed by the evaluation of their deformability, zeta potential and membrane proteins. The adhesion of these enzyme-treated RBCs to cultured human umbilical vein endothelial cells (ECs) is studied in the presence of 70 or 500 kDa dextran with a flow chamber assay.Our results demonstrate that removal of sialic acids from RBC surface can induce erythrocyte adhesion to endothelial cells and that such adhesion is significantly enhanced in the presence of high-molecular weight dextran. The adhesion-promoting effect of dextran exhibits a strong dependence on dextran concentration and molecular mass, and it is concluded to originate from macromolecular depletion interaction.These results suggest that elevated levels of non-adsorbing macromolecules in plasma might play a significant role in promoting endothelial adhesion of erythrocytes with reduced sialic acids.Our findings should therefore be of great value in understanding abnormal RBC–EC interactions in pathophysiological conditions (e.g., sickle cell disease and diabetes) and after blood transfusions.Display Omitted
Keywords: Sialic acids; Neuraminidase; Red blood cell adhesion; Dextran; Macromolecular depletion interaction;

Orlistat, a fatty acid synthase (FASN) inhibitor, has been demonstrated to inhibit tumor cell survival. However, the mechanism(s) of its tumor growth retarding action against malignancies of hematological origin remains unclear. It is also not understood if the antitumor action of orlistat implicates modulated susceptibility of tumor cell to anticancer drugs. Therefore, the present investigation focuses to study the antitumor and chemosensitizing action of orlistat in a murine host bearing a progressively growing T cell lymphoma.Tumor-bearing mice were administered with vehicle alone or containing orlistat followed by administration of PBS with or without cisplatin. Tumor progression and survival of tumor-bearing host were monitored along with analysis of tumor cell survival and apoptosis. Tumor ascitic fluid was examined for pH, NO and cytokines. Expression of genes and proteins was investigated by RT-PCR and western blot respectively. ROS was analyzed by DCFDA staining and FASN activity by spectrophotometry.Orlistat administration to tumor-bearing mice resulted in tumor growth retardation, prolonged life span, declined tumor cell survival and chemosensitization to cisplatin. It was accompanied by increased osmotic fragility, modulated acidosis, expression of ROS, NO, cytokines, MCT-1 and VH+ ATPase, Bcl2, Caspase-3, P53, inhibited FASN activity and declined expression of MDR and MRP-1 proteins.Orlistat manifests antitumor and chemosensitizing action implicating modulated regulation of cell survival, reconstituted-tumor microenvironment and altered MDR phenotype.These observations indicate that orlistat could be utilized as an adjunct regimen for improving antitumor efficacy of cisplatin.
Keywords: Apoptosis; Chemosensitization; Orlistat; T cell lymphoma; Fatty acid synthase;

The thioredoxin system maintains redox balance through the action of thioredoxin and thioredoxin reductase. Thioredoxin regulates the activity of various substrates, including those that function to counteract cellular oxidative stress. These include the peroxiredoxins, methionine sulfoxide reductase A and specific transcription factors. Of particular relevance is Redox Factor-1, which in turn activates other redox-regulated transcription factors.Experimentally defined transcription factor binding sites in the human thioredoxin and thioredoxin reductase gene promoters together with promoters of the major thioredoxin system substrates involved in regulating cellular redox status are discussed. An in silico approach was used to identify potential putative binding sites for these transcription factors in all of these promoters.Our analysis reveals that many redox gene promoters contain the same transcription factor binding sites. Several of these transcription factors are in turn redox regulated. The ARE is present in several of these promoters and is bound by Nrf2 during various oxidative stress stimuli to upregulate gene expression. Other transcription factors also bind to these promoters during the same oxidative stress stimuli, with this redundancy supporting the importance of the antioxidant response. Putative transcription factor sites were identified in silico, which in combination with specific regulatory knowledge for that gene promoter may inform future experiments.Redox proteins are involved in many cellular signalling pathways and aberrant expression can lead to disease or other pathological conditions. Therefore understanding how their expression is regulated is relevant for developing therapeutic agents that target these pathways.
Keywords: Redox control; Transcription factor; Antioxidant responsive element; Oxidative stress; Gene promoter; Regulatory element;

A mutation associated with centronuclear myopathy enhances the size and stability of dynamin 2 complexes in cells by Nicholas G. James; Michelle A. Digman; Justin A. Ross; Barbara Barylko; Lei Wang; Jinhui Li; Yan Chen; Joachim D. Mueller; Enrico Gratton; Joseph P. Albanesi; David M. Jameson (315-321).
Dynamin 2 (Dyn2) is a ~ 100 kDa GTPase that assembles around the necks of nascent endocytic and Golgi vesicles and catalyzes membrane scission. Mutations in Dyn2 that cause centronuclear myopathy (CNM) have been shown to stabilize Dyn2 polymers against GTP-dependent disassembly in vitro. Precisely timed regulation of assembly and disassembly is believed to be critical for Dyn2 function in membrane vesiculation, and the CNM mutations interfere with this regulation by shifting the equilibrium toward the assembled state.In this study we use two fluorescence fluctuation spectroscopy (FFS) approaches to show that a CNM mutant form of Dyn2 also has a greater propensity to self-assemble in the cytosol and on the plasma membrane of living cells.Results obtained using brightness analysis indicate that unassembled wild-type Dyn2 is predominantly tetrameric in the cytosol, although different oligomeric species are observed, depending on the concentration of expressed protein. In contrast, an R369W mutant identified in CNM patients forms higher-order oligomers at concentrations above 1 μM. Investigation of Dyn2-R369W by Total Internal Reflection Fluorescence (TIRF) FFS reveals that this mutant forms larger and more stable clathrin-containing structures on the plasma membrane than wild-type Dyn2.These observations may explain defects in membrane trafficking reported in CNM patient cells and in heterologous systems expressing CNM-associated Dyn2 mutants.
Keywords: Dynamin 2; Centronuclear myopathy; R369W mutation; EGFP; Fluorescence fluctuation spectroscopy; TIRF;

Nonsteroidal anti-inflammatory drug sulindac sulfide suppresses structural protein Nesprin-2 expression in colorectal cancer cells by Jason L. Liggett; Chang Kyoung Choi; Robert L. Donnell; Kenneth D. Kihm; Jong-Sik Kim; Kyung-Won Min; Angelika Anna Noegel; Seung Joon Baek (322-331).
Nonsteroidal anti-inflammatory drugs (NSAIDs) are well known for treating inflammatory disease and have been reported to have anti-tumorigenic effects. Their mechanisms are not fully understood, but both cyclooxygenase (COX) dependent and independent pathways are involved. Our goal was to shed further light on COX-independent activity.Human colorectal cancer cells were observed under differential interference contrast microscopy (DICM), fluorescent microscopy, and micro-impedance measurement. Microarray analysis was performed using HCT-116 cells treated with sulindac sulfide (SS). PCR and Western blots were performed to confirm the microarray data and immunohistochemistry was performed to screen for Nesprin-2 expression. Micro-impedance was repeating including Nesprin-2 knock-down by siRNA.HCT-116 cells treated with SS showed dramatic morphological changes under DICM and fluorescent microscopy, as well as weakened cellular adhesion as measured by micro-impedance. Nesprin-2 was selected from two independent microarrays, based on its novelty in relation to cancer and its role in cell organization. SS diminished Nesprin-2 mRNA expression as assessed by reverse transcriptase and real time PCR. Various other NSAIDs were also tested and demonstrated that inhibition of Nesprin-2 mRNA was not unique to SS. Additionally, immunohistochemistry showed higher levels of Nesprin-2 in many tumors in comparison with normal tissues. Further micro-impedance experiments on cells with reduced Nesprin-2 expression showed a proportional loss of cellular adhesion.Nesprin-2 is down-regulated by NSAIDs and highly expressed in many cancers.Our data suggest that Nesprin-2 may be a potential novel oncogene in human cancer cells and NSAIDs could decrease its expression.
Keywords: Nesprin-2; NUANCE; NSAID; Colon cancer; Micro-impedance; NAG-1;

Design and discovery of novel quinazolinedione-based redox modulators as therapies for pancreatic cancer by Divya Pathania; Mario Sechi; Michele Palomba; Vanna Sanna; Francesco Berrettini; Angela Sias; Laleh Taheri; Nouri Neamati (332-343).
Altered cellular bioenergetics and oxidative stress are emerging hallmarks of most cancers including pancreatic cancer. Elevated levels of intrinsic reactive oxygen species (ROS) in tumors make them more susceptible to exogenously induced oxidative stress. Excessive oxidative insults overwhelm their adaptive antioxidant capacity and trigger ROS-mediated cell death. Recently, we have discovered a novel class of quinazolinediones that exert their cytotoxic effects by modulating ROS-mediated signaling.Cytotoxic potential was determined by colorimetric and colony formation assays. An XF24 Extracellular Flux Analyzer, and colorimetric and fluorescent techniques were used to assess the bioenergetics and oxidative stress effects, respectively. Mechanism was determined by Western blots.Compound 3a (6-[(2-acetylphenyl)amino]quinazoline-5,8-dione) was identified through a medium throughput screen of ~ 1000 highly diverse in-house compounds and chemotherapeutic agents for their ability to alter cellular bioenergetics. Further structural optimizations led to the discovery of a more potent analog, 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) that displayed anti-proliferative activities in low micromolar range in both drug-sensitive and drug-resistant cancer cells. Treatment with 3b causes Akt activation resulting in increased cellular oxygen consumption and oxidative stress in pancreatic cancer cells. Moreover, oxidative stress induced by 3b promoted activation of stress kinases (p38/JNK) resulting in cancer cell death. Treatment with antioxidants was able to reduce cell death confirming ROS-mediated cytotoxicity.In conclusion, our novel quinazolinediones are promising lead compounds that selectively induce ROS-mediated cell death in cancer cells and warrant further preclinical studies.Since 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) exerts Akt-dependent ROS-mediated cell death, it might provide potential therapeutic options for chemoresistant and Akt-overexpressing cancers.Display Omitted
Keywords: Redox regulation; Small molecule drug discovery; Anticancer compounds; Oxidative stress in cancer cells; ROS-mediated cell death;

A putative phospholipase C is involved in Pichia fermentans dimorphic transition by Maria Lina Sanna; Giacomo Zara; Severino Zara; Quirico Migheli; Marilena Budroni; Ilaria Mannazzu (344-349).
Pichia fermentans DiSAABA 726 is a dimorphic yeast that reversibly shifts from yeast-like to pseudohyphal morphology. This yeast behaves as a promising antagonist of Monilia spp. in the yeast-like form, but becomes a destructive plant pathogen in the pseudohyphal form thus raising the problem of the biological risk associated with the use of dimorphic yeasts as microbial antagonists in the biocontrol of phytopathogenic fungi. Pichia fermentans DiSAABA 726 was grown in urea- and methionine-containing media in order to induce and separate yeast-like and pseudohyphal morphologies. Total RNA was extracted from yeast-like cells and pseudohyphae and retro-transcribed into cDNA. A rapid subtraction hybridization approach was utilized to obtain the cDNA sequences putatively over-expressed during growth on methionine-containing medium and involved in pseudohyphal transition.Five genes that are over-expressed during yeast-like/pseudohyphal dimorphic transition were isolated. One of these, encoding a putative phospholipase C, is involved in P. fermentans filamentation. In fact, while the inhibition of phospholipase C, by means of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (Et-18), is accompanied by a significant reduction of pseudohyphae formation in P. fermentans, the addition of exogenous cAMP fully restores pseudohyphal growth also in the presence of Et-18.Phospholipase C is part of a putative “methionine sensing machinery” that activates cAMP-PKA signal transduction pathway and controls P. fermentans yeast-like/pseudohyphal dimorphic transition.Phospholipase C is a promising molecular target for further investigations into the link between pseudohyphae formation and pathogenicity in P. fermentans.
Keywords: Dimorphic shift; Phospholipase C; Pichia fermentans; Rapid subtraction hybridization (RaSH); 3′-5′-cyclic adenosine monophosphate (cAMP); Pseudohypha;

Nucleic acid binding properties of allicin: Spectroscopic analysis and estimation of anti-tumor potential by Gunjan Tyagi; Shrikant Pradhan; Tapasya Srivastava; Ranjana Mehrotra (350-356).
Allicin has received much attention due to its anti-proliferative activity and not-well elucidated underlying mechanism of action. This work focuses towards determining the cellular toxicity of allicin and understanding its interaction with nucleic acid at molecular level.MTT assay was used to assess the cell viability of A549 lung cancer cells against allicin. Fourier transform infrared (FTIR) and UV-visible spectroscopy were used to study the binding parameters of nucleic acid-allicin interaction.Allicin inhibits the proliferation of cancer cells in a concentration dependent manner. FTIR spectroscopy exhibited that allicin binds preferentially to minor groove of DNA via thymine base. Analysis of tRNA allicin complex has also revealed that allicin binds primarily through nitrogenous bases. Some amount of external binding with phosphate backbone was also observed for both DNA and RNA. UV visible spectra of both DNA allicin and RNA allicin complexes showed hypochromic shift with an estimated binding constant of 1.2 × 104  M- 1 for DNA and 1.06 × 103  M− 1for RNA binding. No major transition from the B-form of DNA and A-form of RNA is observed after their interaction with allicin.The results demonstrated that allicin treatment inhibited the proliferation of A549 cells in a dose-dependent manner. Biophysical outcomes are suggestive of base binding and helix contraction of nucleic acid structure upon binding with allicin.The results describe cytotoxic potential of allicin and its binding properties with cellular nucleic acid, which could be helpful in deciphering the complete mechanism of cell death exerted by allicin.Display Omitted
Keywords: Deoxyribonucleicacid; Ribonucleicacid; Allicin; Fourier transform infrared spectroscopy; MTT;

Stabilities and structures of islet amyloid polypeptide (IAPP22–28) oligomers: From dimer to 16-mer by Jingjing Guo; Yan Zhang; Lulu Ning; Pingzu Jiao; Huanxiang Liu; Xiaojun Yao (357-366).
The formation of amyloid fibrils is associated with many age-related degenerative diseases. Nevertheless, the molecular mechanism that directs the nucleation of these fibrils is not fully understood.Here, we performed MD simulations for the NFGAILS motif of hIAPP associated with the type II diabetes to estimate the stabilities of hIAPP22–28 protofibrils with different sizes: from 2 to 16 chains. In addition, to study the initial self-assembly stage, 4 and 8 IAPP22–28 chains in explicit solvent were also simulated.Our results indicate that the ordered protofibrils with no more than 16 hIAPP22–28 chains will be structurally stable in two layers, while one-layer or three-layer models are not stable as expected. Furthermore, the oligomerization simulations show that the initial coil structures of peptides can quickly aggregate and convert to partially ordered β-sheet-rich oligomers.Based on the obtained results, we found that the stability of an IAPP22–28 oligomer was not only related with its size but also with its morphology. The driving forces to form and stabilize an oligomer are the hydrophobic effects and backbone H-bond interaction. Our simulations also indicate that IAPP22–28 peptides tend to form an antiparallel strand orientation within the sheet.Our finding can not only enhance the understanding about potential mechanisms of hIAPP nuclei formation and the extensive structural polymorphisms of oligomers, but also provide valuable information to develop potential β-sheet formation inhibitors against type II diabetes.
Keywords: Nucleation; Amyloid fibril; Molecular dynamics simulation; Islet amyloid polypeptide (residues 22-28); Type II diabetes;

The identification and molecular characterization of the first archaeal bifunctional exo-β-glucosidase/N-acetyl-β-glucosaminidase demonstrate that family GH116 is made of three functionally distinct subfamilies by Maria Carmina Ferrara; Beatrice Cobucci-Ponzano; Andrea Carpentieri; Bernard Henrissat; Mosè Rossi; Angela Amoresano; Marco Moracci (367-377).
β-N-acetylhexosaminidases, which are involved in a variety of biological processes including energy metabolism, cell proliferation, signal transduction and in pathogen-related inflammation and autoimmune diseases, are widely distributed in Bacteria and Eukaryotes, but only few examples have been found in Archaea so far. However, N-acetylgluco- and galactosamine are commonly found in the extracellular storage polymers and in the glycans decorating abundantly expressed glycoproteins from different Crenarchaeota Sulfolobus sp., suggesting that β-N-acetylglucosaminidase activities could be involved in the modification/recycling of these cellular components.A thermophilic β-N-acetylglucosaminidase was purified from cellular extracts of S. solfataricus, strain P2, identified by mass spectrometry, and cloned and expressed in E. coli. Glycosidase assays on different strains of S. solfataricus, steady state kinetic constants, substrate specificity analysis, and the sensitivity to two inhibitors of the recombinant enzyme were also reported.A new β-N-acetylglucosaminidase from S. solfataricus was unequivocally identified as the product of gene sso3039. The detailed enzymatic characterization demonstrates that this enzyme is a bifunctional β-glucosidase/β-N-acetylglucosaminidase belonging to family GH116 of the carbohydrate active enzyme (CAZy) classification.This study allowed us to propose that family GH116 is composed of three subfamilies, which show distinct substrate specificities and inhibitor sensitivities.The characterization of SSO3039 allows, for the first time in Archaea, the identification of an enzyme involved in the metabolism β-N-acetylhexosaminide, an essential component of glycoproteins in this domain of life, and substantially increases our knowledge on the functional role and phylogenetic relationships amongst the GH116 CAZy family members.
Keywords: Protein N-glycosylation; Carbohydrate active enzymes; Glycobiology;

T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors: Towards tumor-directed oligoclonal T cell therapy by Fatemeh Rahimi Jamnani; Fatemeh Rahbarizadeh; Mohammad Ali Shokrgozar; Fereidoun Mahboudi; Davoud Ahmadvand; Zahra Sharifzadeh; Ladan Parhamifar; S. Moein Moghimi (378-386).
Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination of costimulatory endodomains for CAR construction to improve the effector functions of the engineered T cells. Camelid single-domain antibodies (VHHs), which are the smallest single domain antibodies, can endow great targeting ability to CAR-engineered T cells.We have developed a method to generate genetically engineered Jurkat T cells armed with a CAR comprising the anti-HER2 VHH as targeting moiety. From an immune camel library, five VHH clones were selected as a set of oligoclonal anti-HER2 VHHs that exhibited diverse binding abilities and joined them to CD28-CD3ζ and CD28-OX40-CD3ζ signaling endodomains. Jurkat T cells expression of VHH-CARs and cell functions were evaluated.The oligoclonal engineered T cells showed higher proliferation, cytokine secretion and cytotoxicity than each individual VHH-CAR-engineered Jurkat T cells.The combination of superior targeting ability of oligoclonal VHHs with the third generation CAR can substantially improve the function of engineered T cells.Antigen-specific directed oligoclonal T cells are alternatively promising, but safer systems, to combat tumor cells.
Keywords: Chimeric antigen receptor; HER2; Oligoclonal T cell therapy; Single domain antibodies (VHH);

Step-by-step mechanism of DNA damage recognition by human 8-oxoguanine DNA glycosylase by Alexandra A. Kuznetsova; Nikita A. Kuznetsov; Alexander A. Ishchenko; Murat K. Saparbaev; Olga S. Fedorova (387-395).
Extensive structural studies of human DNA glycosylase hOGG1 have revealed essential conformational changes of the enzyme. However, at present there is little information about the time scale of the rearrangements of the protein structure as well as the dynamic behavior of individual amino acids.Using pre-steady-state kinetic analysis with Trp and 2-aminopurine fluorescence detection the conformational dynamics of hOGG1 wild-type (WT) and mutants Y203W, Y203A, H270W, F45W, F319W and K249Q as well as DNA–substrates was examined.The roles of catalytically important amino acids F45, Y203, K249, H270, and F319 in the hOGG1 enzymatic pathway and their involvement in the step-by-step mechanism of oxidative DNA lesion recognition and catalysis were elucidated.The results show that Tyr-203 participates in the initial steps of the lesion site recognition. The interaction of the His-270 residue with the oxoG base plays a key role in the insertion of the damaged base into the active site. Lys-249 participates not only in the catalytic stages but also in the processes of local duplex distortion and flipping out of the oxoG residue. Non-damaged DNA does not form a stable complex with hOGG1, although a complex with a flipped out guanine base can be formed transiently.The kinetic data obtained in this study significantly improves our understanding of the molecular mechanism of lesion recognition by hOGG1.
Keywords: Base excision repair; DNA glycosylase; Conformational dynamics; Enzyme kinetics; Human 8-oxoguanine DNA glycosylase;

A phospholipase A2 gene is linked to Jack bean urease toxicity in the Chagas' disease vector Rhodnius prolixus by M.S. Defferrari; D.H. Lee; C.L. Fernandes; I. Orchard; C.R. Carlini (396-405).
Ureases are multifunctional enzymes that display biological activities independent of their enzymatic function, including exocytosis induction and insecticidal effects. The hemipteran Rhodnius prolixus is one of the known susceptible models for this toxicity. It has been shown that Jack bean urease (JBU) has deleterious effects on R. prolixus, and these effects are modulated by eicosanoids, which are synthesized in a cascade involving phospholipase A2 (PLA2) enzymes. R. prolixus genome was screened for putative PLA2s and matching transcripts were cloned. Predicted amino acid sequences were analyzed and transcript distribution among tissues was determined by qPCR. RNAi techniques were used and subsequent JBU toxicity assays were performed.Two PLA2 genes were identified, Rhopr-PLA2III and Rhopr-PLA2XII. The transcripts are widely distributed in the tissues but at different levels. The analyses fit the putative proteins into groups III and XII of secretory PLA2s. After 70% of Rhopr-PLA2XII expression was knocked down, JBU's toxicity was decreased by more than 50% on 5th instars R. prolixus. Rhopr-PLA2XII gene is linked to JBU's toxic effect in R. prolixus and our findings support previous studies demonstrating that eicosanoids modulate this toxicity.Besides identifying and characterizing two PLA2 genes in the major Chagas' disease vector R. prolixus, we have shown that the potent toxicity of JBU is linked to one of these genes. Our results contribute to the general comprehension of urease's mechanisms of action in insects, and, potentially, to studies on the control of the Chagas' disease parasite transmission.
Keywords: Insect; Eicosanoid; qPCR; Transcript cloning; RNAi; Gene knockdown;

Structural and functional characterization of simvastatin-induced myotoxicity in different skeletal muscles by Nihal Simsek Ozek; I. Burak Bal; Yildirim Sara; Rustu Onur; Feride Severcan (406-415).
Statins are the most commonly used drugs for the treatment of hypercholesterolemia. Their most frequent side effect is myotoxicity. To date, it remains unclear whether statins preferentially induce myotoxicity in fast- or in slow-twitch muscles. Therefore, we investigated these effects on fast- (extensor digitorum longus; EDL), slow- (soleus; SOL), and mixed-twitch muscles (diaphragm; DIA) in rats by comparing their contractile and molecular structural properties.Simvastatin-induced functional changes were determined by muscle contraction measurements, and drug-induced molecular changes were investigated using Fourier transform infrared (FTIR) and attenuated total reflectance (ATR) FTIR spectroscopy.With simvastatin administration (30 days, 50 mg/kg), a depression in the force–frequency curves in all muscles was observed, indicating the impairment of muscle contractility; however, the EDL and DIA muscles were affected more severely than the SOL muscle. Spectroscopic findings also showed a decrease in protein, glycogen, nucleic acid, lipid content and an increase in lipid order and lipid dynamics in the simvastatin-treated muscles. The lipid order and dynamics directly affect membrane thickness. Therefore, the kinetics and functions of membrane ion channels were also affected, contributing to the statin-induced impairment of muscle contractility. Furthermore, a reduction in α-helix and β-sheet and an increase in random coil, aggregated and antiparallel β-sheet were observed, indicating the protein denaturation. Spectral studies showed that the extent of molecular structural alterations in the muscles following simvastatin administration was in the order EDL > DIA > SOL.Simvastatin-induced structural and functional alterations are more profound in the fast-twitch than in the slow-twitch muscles.Myotoxic effects of simvastatin are primarily observed in the fast-twitch muscles.
Keywords: Statin; Extensor digitorum longus; Soleus; Diaphragm; Contractility; Fourier Transform Infrared spectroscopy;

Human α1-proteinase inhibitor (α1-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α1-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern.Five variants of plant-expressed recombinant α1-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α1-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy.Urea-induced unfolding of recombinant α1-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4 M to 4.3 M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~ 5-fold at 54 °C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α1-PI but provided enhanced resistance to oxidative inactivation.Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α1-PI protein, without inflicting the inhibitory activity of the protein.Our results demonstrate the significance of engineered modifications in plant-derived recombinant α1-PI protein molecule for further therapeutic development.
Keywords: Human α1-proteinase inhibitor; Recombinant therapeutic protein; Transgenic plant; Single amino acid substitution; Protein stability; Oxidation resistance;

Synthesizing and staining manganese oxide nanoparticles for cytotoxicity and cellular uptake investigation by Hamed Omid; Mohammad Ali Oghabian; Reza Ahmadi; Narges Shahbazi; Hamid Reza Madaah Hosseini; Saeed Shanehsazzadeh; Rashin Namivandi Zangeneh (428-433).
For decades, contrast agents have been used to reduce longitudinal (T1) or transverse (T2) relaxation times. High toxicity of gadolinium-based contrast agents leads researchers to new T1 contrast agents. Manganese oxide (MnO) nanoparticle (NP) with the lower peril and good enough signal change ability has been offered as a new possibility for magnetic resonance imaging (MRI).The synthesized NPs were investigated for physicochemical and biological properties by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscope, dynamic light scattering (DLS), inductively coupled plasma, enzyme-linked immunosorbent assay, and 3 T magnetic resonance imaging.Due to physical contact importance of T1 contrast agents with tissues' protons, extremely thin layer of the surfactant, less than 2 nm, was coated on NPs for aqueous stabilizing. The hydrophilic gentisic acid with low Dalton, around 154, did that role truly. Moreover, decreasing NP size to 5 nm which increases available surface for the proton relaxation is another important parameter to reach an appropriate longitudinal relaxation rate. The NPs didn't reveal any side effects on the cells, and cellular uptake was considerable.The synthesized NPs represented a promising result in comparison to clinical gadolinium chelates, due to higher r1 relaxivity and lower toxicity.In addition to considerable signal change and cellular uptake, Prussian blue was tried on MnO NPs for the initial time, which can be observed within cells by pale blue color.
Keywords: Manganese oxide nanoparticle; Prussian blue staining; MRI contrast agent; Cell uptake; Cytotoxicity;

-Acetylhistidine (NAH) is present in very high concentrations exclusively in the brain and lens of ectothermic vertebrates, including ray-finned fishes, amphibians and reptiles, and not in those of endothermic birds and mammals. Although NAH is known to be synthesized from l-His and acetyl-CoA by histidine N-acetyltransferase (HISAT; EC 2.3.1.33), the gene encoding HISAT has remained unknown for any organism.HISAT was purified from the blue mackerel brain, and its partial amino acid sequences were analyzed using mass spectrometry and Edman degradation. Using the sequence information, the corresponding gene was cloned and sequenced. Recombinant proteins encoded by the fish gene and its human homologue were expressed in a cell-free translation system.HISAT was identified to be a protein encoded by a fish homologue of the human predicted gene NAT16 (N-acetyltransferase 16). HISAT is an unstable enzyme that is rapidly and irreversibly inactivated during preincubation at 37 °C in the absence of acetyl-CoA. In fish brain, the HISAT gene is expressed as two splice variants containing an identical ORF but differing lengths of 5′-UTR. Both variants are expressed exclusively in the fish brain and lens. Interestingly, the recombinant human NAT16 protein, unlike the recombinant fish HISAT, has only trace enzyme activity for NAH synthesis.These results propose that the function of mammalian NAT16 has been altered from l-His acetylation (NAH synthesis) to another different biological role.The molecular identification of HISAT will allow progress in the understanding of the physiological function of NAH in ectothermic vertebrates.
Keywords: C7orf52; Ectotherm; Histidine N-acetyltransferase; NAT16; Nα-acetylhistidine;

The expanding roles of the Sda/Cad carbohydrate antigen and its cognate glycosyltransferase B4GALNT2 by Fabio Dall'Olio; Nadia Malagolini; Mariella Chiricolo; Marco Trinchera; Anne Harduin-Lepers (443-453).
The histo-blood group antigens are carbohydrate structures present in tissues and body fluids, which contribute to the definition of the individual immunophenotype. One of these, the Sda antigen, is expressed on the surface of erythrocytes and in secretions of the vast majority of the Caucasians and other ethnic groups.We describe the multiple and unsuspected aspects of the biology of the Sda antigen and its biosynthetic enzyme β1,4-N-acetylgalactosaminyltransferase 2 (B4GALNT2) in various physiological and pathological settings.The immunodominant sugar of the Sda antigen is a β1,4-linked N-acetylgalactosamine (GalNAc). Its cognate glycosyltransferase B4GALNT2 displays a restricted pattern of tissue expression, is regulated by unknown mechanisms - including promoter methylation, and encodes at least two different proteins, one of which with an unconventionally long cytoplasmic portion. In different settings, the Sda antigen plays multiple and unsuspected roles. 1) In colon cancer, its dramatic down-regulation plays a potential role in the overexpression of sialyl Lewis antigens, increasing metastasis formation. 2) It is involved in the lytic function of murine cytotoxic T lymphocytes. 3) It prevents the development of muscular dystrophy in various dystrophic murine models, when overexpressed in muscular fibers. 4) It regulates the circulating half-life of the von Willebrand factor (vWf), determining the onset of a bleeding disorder in a murine model.The expression of the Sda antigen has a wide impact on the physiology and the pathology of different biological systems.
Keywords: Histo-blood group antigen; Glycosyltransferase; Epigenetic control; Muscular dystrophy; Bleeding disorder; Sialyl Lewis antigen;

The impact of the receptor binding profiles of the vascular endothelial growth factors on their angiogenic features by Tiina Nieminen; Pyry I. Toivanen; Nina Rintanen; Tommi Heikura; Suvi Jauhiainen; Kari J. Airenne; Kari Alitalo; Varpu Marjomäki; Seppo Ylä-Herttuala (454-463).
Vascular endothelial growth factors (VEGFs) are potential therapeutic agents for treatment of ischemic diseases. Their angiogenic effects are mainly mediated through VEGF receptor 2 (VEGFR2).Receptor binding, signaling, and biological efficacy of several VEGFR2 ligands were compared to determine their characteristics regarding angiogenic activity and vascular permeability.Tested VEGFR2 ligands induced receptor tyrosine phosphorylation with different efficacy depending on their binding affinities. However, the tyrosine phosphorylation pattern and the activation of the major downstream signaling pathways were comparable. The maximal angiogenic effect stimulated by different VEGFR2 ligands was dependent on their ability to bind to co-receptor Neuropilin (Nrp), which was shown to form complexes with VEGFR2. The ability of these VEGFR2 ligands to induce vascular permeability was dependent on their concentration and VEGFR2 affinity, but not on Nrp binding.VEGFR2 activation alone is sufficient for inducing endothelial cell proliferation, formation of tube-like structures and vascular permeability. The level of VEGFR2 activation is dependent on the binding properties of the ligand used. However, closely similar activation pattern of the receptor kinase domain is seen with all VEGFR2 ligands. Nrp binding strengthens the angiogenic potency without increasing vascular permeability.This study sheds light on how different structurally closely related VEGFR2 ligands bind to and signal via VEGFR2/Nrp complex to induce angiogenesis and vascular permeability. The knowledge of this study could be used for designing VEGFR2/Nrp ligands with improved therapeutic properties.
Keywords: Vascular endothelial growth factor; Neuropilin; Receptor activation; Angiogenesis;

Dissecting cobamide diversity through structural and functional analyses of the base-activating CobT enzyme of Salmonella enterica by Chi Ho Chan; Sean A. Newmister; Keenan Talyor; Kathy R. Claas; Ivan Rayment; Jorge C. Escalante-Semerena (464-475).
Cobamide diversity arises from the nature of the nucleotide base. Nicotinate mononucleotide (NaMN):base phosphoribosyltransferases (CobT) synthesize α-linked riboside monophosphates from diverse nucleotide base substrates (e.g., benzimidazoles, purines, phenolics) that are incorporated into cobamides.Structural investigations of two members of the CobT family of enzymes in complex with various substrate bases as well as in vivo and vitro activity analyses of enzyme variants were performed to elucidate the roles of key amino acid residues important for substrate recognition.Results of in vitro and in vivo studies of active-site variants of the Salmonella enterica CobT (SeCobT) enzyme suggest that a catalytic base may not be required for catalysis. This idea is supported by the analyses of crystal structures that show that two glutamate residues function primarily to maintain an active conformation of the enzyme. In light of these findings, we propose that proper positioning of the substrates in the active site triggers the attack at the C1 ribose of NaMN.Whether or not a catalytic base is needed for function is discussed within the framework of the in vitro analysis of the enzyme activity. Additionally, structure-guided site-directed mutagenesis of SeCobT broadened its substrate specificity to include phenolic bases, revealing likely evolutionary changes needed to increase cobamide diversity, and further supporting the proposed mechanism for the phosphoribosylation of phenolic substrates.Results of this study uncover key residues in the CobT enzyme that contribute to the diversity of cobamides in nature.Display Omitted
Keywords: B12 biosynthesis; Lower base activation; Directed enzyme evolution; Cobamide diversity;

Evaluation of chitosan nanoformulations as potent anti-HIV therapeutic systems by Lakshmi Narashimhan Ramana; Shilpee Sharma; Swaminathan Sethuraman; Udaykumar Ranga; Uma Maheswari Krishnan (476-484).
Antiretroviral Therapy (ART) is currently the major therapeutic intervention in the treatment of AIDS. ART, however, is severely limited due to poor availability, high cytotoxicity, and enhanced metabolism and clearance of the drug molecules by the renal system. The use of nanocarriers encapsulating the anti-retroviral drugs may provide a solution to the aforementioned problems. Importantly, the application of mildly immunogenic polymeric carrier confers the advantage of making the nanoparticles more visible to the immune system leading to their efficient uptake by the phagocytes.The saquinavir-loaded chitosan nanoparticles were characterized by transmission electron microscopy and differential scanning calorimetry and analyzed for the encapsulation efficiency, swelling characteristics, particle size properties, and the zeta potential. Furthermore, cellular uptake of the chitosan nanocarriers was evaluated using confocal microscopy and Flow cytometry. The antiviral efficacy was quantified using viral infection of the target cells.Using novel chitosan carriers loaded with saquinavir, a protease inhibitor, we demonstrate a drug encapsulation efficiency of 75% and cell targeting efficiency greater than 92%. As compared to the soluble drug control, the saquinavir-loaded chitosan carriers caused superior control of the viral proliferation as measured by using two different viral strains, NL4-3 and Indie-C1, and two different target T-cells, Jurkat and CEM-CCR5.Chitosan nanoparticles loaded with saquinavir were characterized and they demonstrated superior drug loading potential with greater cell targeting efficiency leading to efficient control of the viral proliferation in target T-cells.Our data ascertain the potential of chitosan nanocarriers as novel vehicles for HIV-1 therapeutics.
Keywords: Nanocarrier; Chitosan; Saquinavir; Antiretroviral;

Differential response of DU145 and PC3 prostate cancer cells to ionizing radiation: Role of reactive oxygen species, GSH and Nrf2 in radiosensitivity by Sundarraj Jayakumar; Amit Kunwar; Santosh K. Sandur; Badri N. Pandey; Ramesh C. Chaubey (485-494).
Radioresistance is the major impediment in radiotherapy of many cancers including prostate cancer, necessitating the need to understand the factors contributing to radioresistance in tumor cells. In the present study, the role of cellular redox and redox sensitive transcription factor, Nrf2 in the radiosensitivity of prostate cancer cell lines PC3 and DU145, has been investigated.Differential radiosensitivity of PC3 and DU145 cells was assessed using clonogenic assay, flow cytometry, and comet assay. Their redox status was measured using DCFDA and DHR probes. Expression of Nrf2 and its dependent genes was measured by EMSA and real time PCR. Knockdown studies were done using shRNA transfection.PC3 and DU145 cells differed significantly in their radiosensitivity as observed by clonogenic survival, apoptosis and neutral comet assays. Both basal and inducible levels of ROS were higher in PC3 cells than that of DU145 cells. DU145 cells showed higher level of basal GSH content and GSH/GSSG ratio than that of PC3 cells. Further, significant increase in both basal and induced levels of Nrf2 and its dependent genes was observed in DU145 cells. Knock-down experiments and pharmacological intervention studies revealed the involvement of Nrf2 in differential radio-resistance of these cells.Cellular redox status and Nrf2 levels play a causal role in radio-resistance of prostate cancer cells.The pivotal role Nrf2 has been shown in the radioresistance of tumor cells and this study will further help in exploiting this factor in radiosensitization of other tumor cell types.Display Omitted
Keywords: Ionizing radiation; Radiosensitivity; Prostate cancer; Nrf2; PC3; DU145;

Thanks to their impressive catalytic properties, cerium oxide nanoparticles (nanoceria) are able to mimic the activity of superoxide dismutase and of catalase, therefore acting as reactive oxygen species (ROS) scavengers in many biological contexts, for instance offering neuroprotection and reduction of apoptosis rate in many types of cells exposed to oxidative stress (stem cells, endothelial cells, epithelial cells, osteoblasts, etc.).We report on the investigation at gene level, through quantitative real time RT-PCR, of the effects of cerium oxide nanoparticles on ROS mechanisms in neuron-like PC12 cells. After three days of treatment, transcription of 84 genes involved in antioxidant defense, in ROS metabolism, and coding oxygen transporters is evaluated, and its relevance to central nervous system degenerative diseases is considered.Experimental evidences reveal intriguing differences in transcriptional profiles of cells treated with cerium oxide nanoparticles with respect to the controls: nanoceria acts as strong exogenous ROS scavenger, modulating transcription of genes involved in natural cell defenses, down-regulating genes involved in inflammatory processes, and up-regulating some genes involved in neuroprotection.Our findings are extremely promising for future biomedical applications of cerium oxide nanoparticles, further supporting their possible exploitation in the treatment of neurodegenerative diseases.This work represents the first documented step to the comprehension of mechanisms underlying the anti-oxidant action of cerium oxide nanoparticles. Our findings allow for a better comprehension of the phenomena of ROS scavenging and neuroprotection at a gene level, suggesting future therapeutic approaches even at a pre-clinical level.Display Omitted
Keywords: PC12 cells; Cerium oxide nanoparticles; Oxidative stress; Anti-oxidant defenses; Neuroprotection;

Photodamage of lipid bilayers by irradiation of a fluorescently labeled cell-penetrating peptide by Igor Meerovich; Nandhini Muthukrishnan; Gregory A. Johnson; Alfredo Erazo-Oliveras; Jean-Philippe Pellois (507-515).
Fluorescently labeled cell-penetrating peptides can translocate into cells by endocytosis and upon light irradiation, lyse the endocytic vesicles. This photo-inducible endosomolytic activity of Fl–CPPs can be used to efficiently deliver macromolecules such as proteins and nucleic acids and other small organic molecules into the cytosol of live cells. The requirement of a light trigger to induce photolysis provides a more spatial and temporal control to the intracellular delivery process.In this report, we examine the molecular level mechanisms by which cell-penetrating peptides such as TAT when labeled with small organic fluorophore molecules acquire a photo-induced lytic activity using a simplified model of lipid vesicles.The peptide TAT labeled with 5(6)-carboxytetramethylrhodamine binds to negatively charged phospholipids, thereby bringing the fluorophore in close proximity to the membrane of liposomes. Upon light irradiation, the excited fluorophore produces reactive oxygen species at the lipid bilayer and oxidation of the membrane is achieved. In addition, the fluorescent peptide causes aggregation of photo-oxidized lipids, an activity that requires the presence of arginine residues in the peptide sequence.These results suggest that the cell-penetrating peptide plays a dual role. On one hand, TAT targets a conjugated fluorophore to membranes. On the other hand, TAT participates directly in the destabilization of photosensitized membranes. Peptide and fluorophore therefore appear to act in synergy to destroy membranes efficiently.Understanding the mechanism behind Fl–CPP mediated membrane photodamage will help to design optimally photo-endosomolytic compounds.Display Omitted
Keywords: Cell-penetrating peptide; Photochemical internalization; Photolysis; Liposome;

Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose by Peter Biely; Mária Cziszárová; Jane W. Agger; Xin-Liang Li; Vladimír Puchart; Mária Vršanská; Vincent G.H. Eijsink; Bjorge Westereng (516-525).
Trichoderma reesei CE16 acetyl esterase (AcE) is a component of the plant cell wall degrading system of the fungus. The enzyme behaves as an exo-acting deacetylase removing acetyl groups from non-reducing end sugar residues.In this work we demonstrate this exo-deacetylating activity on natural acetylated xylooligosaccharides using MALDI ToF MS.The combined action of GH10 xylanase and acetylxylan esterases (AcXEs) leads to formation of neutral and acidic xylooligosaccharides with a few resistant acetyl groups mainly at their non-reducing ends. We show here that these acetyl groups serve as targets for TrCE16 AcE. The most prominent target is the 3-O-acetyl group at the non-reducing terminal Xylp residues of linear neutral xylooligosaccharides or on aldouronic acids carrying MeGlcA at the non-reducing terminus. Deacetylation of the non-reducing end sugar may involve migration of acetyl groups to position 4, which also serves as substrate of the TrCE16 esterase.Concerted action of CtGH10 xylanase, an AcXE and TrCE16 AcE resulted in close to complete deacetylation of neutral xylooligosaccharides, whereas substitution with MeGlcA prevents removal of acetyl groups from only a small fraction of the aldouronic acids. Experiments with diacetyl derivatives of methyl β-d-xylopyranoside confirmed that the best substrate of TrCE16 AcE is 3-O-acetylated Xylp residue followed by 4-O-acetylated Xylp residue with a free vicinal hydroxyl group.This study shows that CE16 acetyl esterases are crucial enzymes to achieve complete deacetylation and, consequently, complete the saccharification of acetylated xylans by xylanases, which is an important task of current biotechnology.
Keywords: Acetyl esterase; Carbohydrate esterase family; Positional specificity; Acetyl glucuronoxylan; MALDI ToF MS; NMR;

Carbon-14 decay as a source of non-canonical bases in DNA by Michel Sassi; Damien J. Carter; Blas P. Uberuaga; Chris R. Stanek; Nigel A. Marks (526-534).
Significant experimental effort has been applied to study radioactive beta-decay in biological systems. Atomic-scale knowledge of this transmutation process is lacking due to the absence of computer simulations. Carbon-14 is an important beta-emitter, being ubiquitous in the environment and an intrinsic part of the genetic code. Over a lifetime, around 50 billion 14C decays occur within human DNA.We apply ab initio molecular dynamics to quantify 14C-induced bond rupture in a variety of organic molecules, including DNA base pairs.We show that double bonds and ring structures confer radiation resistance. These features, present in the canonical bases of the DNA, enhance their resistance to 14C-induced bond-breaking. In contrast, the sugar group of the DNA and RNA backbone is vulnerable to single-strand breaking. We also show that Carbon-14 decay provides a mechanism for creating mutagenic wobble-type mispairs.The observation that DNA has a resistance to natural radioactivity has not previously been recognized. We show that 14C decay can be a source for generating non-canonical bases.Our findings raise questions such as how the genetic apparatus deals with the appearance of an extra nitrogen in the canonical bases. It is not obvious whether or not the DNA repair mechanism detects this modification nor how DNA replication is affected by a non-canonical nucleobase. Accordingly, 14C may prove to be a source of genetic alteration that is impossible to avoid due to the universal presence of radiocarbon in the environment.Display Omitted
Keywords: Carbon-14; Beta-decay; DNA; Bond-breaking; Non-canonical base; Mutation;

The Salmonella enterica ZinT structure, zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc by Andrea Ilari; Flaminia Alaleona; Giancarlo Tria; Patrizia Petrarca; Andrea Battistoni; Carlotta Zamparelli; Daniela Verzili; Mattia Falconi; Emilia Chiancone (535-544).
In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT–ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim.ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy).The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a KD value of 22 ± 2 nM for ZinT, while those with ZnuA point to one high affinity (KD  < 20 nM) and one low affinity Zn(II) binding site (KD in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT–ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other.ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA.Knowledge of the ZinT–ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.Display Omitted
Keywords: Salmonella enterica; Zinc transport; Zinc affinity; Protein-protein interaction; SAXS; Analytical ultracentrifugation;

Carotenoids have been found to play roles in the prevention and therapy of some cancers which PPARγ was also discovered to be involved in. The present studies were directed to determine the inhibitory effects of carotenoids in combination with rosiglitazone, a synthetic PPARγ agonist, on K562 cell proliferation and elucidate the contribution of PPARγ-dependent pathway to cell proliferation suppression.The effects of carotenoid and rosiglitazone combination on K562 cell proliferation were evaluated by trypan blue dye exclusion assay and MTT assay. When PPARγ has been inhibited by GW9662 and siRNA, cycle-related regulator expression in K562 cells treated with carotenoid and rosiglitazone combination was analyzed by Western blotting.Rosiglitazone inhibited K562 cell proliferation and augmented the inhibitory effects of carotenoids on the cell proliferation greatly. Specific PPARγ inhibition attenuated the cell growth suppression induced by carotenoid and rosiglitazone combination. GW9662 pre-treatment attenuated the enhanced up-regulation of PPARγ expression caused by the combination treatment. Moreover, GW9662 and PPARγ siRNA also significantly attenuated the up-regulation of p21 and down-regulation of cyclin D1 caused by carotenoids and rosiglitazone.PPARγ signaling pathway, via stimulating p21 and inhibiting cyclin D1, may play an important role in the anti-proliferative effects of carotenoid and rosiglitazone combination on K562 cells.Carotenoids in combination with rosiglitazone are hopeful to provide attractive dietary or supplementation-based and pharmaceutical strategies to treat cancer diseases.
Keywords: Carotenoid; Rosiglitazone; PPARγ; K562 cell;

In the Crabtree-negative Kluyveromyces lactis yeast the rag8 mutant is one of nineteen complementation groups constituting the fermentative-deficient model equivalent to the Saccharomyces cerevisiae respiratory petite mutants. These mutants display pleiotropic defects in membrane fatty acids and/or cell walls, osmo-sensitivity and the inability to grow under strictly anaerobic conditions (Rag phenotype). RAG8 is an essential gene coding for the casein kinase I, an evolutionary conserved activity involved in a wide range of cellular processes coordinating morphogenesis and glycolytic flux with glucose/oxygen sensing.A metabolomic approach was performed by NMR spectroscopy to investigate how the broad physiological roles of Rag8, taken as a model for all rag mutants, coordinate cellular responses.Statistical analysis of metabolomic data showed a significant increase in the level of metabolites in reactions directly involved in the reoxidation of the NAD(P)H in rag8 mutant samples with respect to the wild type ones. We also observed an increased de novo synthesis of nicotinamide adenine dinucleotide. On the contrary, the production of metabolites in pathways leading to the reduction of the cofactors was reduced.The changes in metabolite levels in rag8 showed a metabolic adaptation that is determined by the intracellular NAD(P)+/NAD(P)H redox balance state.The inadequate glycolytic flux of the mutant leads to a reduced/asymmetric distribution of acetyl-CoA to the different cellular compartments with loss of the fatty acid dynamic respiratory/fermentative adaptive balance response.
Keywords: Yeast; Metabolomics; Casein kinase; NAD(P)+/NAD(P)H balance; NMR spectroscopy;

Physico-chemical characterization and the in vitro genotoxicity of medical implants metal alloy (TiAlV and CoCrMo) and polyethylene particles in human lymphocytes by Goran Gajski; Želimir Jelčić; Višnja Oreščanin; Marko Gerić; Robert Kollar; Vera Garaj-Vrhovac (565-576).
The main objective of the present study was to investigate chemical composition and possible cyto/genotoxic potential of several medical implant materials commonly used in total hip joint replacement.Medical implant metal alloy (Ti6Al4V and CoCrMo) and high density polyethylene particles were analyzed by energy dispersive X-ray spectrometry while toxicological characterization was done on human lymphocytes using multi-biomarker approach.Energy dispersive X-ray spectrometry showed that none of the elements identified deviate from the chemical composition defined by appropriate ISO standard. Toxicological characterization showed that the tested materials were non-cyto/genotoxic as determined by the comet and cytokinesis-block micronucleus (CBMN) assay. Particle morphology was found (by using scanning electron and optical microscope) as flat, sharp-edged, irregularly shaped fiber-like grains with the mean particle size less than 10 µm; this corresponds to the so-called "submicron wear". The very large surface area per wear volume enables high reactivity with surrounding media and cellular elements.Although orthopedic implants proved to be non-cyto/genotoxic, in tested concentration (10 μg/ml) there is a constant need for monitoring of patients that have implanted artificial hips or other joints, to minimize the risks of any unwanted health effects.The fractal and multifractal analyses, performed in order to evaluate the degree of particle shape effect, showed that the fractal and multifractal terms are related to the "remnant" level of the particles' toxicity especially with the cell viability (trypan blue method) and total number of nucleoplasmic bridges and nuclear buds as CBMN assay parameters.
Keywords: Joint replacement; Human lymphocyte; Cytogenotoxicity; Scanning electron microscope; Fractal analysis; Multifractal analysis;

Acute exposure of beta-cells to troglitazone decreases insulin hypersecretion via activating AMPK by Ruyuan Deng; Aifang Nie; Fangfang Jian; Yun Liu; Hongju Tang; Juan Zhang; Yuqing Zhang; Li Shao; Fengying Li; Libin Zhou; Xiao Wang; Guang Ning (577-585).
It has been recognized that insulin hypersecretion can lead to the development of insulin resistance and type 2 diabetes mellitus. There is substantial evidence demonstrating that thiazolidinediones are able to delay and prevent the progression of pancreatic β-cell dysfunction. However, the mechanism underlying the protective effect of thiazolidinediones on β-cell function remains elusive.We synchronously detected the effects of troglitazone on insulin secretion and AMP-activated protein kinase (AMPK) activity under various conditions in isolated rat islets and MIN6 cells.Long-term exposure to high glucose stimulated insulin hypersecretion and inhibited AMPK activity in rat islets. Troglitazone-suppressed insulin hypersecretion was closely related to the activation of AMPK. This action was most prominent at the moderate concentration of glucose. Glucose-stimulated insulin secretion was decreased by long-term troglitazone treatment, but significantly increased after the drug withdrawal. Compound C, an AMPK inhibitor, reversed troglitazone-suppressed insulin secretion in MIN6 cells and rat islets. Knockdown of AMPKα2 showed a similar result. In MIN6 cells, troglitazone blocked high glucose-closed ATP-sensitive K+ (KATP) channel and decreased membrane potential, along with increased voltage-dependent potassium channel currents. Troglitazone suppressed intracellular Ca2 + response to high glucose, which was abolished by treatment with compound C.Our results suggest that troglitazone provides β-cell “a rest” through activating AMPK and inhibiting insulin hypersecretion, and thus restores its response to glucose.These data support that AMPK activation may be an important mechanism for thiazolidinediones preserving β-cell function.
Keywords: Troglitazone; Insulin hyperscretion; AMP-activated protein kinase; Beta-cell;

Anti-inflammatory action of lipid nanocarrier-delivered myriocin: therapeutic potential in cystic fibrosis by Anna Caretti; Alessandra Bragonzi; Marcella Facchini; Ida De Fino; Camilla Riva; Paolo Gasco; Claudia Musicanti; Josefina Casas; Gemma Fabriàs; Riccardo Ghidoni; Paola Signorelli (586-594).
Sphingolipids take part in immune response and can initiate and/or sustain inflammation. Various inflammatory diseases have been associated with increased ceramide content, and pharmacological reduction of ceramide diminishes inflammation damage in vivo. Inflammation and susceptibility to microbial infection are two elements in a vicious circle. Recently, sphingolipid metabolism inhibitors were used to reduce infection. Cystic fibrosis (CF) is characterized by a hyper-inflammation and an excessive innate immune response, which fails to evolve into adaptive immunity and to eradicate infection. Chronic infections result in lung damage and patient morbidity. Notably, ceramide content in mucosa airways is higher in CF mouse models and in patients than in control mice or healthy subjects.The therapeutic potential of myriocin, an inhibitor of the sphingolipid de novo synthesis rate limiting enzyme (Serine Palmitoyl Transferase, SPT),was investigated in CF cells and mice models.We treated CF human respiratory epithelial cells with myriocin, This treatment resulted in reduced basal, as well as TNFα-stimulated, inflammation. In turn, TNFα induced an increase in SPT in these cells, linking de novo synthesis of ceramide to inflammation. Furthermore, myriocin-loaded nanocarrier, injected intratrachea prior to P. aeruginosa challenge, enabled a significant reduction of lung infection and reduced inflammation.The presented data suggest that de novo ceramide synthesis is constitutively enhanced in CF mucosa and that it can be envisaged as pharmacological target for modulating inflammation and restoring effective innate immunity against acute infection.Myriocin stands as a powerful immunomodulatory agent for inflammatory and infectious diseases.
Keywords: Ceramide; Sphingolipids; Inflammation; Innate immune responses; Cystic fibrosis; Nanocarriers;

Vacancy of occupied N-glycosylation sites of glycoproteins is quite disruptive to a multicellular organism, as underlined by congenital disorders of glycosylation. Since a neuronal component is typically associated with this disease, we evaluated the impact of N-glycosylation processing of a neuronal voltage gated potassium channel, Kv3.1b, expressed in a neuronal-derived cell line, B35 neuroblastoma cells.Total internal reflection fluorescence and differential interference contrast microscopy measurements of live B35 cells expressing wild type and glycosylation mutant Kv3.1b proteins were used to evaluate the distribution of the various forms of the Kv3.1b protein in the cell body and outgrowths. Cell adhesion assays were also employed.Microscopy images revealed that occupancy of both N-glycosylation sites of Kv3.1b had relatively similar amounts of Kv3.1b in the outgrowth and cell body while vacancy of one or both sites led to increased accumulation of Kv3.1b in the cell body. Further both the fully glycosylated and partially glycosylated N229Q Kv3.1b proteins formed higher density particles in outgrowths compared to cell body. Cellular assays demonstrated that the distinct spatial arrangements altered cell adhesion properties.Our findings provide direct evidence that occupancy of the N-glycosylation sites of Kv3.1b contributes significantly to its lateral heterogeneity in membranes of neuronal-derived cells, and in turn alters cellular properties.Our study demonstrates that N-glycans of Kv3.1b contain information regarding the association, clustering, and distribution of Kv3.1b in the cell membrane, and furthermore that decreased occupancy caused by congenital disorders of glycosylation may alter the biological activity of Kv3.1b.Display Omitted
Keywords: N-glycosylation; Potassium channel; Membrane glycoprotein; Membrane trafficking; High density potassium channel cluster; Excitable membrane;

Decreased solute adsorption onto cracked surfaces of mechanically injured articular cartilage: Towards the design of cartilage-specific functional contrast agents by Mohammad Moeini; Sarah G.A. Decker; Hooi Chuan Chin; Yousef Shafieyan; Derek H. Rosenzweig; Thomas M. Quinn (605-614).
Currently available methods for contrast agent-based magnetic resonance imaging (MRI) and computed tomography (CT) of articular cartilage can only detect cartilage degradation after biochemical changes have occurred within the tissue volume. Differential adsorption of solutes to damaged and intact surfaces of cartilage may be used as a potential mechanism for detection of injuries before biochemical changes in the tissue volume occur.Adsorption of four fluorescent macromolecules to surfaces of injured and sliced cartilage explants was studied. Solutes included native dextran, dextrans modified with aldehyde groups or a chondroitin sulfate (CS)-binding peptide and the peptide alone.Adsorption of solutes to fissures was significantly less than to intact surfaces of injured and sliced explants. Moreover, solute adsorption at intact surfaces of injured and sliced explants was less reversible than at surfaces of uninjured explants. Modification of dextrans with aldehyde or the peptide enhanced adsorption with the same level of differential adsorption to cracked and intact surfaces. However, aldehyde–dextran exhibited irreversible adsorption. Equilibration of explants in solutes did not decrease the viability of chondrocytes.Studied solutes showed promising potential for detection of surface injuries based on differential interactions with cracked and intact surfaces. Additionally, altered adsorption properties at surfaces of damaged cartilage which visually look healthy can be used to detect micro-damage or biochemical changes in these regions. Studied solutes can be used in in vivo fluorescence imaging methods or conjugated with MRI or CT contrast agents to develop functional imaging agents.
Keywords: Articular cartilage; Contrast agent; Dextrans; FITC; Injury; Solute adsorption;

Resveratrol enhances chemosensitivity of doxorubicin in multidrug-resistant human breast cancer cells via increased cellular influx of doxorubicin by Tae Hyung Kim; Yu Jin Shin; A. Jin Won; Byung Mu Lee; Wahn Soo Choi; Jee H. Jung; Hae Young Chung; Hyung Sik Kim (615-625).
Multidrug resistance is a major problem in the treatment of breast cancer, and a number of studies have attempted to find an efficient strategy with which to overcome it. In this study, we investigate the synergistic anticancer effects of resveratrol (RSV) and doxorubicin (Dox) against human breast cancer cell lines.The synergistic effects of RSV on chemosensitivity were examined in Dox-resistant breast cancer (MCF-7/adr) and MDA-MB-231 cells. In vivo experiments were performed using a nude mouse xenograft model to investigate the combined sensitization effect of RSV and Dox.RSV markedly enhanced Dox-induced cytotoxicity in MCF-7/adr and MDA-MB-231 cells. Treatment with a combination of RSV and Dox significantly increased the cellular accumulation of Dox by down-regulating the expression levels of ATP-binding cassette (ABC) transporter genes, MDR1, and MRP1. Further in vivo experiments in the xenograft model revealed that treatment with a combination of RSV and Dox significantly inhibited tumor volume by 60%, relative to the control group.These results suggest that treatment with a combination of RSV and Dox would be a helpful strategy for increasing the efficacy of Dox by promoting an intracellular accumulation of Dox and decreasing multi-drug resistance in human breast cancer cells.
Keywords: Resveratrol; Doxorubicin; Drug-resistant; Breast cancer;

Mutation of a pH-modulating residue in a GH51 α-l-arabinofuranosidase leads to a severe reduction of the secondary hydrolysis of transfuranosylation products by Bastien Bissaro; Olivier Saurel; Faten Arab-Jaziri; Luc Saulnier; Alain Milon; Maija Tenkanen; Pierre Monsan; Michael J. O'Donohue; Régis Fauré (626-636).
The development of enzyme-mediated glycosynthesis using glycoside hydrolases is still an inexact science, because the underlying molecular determinants of transglycosylation are not well understood. In the framework of this challenge, this study focused on the family GH51 α-l-arabinofuranosidase from Thermobacillus xylanilyticus, with the aim to understand why the mutation of position 344 provokes a significant modification of the transglycosylation/hydrolysis partition.Detailed kinetic analysis (k cat, K M, pK a determination and time-course NMR kinetics) and saturation transfer difference nuclear magnetic resonance spectroscopy was employed to determine the synthetic and hydrolytic ability modification induced by the redundant N344 mutation disclosed in libraries from directed evolution.The mutants N344P and N344Y displayed crippled hydrolytic abilities, and thus procured improved transglycosylation yields. This behavior was correlated with an increased pK a of the catalytic nucleophile (E298), the pK a of the acid/base catalyst remaining unaffected. Finally, mutations at position 344 provoked a pH-dependent product inhibition phenomenon, which is likely to be the result of a significant modification of the proton sharing network in the mutants.Using a combination of biochemical and biophysical methods, we have studied TxAbf-N344 mutants, thus revealing some fundamental details concerning pH modulation. Although these results concern a GH51 α-l-arabinofuranosidase, it is likely that the general lessons that can be drawn from them will be applicable to other glycoside hydrolases. Moreover, the effects of mutations at position 344 on the transglycosylation/hydrolysis partition provide clues as to how TxAbf can be further engineered to obtain an efficient transfuranosidase.Display Omitted
Keywords: Pentoses/furanoses; Transglycosylation; pK a modulation; pH-dependent inhibition; STD NMR;

The level of circulating interleukin-6 receptor in human blood varies depending on the genetic and/or physiological causes, and has been implicated in the development of chronic inflammatory diseases.The cis-regulatory effects of genetic variations on the transcription of interleukin-6 receptor gene, IL6R, were studied by assessing allelic transcriptions in the immortalized lymphocytes derived from unrelated and familial samples.The assays for allelic transcription in the cells from unrelated subjects demonstrated an extensive and variable range of allelic transcriptional imbalances, suggesting an operation of multiple cis-regulations with varying degrees on the locus. Analysis of the familial samples illustrated the Mendelian inheritance of allelic transcriptions, enabling us to assign each haplotype allele into one of the 3 transcriptional strengths. A comparison of the allele structures based on the transcriptional attributes highlighted 2 SNP variations, rs952146 and rs4845617, as being associated with higher allelic transcription. Consistently, lymphocytes that were homozygous for the 2 SNPs exhibited differences in their transcript levels depending on the haplotypes.Inheritance assessment of allelic transcription of IL6R identified 2 SNPs that are associated with transcriptional variation in cis.Our results not only demonstrate genetic variations that are associated with IL6R transcription in cis but also demonstrate an effective genetic approach for isolating cis-regulatory variations.
Keywords: IL6R; Regulatory variation; Allelic transcription; Single Nucleotide Polymorphism;

Discovery of novel differentiation markers in the early stage of chondrogenesis by glycoform-focused reverse proteomics and genomics by Takeshi Ishihara; Kiyoshi Kakiya; Koji Takahashi; Hiroto Miwa; Masatomo Rokushima; Tomoyo Yoshinaga; Yoshikazu Tanaka; Takaomi Ito; Hiroko Togame; Hiroshi Takemoto; Maho Amano; Norimasa Iwasaki; Akio Minami; Shin-Ichiro Nishimura (645-655).
Osteoarthritis (OA) is one of the most common chronic diseases among adults, especially the elderly, which is characterized by destruction of the articular cartilage. Despite affecting more than 100 million individuals all over the world, therapy is currently limited to treating pain, which is a principal symptom of OA. New approaches to the treatment of OA that induce regeneration and repair of cartilage are strongly needed.To discover potent markers for chondrogenic differentiation, glycoform-focused reverse proteomics and genomics were performed on the basis of glycoblotting-based comprehensive approach.Expression levels of high-mannose type N-glycans were up-regulated significantly at the late stage of differentiation of the mouse chondroprogenitor cells. Among 246 glycoproteins carrying this glycotype identified by ConA affinity chromatography and LC/MS, it was demonstrated that 52% are classified as cell surface glycoproteins. Gene expression levels indicated that mRNAs for 15 glycoproteins increased distinctly in the earlier stages during differentiation compared with Type II collagen. The feasibility of mouse chondrocyte markers in human chondrogenesis model was demonstrated by testing gene expression levels of these 15 glycoproteins during differentiation in human mesenchymal stem cells.The results showed clearly an evidence of up-regulation of 5 genes, ectonucleotide pyrophosphatase/phosphodiesterase family member 1, collagen alpha-1(III) chain, collagen alpha-1(XI) chain, aquaporin-1, and netrin receptor UNC5B, in the early stages of differentiation.These cell surface 5 glycoproteins become highly sensitive differentiation markers of human chondrocytes that contribute to regenerative therapies, and development of novel therapeutic reagents.
Keywords: Osteoarthritis; Chondrogenic differentiation; Glycomics; Glycoform-focused reverse proteomics and genomics; Mass spectrometry;

Palmitate interaction with physiological states of myoglobin by Lifan Shih; Youngran Chung; Renuka Sriram; Thomas Jue (656-666).
Previous studies have shown that palmitate (PA) can bind specifically and non-specifically to Fe(III) MbCN. The present study has observed PA interaction with physiological states of Fe(II) Mb, and the observations support the hypothesis that Mb may have a potential role in facilitating intracellular fatty acid transport. 1H NMR spectra measurements of the Mb signal during PA titration show signal changes consistent with specific and non-specific binding.Palmitate (PA) interacts differently with physiological states of Mb. Deoxy Mb does not interact specifically or non-specifically with PA, while the carbonmonoxy myoglobin (MbCO) interaction with PA decreases the intensity of selective signals and produces a 0.15 ppm upfield shift of the PA methylene peak. The selective signal change upon PA titration provides a basis to determine an apparent PA binding constant, which serves to create a model comparing the competitive PA binding and facilitated fatty acid transport of Mb and fatty acid binding protein (FABP).Given contrasting PA interaction of ligated vs. unligated Mb, the cellular fatty acid binding protein (FABP) and Mb concentration in the cell, the reported cellular diffusion coefficients, the PA dissociation constants from ligated Mb and FABP, a fatty acid flux model suggests that Mb can compete with FABP transporting cellular fatty acid.Under oxygenated conditions and continuous energy demand, Mb dependent fatty acid transport could influence the cell's preference for carbohydrate or fatty acid as a fuel source and regulate fatty acid metabolism.
Keywords: Lipid; Fatty acid; NMR; Metabolism; Bioenergetics;

CD1d favors MHC neighborhood, GM1 ganglioside proximity and low detergent sensitive membrane regions on the surface of B lymphocytes by Dilip Shrestha; Mark A. Exley; György Vereb; János Szöllősi; Attila Jenei (667-680).
Cluster of differentiation 1 (CD1) represents a family of proteins which is involved in lipid-based antigen presentation. Primarily, antigen presenting cells, like B cells, express CD1 proteins. Here, we examined the cell-surface distribution of CD1d, a subtype of CD1 receptors, on B lymphocytes.Fluorescence labeling methods, including fluorescence resonance energy transfer (FRET), were employed to investigate plasma membrane features of CD1d receptors.High FRET efficiency was observed between CD1d and MHC I heavy chain (MHC I-HC), β2-microglobulin (β2m) and MHC II proteins in the plasma membrane. In addition, overexpression of CD1d reduced the expression of MHC II and increased the expression of MHC I-HC and β2m proteins on the cell-surface. Surprisingly, β2m dependent CD1d isoform constituted only ~ 15% of the total membrane CD1d proteins. Treatment of B cells with methyl-β-cyclodextrin (MβCD) / simvastatin caused protein rearrangement; however, FRET demonstrated only minimal effect of these chemicals on the association between CD1d and GM1 ganglioside on cell-surface. Likewise, a modest effect was only observed in a co-culture assay between MβCD/simvastatin treated C1R–CD1d cells and invariant natural killer T cells on measuring secreted cytokines (IFNγ and IL4). Furthermore, CD1d rich regions were highly sensitive to low concentration of Triton X-100. Physical proximity between CD1d, MHC and GM1 molecules was also detected in the plasma membrane.An intricate relationship between CD1d, MHC, and lipid species was found on the membrane of human B cells.Organization of CD1d on the plasma membrane might be critical for its biological functions.
Keywords: CD1d; MHC; Rafts; FRET; Methyl-β-cyclodextrin; Simvastatin;

Ochratoxin A (OTA), a mycotoxin, causes extensive cell damage, affecting liver and kidney cells. OTA toxicity is fairly well characterized where oxidative stress is believed to play a role, however, the sequence of molecular events after OTA-exposure, have not been characterized in literature. Further, antidotes for alleviating the toxicity are sparsely reported. The aim of this study was to understand the sequence of some molecular mechanisms for OTA-induced toxicity and the cytoprotective effect of quercetin on OTA-induced toxicity.Time course studies to evaluate the time of intracellular calcium release and ROS induction were carried out. The time of activation and induction of two key redox- sensitive transcription factors, NF-κB and Nrf-2 were determined by nuclear localization and expression respectively. The time of expression of inflammatory marker COX-2 was determined. Oxidative DNA damage by comet assay and micronucleus formation was studied. The ameliorative effect of quercetin on OTA-induced toxicity was also determined on all the above-mentioned parameters.OTA-induced calcium release, ROS generation and activated NF-κB nuclear translocation and expression. Pre-treatment with quercetin ameliorated ROS and calcium release as well as NF-κB induction and expression. Quercetin induced Nrf-2 nuclear translocation and expression. Quercetin's anti-inflammatory property was exhibited as it down regulated COX-2. Anti-genotoxic effect of quercetin was evident in prevention of DNA damage and micronucleus formation.Quercetin modulated OTA-induced oxidative stress and redox-signaling in HepG2 cells.The results of the study demonstrate for the first time that quercetin prevents OTA-induced toxicity in HepG2 cells.
Keywords: Ochratoxin; Oxidative stress; Quercetin; Inflammation; DNA damage;

Inactivation of glucocorticoid hormones and p44/42 mitogen-activated protein kinase (MAPK) is thought to be important in small intestinal maturation and expression of genes related to intestinal differentiation and functions.We investigated target genes induced by co-treatment for 48 h with a glucocorticoid hormone agonist, dexamethasone (Dex), and a p44/42 MAPK inhibitor, PD98059 (PD), in a small intestine-like cell line (Caco-2) using microarray analysis. We also investigated whether expression changes of the target genes induced by the co-treatment are associated with histone modifications around these genes.Co-treatment of Caco-2 cells with Dex and PD enhanced several genes related to intestinal differentiation and functions such as SCNN1A, FXYD3, LCT and LOX. Induction of the SCNN1A gene was associated with increased presence of acetylated histone H3 and H4 and di-methylated histone H3 at lysine (K) 4 around the transcribed region of the gene, and induction of the FXYD3 gene was associated with increased presence of acetylated histones H3 and H4 from the promoter/enhancer to the transcribed region of the gene. Induction of LCT and LOX genes was associated with increased presence of acetylated histone H4 on the promoter/enhancer region of the genes.Histone acetylation and/or histone H3 K4 methylation around the promoter/enhancer or/and transcribed regions of target genes are associated with induction of the genes by co-treatment with Dex and PD in Caco-2 cells.The histone code is specific to each gene with respect to induction by glucocorticoid hormone and inhibition of p44/42 MAPK in Caco-2 cells.
Keywords: Histone code; Differentiation; Caco-2 cells; Glucocorticoid hormone; p44/42 MAPK;

Trypanosoma brucei, responsible for African sleeping sickness, is a lethal parasite against which there is need for new drug protocols. It is therefore relevant to attack possible biomedical targets with specific preparations and since arginine kinase does not occur in humans but is present in the parasite it becomes a suitable target.Fluorescence quenching, thermodynamic analysis and FRET have shown that arginine kinase from T. brucei interacted with silver or gold nanoparticles.The enzyme only had one binding site. At 25 °C the dissociation (Kd) and Stern–Volmer constants (KSV) were 15.2 nM, 0.058 nM− 1 [Ag]; and 43.5 nM, 0.052 nM− 1 [Au] and these decreased to 11.2 nM, 0.041 nM− 1 [Ag]; and 24.2 nM, 0.039 nM− 1 [Au] at 30 °C illustrating static quenching and the formation of a non-fluorescent fluorophore–nanoparticle complex. Silver nanoparticles bound to arginine kinase with greater affinity, enhanced fluorescence quenching and easier access to tryptophan molecules than gold. Negative ΔH and ΔG values implied that the interaction of both Ag and Au nanoparticles with arginine kinase was spontaneous with electrostatic forces. FRET confirmed that the nanoparticles were bound 2.11 nm [Ag] and 2.26 nm [Au] from a single surface tryptophan residue.The nanoparticles bind close to the arginine substrate through a cysteine residue that controls the electrophilic and nucleophilic characters of the substrate arginine–guanidinium group crucial for enzymatic phosphoryl transfer between ADP and ATP.The nanoparticles of silver and gold interact with arginine kinase from T. brucei and may prove to have far reaching consequences in clinical trials.Proposed structure of the binding sites for TbAK showing the interaction of silver/gold nanoparticles through Cys271, interfering with N1 of the arginine substrate. The interatomic distance between the thiolate atom of Cys271 and N1 of the arginine substrate is 3.3 Å. The interatomic distance between Trp104 and N1 is 22.2 Å while that distance between Trp104 and bound Ag/Au nanoparticles is 21.1 Å and 22.6 Å respectively. Nitrate not shown.Display Omitted
Keywords: Metal nanoparticle; Thermodynamic fluorimetric analysis; Trypanosomiasis; Arginine kinase;