BBA - General Subjects (v.1850, #6)

Investigation of the heparin–thrombin interaction by dynamic force spectroscopy by Congzhou Wang; Yingzi Jin; Umesh R. Desai; Vamsi K. Yadavalli (1099-1106).
The interaction between heparin and thrombin is a vital step in the blood (anti)coagulation process. Unraveling the molecular basis of the interactions is therefore extremely important in understanding the mechanisms of this complex biological process.In this study, we use a combination of an efficient thiolation chemistry of heparin, a self-assembled monolayer-based single molecule platform, and a dynamic force spectroscopy to provide new insights into the heparin–thrombin interaction from an energy viewpoint at the molecular scale.Well-separated single molecules of heparin covalently attached to mixed self-assembled monolayers are demonstrated, whereby interaction forces with thrombin can be measured via atomic force microscopy-based spectroscopy. Further these interactions are studied at different loading rates and salt concentrations to directly obtain kinetic parameters.An increase in the loading rate shows a higher interaction force between the heparin and thrombin, which can be directly linked to the kinetic dissociation rate constant (koff ). The stability of the heparin/thrombin complex decreased with increasing NaCl concentration such that the off-rate was found to be driven primarily by non-ionic forces.These results contribute to understanding the role of specific and nonspecific forces that drive heparin–thrombin interactions under applied force or flow conditions.
Keywords: Heparin; Thrombin; Self-assembled monolayers; Atomic force microscopy; Force spectroscopy;

Naturally occurring single mutants, I56T, F57I, W64R and D67H of lysozyme in human, have been known to form abnormal protein aggregates (amyloid fibrils) and to accumulate in several organs, including the liver, spleen and kidney, resulting in familial systemic amyloidosis. These human pathogenic lysozyme variants are considered to raise subtle conformational changes compared to the wild type.Here we examined the effects of the aberrant mutant lysozymes I56T, F57I, W64R and D67H, each of which possesses a point mutation in its molecule, on a cultured human cell line, HEK293, in which the genes were individually integrated and overexpressed.Western blot analyses showed lesser amounts of these variant proteins in the medium compared to the wild type, but they were abundant in the cell pellets, indicating that the modified lysozyme proteins were scarcely secreted into the medium but were retained in the cells. Immunocytochemistry revealed that these proteins resided in restricted regions which were stained by an endoplasmic reticulum (ER) marker. Moreover, the overexpression of the mutant lysozymes were accompanied by marked increases in XBP-1s and GRP78/BiP, which are downstream agents of the IRE1α signaling pathway responding to the unfolded protein response (UPR) upon ER stress. RNAi for the mutant lysozymes' expression greatly suppressed the increases of these agents.Our results suggest that the accumulation of pathogenic lysozymes in the ER caused ER stress and the UPR response mainly via the IRE1α pathway.
Keywords: Lysozyme; Mutants; Amyloidosis; Endoplasmic reticulum; ER stress;

Membrane-associated respiratory complexes, purinosome and many intracellular soluble activities have reported to be organized in dynamic multi-component macromolecular complexes using native PAGE, 2D SDS-PAGE, electron and systematic microscopy and genome-wide GFP fusion library.In-gel staining assays, SDS-PAGE and LC–MSMS techniques were performed on cellular extracts to analyze, isolate and identify the proteins associated with glucose 6-phosphate dehydrogenase (G6PDH) and fermentative alcohol dehydrogenase (ADH) I isoform in both Kluyveromyces lactis and Saccharomyces cerevisiae yeasts.Analysis of LC–MSMS data showed that a large number of components, belonging to glycolysis, pentose phosphate, folding and stress response pathways, were associated with G6PDH and Adh1 putative complexes and that a number of these proteins were identical in either network in both yeasts. However, comparison of in-gel staining assays for hexokinase, phosphoglucoisomerase, acetaldehyde dehydrogenase, ADH and G6PDH showed that, despite their identification in these structures, functional localization of these activities varied according to growth conditions and to NAD(P)+/NAD(P)H redox ratio.Reported data show that intracellular proteins are organized in large dynamic ‘depots’ and the NAD(P)+/NAD(P)H redox balance is one of the major factors regulating the assembly and the re-assortment of components inside the different metabolic structures.The aim of this work is directed towards the comprehension of the mechanisms involved in the assembly, organization, functioning and dynamic re-assortment of cellular components according to physiological and/or pathological conditions.
Keywords: In gel-native assay; Mass spectrometry; Depots; NAD(P)+/NAD(P)H redox balance;

The oligomeric state of the human DNMT3A is functionally important and cancer cells are known to undergo changes in pH (intracellular).Light scattering, gel filtration, and fluorescence anisotropy. Also, methylation and processivity assays.Physiologically relevant changes in pH result in changes in DNMT3A oligomer composition which have dramatic consequences on DNMT3A function.The pH changes which occur within cancer cells alter the oligomeric state and function of DNMT3A which could contribute to changes in genomic DNA methylation observed in vivo.
Keywords: DNA methylation; pH; Oligomers; Processivity; Cancer; DNMT3A;

Tat PTD–endostatin: A novel anti-angiogenesis protein with ocular barrier permeability via eye-drops by Xinke Zhang; Yan Li; Yanna Cheng; Haining Tan; Zhiwei Li; Yi Qu; Guoying Mu; Fengshan Wang (1140-1149).
Endostatin, a specific inhibitor of endothelial cell proliferation and angiogenesis, has been proved to have effects on ocular neovascular diseases by intraocular injection. In order to increase its permeability to ocular barriers and make it effective on fundus oculi angiogenesis diseases via non-invasive administration (eye drops), endostatin was fused to Tat PTD via a genetic engineering method.Most of the Tat PTD– endostatin was expressed as inclusion bodies in Escherichia coli, so pure and active Tat PTD–endostatin was prepared by a series of operations, including inclusion body denaturation, refolding and chromatography. The anti-angiogenesis activity of Tat PTD–endostatin was investigated by cell proliferation experiments and chick embryo chorioallantoic membrane assay. In addition, its translocating ability and concrete entry mechanism into cells were also investigated by fluorescence microscope and flow cytometry. The penetrating ability to ocular barriers was also studied by immunohistochemistry. A mouse choroidal neovascularization model was established to investigate the pharmacodynamics of Tat PTD–endostatin.The obtained Tat PTD–endostatin had excellent anti-angiogenesis activity and was superior to Es in cellular translocating. Macropinocytosis may be the dominant route of entry of Tat PTD–endostatin into cells. Tat PTD–endostatin could cross ocular barriers and arrive at the retina after eye-drop administration. In addition, it displayed inhibitory effects on choroidal neovascularization via eye drops.Tat PTD–endostatin possessed excellent ocular penetrating ability and anti-angiogenesis effects.Tat PTD is a promising ocular delivery tool, and Tat PTD–endostatin is a potential drug for curing fundus oculi angiogenesis diseases.
Keywords: Tat PTD; Endostatin; Fusion protein; Ocular barrier; CNV;

Photocytotoxicity of a cyanine dye with two chromophores toward melanoma and normal cells by L.S. Murakami; L.P. Ferreira; J.S. Santos; R.S. da Silva; A. Nomizo; V.A. Kuz'min; I.E. Borissevitch (1150-1157).
Due to high optical absorption, triplet quantum yield and affinity to biological structures bichromophoric cyanine dyes (BCDs) can be considered promising sensitizers for application in photodynamic therapy (PDT). In this work, we report on the study of the BCD photocytotoxicity toward melanoma and normal cells in comparison with that of commercial photosensitizer Photogem®.The cytotoxic and phototoxic effects were measured by standard tests of cell viability. The drug uptake was obtained by the flow cytometry and optical absorption techniques. The BCD intracellular distribution was obtained by the fluorescence image microscopy using specific organelle markers.Both drugs demonstrated increased cytotoxicity under irradiation, while in darkness their cytotoxic effect at concentrations lower than 20 μM after 24 h of incubation did not exceed 20%. For 5 h of incubation, BCD photocytotoxicity in relation to melanoma cells reached 100% already at concentrations below 5 μM, while for normal cells the effect did not exceed 70% even for the 20 μM concentration. It is shown that BCD penetrates into the cells and is located predominantly in perinuclear cytoplasmic structures.The BCD photosensitizing characteristics appear more adequate for application in PDT than that of the actually applied commercial photosensitizer Photogem®. Higher light absorption by BCD in the near IR region and its preferential localization in mitochondria can explain its high photocytotoxicity.BCD can be considered as a new promising photosensitizer class for cancer PDT.
Keywords: Bichromophoric cyanine dye; Photocytotoxicity; Melanoma; Normal cell;

The use of conjugated polymers allows versatile interactions between cells and flexible processable materials, while providing a platform for electrical stimulation, which is particularly relevant when targeting differentiation of neural stem cells and further application for therapy or drug screening.Materials were tested for cytotoxicity following the ISO10993-5. PEDOT:PSS was cross-linked. ReNcellVM neural stem cells (NSC) were seeded in laminin coated surfaces, cultured for 4 days in the presence of EGF (20 ng/mL), FGF-2 (20 ng/mL) and B27 (20 μg/mL) and differentiated over eight additional days in the absence of those factors under 100 Hz pulsed DC electrical stimulation, 1 V with 10 ms pulses. NSC and neuron elongation aspect ratio as well as neurite length were assessed using ImageJ. Cells were immune-stained for Tuj1 and GFAP.F8T2, MEH-PPV, P3HT and cross-linked PEDOT:PSS (xPEDOT:PSS) were assessed as non-cytotoxic. L929 fibroblast population was 1.3 higher for xPEDOT:PSS than for glass control, while F8T2 presents moderate proliferation. The population of neurons (Tuj1) was 1.6 times higher with longer neurites (73 vs 108 μm) for cells cultured under electrical stimulus, with cultured NSC. Such stimulus led also to longer neurons.xPEDOT:PSS was, for the first time, used to elongate human NSC through the application of pulsed current, impacting on their differentiation towards neurons and contributing to longer neurites.The range of conductive conjugated polymers known as non-cytotoxic was expanded. xPEDOT:PSS was introduced as a stable material, easily processed from solution, to interface with biological systems, in particular NSC, without the need of in-situ polymerization.
Keywords: Conjugated conductive polymer; Cross-linked PEDOT:PSS; Electrical stimulation; Human neural stem cell; Neuron;

Anti-angiogenic activities of snake venom CRISP isolated from Echis carinatus sochureki by Shimon Lecht; Rachel A. Chiaverelli; Jonathan Gerstenhaber; Juan J. Calvete; Philip Lazarovici; Nicholas R. Casewell; Robert Harrison; Peter I. Lelkes; Cezary Marcinkiewicz (1169-1179).
Cysteine-rich secretory protein (CRISP) is present in majority of vertebrate including human. The physiological role of this protein is not characterized. We report that a CRISP isolated from Echis carinatus sochureki venom (ES-CRISP) inhibits angiogenesis.The anti-angiogenic activity of purified ES-CRISP from snake venom was investigated in vitro using endothelial cells assays such as proliferation, migration and tube formation in Matrigel, as well as in vivo in quail embryonic CAM system. The modulatory effect of ES-CRISP on the expression of major angiogenesis factors and activation of angiogenesis pathways was tested by qRT-PCR and Western blot.The amino acid sequence of ES-CRISP was found highly similar to other members of this snake venom protein family, and shares over 50% identity with human CRISP-3. ES-CRISP supported adhesion to endothelial cells, although it was also internalized into the cytoplasm in a granule-like manner. It blocked EC proliferation, migration and tube formation in Matrigel. In the embryonic quail CAM system, ES-CRISP abolished neovascularization process induced by exogenous growth factors (bFGF, vpVEGF) and by developing gliomas. CRISP modulates the expression of several factors at the mRNA level, which were characterized as regulators of angiogenesis and blocked activation of MAPK Erk1/2 induced by VEGF.ES-CRISP was characterized as a negative regulator of the angiogenesis, by direct interaction with endothelial cells.The presented work may lead to the development of novel angiostatic therapy, as well as contribute to the identification of the physiological relevance of this functionally uncharacterized protein.
Keywords: Angiogenesis; CRISP; Endothelial cells; Cell proliferation; Migration;

New proline-rich oligopeptides from the venom of African adders: Insights into the hypotensive effect of the venoms by Roberto T. Kodama; Daniela Cajado-Carvalho; Alexandre K. Kuniyoshi; Eduardo S. Kitano; Alexandre K. Tashima; Barbara F. Barna; Ana Carolina Takakura; Solange M.T. Serrano; Wilmar Dias-Da-Silva; Denise V. Tambourgi; Fernanda V. Portaro (1180-1187).
The snakes from the Bitis genus are some of the most medically important venomous snakes in sub Saharan Africa, however little is known about the composition and effects of these snake venom peptides. Considering that the victims with Bitis genus snakes have exacerbate hypotension and cardiovascular disorders, we investigated here the presence of angiotensin-converting enzyme modulators on four different species of venoms.The peptide fractions from Bitis gabonica gabonica, Bitis nasicornis, Bitis gabonica rhinoceros and Bitis arietans which showed inhibitory activity on angiotensin-converting enzyme were subjected to mass spectrometry analysis. Eight proline-rich peptides were synthetized and their potencies were evaluated in vitro and in vivo.The MS analysis resulted in over 150 sequences, out of which 32 are new proline-rich oligopeptides, and eight were selected for syntheses. For some peptides, inhibition assays showed inhibitory potentials of cleavage of angiotensin I ten times greater when compared to bradykinin. In vivo tests showed that all peptides decreased mean arterial pressure, followed by tachycardia in 6 out of 8 of the tests.We describe here some new and already known proline-rich peptides, also known as bradykinin-potentiating peptides. Four synthetic peptides indicated a preferential inhibition of angiotensin-converting enzyme C-domain. In vivo studies show that the proline-rich oligopeptides are hypotensive molecules.Although proline-rich oligopeptides are known molecules, we present here 32 new sequences that are inhibitors of the angiotensin-converting enzyme and consistent with the symptoms of the victims of Bitis spp, who display severe hypotension.
Keywords: Bitis; Venom; Proline-rich oligopeptide (PRO); Bradykinin-potentiating peptide (BPP); Angiotensin-converting enzyme (ACE); Hypotension;

Turtle anoxia tolerance: Biochemistry and gene regulation by Anastasia Krivoruchko; Kenneth B. Storey (1188-1196).
While oxygen limitation can be extremely damaging for many animals, some vertebrates have perfected anaerobic survival. Freshwater turtles belonging to the Trachemys and Chrysemys genera, for example, can survive many weeks without oxygen, and as such are commonly used as model animals for vertebrate anoxia tolerance.In the present review we discuss the recent advances made in understanding the biochemical and molecular nature of natural anoxia tolerance of freshwater turtles.Research in recent years has shown that activation of several important pathways occurs in response to anoxia in turtles, including those that function in the stress response, cell cycle arrest, inhibition of gene expression and metabolism. These likely contribute to anoxia tolerance in turtle tissues by minimizing cell damage in response to anoxia, as well as facilitating metabolic rate depression.The research discussed in the present review contributes to the understanding of how freshwater turtles can survive without oxygen for prolonged periods of time. This could also improve understanding of the molecular nature of hypoxic/ischemic injuries in mammalian tissues and suggest potential ways to avoid these.
Keywords: Heat shock response; NF-kappaB; Unfolded protein response; FoxO; p53; HDAC;

Oct-3/4 modulates the drug-resistant phenotype of glioblastoma cells through expression of ATP binding cassette transporter G2 by Yuki Hosokawa; Hisaaki Takahashi; Akihiro Inoue; Yuya Kawabe; Yu Funahashi; Kenji Kameda; Kana Sugimoto; Hajime Yano; Hironobu Harada; Shohei Kohno; Shiro Ohue; Takanori Ohnishi; Junya Tanaka (1197-1205).
Drug resistance is a major obstacle for the efficacy of chemotherapeutic treatment of tumors. Oct-3/4, a self-renewal regulator in stem cells, is expressed in various kinds of solid tumors including glioblastoma. Although Oct-3/4 expression has been implicated in the malignancy and prognosis of glioblastomas, little is known of its involvement in drug resistances of glioblastoma.The involvement of Oct-3/4 in drug resistance of glioblastoma cells was assessed by lactate dehydrogenase assay, efflux assay of an anticancer drug, poly ADP-ribose polymerase cleavage, and in vivo xenograft experiments. Involvement of a drug efflux pump ATP binding cassette transporter G2 in Oct-3/4-induced drug resistance was evaluated by quantitative PCR analysis and knockdown by shRNA.Oct-3/4 decreased the susceptibility to chemotherapeutic drugs by enhancing excretion of drugs through a drug efflux pump gene, ATP binding cassette transporter G2. Moreover, the expression of Oct-3/4 was well correlated to ATP binding cassette transporter G2 expression in clinical GB tissues.Oct-3/4 elevated the ATP binding cassette transporter G2 expression, leading to acquisition of a drug-resistant phenotype by glioblastoma cells.If the drug-resistance of glioblastoma cells could be suppressed, it should be a highly ameliorative treatment for glioblastoma patients. Therefore, signaling pathways from Oct-3/4 to ATP binding cassette transporter G2 should be intensively elucidated to develop new therapeutic interventions for better efficacy of anti-cancer drugs.
Keywords: Glioblastoma; Oct-3/4; Drug resistance; ABC transporter;

Functional GIP receptors play a major role in islet compensatory response to high fat feeding in mice by R. Charlotte Moffett; Srividya Vasu; Peter R. Flatt (1206-1214).
Consumption of high fat diet and insulin resistance induce significant changes in pancreatic islet morphology and function essential for maintenance of normal glucose homeostasis. We have used incretin receptor null mice to evaluate the role of gastric inhibitory polypeptide (GIP) in this adaptive response.C57BL/6 and GIPRKO mice were fed high fat diet for 45 weeks from weaning. Changes of pancreatic islet morphology were assessed by immunohistochemistry. Body fat, glucose, insulin, glucagon, glucagon-like peptide 1 (GLP-1) and GIP were assessed by routine assays.Compared with normal diet controls, high fat fed C57BL/6 mice exhibited increased body fat, hyperinsulinaemia and insulin resistance, associated with decreased pancreatic glucagon, unchanged pancreatic GLP-1 and marked increases of insulin, islet number, islet size and both beta- and alpha-cell areas. Beta cell proliferation and apoptosis were increased under high fat feeding, but the overall effect favoured enhanced beta cell mass. A broadly similar pattern of change was observed in high fat fed GIPRKO mice but islet compensation was severely impaired in every respect. The inability to enhance beta cell proliferation was associated with the depletion of pancreatic GLP-1 and lack of hyperinsulinaemic response, resulting in non-fasting hyperglycaemia. GIP and GLP-1 were expressed in islets of all groups of mice but high fat fed GIPRKO mice displayed decreased numbers of GLP-1 containing alpha cells plus non-functional enhancement of pancreatic GIP content.These data suggest that GIP released from islet alpha-cells and intestinal K-cells plays an important role in islet adaptations to high fat feeding.
Keywords: Gastric inhibitory polypeptide; Alpha-cell; K-cell; Immunohistochemistry;

Nav1.5 channels can reach the plasma membrane through distinct N-glycosylation states by Aurélie Mercier; Romain Clément; Thomas Harnois; Nicolas Bourmeyster; Patrick Bois; Aurélien Chatelier (1215-1223).
Like many voltage-gated sodium channels, the cardiac isoform Nav1.5 is well known as a glycoprotein which necessarily undergoes N-glycosylation processing during its transit to the plasma membrane. In some cardiac disorders, especially the Brugada syndrome (BrS), mutations in Nav1.5 encoding gene lead to intracellular retention and consequently trafficking defect of these proteins. We used two BrS mutants as tools to clarify both Nav1.5 glycosylation states and associated secretory behaviors.Patch-clamp recordings and surface biotinylation assays of HEK293T cells expressing wild-type (WT) and/or mutant Nav1.5 proteins were performed to assess the impact of mutant co-expression on the membrane activity and localization of WT channels. Enzymatic deglycosylation assays and brefeldin A (BFA) treatments were also employed to further characterize recombinant and native Nav1.5 maturation.The present data demonstrate that Nav1.5 channels mainly exist as two differentially glycosylated forms. We reveal that dominant negative effects induced by BrS mutants upon WT channel current result from the abnormal surface expression of the fully-glycosylated forms exclusively. Furthermore, we show that core-glycosylated channels can be found at the surface membrane of BFA-treated or untreated cells, but obviously without generating any sodium current.Our findings provide evidence that native and recombinant Nav1.5 subunits are expressed as two distinct matured forms. Fully-glycosylated state of Nav1.5 seems to determine its functionality whereas core-glycosylated forms might be transported to the plasma membrane through an unconventional Golgi-independent secretory route.This work highlights that N-linked glycosylation processing would be critical for Nav1.5 membrane trafficking and function.
Keywords: Voltage-gated sodium channel; Nav1.5; N-glycosylation; Secretory pathway; Negative dominance; Brugada syndrome;

Cigarette smoke exposure reveals a novel role for the MEK/ERK1/2 MAPK pathway in regulation of CFTR by Xiaohua Xu; Robert Balsiger; Jean Tyrrell; Prosper N. Boyaka; Robert Tarran; Estelle Cormet-Boyaka (1224-1232).
Cystic fibrosis transmembrane conductance regulator plays a key role in maintenance of lung fluid homeostasis. Cigarette smoke decreases CFTR expression in the lung but neither the mechanisms leading to CFTR loss, nor potential ways to prevent its loss have been identified to date.The molecular mechanisms leading to down-regulation of CFTR by cigarette smoke were determined using pharmacologic inhibitors and silencing ribonucleic acids (RNAs).Using human bronchial epithelial cells, here we show that cigarette smoke induces degradation of CFTR that is attenuated by lysosomal inhibitors, but not proteasome inhibitors. Cigarette smoke can activate multiple signaling pathways in airway epithelial cells, including the MEK/Erk1/2 MAPK (MEK: mitogen-activated protein kinase/ERK kinase Erk1/2: extracellular signal-regulated kinase 1/2 MAPK: Mitogen-activated protein kinase) pathway regulating cell survival. Interestingly, pharmacological inhibition of the MEK/Erk1/2 MAPK pathway prevented the loss of plasma membrane CFTR upon cigarette smoke exposure. Similarly, decreased expression of Erk1/2 using silencing RNAs prevented the suppression of CFTR protein by cigarette smoke. Conversely, specific inhibitors of the c-Jun N-terminal kinase (JNK) or p38 MAPK pathways had no effect on CFTR decrease after cigarette smoke exposure. In addition, inhibition of the MEK/Erk1/2 MAPK pathway prevented the reduction of the airway surface liquid observed upon cigarette smoke exposure of primary human airway epithelial cells. Finally, addition of the antioxidant N-acetylcysteine inhibited activation of Erk1/2 by cigarette smoke and precluded the cigarette smoke-induced decrease of CFTR.These results show that the MEK/Erk1/2 MAPK pathway regulates plasma membrane CFTR in human airway cells.The MEK/Erk1/2 MAPK pathway should be considered as a target for strategies to maintain/restore CFTR expression in the lung of smokers.
Keywords: CFTR; Cigarette smoke; Airway epithelial cell; MAPK pathway;

New enzymatic pathways for the reduction of reactive oxygen species in Entamoeba histolytica by Matías S. Cabeza; Sergio A. Guerrero; Alberto A. Iglesias; Diego G. Arias (1233-1244).
Entamoeba histolytica, an intestinal parasite that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. A flavodiiron protein and a rubrerythrin have been characterized in this human pathogen, although their physiological reductants have not been identified.The present work deals with biochemical studies performed to reach a better understanding of the kinetic and structural properties of rubredoxin reductase and two ferredoxins from E. histolytica.We complemented the characterization of two different metabolic pathways for O2 and H2O2 detoxification in E. histolytica. We characterized a novel amoebic protein with rubredoxin reductase activity that is able to catalyze the NAD(P)H-dependent reduction of heterologous rubredoxins, amoebic rubrerythrin and flavodiiron protein but not ferredoxins. In addition, the protein exhibited an NAD(P)H oxidase activity, which generates hydrogen peroxide from molecular oxygen. We describe how different ferredoxins were also efficient reducing substrates for both flavodiiron protein and rubrerythrin.The enzymatic systems herein characterized could contribute to the in vivo detoxification of O2 and H2O2, playing a key role for the parasite defense against reactive oxidant species.To the best of our knowledge this is the first characterization of a eukaryotic rubredoxin reductase, including a novel kinetic study on ferredoxin-dependent reduction of flavodiiron and rubrerythrin proteins.
Keywords: Entamoeba; Rubredoxin; Antioxidant; Flavodiiron-protein; Ferredoxin; Rubrerythrin;

Action mechanism of 6, 6′-dihydroxythiobinupharidine from Nuphar japonicum, which showed anti-MRSA and anti-VRE activities by Shinya Okamura; Eri Nishiyama; Tomohiro Yamazaki; Nao Otsuka; Shoko Taniguchi; Wakano Ogawa; Tsutomu Hatano; Tomofusa Tsuchiya; Teruo Kuroda (1245-1252).
Multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), cause serious infections at clinical sites, for which the development of new drugs is necessary. We screened candidates for new antibiotics and investigated its action mechanism.An antimicrobial compound was isolated from an extract of Nuphar japonicum. Its chemical structure was determined by NMR, MS, and optical rotation. We measured its minimum inhibitory concentration (MIC) using the microdilution method. The effects of the compound on DNA gyrase and DNA topoisomerase IV were investigated with DNA supercoiling, decatenation, and cleavage assay.We isolated and identified 6,6′-dihydroxythiobinupharidine as the antimicrobial compound. The MIC of this compound was 1–4 μg/mL against various MRSA and VRE strains. We also demonstrated that this compound inhibited DNA topoisomerase IV (IC50 was 10–15 μM), but not DNA gyrase in S. aureus, both of which are known to be the targets of quinolone antibiotics and necessary for DNA replication. However, this compound only exhibited slight cross-resistance to norfloxacin-resistant S. aureus, which indicated that DTBN might inhibit other targets besides topoisomerase IV. These results suggest that 6,6′-dihydroxythiobinupharidine may be a potent candidate or seed for novel antibacterial agents.DTBN from N. japonicum showed anti-MRSA and anti-VRE activities. DTBN might be involved in the inhibition of DNA topoisomerase IV.DTBN might be useful as a seed compound. The information on the inhibition mechanism of DTBN will be useful for the modification of DTBN towards developing novel anti-MRSA and anti-VRE drug.Display Omitted
Keywords: MRSA; VRE; Nuphar japonicum; DNA topoisomerase IV;

Rhodamine B-conjugated encrypted vipericidin nonapeptide is a potent toxin to zebrafish and associated with in vitro cytotoxicity by Liang Wang; Judy Y.W. Chan; Juciane V. Rêgo; Cheong-Meng Chong; Nana Ai; Cláudio B. Falcão; Gandhi Rádis-Baptista; Simon M.Y. Lee (1253-1260).
Animal venoms contain a diverse array of proteins and enzymes that are toxic toward various physiological systems. However, there are also some practical medicinal uses for these toxins including use as anti-bacterial and anti-tumor agents.In this study, we identified a nine-residue cryptic oligopeptide, KRFKKFFKK (EVP50) that is repeatedly encoded in tandem within vipericidin sequences.EVP50 displayed in vivo potent lethal toxicity to zebrafish larvae (LD50  = 6 μM) when the peptide's N-terminus was chemically conjugated to rhodamine B (RhoB). In vitro, RhoB-conjugated EVP50 (RhoB-EVP50) exhibited a concentration-dependent cytotoxic effect toward MCF-7 and MDA-MB-231 breast cancer cells. In MCF-7 cells, the RhoB-EVP50 nonapeptide accumulated inside the cells within minutes. In the cytoplasm, the RhoB-EVP50 induced extracellular calcium influx and intracellular calcium release. Membrane budding was also observed after incubation with micromolar concentrations of the fluorescent EVP50 conjugate.The conjugate's interference with calcium homeostasis, its intracellular accumulation and its induced membrane dysfunction (budding and vacuolization) seem to act in concert to disrupt the cell circuitry. Contrastively, unconjugated EVP50 peptide did not display neither toxic nor cytotoxic activities in our in vivo and in vitro models.The synergic mechanism of toxicity was restricted to the structurally modified encrypted vipericidin nonapeptide.Display Omitted
Keywords: Vipericidins; Encrypted peptides; Cell-penetrating peptide; Cytotoxin; Zebra-fish model; Rhodamine B-conjugated peptide;

Physical cues of cellular environment affect cell fate and differentiation. For example, an environment with high stiffness drives mesenchymal stem cells (MSCs) to undergo osteogenic differentiation, while low stiffness leads to lipogenic differentiation. Such effects could be independent of chemical/biochemical inducers.Stiffness and/or topography of cellular environment can control MSC differentiation and fate determination. In addition, physical factors such as tension, which resulted from profound cytoskeleton reorganization during MSC differentiation, affect the gene expression essential for the differentiation. Although physical cues control MSC lineage specification probably by reorganizing and tuning cytoskeleton, the full mechanism is largely unclear. It also remains elusive how physical signals are sensed by cells and transformed into biochemical and biological signals. More importantly, it becomes pivotal to define explicitly the physical cue(s) essential for cell differentiation and fate decision. With a focus on MSC, we present herein current understanding of the interplay between i) physical cue and factors and ii) MSC differentiation and fate determination.Biophysical cues can initiate or strengthen the biochemical signaling for MSC fate determination and differentiation. Physical properties of cellular environment direct the structural adaptation and functional coupling of the cells to their environment.These observations not only open a simple avenue to engineer cell fate in vitro, but also start to reveal the physical elements that regulate and determine cell fate.
Keywords: Mesenchymal stem cell; Topography; Stiffness; Cytoskeleton; Physical cue;

The Ferritin-Heavy-Polypeptide-Like-17 (FTHL17) gene encodes a ferritin with low stability and no ferroxidase activity and with a partial nuclear localization by Paola Ruzzenenti; Michela Asperti; Stefania Mitola; Elisabetta Crescini; Federica Maccarinelli; Magdalena Gryzik; Maria Regoni; Dario Finazzi; Paolo Arosio; Maura Poli (1267-1273).
Three functional ferritin genes have been identified so far in mammals, and they encode the cytosolic Heavy (FTH) and Light chain (FTL) and the mitochondrial ferritin. The expression of a transcript by a fourth ferritin-like gene (Ferritin-Heavy-Polypeptide-Like-17, FTHL17) on the X chromosome was reported in mouse spermatogonia and in early embryonic cells.The intronless human FTHL17 gene encodes a protein with 64% identity to human FTH with substitution of key residues of the ferroxidase center. The gene was cloned into vectors for expression in Escherichia coli and mammalian cells, linked to a flag-tag.The recombinant FTHL17 from E. coli purified as an assembled 24-mer ferritin devoid of ferroxidase activity and with a reduced physical stability. When transiently expressed in mammalian cells the flag-FTHL17 assembled in ferritin shells that showed reduced stability to denaturants compared with flag H and L ferritins. Immunocytochemistry with anti-flag antibody decorated the nuclei of flag-FTHL17 transfected COS cells, but not those of the cells transfected with flag-FTH or flag-FTL.We concluded that FTHL17 encodes a ferritin-like protein without ferroxidase activity. Its restricted embryonic expression and partial nuclear localization suggest that this novel ferritin type may have functions other than iron storage.The work confirms the presence of a fourth functional human ferritin gene with properties distinct from the canonical cytosolic ones.
Keywords: Ferritin; Ferroxidase center; Protein stability; Embryonic cell;

Thioredoxin 1 and glutaredoxin 2 contribute to maintain the phenotype and integrity of neurons following perinatal asphyxia by Juan Ignacio Romero; Eva-Maria Hanschmann; Manuela Gellert; Susanne Eitner; Mariana Inés Holubiec; Eduardo Blanco-Calvo; Christopher Horst Lillig; Francisco Capani (1274-1285).
Thioredoxin (Trx) family proteins are crucial mediators of cell functions via regulation of the thiol redox state of various key proteins and the levels of the intracellular second messenger hydrogen peroxide. Their expression, localization and functions are altered in various pathologies. Here, we have analyzed the impact of Trx family proteins in neuronal development and recovery, following hypoxia/ischemia and reperfusion.We have analyzed the regulation and potential functions of Trx family proteins during hypoxia/ischemia and reoxygenation of the developing brain in both an animal and a cellular model of perinatal asphyxia. We have analyzed the distribution of 14 Trx family and related proteins in the cerebellum, striatum, and hippocampus, three areas of the rat brain that are especially susceptible to hypoxia. Using SH-SY5Y cells subjected to hypoxia and reoxygenation, we have analyzed the functions of some redoxins suggested by the animal experiment.We have described/discovered a complex, cell-type and tissue-specific expression pattern following the hypoxia/ischemia and reoxygenation. Particularly, Grx2 and Trx1 showed distinct changes during tissue recovery following hypoxia/ischemia and reoxygenation. Silencing of these proteins in SH-SY5Y cells subjected to hypoxia-reoxygenation confirmed that these proteins are required to maintain the normal neuronal phenotype.These findings demonstrate the significance of redox signaling in cellular pathways. Grx2 and Trx1 contribute significantly to neuronal integrity and could be clinically relevant in neuronal damage following perinatal asphyxia and other neuronal disorders.
Keywords: Common carotid artery occlusion; Thioredoxin family of proteins; Hypoxia; Reoxygenation; Perinatal asphyxia;

Band keratopathy, an ocular disease that is characterized by hypercalcemia and opaque bands across the cornea, has been associated with kidney disease. Type-II renal tubular acidosis (RTA), a condition in which the kidneys fail to recover bicarbonate (HCO3 ) in the proximal tubule of the nephron, results in HCO3 wastage in the urine and low blood pH. The development of these diseases is associated with autosomal-recessive mutations in the Na+-coupled HCO3 cotransporter NBCe1-A located at the basolateral membranes of either cell type.We provide insight into the devastating R298S mutation found in type-II RTA-afflicted individuals using confocal-microscopy imaging of fluorescently-tagged NBCe1-A and NBCe1-A-R298S molecules expressed in human corneal endothelial and proximal tubule cells and from in-depth biophysical studies of their cytoplasmic N-terminal domains (Nt and Nt-R298S), including Nt crystal structure, melting-temperature, and homodimer dissociation constant (KD) analyses.We illuminate and rescue trafficking defects of the R298S mutation of NBCe1-A. The KD for Nt monomer–dimer equilibrium is established. The KD for Nt-R298S is significantly higher, but immeasurable due to environmental factors (pH, temperature, concentration) that result in dimer instability leading to precipitation. The crystal structure of Nt-dimer shows that R298 is part of a putative substrate conduit and resides near the dimer interface held together by hydrogen-bond networks.The R298S is a temperature-sensitive mutation in Nt that results in instability of the colloidal system leading to abnormal aggregation.Our findings provide new perspectives to the aberrant mechanism of certain ocular pathologies and type-II RTA associated with the R298S mutation.
Keywords: Protein aggregation; Composition-gradient light scattering; X-ray crystallography; Confocal microscopy;

The role of His-83 of yeast apurinic/apyrimidinic endonuclease Apn1 in catalytic incision of abasic sites in DNA by Elena S. Dyakonova; Vladimir V. Koval; Alexander A. Lomzov; Alexander A. Ishchenko; Olga S. Fedorova (1297-1309).
The apurinic/apyrimidinic (AP) endonuclease Apn1 from Saccharomyces cerevisiae is a key enzyme involved in the base excision repair (BER) at the cleavage stage of abasic sites (AP sites) in DNA. The crystal structure of Apn1 from S. cerevisiae is unresolved. Based on its high amino acid homology to Escherichia coli Endo IV, His-83 is believed to coordinate one of three Zn2 + ions in Apn1's active site similar to His-69 in Endo IV. Substituting His-83 with Ala is proposed to decrease the AP endonuclease activity of Apn1 owing to weak coordination of Zn2 + ions involved in enzymatic catalysis.The kinetics of recognition, binding, and incision of DNA substrates with the H83A Apn1 mutant was investigated. The stopped-flow method detecting fluorescence intensity changes of 2-aminopurine (2-aPu) was used to monitor the conformational dynamics of DNA at pre-steady-state conditions.We found substituting His-83 with Ala influenced catalytic complex formation and further incision of the damaged DNA strand. The H83A Apn1 catalysis depends not only on the location of the mismatch relative to the abasic site in DNA, but also on the nature of damage.We consider His-83 properly coordinates the active site Zn2 + ion playing a crucial role in catalytic incision stage. Our data prove suppressed enzymatic activity of H83A Apn1 results from the reduced number of active site Zn2 + ions.Our study provides insights into mechanistic specialty of AP site repair by yeast AP endonuclease Apn1 of Endo IV family, which members are not found in mammals, but are present in many microorganisms. The results will provide useful guidelines for design of new anti-fungal and anti-malarial agents.
Keywords: AP endonuclease; Abasic site; DNA repair; Base excision repair; 2-Aminopurine; MD simulation;

Enhancement of dynamin polymerization and GTPase activity by Arc/Arg3.1 by Christopher E. Byers; Barbara Barylko; Justin A. Ross; Daniel R. Southworth; Nicholas G. James; Clinton A. Taylor; Lei Wang; Katie A. Collins; Armando Estrada; Maggie Waung; Tara C. Tassin; Kimberly M. Huber; David M. Jameson; Joseph P. Albanesi (1310-1318).
The Activity-regulated cytoskeleton-associated protein, Arc, is an immediate-early gene product implicated in various forms of synaptic plasticity. Arc promotes endocytosis of AMPA type glutamate receptors and regulates cytoskeletal assembly in neuronal dendrites. Its role in endocytosis may be mediated by its reported interaction with dynamin 2, a 100 kDa GTPase that polymerizes around the necks of budding vesicles and catalyzes membrane scission.Enzymatic and turbidity assays are used in this study to monitor effects of Arc on dynamin activity and polymerization. Arc oligomerization is measured using a combination of approaches, including size exclusion chromatography, sedimentation analysis, dynamic light scattering, fluorescence correlation spectroscopy, and electron microscopy.We present evidence that bacterially-expressed His6-Arc facilitates the polymerization of dynamin 2 and stimulates its GTPase activity under physiologic conditions (37 °C and 100 mM NaCl). At lower ionic strength Arc also stabilizes pre-formed dynamin 2 polymers against GTP-dependent disassembly, thereby prolonging assembly-dependent GTP hydrolysis catalyzed by dynamin 2. Arc also increases the GTPase activity of dynamin 3, an isoform of implicated in dendrite remodeling, but does not affect the activity of dynamin 1, a neuron-specific isoform involved in synaptic vesicle recycling. We further show in this study that Arc (either His6-tagged or untagged) has a tendency to form large soluble oligomers, which may function as a scaffold for dynamin assembly and activation.The ability of Arc to enhance dynamin polymerization and GTPase activation may provide a mechanism to explain Arc-mediated endocytosis of AMPA receptors and the accompanying effects on synaptic plasticity.
Keywords: Arc/Arg3.1; Dynamin; GTPase; Self-assembly;

N-acetylglucosamine modification in the lumen of the endoplasmic reticulum by Mitsutaka Ogawa; Shogo Sawaguchi; Koichi Furukawa; Tetsuya Okajima (1319-1324).
O-linked β-N-acetylglucosamine (O-GlcNAc) modification of epidermal growth factor (EGF) domains catalyzed by EGF domain O-GlcNAc transferase (EOGT) is the first example of GlcNAc modification in the lumen of the endoplasmic reticulum (ER).This review summarizes current knowledge on the EOGT-catalyzed O-GlcNAc modification of EGF domains obtained through biochemical characterization, genetic analysis in Drosophila, and identification of human EOGT mutation. Additionally, this review discusses GTDC2—another ER protein homologous to EOGT that catalyzes the GlcNAc modification of O-mannosylated α-dystroglycan—and other components of the biosynthetic pathway involved in GlcNAc modification in the ER lumen.GlcNAc modification in the ER lumen has been identified as a novel type of protein modification that regulates specific protein function. Moreover, abnormal GlcNAc modification in the ER lumen is responsible for Adams–Oliver syndrome and Walker–Warburg syndrome.Elucidation of the biological function of GlcNAc modification in the ER lumen will provide new insights into the unique roles of O-glycans, whose importance has been demonstrated in multifunctional glycoproteins such as Notch receptors and α-dystroglyan.
Keywords: Notch; EOGT; O-GlcNAc; UDP-GlcNAc transporter; α-Dystroglycan; GTDC2;

Combining polyethylenimine and Fe(III) for mediating pDNA transfection by Andreia F. Jorge; Ruth Röder; Petra Kos; Rita S. Dias; Ernst Wagner; Alberto A.C.C. Pais (1325-1335).
The potential use of Fe(III) ions in biomedical applications may predict the interest of its combination with pDNA–PEI polyplexes. The present work aims at assessing the impact of this metal on pDNA complex properties.Variations in the formation of complexes were imposed by using two types of biological buffers at different salt conditions. The incorporation of pDNA in complexes was characterised by gel electrophoresis and dynamic light scattering. Transfection efficiency and cytotoxicity were evaluated in HeLa and HUH-7 cell lines, supported by flow cytometry assays.Fe(III) enhances pDNA incorporation in the complex, irrespective of the buffer used. Transfection studies reveal that the addition of Fe(III) to complexes at low ionic strength reduces gene transfection, while those prepared under high salt content do not affect or, in a specific case, increase gene transfection up to 5 times. This increase may be a consequence of a favoured interaction of polyplexes with cell membrane and uptake. At low salt conditions, results attained with chloroquine indicate that the metal may inhibit polyplex endosomal escape. A reduction on the amount of PEI (N/P 5) formed at intermediary ionic strength, complemented by Fe(III), reduces the size of complexes while maintaining a transfection efficiency similar to that obtained to N/P 6.Fe(III) emerges as a good supporting condensing agent to modulate pDNA–PEI properties, including condensation, size and cytotoxicity, without a large penalty on gene transfection.This study highlights important aspects that govern pDNA transfection and elucidates the benefits of incorporating the versatile Fe(III) in a gene delivery system.A complete study of the impact of a second condensing agent, Fe(III) ions, on pDNA–PEI complexes properties, cytotoxicity and transfection efficiency.Display Omitted
Keywords: pDNA; Polyplexes; Transfection; Polyethylenimine; Fe(III);

C1-inhibitor polymers activate the FXII-dependent kallikrein–kinin system: Implication for a role in hereditary angioedema by Daniel Elenius Madsen; Johannes Jakobsen Sidelmann; Daniel Biltoft; Jørgen Gram; Soren Hansen (1336-1342).
The FXII-dependent kallikrein–kinin system (KKS) is tightly regulated by the serine protease inhibitor (serpin) C1-inhibitor (C1-inh). When regulation of the FXII-dependent KKS fails, which is the case in hereditary angioedema (HAE), patients consequently experience invalidating edema attacks. HAE is caused by mutations in the C1-inh encoding gene, and we recently demonstrated that some mutations give rise to the presence of polymerized C1-inh in the plasma of HAE patients.C1-inh polymers corresponding to the size of polymers observed in vivo were produced using heat denaturation and gel filtration. The ability of these polymers to facilitate FXII activation was assessed in vitro in an FXII activation bandshift assay. After spiking of plasma with C1-inh polymers, kallikrein generation was analyzed in a global kallikrein generation method. Prekallikrein consumption in the entire Danish HAE cohort was analyzed using an ELISA method.C1-inh polymers mediated FXII activation, and a dose dependent kallikrein generation in plasma spiked with C1-inh polymers. An increased (pre)kallikrein consumption was observed in plasma samples from HAE patients presenting with C1-inh polymers in vivo.Polymerization of the C1-inh transforms the major inhibitor of the FXII-dependent KKS, into a potent activator of the very same system.The C1-inh polymers might play a role in the pathophysiology of HAE, but several diseases are characterized by the presence of serpin polymers. The role of serpin polymers has so far remained elusive, but our results indicate that such polymers can play a role as inflammatory mediators through the FXII-dependent KKS.
Keywords: Hereditary angioedema; Kallikrein-kinin system; Complement C1 esterase inhibitor; Serpinopathy; Coagulation factor XII; Polymerization;