BBA - General Subjects (v.1840, #3)

Structure–function studies on jaburetox, a recombinant insecticidal peptide derived from jack bean (Canavalia ensiformis) urease by Anne H.S. Martinelli; Karine Kappaun; Rodrigo Ligabue-Braun; Marina S. Defferrari; Angela R. Piovesan; Fernanda Stanisçuaski; Diogo R. Demartini; Chariston A. Dal Belo; Carlos G.M. Almeida; Cristian Follmer; Hugo Verli; Celia R. Carlini; Giancarlo Pasquali (935-944).
Ureases are metalloenzymes involved in defense mechanisms in plants. The insecticidal activity of Canavalia ensiformis (jack bean) ureases relies partially on an internal 10 kDa peptide generated by enzymatic hydrolysis of the protein within susceptible insects. A recombinant version of this peptide, jaburetox, exhibits insecticidal, antifungal and membrane-disruptive properties. Molecular modeling of jaburetox revealed a prominent β-hairpin motif consistent with either neurotoxicity or pore formation.Aiming to identify structural motifs involved in its effects, mutated versions of jaburetox were built: 1) a peptide lacking the β-hairpin motif (residues 61–74), JbtxΔ-β; 2) a peptide corresponding the N-terminal half (residues 1–44), Jbtx N-ter, and 3) a peptide corresponding the C-terminal half (residues 45–93), Jbtx C-ter.1) JbtxΔ-β disrupts liposomes, and exhibited entomotoxic effects similar to the whole peptide, suggesting that the β-hairpin motif is not a determinant of these biological activities; 2) both Jbtx C-ter and Jbtx N-ter disrupted liposomes, the C-terminal peptide being the most active; and 3) while Jbtx N-ter persisted to be biologically active, Jbtx C-ter was less active when tested on different insect preparations. Molecular modeling and dynamics were applied to the urease-derived peptides to complement the structure–function analysis.The N-terminal portion of the Jbtx carries the most important entomotoxic domain which is fully active in the absence of the β-hairpin motif. Although the β-hairpin contributes to some extent, probably by interaction with insect membranes, it is not essential for the entomotoxic properties of Jbtx.Jbtx represents a new type of insecticidal and membrane-active peptide.Display Omitted
Keywords: Site-directed mutagenesis; β-hairpin; Urease-derived peptide; Molecular modeling; Membrane-disturbing; Insect;

Membrane-modifying properties of crotamine, a small peptide-toxin from Crotalus durissus terifficus venom by Michaela Sieber; Bettina Bosch; Wolfgang Hanke; Vera Maura Fernandes de Lima (945-950).
Crotamine is a small, highly basic myotoxin from the venom of the South American rattlesnake Crotalus durissus terifficus. It is structurally well defined and exhibits some similarities with the β-defensins of vertebrates. An amazing variety of functions and targets that range from analgesia and tumor-related activity to cell penetration have been associated with crotamine. Similar to defensins, it had been argued that crotamine has antimicrobial activity, and this supposition was recently proven. Moreover, it has been argued that the antimicrobial activity of crotamine is due to the membrane permeabilizing properties of the peptide. However, until now, the detailed mechanism of this postulated membrane permeabilization was still unclear.In this paper, we used gradient SDS-gels, mass spectroscopy (MALDI-TOF), and monolayer and planar lipid bilayer experiments to investigate the membrane modifying properties of crotamine.We showed that crotamine itself forms stable monolayers because of its amphipathic structure, is easily incorporated into lipid monolayers and forms well-defined pores with low cationic selectivity in planar lipid bilayers; these properties might account for the antimicrobial activity of crotamine. The pores are probably oligomeric aggregates of crotamine molecules, as suggested by the tendency of crotamine to form oligomers in aqueous solution and the fact that the structure of crotamine does not allow pore formation by monomers.The membrane modifying and antimicrobial properties of crotamine are probably due to homo-oligomeric pore formation in membranes.The results should be highly interesting to researchers in the fields of biophysics, pharmacology, toxicology and antibiotics.
Keywords: Crotamine; Crotalus venom; Monolayer; Planar lipid bilayer; Pore formation; Antimicrobial peptides;

Genetically encoded fluorescent indicator for imaging NAD+/NADH ratio changes in different cellular compartments by Dmitry S. Bilan; Mikhail E. Matlashov; Andrey Yu. Gorokhovatsky; Carsten Schultz; Grigori Enikolopov; Vsevolod V. Belousov (951-957).
The ratio of NAD+/NADH is a key indicator that reflects the overall redox state of the cells. Until recently, there were no methods for real time NAD+/NADH monitoring in living cells. Genetically encoded fluorescent probes for NAD+/NADH are fundamentally new approach for studying the NAD+/NADH dynamics.We developed a genetically encoded probe for the nicotinamide adenine dinucleotide, NAD(H), redox state changes by inserting circularly permuted YFP into redox sensor T-REX from Thermus aquaticus. We characterized the sensor in vitro using spectrofluorometry and in cultured mammalian cells using confocal fluorescent microscopy.The sensor, named RexYFP, reports changes in the NAD+/NADH ratio in different compartments of living cells. Using RexYFP, we were able to track changes in NAD+/NADH in cytoplasm and mitochondrial matrix of cells under a variety of conditions. The affinity of the probe enables comparison of NAD+/NADH in compartments with low (cytoplasm) and high (mitochondria) NADH concentration. We developed a method of eliminating pH-driven artifacts by normalizing the signal to the signal of the pH sensor with the same chromophore.RexYFP is suitable for detecting the NAD(H) redox state in different cellular compartments.RexYFP has several advantages over existing NAD+/NADH sensors such as smallest size and optimal affinity for different compartments. Our results show that normalizing the signal of the sensor to the pH changes is a good strategy for overcoming pH-induced artifacts in imaging.
Keywords: NAD+/NADH ratio; Fluorescent probe; Redox sensor;

Engineering a switch-on peptide to ricin A chain for increasing its specificity towards HIV-infected cells by Ka-Yee Au; Rui-Rui Wang; Yuen-Ting Wong; Kam-Bo Wong; Yong-Tang Zheng; Pang-Chui Shaw (958-963).
Ricin is a type II ribosome-inactivating protein (RIP) that potently inactivates eukaryotic ribosomes by removing a specific adenine residue at the conserved α-sarcin/ricin loop of 28S ribosomal RNA (rRNA). Here, we try to increase the specificity of the enzymatically active ricin A chain (RTA) towards human immunodeficiency virus type 1 (HIV-1) by adding a loop with HIV protease recognition site to RTA.HIV-specific RTA variants were constructed by inserting a peptide with HIV-protease recognition site either internally or at the C-terminal region of wild type RTA. Cleavability of variants by viral protease was tested in vitro and in HIV-infected cells. The production of viral p24 antigen and syncytium in the presence of C-terminal variants was measured to examine the anti-HIV activities of the variants.C-terminal RTA variants were specifically cleaved by HIV-1 protease both in vitro and in HIV-infected cells. Upon proteolysis, the processed variants showed enhanced antiviral effect with low cytotoxicity towards uninfected cells.RTA variants with HIV protease recognition sequence engineered at the C-terminus were cleaved and the products mediated specific inhibitory effect towards HIV replication.Current cocktail treatment of HIV infection fails to eradicate the virus from patients. Here we illustrate the feasibility of targeting an RIP towards HIV-infected cells by incorporation of HIV protease cleavage sequence. This approach may be generalized to other RIPs and is promising in drug design for combating HIV.
Keywords: Ribosome-inactivating protein; Ricin; HIV; Protease;

Frequency decoding of calcium oscillations by Erik Smedler; Per Uhlén (964-969).
Calcium (Ca2 +) oscillations are ubiquitous signals present in all cells that provide efficient means to transmit intracellular biological information. Either spontaneously or upon receptor ligand binding, the otherwise stable cytosolic Ca2 + concentration starts to oscillate. The resulting specific oscillatory pattern is interpreted by intracellular downstream effectors that subsequently activate different cellular processes. This signal transduction can occur through frequency modulation (FM) or amplitude modulation (AM), much similar to a radio signal. The decoding of the oscillatory signal is typically performed by enzymes with multiple Ca2 + binding residues that diversely can regulate its total phosphorylation, thereby activating cellular program. To date, NFAT, NF-κB, CaMKII, MAPK and calpain have been reported to have frequency decoding properties.The basic principles and recent discoveries reporting frequency decoding of FM Ca2 + oscillations are reviewed here.A limited number of cellular frequency decoding molecules of Ca2 + oscillations have yet been reported. Interestingly, their responsiveness to Ca2 + oscillatory frequencies shows little overlap, suggesting their specific roles in cells.Frequency modulation of Ca2 + oscillations provides an efficient means to differentiate biological responses in the cell, both in health and in disease. Thus, it is crucial to identify and characterize all cellular frequency decoding molecules to understand how cells control important cell programs.
Keywords: Calcium signaling; Frequency modulation; Frequency decoding;

Tyrosine residues play an important role in heme detoxification by serum albumin by Yi Huang; Youxia Shuai; Hailing Li; Zhonghong Gao (970-976).
Serum albumin binds avidly to heme to form heme–serum albumin complex, also called methemalbumin, and this binding is thought to protect against the potentially toxic effects of heme. However, the mechanism of detoxification has not been fully elucidated.SDS-PAGE and Western blot were used to determine the efficiency of methemalbumin on catalyzing protein carbonylation and nitration. HPLC was used to test the formation of heme to protein cross-linked methemalbumin.The peroxidase activity of heme increased upon human serum albumin (HSA) binding. Methemalbumin showed higher efficiency in catalyzing tyrosine oxidation than free heme in the presence of H2O2. Methemalbumin catalyzed self-nitration and significantly promoted the nitration of tyrosine in coexistent protein, but decreased the carbonylation of coexistent protein compared with heme. The heme to protein cross-linked form of methemalbumin suggested that HSA trapped the free radical accompanied by the formation of ferryl heme. When tyrosine residues in HSA were modified by iodination, HSA lost of protection effect on protein carbonylation. The low concentration of glutathione could effectively inhibit tyrosine nitration, but had no effect on protein carbonylation.HSA protects against the toxic effect of heme by transferring the free radical to tyrosine residues in HSA, therefore protecting surrounding proteins from irreversible oxidation, rather than by direct inhibiting the peroxidase activity. The increased tyrosine radicals can be reduced by endogenic antioxidants such as GSH.This investigation indicated the important role of tyrosine residues in heme detoxification by HSA and suggested a possible novel mechanism.Display Omitted
Keywords: Human serum albumin; Heme; Peroxidative activity; Tyrosine;

Asteropsins B–D, sponge-derived knottins with potential utility as a novel scaffold for oral peptide drugs by Huayue Li; John J. Bowling; Mingzhi Su; Jongki Hong; Bong-Jin Lee; Mark T. Hamann; Jee H. Jung (977-984).
Known linear knottins are unsuitable as scaffolds for oral peptide drug due to their gastrointestinal instability. Herein, a new subclass of knottin peptides from Porifera is structurally described and characterized regarding their potential for oral peptide drug development.Asteropsins B–D (ASPB, ASPC, and ASPD) were isolated from the marine sponge Asteropus sp. The tertiary structures of ASPB and ASPC were determined by solution NMR spectroscopy and that of ASPD by homology modeling.The isolated asteropsins B–D, together with the previously reported asteropsin A (ASPA), compose a new subclass of knottins that share a highly conserved structural framework and remarkable stability against the enzymes in gastrointestinal tract (chymotrypsin, elastase, pepsin, and trypsin) and human plasma.Asteropsins can be considered as promising peptide scaffolds for oral bioavailability.The structural details of asteropsins provide essential information for the engineering of orally bioavailable peptides.
Keywords: Knottin; Marine sponge; Asteropus sp.; NMR; Solution structure; Oral peptide drug delivery;

When cells become stressed, they form stress granules (SGs) and show an increase of the molecular chaperone HSP70. The translational regulator YB-1 is a component of SGs, but it is unclear whether it contributes to the translational induction of HSP70 mRNA. Here we examined the roles of YB-1 in SG assembly and translational regulation of HSP70 mRNA under arsenite-induced stress.Using arsenite-treated NG108-15 cells, we examined whether YB-1 was included in SGs with GluR2 mRNA, a target of YB-1, and investigated the interaction of YB-1 with HSP70 mRNA and its effect on translation of the mRNA. We also investigated the distribution of these mRNAs to SGs or polysomes, and evaluated the role of YB-1 in SG assembly.Arsenite treatment reduced the translation level of GluR2 mRNA; concomitantly, YB-1-bound HSP70 mRNA was increased and its translation was induced. Sucrose gradient analysis revealed that the distribution of GluR2 mRNA was shifted from heavy-sedimenting to much lighter fractions, and also to SG-containing non-polysomal fractions. Conversely, HSP70 mRNA was shifted from the non-polysomal to polysome fractions. YB-1 depletion abrogated the arsenite-responsive activation of HSP70 synthesis, but SGs harboring both mRNAs were still assembled. The number of SGs was increased by YB-1 depletion and decreased by its overexpression.In arsenite-treated cells, YB-1 mediates the translational activation of HSP70 mRNA and also controls the number of SGs through inhibition of their assembly.Under stress conditions, YB-1 exerts simultaneous but opposing actions on the regulation of translation via SGs and polysomes.
Keywords: YB-1; Stress granule assembly; GluR2 mRNA; HSP70 mRNA; Translational regulation;

Contribution of proline to the pre-structuring tendency of transient helical secondary structure elements in intrinsically disordered proteins by Chewook Lee; Lajos Kalmar; Bin Xue; Peter Tompa; Gary W. Daughdrill; Vladimir N. Uversky; Kyou-Hoon Han (993-1003).
IDPs function without relying on three-dimensional structures. No clear rationale for such a behavior is available yet. PreSMos are transient secondary structures observed in the target-free IDPs and serve as the target-binding “active” motifs in IDPs. Prolines are frequently found in the flanking regions of PreSMos. Contribution of prolines to the conformational stability of the helical PreSMos in IDPs is investigated.MD simulations are performed for several IDP segments containing a helical PreSMo and the flanking prolines. To measure the influence of flanking-prolines on the structural content of a helical PreSMo calculations were done for wild type as well as for mutant segments with Pro → Asp, His, Lys, or Ala. The change in the helicity due to removal of a proline was measured both for the PreSMo region and for the flanking regions.The α-helical content in ~ 70% of the helical PreSMos at the early stage of simulation decreases due to replacement of an N-terminal flanking proline by other residues whereas the helix content in nearly all PreSMos increases when the same replacements occur at the C-terminal flanking region. The helix destabilizing/terminating role of the C-terminal flanking prolines is more pronounced than the helix promoting effect of the N-terminal flanking prolines.This work represents a novel example demonstrating that a proline is encoded in an IDP with a defined purpose. The helical PreSMos presage their target-bound conformations. As they most likely mediate IDP-target binding via conformational selection their helical content can be an important feature for IDP function.
Keywords: Intrinsically disordered protein (IDP); PreSMo (Pre-Structured Motif); Flanking proline; Molecular dynamics simulation;

Autocrine GM-CSF transcription in the leukemic progenitor cell line KG1a is mediated by the transcription factor ETS1 and is negatively regulated through SECTM1 mediated ligation of CD7 by Christina Bade-Döding; Wiebke Göttmann; Anja Baigger; Matthew Farren; Kelvin P. Lee; Rainer Blasczyk; Trevor Huyton (1004-1013).
CD7 expression is found on ~ 30% of acute myeloblastic leukemias (AML). The leukemic progenitor cell line KG1a (CD7 +) constitutively expresses GM-CSF while the parental KG1 (CD7-) cell line does not. This study focuses on the molecular basis of CD7 mediated GM-CSF regulation.KG1a cells were treated with recombinant SECTM1-Fc protein, the PI3K kinase inhibitors wortmannin, LY292004, or PI4K activator spermine. Stable KG1-CD7 +, KG1a-shCD7, KG1a-shETS1 as well as KG1a-GFP, KG1a-PKCβII-GFP cell lines were generated and the levels of CD7, GM-CSF and ETS-1 mRNA and protein were compared by real-time-PCR, western blotting, flow cytometry and ELISA.SECTM1 is expressed in Human Bone Marrow Endothelial Cells (HBMEC) and its expression can be upregulated by both IFN-γ. KG1a cells demonstrated high expression levels of CD7 and ETS-1 allowing a constitutative signaling through the PI3K/Atk pathway to promote GM-CSF expression, while KG1 cells with low expression of CD7 and ETS-1 showed low GM-CSF expression. On KG1a cells GM-CSF expression could be negatively regulated by PI3K inhibitors or by recombinant SECTM1-Fc. Overexpression of CD7 in KG1 cells was insufficient to promote GM-CSF expression, while silencing of CD7 or ETS-1 resulted in reduced GM-CSF expression levels. Differentiation capable KG1a cells overexpressing PKCβII illustrated complete loss of CD7, but maintained normal levels of both ETS-1 and GM-CSF expression.These findings add an additional layer to the previously described autocrine/paracrine signaling between leukemic progenitor cells and the bone marrow microenvironment and highlight a role for SECTM1 in both normal and malignant hematopoiesis.This work shows that SECTM1 secreted from bone marrow stromal cells may interact with CD7 to influence GM-CSF expression in leukemic cells.
Keywords: CD7; SECTM1; GM-CSF; ETS1; Hematopoietic progenitors; Acute myeloid leukemia;

DNA damage dependent activation of checkpoint kinase-1 and mitogen-activated protein kinase-p38 are required in malabaricone C-induced mitochondrial cell death by Mrityunjay Tyagi; Rahul Bhattacharyya; Ajay Kumar Bauri; Birija Sankar Patro; Subrata Chattopadhyay (1014-1027).
Given that lung cancer is the second leading cause of cancer-related deaths with low survival rates, the project was aimed to formulate an efficient drug with minimum side effects, and rationalize its action mechanistically.Mitochondria deficient cells, shRNA-mediated BCL2 and ATM depleted cells and pharmacological inhibition of DNA-damage response proteins were employed to explore the signaling mechanism governed between nucleus and mitochondria in response to mal C.Mal C decreased cell viability in three lung carcinoma cells, associated with DNA damage, p38-MAPK activation, imbalance in BAX/BCL2 expression, mitochondrial dysfunction and cytochrome-c release. Mitochondria depletion and p38-MAPK inhibition made A549 cells extremely resistant, but BCL2 knock-down partially sensitized the cells to mal C treatment. The mal C-induced apoptosis in A549 cells was initiated by DNA single strand breaks that led to double strand breaks (DSBs). DSB generation paralleled the induction of ATM- and ATR-mediated CHK1 phosphorylation. ATM silencing and ATR inhibition partially attenuated the mal C-induced p38-MAPK activation, CHK1 phosphorylation and apoptosis, which were completely suppressed by CHK1 inhibition.Mal C activates the ATM-CHK1-p38 MAPK cascade to cause mitochondrial cell death in lung carcinoma cells.Given that mal C has appreciable natural abundance and is non-toxic to mice, further in vivo evaluation would help in establishing its anti-cancer property.
Keywords: ATM/ATR; DNA strand break; Lung cancer cell; Malabaricone C; MAPK; Mitochondrial dysfunction;

Atomic Force Microscopy and pharmacology: From microbiology to cancerology by Flavien Pillet; Louise Chopinet; Cécile Formosa; Étienne Dague (1028-1050).
Atomic Force Microscopy (AFM) has been extensively used to study biological samples. Researchers take advantage of its ability to image living samples to increase our fundamental knowledge (biophysical properties/biochemical behavior) on living cell surface properties, at the nano-scale.AFM, in the imaging modes, can probe cells morphological modifications induced by drugs. In the force spectroscopy mode, it is possible to follow the nanomechanical properties of a cell and to probe the mechanical modifications induced by drugs. AFM can be used to map single molecule distribution at the cell surface. We will focus on a collection of results aiming at evaluating the nano-scale effects of drugs, by AFM. Studies on yeast, bacteria and mammal cells will illustrate our discussion. Especially, we will show how AFM can help in getting a better understanding of drug mechanism of action.This review demonstrates that AFM is a versatile tool, useful in pharmacology. In microbiology, it has been used to study the drugs fighting Candida albicans or Pseudomonas aeruginosa. The major conclusions are a better understanding of the microbes' cell wall and of the drugs mechanism of action. In cancerology, AFM has been used to explore the effects of cytotoxic drugs or as an innovative diagnostic technology. AFM has provided original results on cultured cells, cells extracted from patient and directly on patient biopsies.This review enhances the interest of AFM technologies for pharmacology. The applications reviewed range from microbiology to cancerology.
Keywords: Atomic Force Microscopy; Pharmacology; Yeast; Bacterium; Cancer;

Syntheses and characterization of non-bisphosphonate quinoline derivatives as new FPPS inhibitors by Jinggong Liu; Weilin Liu; Hu Ge; Jinbo Gao; Qingqing He; Lijuan Su; Jun Xu; Lian-quan Gu; Zhi-shu Huang; Ding Li (1051-1062).
Farnesyl pyrophosphate synthase (FPPS) is a key regulatory enzyme in the biosynthesis of cholesterol and in the post-translational modification of signaling proteins. It has been reported that non-bisphosphonate FPPS inhibitors targeting its allosteric binding pocket are potentially important for the development of promising anti-cancer drugs.The following methods were used: organic syntheses of non-bisphosphonate quinoline derivatives, enzyme inhibition studies, fluorescence titration assays, synergistic effect studies of quinoline derivatives with zoledronate, ITC studies for the binding of FPPS with quinoline derivatives, NMR-based HAP binding assays, molecular modeling studies, fluorescence imaging assay and MTT assays.We report our syntheses of a series of quinoline derivatives as new FPPS inhibitors possibly targeting the allosteric site of the enzyme. Compound 6b showed potent inhibition to FPPS without significant hydroxyapatite binding affinity. The compound showed synergistic inhibitory effect with active-site inhibitor zoledronate. ITC experiment confirmed the good binding effect of compound 6b to FPPS, and further indicated the binding ratio of 1:1. Molecular modeling studies showed that 6b could possibly bind to the allosteric binding pocket of the enzyme. The fluorescence microscopy indicated that these compounds could get into cancer cells.Our results showed that quinoline derivative 6b could become a new lead compound for further optimization for cancer treatment.The traditional FPPS active-site inhibitors bisphosphonates show poor membrane permeability to tumor cells, due to their strong polarity. The development of new non-bisphosphonate FPPS inhibitors with good cell membrane permeability is potentially important.
Keywords: Quinoline derivative; Bisphosphonate; Farnesyl pyrophosphate synthase; Allosteric site; Cancer;

Evolutionary conservation of long non-coding RNAs; sequence, structure, function by Per Johnsson; Leonard Lipovich; Dan Grandér; Kevin V. Morris (1063-1071).
Recent advances in genomewide studies have revealed the abundance of long non-coding RNAs (lncRNAs) in mammalian transcriptomes. The ENCODE Consortium has elucidated the prevalence of human lncRNA genes, which are as numerous as protein-coding genes. Surprisingly, many lncRNAs do not show the same pattern of high interspecies conservation as protein-coding genes. The absence of functional studies and the frequent lack of sequence conservation therefore make functional interpretation of these newly discovered transcripts challenging. Many investigators have suggested the presence and importance of secondary structural elements within lncRNAs, but mammalian lncRNA secondary structure remains poorly understood. It is intriguing to speculate that in this group of genes, RNA secondary structures might be preserved throughout evolution and that this might explain the lack of sequence conservation among many lncRNAs.Here, we review the extent of interspecies conservation among different lncRNAs, with a focus on a subset of lncRNAs that have been functionally investigated. The function of lncRNAs is widespread and we investigate whether different forms of functionalities may be conserved.Lack of conservation does not imbue a lack of function. We highlight several examples of lncRNAs where RNA structure appears to be the main functional unit and evolutionary constraint. We survey existing genomewide studies of mammalian lncRNA conservation and summarize their limitations. We further review specific human lncRNAs which lack evolutionary conservation beyond primates but have proven to be both functional and therapeutically relevant.Pioneering studies highlight a role in lncRNAs for secondary structures, and possibly the presence of functional “modules”, which are interspersed with longer and less conserved stretches of nucleotide sequences. Taken together, high-throughput analysis of conservation and functional composition of the still-mysterious lncRNA genes is only now becoming feasible.
Keywords: Long non-coding RNA; Antisense RNA; Evolution; Secondary structure; Polypurines; Epigenetic;

The special geometry of the steroid skeleton causes that bile acid anions, in contrast to aliphatic amphiphiles, form micelles with a small aggregation number.The number-average aggregation numbers ( n ¯ ) are determined using Moroi–Matsuoka–Sugioka thermodynamic method. Also, for analysed bile acid sodium salts functions between spin–lattice relaxation time (T 1) and concentration of monomers ( c B A − ) are determined.For 7-oxodeoxicholic (7-ODC) acid and hyodeoxicholic acid (HD) monomers, curve T 1 = f c BA − contains two inflexion points. Mentioned monomers and cholic acid anion (C) are influential observations in relation to a line of linear regression between n ¯ and parameter of monomer hydrophobicity (lnk, retention capacity from RPHPLC). This suggests that, in micelles of bile acid anions: 7-ODC, HD and C, beside main, hydrophobic interactions, hydrogen bonds are also possible between building units.The increase in the number of oxo groups in the molecule is accompanied with a decrease in the hydrophobicity of the convex side of the steroid skeleton of the bile acid anion, resulting in a lower aggregation number. Obtained results indicate that C12 and C7 α-axial OH and oxo groups on the same C atoms of the investigated bile acid molecules have different spatial environment, which is confirmed by conformational analysis.Deviation from the linear model: number-average aggregation numbers with hydrophobicity of monomers, suggests the existence of additional, intermolecular interactions beside hydrophobic in micelles.Display Omitted
Keywords: micelle of bile salt; aggregation number; steroid skeleton; conformational analysis; spin–lattice relaxation time;

17α-ethinyl-estradiol (17α-EE), a synthetic estrogen is the world's most widely and commonly used orally bioactive estrogen. Currently, 17α-EE is in use in all formulations of contraceptive pills and is implicated in the complication of breast cancer. Receptor for advanced glycation end products (RAGE) is a cell surface immunoglobulin class of molecule. RAGE is involved in the complication of various cancers.This study indicates that treatment of MCF-7 breast cancer cells with 17α-EE enhances the expression of estrogen receptor related receptor gamma (ERRγ), followed by enhanced level of oxidative stress and subsequent activation of the transcription factor, nuclear factor kappa-B (NF-кB), leading to increase in RAGE expression. RAGE thus expressed by 17α-EE treatment causes further enhancement of the oxidative stress which, in turn, activates expression of cell cycle protein cyclin D1 and subsequent induction of MCF-7 breast cancer cell proliferation. RAGE also enhanced phosphorylation of prosurvival protein AKT and increased expression of Bcl2, an antiapoptotic protein.In MCF-7 breast cancer cells, 17α-EE-ERRγ interaction induces the expression of RAGE, which in turn, enhances the number of MCF-7 breast cancer cells through a multiprong action on the divergent molecules like cyclin D1, AKT and Bcl2.This is the first report which explains the intermediate role of ERRγ in the 17α-EE dependent RAGE expression in MCF-7 breast cancer cells. This report for the first time explains that RAGE is important not only for MCF-7 breast cancer cell proliferation but also for its survival and anti-apoptotic activities.
Keywords: MCF-7 breast cancer cells proliferation and survival; 17-alpha-ethinyl estradiol; Estrogen receptor related receptor gamma; Receptor for advanced glycation product; Reactive oxygen species;

Localized radiotherapy is long known to cause damages to not only targeted but also non-targeted cells, the so-called bystander (BS) effect. Recently, BS effect was demonstrated in response to chemotherapy. To get further insight into the mechanism of chemotherapy-induced BS effect in vivo, we investigated the response of normal tissues and untreated BS melanomas, at distance from localized chemotherapy-treated melanomas.B16 melanoma cells were inoculated sc in one flank, in mice. Chemotherapy was administered intratumorally. After 3 weeks, untreated melanomas were implanted into the other flank. Tumors were analyzed morphologically, and using metabolomics and transcriptomics.Locally-treated melanomas showed growth inhibition and pleiotropic metabolic and transcriptional alterations. Tumors recovered slow proliferation while exhibiting prominent oxidative stress response (decreased glutathione level, and increased expression of genes including Mt1, Gpx3, Sod3, and Hmox1). Plasma contained increased levels of oxidative stress products. However, liver and soleus muscle displayed unaltered morphological characteristics. In contrast, untreated BS melanomas induced from naive B16 cells showed reduced growth, marked oxidative stress response (decreased glutathione level, and increased expression of genes including Sod2, Gpx1 and Gsr), and ras oncogene expression alterations. Furthermore, metabolomics and transcriptomics enabled to estimate the proportion of cells undergoing the BS effect within treated tumors.Treatment of tumors with chemotherapy induces BS effects, underpinned by oxidative stress, in abnormal proliferating tissues in vivo, not in normal tissue, that significantly contribute to overall tumor response. General significance BS effect significantly contributes to response to chemotherapy, and may be exploited to improve overall response to cancer treatment.
Keywords: Chemotherapy; Bystander tumor; Metabolomics; Transcriptomics; Oxidative stress;

A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides by Takahiko Matsushita; Wataru Takada; Kota Igarashi; Kentaro Naruchi; Risho Miyoshi; Fayna Garcia-Martin; Maho Amano; Hiroshi Hinou; Shin-Ichiro Nishimura (1105-1116).
Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear.A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies.Selective imine-coupling between aminooxy-functionalized methacrylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Acα2,3Galβ1,3GalNAcα1→) at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUC1 monoclonal antibodies such as VU-3D1, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUC1 tandem repeats.We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption.The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes.
Keywords: MUC1 glycopeptide; Microarray; Epitope mapping; Non-specific protein adsorption; Autoantibody;

Der p 1 is the primary activator of Der p 3, Der p 6 and Der p 9 the proteolytic allergens produced by the house dust mite Dermatophagoides pteronyssinus by Julie Herman; Nicolas Thelen; Nicolas Smargiasso; Anne-Catherine Mailleux; André Luxen; Marie Cloes; Edwin De Pauw; Andy Chevigné; Moreno Galleni; Marie-Eve Dumez (1117-1124).
The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation cascade of their corresponding precursor forms and particularly to highlight the interconnection between proteases during this cascade.The cleavage of the four peptides corresponding to the mite zymogen activation sites was studied on the basis of the Förster Resonance Energy Transfer method. The proDer p 6 zymogen was then produced in Pichia pastoris to elucidate its activation mechanism by mite proteases, especially Der p 1. The role of the propeptide in the inhibition of the enzymatic activity of Der p 6 was also examined. Finally, the Der p 1 and Der p 6 proteases were localised via immunolocalisation in D. pteronyssinus.All peptides were specifically cleaved by Der p 1, such as proDer p 6. The propeptide of proDer p 6 inhibited the proteolytic activity of Der p 6, but once cleaved, it was degraded by the protease. The Der p 1 and Der p 6 proteases were both localised to the midgut of the mite.Der p 1 in either its recombinant form or in the natural context of house dust mite extracts specifically cleaves all zymogens, thus establishing its role as a major activator of both mite cysteine and serine proteases.This finding suggests that Der p 1 may be valuable target against mites.
Keywords: Mite allergen; Protease; Digestion; Dermatophagoides pteronyssinus;

Cellular plasticity and complex functional requirements of the periodontal ligament (PDL) assume a local stem cell (SC) niche to maintain tissue homeostasis and repair. Here, pathological alterations caused by inflammatory insults might impact the regenerative capacities of these cells. As bone homeostasis is fundamentally controlled by Wnt-mediated signals, it was the aim of this study to characterize the SC-like capacities of cells derived from PDL and to investigate their involvement in bone pathophysiology especially regarding the canonical Wnt pathway.PDLSCs were investigated for their SC characteristics via analysis of cell surface marker expression, colony forming unit efficiency, proliferation, osteogenic differentiation and adipogenic differentiation, and compared to bone marrow derived mesenchymal SCs (BMMSCs). To determine the impact of both inflammation and the canonical Wnt pathway on osteogenic differentiation, cells were challenged with TNF-α, maintained with or without Wnt3a or DKK-1 under osteogenic induction conditions and investigated for p-IκBα, p-NF-κB, p-Akt, β-catenin, p-GSK-3β, ALP and Runx2.PDLSCs exhibit weaker adipogenic and osteogenic differentiation capacities compared to BMMSCs. TNF-α inhibited osteogenic differentiation of PDLSCs more than BMMSCs mainly through regulating canonical Wnt pathway. Blocking the canonical Wnt pathway by DKK-1 reconstituted osteogenic differentiation of PDLSCs under inflammatory conditions, whereas activation by Wnt3a increased osteogenic differentiation of BMMSCs.Our results suggest a diverse regulation of the inhibitory effect of TNF-α in BMMSCs and PDLSCs via canonical Wnt pathway modulation.These findings provide novel insights on PDLSC SC-like capacities and their involvement in bone pathophysiology under the impact of the canonical Wnt pathway.
Keywords: Bone marrow derived mesenchymal stem cell; Canonical Wnt signaling; Inflammation; Osteogenesis; Periodontal ligament stem cell;

Powerful tumor cell growth-inhibiting activity of a synthetic derivative of atractyligenin: Involvement of PI3K/Akt pathway and thioredoxin system by Roberta Cotugno; Dario Gallotta; Fabrizio Dal Piaz; Ivana Apicella; Sandro De Falco; Sergio Rosselli; Maurizio Bruno; Maria Antonietta Belisario (1135-1144).
The semi-synthetic ent-kaurane 15-ketoatractyligenin methyl ester (SC2017) has been previously reported to possess high antiproliferative activity against several solid tumor-derived cell lines. Our study was aimed at investigating SC2017 tumor growth-inhibiting activity and the underlying mechanisms in Jurkat cells (T-cell leukemia) and xenograft tumor models.Cell viability was evaluated by MTT assay. Cell cycle progression, reactive oxygen species (ROS) elevation and apoptotic hallmarks were monitored by flow cytometry. Inhibition of thioredoxin reductase (TrxR) by biochemical assays. Levels and/or activation status of signaling proteins were assessed by western blotting. Xenograft tumors were generated with HCT 116 colon carcinoma cells.SC2017 displayed cell growth-inhibiting activity against Jurkat cells (half maximal inhibitory concentration values (IC50) < 2 μM), but low cell-killing potential in human peripheral blood mononuclear cells (PBMC). The primary response of Jurkat cells to SC2017 was an arrest in G2 phase followed by caspase-dependent apoptosis. Inhibition of PI3K/Akt pathway and TrxR activity by SC2017 was demonstrated by biochemical and pharmacological approaches. At least, SC2017 was found to inhibit xenograft tumor growth.Our results demonstrate that SC2017 inhibits tumor cell growth in in vitro and in vivo models, but displays moderate toxicity against PBMC. We also demonstrate that SC2017 promotes caspase-dependent apoptosis in Jurkat cells by affecting Akt activation status and TrxR functionality.Our observations suggest the semi-synthetic ent-kaurane SC2017 as a promising chemotherapeutic compound. SC2017 has, indeed, shown to possess tumor growth inhibiting activity and be able to counteract PI3K/Akt and Trx system survival signaling.
Keywords: Ent-kaurane; Apoptosis; Cell cycle; PI3K/Akt; Thioredoxin system; HCT 116 xenograft;

Lipoic acid prevents fructose-induced changes in liver carbohydrate metabolism: Role of oxidative stress by María C. Castro; Flavio Francini; Juan J. Gagliardino; María L. Massa (1145-1151).
Fructose administration rapidly induces oxidative stress that triggers compensatory hepatic metabolic changes. We evaluated the effect of an antioxidant, R/S-α-lipoic acid on fructose-induced oxidative stress and carbohydrate metabolism changes.Wistar rats were fed a standard commercial diet, the same diet plus 10% fructose in drinking water, or injected with R/S-α-lipoic acid (35 mg/kg, i.p.) (control + L and fructose + L). Three weeks thereafter, blood samples were drawn to measure glucose, triglycerides, insulin, and the homeostasis model assessment-insulin resistance (HOMA-IR) and Matsuda indices. In the liver, we measured gene expression, protein content and activity of several enzymes, and metabolite concentration.Comparable body weight changes and calorie intake were recorded in all groups after the treatments. Fructose fed rats had hyperinsulinemia, hypertriglyceridemia, higher HOMA-IR and lower Matsuda indices compared to control animals. Fructose fed rats showed increased fructokinase gene expression, protein content and activity, glucokinase and glucose-6-phosphatase gene expression and activity, glycogen storage, glucose-6-phosphate dehydrogenase mRNA and enzyme activity, NAD(P)H oxidase subunits (gp91 phox and p22 phox ) gene expression and protein concentration and phosphofructokinase-2 protein content than control rats. All these changes were prevented by R/S-α-lipoic acid co-administration.Fructose induces hepatic metabolic changes that presumably begin with increased fructose phosphorylation by fructokinase, followed by adaptive changes that attempt to switch the substrate flow from mitochondrial metabolism to energy storage. These changes can be effectively prevented by R/S-α-lipoic acid co-administration.Control of oxidative stress could be a useful strategy to prevent the transition from impaired glucose tolerance to type 2 diabetes.
Keywords: Fructokinase; Glucokinase; Glucose metabolism; Glycoxidative stress; R/S-α-lipoic acid;

Albumin fusion renders thioredoxin an effective anti-oxidative and anti-inflammatory agent for preventing cisplatin-induced nephrotoxicity by Azusa Kodama; Hiroshi Watanabe; Ryota Tanaka; Masumi Kondo; Victor Tuan Giam Chuang; Qiong Wu; Masayuki Endo; Yu Ishima; Masafumi Fukagawa; Masaki Otagiri; Toru Maruyama (1152-1162).
A strategy for preventing cisplatin nephrotoxicity due to enhanced oxidative stress and inflammatory response is highly desirable. Thioredoxin-1 (Trx), an endogenous redox-active protein, has a short retention time in the blood. A long acting form of Trx, human serum albumin-Trx (HSA-Trx), was produced by recombinant HSA fusion and its effectiveness in preventing cisplatin nephrotoxicity was examined.HSA-Trx was prepared in Pichia expression system. Cisplatin-induced nephropathy mouse model was established by a single administration of cisplatin.Compared to saline, Trx or N-acetylcysteine, an intravenous administration of HSA-Trx attenuated the cisplatin-induced elevation in serum creatinine, blood urea nitrogen and urinary N-acetyl-β-d-glucosaminidase along with the decrease in creatinine clearance. HSA-Trx caused a substantial reduction in the histological features of renal tubular injuries and the apoptosis-positive tubular cells. Changes in superoxide, 8-OHdG, glutathione and nitrotyrosine levels indicated that HSA-Trx significantly suppressed renal oxidative stress. HSA-Trx also suppressed the elevation of TNF-α, IL-1β and IL-6. Administered fluorescein isothiocyanate-labeled HSA-Trx was found partially localized in the proximal tubular cells whereas majority remained in the blood circulation. Specific cellular uptake and the scavenging of intracellular reactive oxygen species by HSA-Trx were observed in HK-2 cells.HSA-Trx could be a novel and effective approach for preventing cisplatin nephrotoxicity due to its prolonged anti-oxidative and anti-inflammatory action not only in extracellular compartment but also inside the proximal tubular cell.We report the renoprotective effect of HSA-Trx against cisplatin nephrotoxicity. This work would enhance developing therapeutics against acute kidney injuries including cisplatin nephrotoxicity.
Keywords: Acute kidney injury; Cisplatin nephrotoxicity; Oxidative stress; Inflammation; Thioredoxin; Fusion protein;

The nature of the polyamine–DNA interactions at a molecular level is not clearly understood.In order to shed light on the binding preferences of polyamine with nucleic acids, the NMR solution structure of the DNA duplex containing covalently bound spermine was determined.The structure of 4-N-[4,9,13-triazatridecan-1-yl]-2′-deoxycytidine (dCSp) modified duplex was compared to the structure of the reference duplex. Both duplexes are regular right-handed helices with all attributes of the B-DNA form. The spermine chain which is located in a major groove and points toward the 3′ end of the modified strand does not perturb the DNA structure.In our study the charged polyamine alkyl chain was found to interact with the DNA surface. In the majority of converged structures we identified the presumed hydrogen bonding interactions between O6 and N7 atoms of G4 and the first internal –NH2+− amino group. Additional interaction was found between the second internal –NH2+− amino group and the oxygen atom of the phosphate of C3 residue.The knowledge of the location and nature of a structure-specific binding site for spermine in DNA should be valuable in understanding gene expression and in the design of new therapeutic drugs.Display Omitted
Keywords: Spermine; Polyamine; Nucleic acid structure; Polyaminooligonucleotide; Nuclear magnetic resonance spectroscopy;

Amorphous silica particles with the primary dimensions of a few tens of nm, have been widely applied as additives in various fields including medicine and food. Especially, they have been widely applied in powders for making tablets and to coat tablets. However, their behavior and biological effects in the gastrointestinal tracts associated with oral administration remains unknown.Amorphous silica particles with diameters of 50, 100, and 200 nm were incubated in the fasted-state and fed-state simulated gastric and intestinal fluids. The sizes, intracellular transport into Caco-2 cells (model cells for intestinal absorption), the Caco-2 monolayer membrane permeability, and the cytotoxicity against Caco-2 cells were then evaluated for the silica particles.Silica particles agglomerated in fed-state simultaneous intestinal fluids. The agglomeration and increased particles size inhibited the particles' absorption into the Caco-2 cells or particles' transport through the Caco-2 cells. The in vitro cytotoxicity of silica particles was not observed when the average size was larger than 100 nm, independent of the fluid and the concentration.Our study indicated the effect of diet on the agglomeration of silica particles. The sizes of silica particles affected the particles' absorption into or transport through the Caco-2 cells, and cytotoxicity in vitro, depending on the various biological fluids.The findings obtained from our study may offer valuable information to evaluate the behavior of silica particles in the gastrointestinal tracts or safety of medicines or foods containing these materials as additives.Display Omitted
Keywords: Nanomaterial; Silica particle; In vitro model; Simulated gastrointestinal fluid;

Heat shock transcription factor HSF1 regulates the expression of the Huntingtin-interacting protein HYPK by Hiroshi Sakurai; Maki Sawai; Yukio Ishikawa; Azumi Ota; Ei Kawahara (1181-1187).
The Huntingtin-interacting protein HYPK possesses chaperone-like activity. We hypothesized that the expression of HYPK could be regulated by heat shock factor HSF1, a transcriptional regulator of chaperone genes.HYPK expression in HeLa cells was assessed by RT-PCR and Western blot analysis. In vivo binding of HSF1 to the HYPK promoter was analyzed by chromatin immunoprecipitation assays. The requirement for HYPK in heat-shocked cells was examined using HYPK-knockdown cells.Levels of HYPK mRNA were slightly increased by heat treatment; however, the levels decreased in HSF1-silenced cells. The HYPK promoter was bound by HSF1 in a heat-inducible manner; however, its core promoter activity was notably suppressed upon heat shock. When cells were exposed to heat shock, silencing HYPK caused a decrease in cell viability. HYPK is a novel target gene of HSF1. HSF1 maintains HYPK expression in heat-shocked cells.The maintenance of HYPK expression by HSF1 is necessary for the survival of cells under thermal stress conditions.
Keywords: Chaperone; Gene expression; HSF1; HYPK; Stress response;

Platelets do not express the oxidized or reduced forms of tissue factor by Beth A. Bouchard; Matthew T. Gissel; Matthew F. Whelihan; Kenneth G. Mann; Saulius Butenas (1188-1193).
Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms.Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results.Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography.Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF.
Keywords: Hemostasis; Factor VIIa; Platelet; Tissue factor; Flow cytometry;

Allosteric modulators of human A2B adenosine receptor by Maria Letizia Trincavelli; Chiara Giacomelli; Simona Daniele; Sabrina Taliani; Barbara Cosimelli; Sonia Laneri; Elda Severi; Elisabetta Barresi; Isabella Pugliesi; Giovanni Greco; Ettore Novellino; Federico Da Settimo; Claudia Martini (1194-1203).
Among adenosine receptors (ARs) the A2B subtype exhibits low affinity for the endogenous agonist compared with the A1, A2A, and A3 subtypes and is therefore activated when concentrations of adenosine increase to a large extent following tissue damages (e.g. ischemia, inflammation). For this reason, A2B AR represents an important pharmacological target.We evaluated seven 1-benzyl-3-ketoindole derivatives (79) for their ability to act as positive or negative allosteric modulators of human A2B AR through binding and functional assays using CHO cells expressing human A1, A2A, A2B, and A3 ARs.The investigated compounds behaved as specific positive or negative allosteric modulators of human A2B AR depending on small differences in their structures. The positive allosteric modulators 7a,b and 8a increased agonist efficacy without any effect on agonist potency. The negative allosteric modulators 8b,c and 9a,b reduced agonist potency and efficacy.A number of 1-benzyl-3-ketoindole derivatives were pharmacologically characterized as selective positive (7a,b) or negative (8c, 9a,b) allosteric modulators of human A2B AR.The 1-benzyl-3-ketoindole derivatives 79 acting as positive or negative allosteric modulators of human A2B AR represent new pharmacological tools useful for the development of therapeutic agents to treat pathological conditions related to an altered functionality of A2B AR.
Keywords: 1-Benzyl-3-ketoindole derivatives; A2B adenosine receptor; Positive allosteric modulators; Negative allosteric modulators; GPCR allosteric modulators; Ligand–receptor interaction;

Calcium electroporation in three cell lines: a comparison of bleomycin and calcium, calcium compounds, and pulsing conditions by Stine Krog Frandsen; Hanne Gissel; Pernille Hojman; Jens Eriksen; Julie Gehl (1204-1208).
Electroporation with calcium (calcium electroporation) can induce ATP depletion-associated cellular death. In the clinical setting, the cytotoxic drug bleomycin is currently used with electroporation (electrochemotherapy) for palliative treatment of tumors. Calcium electroporation offers several advantages over standard treatment options: calcium is inexpensive and may readily be applied without special precautions, as is the case with cytostatic drugs. Therefore, details on the use of calcium electroporation are essential for carrying out clinical trials comparing calcium electroporation and electrochemotherapy.The effects of calcium electroporation and bleomycin electroporation (alone or in combination) were compared in three different cell lines (DC-3F, transformed Chinese hamster lung fibroblast; K-562, human leukemia; and murine Lewis Lung Carcinoma). Furthermore, the effects of electrical pulsing parameters and calcium compound on treatment efficacy were determined.Electroporation with either calcium or bleomycin significantly reduced cell survival (p < 0.0001), without evidence of a synergistic effect. Cellular death following calcium or bleomycin treatment occurred at similar applied voltages, suggesting that similar parameters should be applied. At equimolar concentrations, calcium chloride and calcium glubionate resulted in comparable decreases in cell viability.Calcium electroporation and bleomycin electroporation significantly reduce cell survival at similar applied voltage parameters. The effect of calcium electroporation is independent of calcium compound.This study strongly supports the use of calcium electroporation as a potential cancer therapy and the results may aid in future clinical trials.
Keywords: Calcium electroporation; Electrochemotherapy; Bleomycin; In vitro; Cancer;

Effect of TNFα on osteoblastogenesis from mesenchymal stem cells by Shigeru Kotake; Yuki Nanke (1209-1213).
Bone destruction and osteoporosis are accelerated in chronic inflammatory diseases, such as rheumatoid arthritis (RA) and periodontitis, in which many studies have shown the proinflammatory cytokines, especially TNFα, play an important role; TNFα causes osteoclast-induced bone destruction as well as the inhibition of osteoblastogenesis.Here we review our current understanding of the mechanism of the effect of TNFα on osteoblastogenesis from mesenchymal stem cells (MSCs). We also highlight the function of MSC in the pathogenesis of autoimmune diseases.Many studies have revealed that TNFα inhibits osteoblastogenesis through several mechanisms. On the other hand, it has been also reported that TNFα promotes osteoblastogenesis. These discrepancies may depend on the cellular types, the model animals, and the timing and duration of TNFα administration.A full understanding of the role and function of TNFα on osteoblastogenesis from MSC may lead to targeted new therapies for chronic inflammation diseases, such as RA and periodontitis.
Keywords: Osteoblastogenesis; TNF-alpha; Wnt signaling pathway;

A thermodynamic assay to test pharmacological chaperones for Fabry disease by Giuseppina Andreotti; Valentina Citro; Antonella Correra; Maria Vittoria Cubellis (1214-1224).
The majority of the disease-causing mutations affect protein stability, but not functional sites and are amenable, in principle, to be treated with pharmacological chaperones. These drugs enhance the thermodynamic stability of their targets. Fabry disease, a disorder caused by mutations in the gene encoding lysosomal alpha-galactosidase, represents an excellent model system to develop experimental protocols to test the efficiency of such drugs.The stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced unfolding followed by limited proteolysis and Western blotting.We measured the concentration of urea needed to obtain half-maximal unfolding because this parameter represents an objective indicator of protein stability.Urea-induced unfolding is a versatile technique that can be adapted to cell extracts containing tiny amounts of wild-type or mutant proteins. It allows testing of protein stability as a function of pH, in the presence or in the absence of drugs. Results are not influenced by the method used to express the protein in transfected cells.Scarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases. This is particularly true for pharmacological chaperones that must be tested on each mutation associated with a given disease. Diverse in vitro tests are needed. We used a method based on chemically induced unfolding as a tool to assess whether a particular Fabry mutation is responsive to pharmacological chaperones, but, by no means is our protocol limited to this disease.Display Omitted
Keywords: Pharmacological chaperone; Lysosomal storage disorder; Urea-induced unfolding; Limited proteolysis; Cell lysate;